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Par
milie Degagn
Dpartement d anatomie et biologie cellulaire
Sherbrooke, Qubec
Mai, 2012
1+1
Bibliothque et
Archives Canada
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978-0-494-93248-3
NOTICE:
AVIS:
Canada
II
RSUM
Rgulation de lexpression du rcepteur P 2 Y 2 dans les maladies inflam m atoires
intestinales par les facteurs de transcription NF-kB et C/EBPp et son implication dans
le processus de rparation de lpithlium intestinal.
Par
milie Degagn
Dpartement d anatomie et biologie cellulaire
Thse prsente la Facult de mdecine et des sciences de la sant en vue de lobtention
du diplme de philosophiae doctor (Ph.D.) en biologie cellulaire, Facult de mdecine et
des sciences de la sant, Universit de Sherbrooke, Sherbrooke, Qubec, Canada, J 1H 5N4.
Les maladies inflammatoires intestinales (M il) sont caractrises par des priodes
d inflammation chronique entrecoupes de priodes de rmission. Elles mobilisent laction
de plusieurs molcules pour favoriser le retour lhomostasie. Il a t dmontr que
lexpression du rcepteur P2 Y 2 (P 2 Y 2) est augmente chez les patients atteints de M il et
dans un modle murin de colite chimique. Cette thse s intresse aux mcanismes de
rgulation transcriptionnelle du gne de P 2 Y 2 et son rle dans les MIL Premirement, la
disponibilit de la chromatine au promoteur de P 2 Y 2 a t caractrise par le statut
d actylation des histones H3 et H4 par immunoprcipitation de la chromatine (ChIP). Le
site d initiation de la transcription du gne de P 2 Y 2 a t identifi par 5 RLM -RACE. NFk B et C/EBPp sont des candidats idals pour rguler lexpression de P 2 Y 2 puisquils sont
impliqus dans la rgulation de gnes lis linflammation. En utilisant le logiciel
TRANSFAC, les sites de liaison potentielle (SLP) de N F - k B et C/EBPp ont t identifis
sur le promoteur du rcepteur P 2 Y 2. Ensuite, des essais lucifrase ont dmontr que N F - kB
et C/EBPP transactivent le promoteur de P 2 Y 2 dans les cellules pithliales intestinales
(CEI) en condition inflammatoire ou non. Lorsque ces deux facteurs de transcription sont
co-exprims dans les CEI, on observe une synergie de la transactivation du prom oteur de
P 2 Y 2 . Par des essais lucifrase avec des constructions du promoteur mutes pour les SLP
des facteurs de transcription ltude ainsi que par des gels de retardement et des essais de
ChIP, nous avons montr que C/EBPp se lie en position -229 -220pb du prom oteur et que
N F - kB se lie en position -181 -172pb. Finalement, la stimulation du rcepteur P 2 Y 2 active
la voie de signalisation PI-3K/Akt qui inactive GSK-3P favorisant la stabilisation des
microtubules permettant la cellule de migrer pour rparer une monocouche de CEI
blesses. De plus, dans les CEI, lexpression de COX-2 et PGE 2 qui sont des molcules
importantes pour la rparation tissulaire, est augmente par la stimulation de P 2 Y 2. In vivo,
la stimulation du rcepteur favorise la rmission de souris atteintes de colite chimique.
Cette thse dmontre donc que lexpression du rcepteur P 2 Y 2 dans les M il est rgule par
N F- k B et C/EBPP et que ce rcepteur joue un rle important dans les M il en favorisant la
phase de rmission.
It doesn't matter how beautiful your theory is, it doesn't matter how sm art yo u are. I f it
doesn't agree with experiment, it's wrong.
Richard P. Feynman
Dans vingt ans vous serez plus dus par les choses que vous n avez pas fa ite s que par
celles que vous avez faites. Alors sortez des sentiers battus. Mettez les voiles. Explorez.
Rvez. Dcouvrez.
M ark Twain
RSUM..........................................................................................................................................II
TABLE DES MA TIRES..............................................................................................................V
LISTE DES TABLEAUX........................................................................................................... VII
LISTE DES FIGURES............................................................................................................ VIII
LISTE DES ABBRVIA TIONS, SIGLES E T SYMBOLES.................................................. X
INTRODUCTION............................................................................................................................1
1.
L i n t e s t i n ........................................................................................................................................................................................l
S tru c tu re et fon ctio n s de l p ith liu m in testin a l ................................................................................................... /
L IMMUNIT DE LA MUQUEUSE INTESTINALE....................................................................................................................4
2.1
L 'im m unit in n e ................................................................................................................................................................ 4
2.2
L 'im m unit a d a p ta tiv e ...................................................................................................................................................... 6
L e s m a l a d ie s i n f l a m m a t o ir e s i n t e s t i n a l e s ............................................................................................................ 9
3. /
La m aladie de C r o h n .........................................................................................................................................................9
3.2
La colite u lc re u se ............................................................................................................................................................11
3.3
La rparation de la m uqueu se in te stin a le .............................................................................................................. 12
3.3.1 Premire tape de la rparation de blessures : la restitution ....................................................................................12
3.3.2 La deuxime tape de la rparation de blessures : la prolifration ........................................................................ 14
3.3.3 La dernire tape de la rparation de blessures : la maturation .............................................................................. 15
3.3.4 Molcules favorisant la rparation de blessures .......................................................................................................... 16
L e s n u c l o t id e s e x t r a c e l l u l a ir e s ..............................................................................................................................17
4 .1
So u rce de nuclotides extra cellu la ires dans l'in te s tin ..................................................................................... 1 7
4.2
C oncentration de nuclotid es dans le m ilieu extra cellu la ire d e l'in te s tin ............................................... 19
4.3
M tabolism e des n u c l o tid e s ....................................................................................................................................... 19
4.4
O rigine de la signalisation p u rin e rg iq u e ............................................................................................................... 2 1
L e s r c e p t e u r s p u r i n e r g i q u e s P 2 .................................................................................................................................21
5.1
Les rcepteurs m tabotro p iq u es P 2 Y ...................................................................................................................... 22
5.2
Le rcep teu r P 2 2 ............................................................................................................................................................ 23
5.2.1
Localisation gnique et polym orphism es g n tiq u es.............................................................................................23
5.2.2
Structure de la protine..................................................................................................................................................24
5.2.3
Signalisation a sso cie.................................................................................................................................................... 26
5.2.4 D istribution et rle dans l'o rg an ism e..............................................................................................................................28
5.2.4
Rgulation transcriptionnelle....................................................................................................................................... 30
L e FACTEUR d e t r a n s c r ip t io n R f. l A / p 6 5 ..................................................................................................................... 31
6. /
Stru ctu re p r o t iq u e .......................................................................................................................................................... 31
6.2
R gulation de l activation d e N F - k B p a r la vo ie ca n o n iq u e ......................................................................... 32
6.3
R gulation de l'a ctiva tio n d e N F - k B p a r les voies a ltern a tives .................................................................. 33
1. I
2.
3.
4.
5.
6.
6.4
R gulation de l activit d e R elA /p 6 5 p a r des m o difications p o s t tra d u ctio n n elles et l'in te ra ctio n
avec des p ro t in e s r g u la tric es .................................................................................................................................................. 33
6.5
R les et fo n c tio n s de NF- k B dans l'in te s tin .......................................................................................................... 34
7.
CHAPITRE 1 .................................................................................................................................39
VI
CHAPITRE 2 ..................................................................................................................................73
P2Y2 RECEPTOR PROMOTES INTESTINAL EPITHELIAL CELL MIGRATION
THROUGH A GO PROTEIN AND INTEGRIN a v PATHWAY ALLOWING
MICROTUBULE STABILIZATION AND MUCOSAL RE-EPITHELIZATION IN
EXPERIMENTAL COLITIS.......................................................................................................73
A u t e u r s d e l a r t i c l e .......................................................................................................................................................................73
St a t u t d e l a r t i c l e
................................................................................................................................................................ 73
A v a n t - p r o p o s ....................................................................................................................................................................................... 73
18.
RSUM DE LARTICLE...........................................................................................................................................................74
19.
A b s t r a c t ................................................................................................................................................................................... 76
20.
In t r o d u c t i o n ......................................................................................................................................................................... 77
21.
M a t e r ia l s a n d m e t h o d s ............................................................................................................................
80
22.
R e s u l t s ......................................................................................................................................................................................84
23.
D i s c u s s i o n ............................................................................................................................................................................... 97
24.
S u p p l e m e n t a l d a t a .......................................................................................................................................................... 101
25.
A c k n o w l e d g m e n t s ........................................................................................................................................................... 102
26.
R e f e r e n c e s ............................................................................................................................................................................ 103
DISCUSSION..............................................................................................................................131
CONCLUSION............................................................................................................................142
VII
REMERCIEMENTS....................................................................................................................145
LISTES DES PUBLICA TIONS................................................................................................ 146
ANNEXES.....................................................................................................................................163
A n n e x e 1 : A r t ic l es
Annexe
2: DAI
p u b l i s en t a n t q u e d e u x i m e a u t e u r
........................................................................................ 163
et t r a n sl o q u a t io n b a c t r ie n n e d a n s la r a i e
de
so u r is
P2Y,"
CO U TE CHIMIQUE............................................................................................................................................................................................... 165
A n n e x e 3 : T r a n s a c t iv a t io n du p r o m o te u r du r c e p t e u r P2Y 2 d a n s le s c e l l u l e s C a c o -2 p a r le s
FACTEURS DE TRANSCRIPTION C /E B P A ET C /E B P a ............................................................................................................. 167
T a b l e a u 1. C a r a c t r i s t i q u e s d e s r c e p t e u r s
T ableau
2.
P2Y.........................................
P2Y2 d a n s l 'o r g a n i s m e
D istr ibu t io n et r le s d u r c e pt e u r
F i g u r e 3 . R g u l a t i o n d e l a m i g r a t i o n c e l l u l a i r e p a r l e c y t o s q u e l e t t e d a c t i n e e t
LES M IC R O T U B U L E S ................................................................................................................................................................. 1 5
F i g u r e 4 . R e p r s e n t a t i o n s c h m a t i q u e d e s e c t o - n u c l o t i d a s e s ................................................. 2 0
F ig u r e 5. R e p r s e n t a t io n de la s t r u c t u r e e n d e u x d im e n s io n s d u r c e p t e u r P 2 Y 2
h u m a in
............................................................................................................................................................................................ 2 6
F ig u r e 6. S c h m a r e p r s e n t a n t l e s v o ie s d e s ig n a l is a t io n a c t iv e s p a r le
RCEPTEUR P 2 Y 2 ...................................................................................................................................................................... 2 7
F ig u r e 7. V o ie s d a c t i v a t i o n d u f a c t e u r d e t r a n s c r i p t i o n
N F-kB ............................................. 3 2
T: T helper cell
TLR: Toll-like receptor
TNF-a: Tumor necrosis factor alpha
TRAF: Tumor necrosis factor-associated factor
Treg: Regulatory T cell
Trem2: Triggering receptor expressed on myeloid cells 2
UDP: Uridine diphosphate
UMP: Uridine monophosphate
UTP: Uridine triphosphate
WASP: W iskott-Aldrich syndrome protein
INTRODUCTION
1. L intestin
Selon Taxe rostro-caudal, lintestin se subdivise en deux entits intimement lies, soit
lintestin grle et le clon (Tortora et Grabowski, 2001). L intestin grle est divis en trois
sections distinctes et complmentaires soit le duodnum, le jjunum et lilon. Le clon est
subdivis en quatre parties soit le clon ascendant, transverse, descendant et sigmode.
souches qui donneront naissance aux cellules diffrencies. La localisation exacte de ces
cellules dans la crypte est encore source de discussion. Des tudes antrieures ont dmontr
que les cellules souches taient localises la base de la crypte (Bjerknes et Cheng, 1981;
Potten et al., 1997). Le rcepteur Lgr5 qui est un rcepteur orphelin coupl aux protines G
est un marqueur spcifique des cellules souches adultes qui a rcemment permis de
confirmer la prsence de cellules souches la base de la crypte (Li et Clevers, 2010; May et
al., 2009; Sato et al., 2009; Scoville et al., 2008). Toutefois, en utilisant des marqueurs de
prolifration comme la thymidine tricie ou le BrdU qui sincorporent lADN des cellules
en division, d autres quipes ont plutt localis les cellules souches la position +4 partir
de la base de la crypte (Batlle, 2008; Potten et al., 2002). Selon certaines tudes, les cellules
en position +4 seraient des cellules souches quiescentes responsables de la rparation
suivant une blessure alors que les cellules souches Lgr5+ du fond de la crypte serait
responsables du renouvellement physiologique des cellules de laxe crypte-villosit (Li et
Clevers, 2010; May et al., 2009; Scoville et al., 2008).
Dans la niche des cellules souches, on retrouve des cellules msenchym ateuses qui
scrtent des facteurs essentiels pour le maintien de la fonction des cellules souches. Elles
scrteront ainsi les ligands de la voie Wnt qui favorise la survie et la prolifration des
cellules souches ainsi que des antagonistes des BM Ps qui permettent aux cellules souches
de rester indiffrencies. Ainsi dans lintestin, on retrouvera un gradient d expression de
Wnt dcroissant vers la villosit et un gradient d expression des BMPs dcroissant vers la
crypte (G regorieff et al., 2005). Lorsque les cellules souches se divisent, elles donneront
naissance aux cellules prognitrices qui en migrant le long de la crypte seront exposes
des concentrations croissantes de BMPs favorisant leurs diffrenciations (Haramis et al.,
2004). Bien que les BMPs soit essentiels pour la diffrentiation des cellules prognitrices,
c est la voie de signalisation Notch qui permettra d effectuer la diffrentiation terminale.
Les cellules prognitrices dans lesquelles la voie Notch sera active perm ettront la
transcription du facteur de transcription Hesl qui est un rpresseur de la transcription du
facteur de transcription M athl (Tamura et al., 1995; Yang et al., 2001). Ces cellules
prognitrices dpourvues de lexpression de M athl deviendront des cellules absorbantes
(Yang et al., 2001). Dans les cellules prognitrices dans lesquelles la voie de signalisation
Notch nest pas active, il y aura expression du facteur de transcription M athl perm ettant la
diffrenciation terminale des cellules en cellules de la ligne scrtrice (M ilano et al.,
2004). Les cellules seront donc pleinement diffrencies latteinte de la jonction cryptevillosit et elles auront une survie denviron trois jours puisque le renouvellement constant
de lpithlium intestinal les pousse migrer vers la pointe de la villosit o elles seront
exfolies par anokose (Hall et al., 1994).
Les cellules diffrencies composant lintestin grle sont les cellules absorbantes nommes
entrocytes et les cellules de la ligne scrtrice soit les cellules caliciformes, les cellules de
Paneth et les cellules entroendocrines.
labsorption et du transport des nutriments contenus dans le bol alimentaire. Les cellules
caliciformes reprsentent 4% 16% des cellules de la muqueuse (Karam, 1999).
Elles
scrtent des mucines et des facteurs en trfles qui ont pour rle de protger lpithlium
contre les assauts des bactries et des molcules dltres pour le maintien de lhomostasie
intestinale (Tortora et Grabowski, 2001).
environ 1% des cellules de la muqueuse (Schonhoff et al., 2004). On compte une quinzaine
de sous-types de cellules entroendocrines qui sont responsables de la scrtion de
diffrentes hormones (Schonhoff et al., 2004). Les cellules de Paneth constituent le dernier
type cellulaire diffrenci qui compose laxe crypte-villosit.
types cellulaires diffrencis prsents, les cellules de Paneth entreprennent une migration
2.
lectrolytes en plus de maintenir une dfense efficace contre les toxines de la lumire
intestinale, des antignes et la microflore. L pithlium maintient sa fonction de barrire
par la formation de diffrents types de jonctions cellulaires qui permettent de lier entre elles
les cellules adjacentes et de sceller lespace intercellulaire (Groschwitz et Hogan, 2009).
Les diffrents types de cellules diffrencies qui composent lpithlium intestinal ont des
rles complmentaires et distincts jo u er dans limmunit inne. Les cellules caliciformes
scrtent diffrentes mucines permettant la formation d une couche de m ucus qui protge la
barrire pithliale en limitant le contact des bactries pathognes et celles de la microflore
sans empcher la diffusion des nutriments, de leau et des lectrolytes (Garrett et al., 2010).
Les cellules de Paneth produisent et stockent dans des granules de scrtions de multiples
molcules antimicrobiennes comme les dfensines, les lysozymes et les cathlicidines
(Garrett et al., 2010; Ito et al., 2012). Les cellules entroendocrines sont positionnes
stratgiquement le long de lpithlium intestinal. Ces cellules expriment, par ailleurs, des
rcepteurs de type Toll et suivant des stimuli microbiens ou alimentaires (par exemple la
prsence de glucose) scrteront diffrentes chimiokines, hormones et dfensines (Garrett
et al., 2010; Selleri et al., 2008). Finalement les entrocytes et les clonocytes expriment
diffrents rcepteurs PAMP (pathogen-associated molecular patterns) comme certains
rcepteurs de type Toll et des rcepteurs de reconnaissance de pathogne intracellulaire
(NOD) qui suivant leurs activations produisent des dfensines, des cytokines et des
chimiokines (Garrett et al., 2010; M uller et al., 2005).
Finalement les cellules du systme
immunitaire rsidentes de
lintestin
soit les
Les macrophages
rsidents sont anergiques et ils se caractrisent par une forte activit phagocytaire, mais par
une faible capacit produire et scrter des cytokines (Smythies et al., 2005). De plus, il
n est pas encore tabli si les macrophages rsidents peuvent faire la prsentation
dantignes et ainsi participer limmunit adaptative (Smith et al., 2011).
On compte deux sous-types dIEL, soit les a(3 IEL et les y IEL (Sheridan et Lefrancois,
2010). Les IEL participent limmunit inne en produisant des facteurs antimicrobiens tel
que Regllly permettant de contrler linvasion de la microflore intestinale (Ismail et al.,
2011). De plus, lors de la prsence d une brche dans lpithlium intestinal du clon, les
Les
Lactivation de ces
diffrentiation en lymphocytes T h 17 qui produiront des cytokines telles lIL 17, lIL-21,
lIL-22 et lIL-2 et permettra de limiter les infections bactriennes et stim ulera le
recrutement des neutrophiles aux sites d inflamm ation (Abraham et Cho, 2009; W eaver et
al., 2007). Finalement, la prsence de TGF-P perm ettra la diffrentiation en lymphocytes
Treg qui scrtent des molcules anti-inflamm atoires telles lIL-10 et le TGF-P et permettra
de diminuer la rponse inflammatoire (Abraham et Cho, 2009; Sakaguchi et Sakaguchi,
2005).
La rgulation fine de la raction immunitaire de lintestin, par exemple contre les bactries
de la microflore, est essentielle pour le m aintien de lhomostasie de cet organe. Un
dsquilibre de la rponse immunitaire peut entraner le dveloppement de maladies
inflammatoires intestinales comme illustr la Figure 2.
prvalence de la maladie de Crohn slevait 223,7 cas par 100 000 habitants alors que
lincidence tait estime 13,4 nouveaux cas par 100 000 habitants faisant du Canada le
pays avec la plus haute prvalence et incidence jam ais rapport (Bernstein et al., 2006). Au
Canada, durant ces mmes annes, la prvalence de la colite ulcreuse tait de 193,7 cas par
100 000 habitants alors que lincidence tait estime 11,8 nouveaux cas par 100 000
habitants (Bernstein et al., 2006).
10
par des douleurs abdominales, des diarrhes chroniques et par une perte de poids
importante (Yamada, 2009).
Les patients atteints de cette maladie prsentent des prdispositions gntiques complexes
pouvant conduire un drglement du systme immunitaire face la m icroflore intestinale
(M acdonald et M onteleone, 2005). Plusieurs patients vont prsenter des polymorphismes
dans un ou plusieurs gnes de susceptibilit pouvant tre regroups en diffrentes
catgories (Tsianos et al., 2011). La premire catgorie reprsente les gnes associs aux
rcepteurs de reconnaissance de patrons molculaires tels les gnes NOD2 (CARD15) et
TLR. Par exemple, dans les cellules de Paneth des mutations dans NOD2 ont t associes
avec une diminution de lexpression de peptides antimicrobiens comme la-dfensines
(W ehkamp et al., 2004). La deuxime catgorie comprend les gnes situs sur le locus
nomm IBD5 et qui seraient impliqus dans lhomostasie de la barrire pithliale. Par
exemple, une mutation dans le gne PDLIM4 pourrait promouvoir la contraction des
filaments d actine dans les cellules pithliales et du coup augmenter la permabilit
paracellulaire (Reinhard et Rioux, 2006).
La troisime catgorie regroupe les gnes lis la rponse immunitaire adaptive et
lapoptose soit CMH et HLA. Des mutations dans le gne de HLADRB1 sont associes la
maladie de Crohn et la colite ulcreuse et pourraient occasionner une rponse immunitaire
inapproprie (Annese et al., 2005). La quatrime catgorie rassemble les gnes impliqus
dans la diffrentiation lymphocytaire tels IL23R et STAT3. Il a t dmontr q u un
polymorphisme dans le gne d IL23R favorise une augmentation de la concentration de la
cytokine pro-inflammatoire IL-22 dans le srum de patients atteints de la maladie de Crohn
(Seiderer et al., 2008).
La dernire catgorie comprend le gne reli lautophagie ATG16L1. L autophagie
permet la dgradation de composants du cytoplasm e en priode o il y a un manque de
nutriments. Durant les infections, lautophagie est employe pour dgrader les pathognes
qui ont pntr lintrieur de la cellule (N aser et al., 2012). Rioux et collaborateurs ont
dmontr le rle essentiel d ATG16Ll dans lautophagie de bactries en utilisant des
cellules HEK-293T dont le gne d A TG 16Ll a t invalid. Ils ont dm ontr que dans ces
11
cellules, il n y avait plus d autophagie dirige envers les bactries pathognes (Rioux et al.,
2007).
De plus, des
12
de PPAR-y par les clonocytes, des anomalies dans la production et la scrtion de mucus
et dans la prsence et lactivation de lymphocytes T auxilliaires de type rgulateur (Treg)
(Danese et Fiocchi, 2011).
incontrle
telle
quobserve
chez
maladies
d une
transition
pithliale-msenchyme
cause
entre
autres
par
une
13
Haller et Imhof, 2003). Ces complexes focaux seront lis au cytosquelette d actine par les
fibres de stress qui sont cres suivant lactivation de RhoA.
focaux qui se sont densifis se localiseront larrire de la cellule o ils porteront le nom
de contacts focaux (W ehrle-Haller et Imhof, 2003). Les microtubules sattacheront ses
contacts focaux et exerceront une force de traction vers lintrieur de la cellule permettant
ainsi le dtachement des contacts focaux de la matrice extracellulaire et lavancement de la
cellule (W ehrle-Haller et Imhof, 2003).
Le rle du cytosquelette d actine dans la migration cellulaire est amplement tudi.
Rcemment, des tudes ont dmontr que les microtubules jouent aussi un rle essentiel
dans la promotion de la migration cellulaire (W atanabe et al., 2005; W ehrle-Haller et
Imhof, 2003).
La dtyrosination est
catalyse par la carboxypeptidase qui est une dtyrosinase cytosolique, (Ham mond et al.,
2008) alors que lactylation est catalyse par le complexe d histone actyltransfrase
Elongator (Creppe et al., 2009).
La premire tude dcrire quil y avait une polarisation et une stabilisation des
microtubules vers le front de migration a t ralise par Gundersen et collaborateurs. Ils
ont dmontr que dans les vingt minutes suivant une blessure d une monocouche de
fibroblastes murins, que les microtubules sorientaient vers le front de migration et q u il y
avait une augmentation de la dtyrosination de ces microtubules. (Gundersen et Bulinski,
1988). Des tudes subsquentes ont dmontr que cette stabilisation des microtubules de la
cellule en migration tait rgul par la protine Rho et son effecteur m Dia (Cook et al.,
1998; Palazzo et al., 2001).
Le rle de lactylation de la-tubuline dans la stabilisation et la promotion de la migration
cellulaire est relativement rcent.
14
Les
cellules souches de lintestin vont gnrer un grand nombre de cellules prognitrices qui
permettra de remplacer le pool de cellules endommages suivant la blessure (Dignass et
Podolsky, 1993).
En effet, les
16
seront responsables de la rtraction des contacts focaux larrire de la cellule perm ettant
celle-ci d avancer alors quau devant de la cellule les microtubules seront responsables de
la livraison de cargo vers les complexes focaux et les lamellipodes. Tir, adapt et modifi
de (W ehrle-Haller et Imhof, 2003).
signaux
extracellulaires.
Plusieurs
facteurs
de
croissance,
cytokines
et
chimiokines tels le TGF-a, le TGF-p, lEGF, lHGF, le KGF, lIGF-I, lIGF-II, lIL -lp,
lIL-2 et les peptides en trfle ont t impliqus dans les tapes de restitution et de
prolifration (Ciacci et al., 1993; Dignass et al., 1994a; Dignass et al., 1994b; Dignass et
Podolsky, 1993; 1996; Dignass et al., 1994c; Housley et al., 1994; Ohneda et al., 1997;
Park et al., 1992). Le TGF-P joue un rle central dans la restitution bien q u il inhibe la
prolifration des cellules pithliales intestinales (Dignass et Podolsky, 1993).
Plusieurs
17
d induire une colite chimique ont dmontr que la prostaglandine E 2 tait le m diateur de
leffet protecteur suivant lactivation du rcepteur de type Toll 4 (Brown et al., 2007;
Fukata et al., 2006). En effet, il a t dm ontr dans un modle de souris traites au DSS
que leffet protecteur du PGE 2 tait d lactivation de son rcepteur de type 4 (EP4)
(Kabashima et al., 2002). Ainsi, des souris dans lesquelles lexpression du rcepteur EP4 a
t invalide, ont montr une susceptibilit beaucoup plus svre la colite induit par le
DSS (Kabashima et al., 2002).
barrire pithliale, des dommages importants des cryptes et une agrgation de neutrophiles
et de lymphocytes dans le clon significativement plus importants que les souris de type
sauvage (Kabashima et a}., 2002).
Le processus de rparation pithliale est astucieusement contrl afin de prom ouvoir le
plus rapidement possible le retour lhomostasie.
de cellules pithliales
intestinales
de 42% et
57%
respectivement
comparativement au contrle (Dignass et al., 1998). Cette tude dmontrait que les
nuclotides extracellulaires jouent un rle important dans la rparation de blessure de
lpithlium intestinal. Cependant les mcanismes et les rcepteurs impliqus dans le rle
de ces nuclotides demeuraient ju sq u ce jour inconnu.
18
fces de ces souris tait de 102 pmole/g (Atarashi et al., 2008). Atarashi et collaborateurs
ont trait ces mmes souris avec deux antibiotiques soit la vancomycine et le mtronidazole
et la concentration d ATP dans les fces chutait sous la limite de dtection de la technique
utilise dmontrant que la majorit de lATP extracellulaire retrouve dans lintestin
provient des bactries. Une autre tude ralise par Iwase et collaborateur a m ontr que la
souche bactrienne Enterococcus gallinarium scrte la majorit de lATP chez ces souris
C57BL/6 SPF (Iwase et al., 2010). De plus, des tudes in vitro ralises sur ces bactries
isoles de fces humaines ont permis de constater que E. gallinarium scrte de lATP
(Iwase et al., 2010).
Les cellules de la muqueuse contribuent elles aussi la relche de nuclotides
extracellulaires. Les travaux de Patel et collaborateur, ont dmontr que la muqueuse
pithliale du clon et de lilon relchait de lATP dans le milieu extracellulaire en
condition physiologique (Patel et al., 2011). Cette relche semble tre associe lhmicanal pannexine-1 puisque lactivation de ce canal est associe la relche d ATP dans le
milieu extracellulaire de cellules pithliales pulmonaires suivant T activation de Rho
(Seminario-Vidal et al., 2011). De plus, en utilisant les cellules pithliales cilies bovines,
Li et collaborateurs ont montr que la relche d ATP dans le milieu extracellulaire par les
cellules pithliales implique une scrtion vsiculaire. L utilisation de bafilomycine, un
inhibiteur de la relche de vsicules a rduit de 25% la relche d ATP dans le milieu
extracellulaire suivant un stress hypotonique (Li et al., 2010).
En condition pathologique o on retrouve de linflammation et/ou une blessure, les
plaquettes sanguines ainsi actives sont une source de nuclotides extracellulaires
importante (M arcus et al., 2003). En effet, les granules des plaquettes sont riches en ATP
et en ADP. Leurs dgranulations perm ettent d lever la concentration de nuclotide
extracellulaire pour une varit de fonctions physiologiques et pathologiques.
Les
neutrophiles recruts au site dinflammation relchent aussi de lATP par la connexine 43,
un hmi-canal (Eltzschig et al., 2006).
19
Il est propos que lATP et lUTP soient relchs de faon sim ilaire puisque la
relche d UTP par les cellules de types non-scrtrices, comme les clonocytes, suit le
mme patron de relche que celui de lATP (Lazarowski et Boucher, 2001). De plus, en
condition physiologique, la muqueuse du clon est expose un stress mcanique lors du
passage des fces. Ce stress mcanique pourrait stim uler la relche d ATP et d UTP par un
facteur de 10 20 fois ce quon retrouve en condition basale (Lazarowski et Boucher,
2001; Lazarowski et al., 2003; Lazarowski et Harden, 1999; Lazarowski et al., 1997).
Comme mentionn prcdemment, les nuclotides extracellulaires peuvent aussi provenir
des bactries de la flore intestinale, des cellules apoptotiques et des cellules du systme
immunitaire. En effet, T activation des leucocytes et des plaquettes sanguines ainsi que les
microenvironnements acide et hypoxique retrouve lors dune inflammation vont favoriser
la relche de nuclotides extracellulaires (Di Virgilio et al., 2001; Gordon, 1986;
Luttikhuizen et al., 2004).
21
5.
Les rcepteurs P2 sont diviss en deux familles soit les rcepteurs ionotropiques P2X et les
rcepteurs mtabotropiques P2Y. La famille de rcepteurs P2X compte sept membres chez
lhumain soit P2X1 P2X7. Contrairement aux rcepteurs P2Y, les rcepteurs P2X ne
possdent quun seul agoniste naturel: lATP.
22
agonistes endognes ainsi que les protines G auxquels chacun des rcepteurs de la famille
P2Y se couplent.
23
Agonistes
Rcepteur
endognes
Mcanismes de
Rfrences
transduction
y ii
3BwMH H Wm
ATP, UTP
P2Y2
Famille Gq/G n
Famille Gi/G0
Famille G |2/G |3
j g g g lg
P2Y6
t a
hh
UDP>UTP
Famille Gq/Gn
BflS9jHHBRS9
|
P2Y,
ADP
1Bminmi
(Communi et al., 1996)
H
Famille Gj/G0
P2Y,
UDP-
Famille G,/G0
glucose
UDPgalactose
24
5.2.2
Structure de la protine
La protine encode par lARNm du rcepteur P 2 Y 2 est compose de 377 acides amins
(voir Figure 5). Le site de liaison des agonistes est situ entre les boucles extracellulaires
des domaines transmembranaires six et sept.
25
dirige que le remplacement de lhistidine en position 262 et des arginines en position 265
et 292 par des leucines qui sont des acides amins neutres rsultaient en une diminution
d environ 100 850 fois la capacit de lATP et de lUTP d induire une rponse calcique
normale (Erb et al., 1995).
Dans la premire boucle extracellulaire du rcepteur P 2 Y 2, on retrouve un m o tif arginineglycine-asparagine (RGD) qui permet la colocalisation du rcepteur P 2 Y 2 avec les
intgrines a v(V P 5 (Erb et al., 2001).
intgrines dyf^/Ps puisque le remplacement du m otif RGD par une squence RGE abolie cet
interaction (Erb et al., 2001).
Le domaine intracellulaire en C-terminal de la protine contient un site de phosphorylation
pour les kinases de rcepteurs coupls aux protines G (GPCR kinases) qui permet la
dsensibilisation et la squestration du rcepteur suivant sa stimulation.
En effet, des
formes tronques pour diffrentes portions du domaine C-terminal ont permis de dmontrer
que la dltion des dix-huit derniers acides amins entranait une dim inution de la
dsensibilisation du rcepteur P 2 Y 2 d environ 30% suivant la stimulation avec les agonistes
(Garrad et al., 1998). La squestration du rcepteur aprs trois heures de stimulation tait
diminue d autant plus que la partie en C-terminale tait tronque (Garrad et al., 1998).
Ces rsultats dmontrent que la portion en C-terminale de la protine est importante pour
rguler la dsensibilisation et la squestration du rcepteur P 2 Y 2.
En plus de son rle important dans la dsenbilisation du rcepteur, la rgion C-terminal de
P 2 Y 2 est implique dans le recrutement et lactivation de Src (Liu et al., 2004).
Le
recrutement de Src se fait grce la prsence de deux domaines de liaison SH3 retrouvs
dans le domaine carboxi-terminal de P 2 Y 2 (Liu et al., 2004).
28
Suivant la
liaison de GTP sur la sous-unit a, il y aura dissociation des sous-units P et y. La sousunit a peut ensuite activer diffrents effecteurs ju sq u ce que la molcule de GTP
laquelle elle est lie soit hydrolyse.
Le tableau 2 rsume la
29
Cellules
Systmes
Rles
Rfrences
......... hI!ni mu wiiBrargfs^w
Cerveau
Astrocytes
HHHH
M igration cellulaire
(Bagchi et al.,
2005)
Systme rnal
BaK& BSSBaBHBBBBSBSmKS^^SsSt^M
Scrtion d ions
Cellules pithliales
(Rajagopal et al.,
2011)
du rein
1181
im i
Systme osseux
Ostoclastes
*,,2007)
Prolifration
Sll
(K a tz et al., 2011)
cellulaire
Peau
Cellules pithliales
M igration cellulaire
Rparation de
(Braun et al.,
2006)
blessure
Tableau 2. Distribution et rles du rcepteur P2Y2 dans lorganisme
Systmes et types cellulaires dans lesquels le rcepteur P2Y2 est exprim suivi du rle
asssoci ces types cellulaires.
30
Finalement, le rcepteur P 2 Y 2 est aussi exprim dans lintestin o il joue plusieurs rles
dans lpithlium intestinal. Ainsi en condition physiologique, le rcepteur P 2 Y 2 participe
labsorption et la scrtion d lectrolytes par les cellules pithliales. Il rgule entre autre
la scrtion de potassium et de chlore ainsi que lactivation du canal ENaC qui rgule la
scrtion de sodium (Dong et al., 2009; Ghanem et al., 2005; Kerstan et al., 1998; M atos et
al., 2005; Matos et al., 2007).
5.2.4
Rgulation transcriptionnelle
Il est connu que suivant la stimulation de diffrents types cellulaires avec des molcules
pro-inflammatoires quil y a une augmentation de lexpression du rcepteur P2Y 2 (Ballerini
et al., 2006; Garcia-Verdugo et al., 2008; Hou et al., 2000; Kong et al., 2009).
Un des
31
Cette famille comprend cinq membres nom mes RelA/p65, c-Rel, RelB, N F- k B 1
(p50/pl05) et N F- kB2 (p52/pl00). RelA, RelB et c-Rel sont actifs constitutivem ent alors
que N F- kB 1 (p50/pl05) et N F- kB2 (p52/pl00) sont synthtiss en un long prcurseur qui
doit tre cliv pour permettre la translocation au noyau et la liaison leurs gnes cibles.
Ces facteurs de transcription forment des dimres dont presque toutes les combinaisons
entre les membres de cette famille sont possibles (Neumann et Naum ann, 2007).
Nanmoins, ce ne sont pas toutes les com binaisons qui pourront induire la transcription
ainsi les dimres p52/p52, p50/p50 ainsi que p50/p52 sont transcriptionnellem ent inactifs
(O'Dea et Hoffmann, 2009). Ces dimres peuvent se lier lADN mais ne peuvent
transactiver leurs gnes cibles (O'Dea et Hoffmann, 2009).
scientifique, la majorit des tudes portent sur le dimre form de RelA/p65 et N F- kB 1 p50
qui est en fait le complexe principal impliqu dans la rponse inflammatoire (Neum ann et
Naumann, 2007).
33
Cette voie
inhibitrice
IicBa,
des
modifications
post-traductionnelles
im pliquant
des
nuclaire qui lui permet de transloquer au noyau et de se lier avec N F- kB (Nelson et al.,
2004).
34
liaison avec IicBa nuclaire et le retour du com plexe dans le cytosol (Chen et al., 2001).
Finalement, lubiquinitation de la lysine 195 de RelA/p65 est associe sa dgradation par
le protasome (Fan et al., 2009).
35
accrue aux modles de blessures induits par les radiations (Egan et al., 2004). Le rle de
RelA/p65 a t dmontr dans un modle murin de colite chimique induit au DSS dans
lequel linvalidation de lexpression de RelA/p65 dans les cellules pithliales a entran
une diminution de lexpression de la molcule anti-apoptotique Bcl-XL (Karrasch et Jobin,
2008).
la lumire de ces tudes et de plusieurs autres, Karrasch et Jobin (Karrasch et Jobin,
2008) ont propos le modle suivant qui implique que lactivation de N F- kB dans les
cellules pithliales intestinales entranerait une rponse anti-inflamm atoire caractrise par
la restitution et la rparation de blessure ainsi q u une activit anti-apoptotique. Cependant,
lactivation des cellules mononucles de la lamina propria induit une augm entation de la
production
de
molcules
pro-inflammatoires
qui
chez
un
individu
susceptible
36
contiennent les domaines d activation de la transcription. Les formes prdom inantes sont
les formes LAP et LIP (Ramji et Foka, 2002).
Par exemple, la
mutation de la lysine 173 de C/EBPp qui est normalement un site de sumoylation levait la
rpression exerce par C/EBPP sur le promoteur de la cycline D1 (Eaton et Sealy, 2003).
37
intestinales avec lIL-1 (3. active C/EBPP par les MAPK et permet la production d IL-6,
cytokine de la phase aigu de linflammation (Hungness et al., 2002a; Hungness et al.,
2002b).
8. Hypothse, objectifs et mthodes
L inflammation augmente lactivit des facteurs de transcription NF-kB et C/EBPp.
De
38
transcription N F- kB et C/EBPP et que suivant son activation par ses agonistes favorisera la
restitution pithliale intestinale.
Deux objectifs gnraux ont t fixs.
O bjectif 1 : Caractriser la rgion du prom oteur du rcepteur P2Y2 et limplication des
facteurs de transcription NF-kB et C/EBPp dans la transactivation du prom oteur du
rcepteur P 2 Y 2.
Les sous-objectifs sont de :
rmission
de
souris
traites
au
DSS.
CHAPITRE 1
P 2 Y 2 receptor transcription is increased by NF-kB and stimulates COX-2 expression
and PGE 2 released by intestinal epithelial cells.
Auteurs de Particle: Emilie Degagn, Djordje M. Grbic, Andre-Anne Dupuis, Elise G.
Lavoie, Christine Langlois, Nishant Jain, Gary A. W eisman, Jean Svigny et FemandPierre Gendron.
Statut de Particle : Publi dans The Journal o f Immunology, volume 183, pages 4521 4529, 2009.
Avant-propos : J ai crit le manuscrit et j ai mis en forme les figures dans la premire
version de la publication. J ai effectu la majorit du travail exprimental lexception de
la figure 4 C et D ainsi que la figure supplmentaire 1.
40
9. Rsum de larticle
Suivant la stimulation avec des molcules pro-inflammatoires, il y a une augmentation de
lexpression de lARNm du rcepteur P 2 Y 2 (P 2 Y 2R) dans les lignes cellulaires Caco-2 et
IEC-6. De plus, on observe une augmentation de lexpression du P 2 Y 2R dans les tissus de
clon provenant de patients atteints de la maladie de Crohn et de la colite ulcreuse.
Cependant, les mcanismes transcriptionnels de la rgulation de lexpression du P 2 Y 2R
sont encore mconnus. Nous avons identifi le site d initiation de la transcription du gne
du P 2 Y 2R et dmontr que les histones H3 et H4 sont actyles respectivement sur la lysine
14 et la lysine 8 suggrant que la chromatine associe au P 2 Y 2R est accessible aux facteurs
de transcription.
41
P2Y2 Receptor Transcription is Increased by NF-kB and Stimulates COX-2 Expression and
PGE 2 Released by Intestinal Epithelial Cells.
42
10. Abstract
Inflammatory stresses associated with inflammatory bowel diseases upregulate P 2 Y 2
mRNA receptor expression in the human colon adenocarcinom a cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues o f patient suffering from C rohns disease and
ulcerative colitis. However, the transcriptional events regulating P2Y 2 receptor (P2Y 2R)
expression are not known.
A NF-kB-
responsive element was identified at -181 to -172bp in the promoter region o f P2Y2.
Hence, activation o f P2Y2R by ATP and UTP stimulated COX-2 expression and PGE2
secretion by IEC. These findings demonstrate that P2Y 2R expression is regulated during
intestinal inflammation through an NF-kB p65-dependent mechanism and could contribute
not only to IBD but to other inflammatory diseases by regulating prostaglandin released.
11. Introduction
Intestinal inflammation is often sees as a pernicious manifestation o f lost o f homeostasis
that can cause significant damage to the host tissue. In fact, inflammation is a key element
o f mucosal defence. It is aimed at limiting entry o f foreign material and microbes in the
blood stream and to facilitate the repair o f damaged tissues (1). The intestinal epithelium
constitutes the first defensive frontline o f the mucosal immune system (2, 3). Despite the
fact that intestinal epithelial cells (IECs) are continually exposed to intraluminal bacteria
and their products, the intestinal mucosa maintains a controlled state o f inflam mation (2, 4).
IECs play an active role in cellular responses to inflammatory stimuli by secreting
cytokines as well as sending cellular messages to immune cells o f the intestinal m ucosa and
submucosa (5, 6).
For many years, cytokines and chemokines and their receptors have
been accepted as regulators o f cross-talk between the intestinal epithelium, immune cells
and the vascular endothelium.
43
Hence,
inflammation likely promotes the release o f nucleotides, such as ATP and UTP or UDP, not
only from IECs, as we recently showed (10), but also from other cell types, such as
leukocytes, platelets and smooth muscle cells as well as from intestinal bacteria (12).
Another important source o f nucleotides is damaged or dead cells that release nucleotides
present at high concentrations in the cytoplasm (7, 10, 11).
extracellular nucleotide concentration in the environment o f inflamed tissues has also been
associated with an increased expression o f P2Y2 and P2Y6 receptor mRNA in colonic
epithelium o f patients with C rohns disease and ulcerative colitis and in mice with
chemical-induced colitis (10). Upregulation o f P 2 Y 2 receptor (P 2 Y2R) expression was also
observed in a variety o f pathophysiological conditions associated with inflammation and/or
tissue damage (13-15).
More
surprisingly, there is no data demonstrating how P2Y 2R gene expression is regulated. The
characterization o f the promoter region o f the P 2 Y 2R gene could help to understand the
transcriptional regulation o f P2 Y 2R expression in IBD and in other inflamm ation process.
Among the various transcription factors previously described, the p65 subunit o f nuclear
factor-kappa B (NF-kB p65) has been shown to regulate the transcription o f a broad range
o f genes related to inflammation in C rohns disease and ulcerative colitis (25). The current
study describes the cloning and sequence analysis o f the proximal prom oter region o f the
P 2 Y 2R gene in which we have detected the presence o f three consensus NF-kB binding
sites. Our results also demonstrate that NF-kB p65 stimulates P 2 Y 2R transcription in
colonic epithelial cells under pro-inflammatory conditions. Finally, we dem onstrated that
upregulation and activation o f the P 2 Y 2R lead both to an increase in COX-2 expression and
in PGE 2 released by IECs.
44
respectively from Calbiochem (San Diego, CA) and BioShop Canada, Inc. (Burlington,
ON).
Mouse monoclonal anti-NF-xB p65 antibody was purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-acetyl-Flistone H3 (lys 14) polyclonal
antibody and rabbit anti-acetyl-Histone H4 (lys 8) polyclonal antibody were from Millipore
(M illipore, Mississauga, ON).
from Caymen Chemical Co. (Burlington, ON). Prostaglandin E 2 (PGE 2) assay kits were
obtained from R&D system (Burlington, ON). The pGL4.10 and pcDNA3.1 vectors were
purchased, respectively, from Promega (M adison, WI) and Invitrogen (Burlington, ON).
The pcDNA3.1/NF-KB p65 construct was kindly provided by Dr. Marc Servant (Universit
de Montreal, Facult de Pharmacie, Quebec, Canada).
Cell Culture
Human coronary artery endothelial cells (HCAEC3; ATCC# CRL-2266) were cultured, as
previously described (26). The human colon carcinoma cell line Caco-2 (ATCC #HTB-37)
and the non-cancerous rat intestinal epithelial cell line IEC-6 (ATCC, CRL-1592) were
grown as previously described (10, 27). Cells were incubated in serum free medium for 24
h at 37C prior to experiments. W hen indicated, 0.5% (w/v) DSS, (10 ng/m l) IL-6 or (12.5
pg/ml) LPS were added to Caco-2 or IEC-6 cells for the specified time period to induce a
pro-inflammatory response, as previously described (10, 28, 29).
45
Total RNA was isolated from Caco-2 and IEC-6 cells with the Totally RNA Kit
(Ambion, Auston, TX) according to the m anufacturers instructions. Com plem entary DNA
(cDNA) was then synthesized from 2 pg o f purified RNA by reverse transcription using the
Superscript II system (Invitrogen). Five percent o f the synthesized cDNA was used as a
template for real-time PCR using the QuantiTect SYBR green PCR Kit (Qiagen,
Mississauga, ON) and the Stratagene M x3000P QPCR System. The sequence-specific
primers for P 2 Y 2R, COX-2 and TBP3 (TATA-binding protein) are listed in Table 1
(supplemental).
43-m er
CTCTCGCCACTGCGCTG
anti-sense
oligonucleotide
(10
pm ol)
5-
CGCTTCTCCTCTCAGGGTGCCGTCGC-3 (Tm=75.3C)
primer
as
positive
control.
The
negative
control
included
5 RLM-RACE
The transcription start site (TSS3) o f the human P 2 Y 2R gene was determ ined using the
RNA ligase-mediated rapid amplification o f 5 cDNA ends (5' RLM -RACE) with the
FirstChoice RLM-RACE kit following the manufacturer's instructions (Ambion). Briefly,
total RNA was isolated from Caco-2 cells using the RNeasy kit (Qiagen) according to the
manufacturers instructions and 10 pg o f RNA was treated with alkaline phosphatase to
remove phosphate groups on degraded mRNA, rRNA, tRNA and DNA. RNA was then
46
treated with tobacco acid pyrophosphatase to remove the cap from full-length nascent
transcripts.
Reaction products were reverse transcribed using Superscript III reverse transcriptase
(Invitrogen) and the oligonucleotide dT20. M odified cDNA was subjected to PCR analysis
using the PCR primer hY2R4 (supplemental data - Table 2) and outer prim er detecting the
5-RACE adapter (Ambion). The generated PCR products were reamplified in a second,
nested PCR using the inner prim er detecting the 5RACE adapter and one o f the hY 2R l
and hY2R3 primers (Table 2, supplemental).
using the Expand High Fidelity PCR system (Roche, Laval, QC).
cloned into pCRII-TOP using the TOPO cloning kit (Invitrogen).
fragments were identified by automatic
DNA
vector (Promega) upstream of the luciferase gene (prP2Y2-luc). Deletion mutants o f the
P 2 Y 2R promoter were generated by PCR amplification with the following oligonucleotides
(Tables 3 and 4, supplemental): prP2Y2A-350bp and prP2Y2/BglII prim ers for deletion o f 1572 bp to -351 bp; prP2Y2A-784bp and prP2Y2/BglII prim ers for deletion o f -1572 bp to 785 bp, and prP2Y2A-l 165bp and prP2Y2/BglII prim ers for deletion o f -1572 bp to -1166
bp.
The PCR fragments were cloned into the pGL4.10 expression vector (Promega)
generated by restriction enzyme digests with StuI (-273 bp) (prP2Y22A-350bp/StuI), Pmll
(-33 bp) (prP2Y2A-350bp/PmlI) and Nhel (-350 bp). Deletion o f the putative N F-kB p65
binding m otif (NBM3) in the prP2Y2A-350bp construct was done by overlap extension
47
Products resulting
from these two PCRs were used DNA templates for the final PCR using the prP2Y2/NheI
and prP2Y2/BglII oligonucleotides. The PCR fragments were then cloned into the pGL4.10
luciferase vector. The presence o f the mutations was verified by sequence analysis (McGill
University and Genome Quebec Innovation Center).
48
antibody (M illipore). Five pg o f normal mouse or normal rabbit IgG (Upstate) was used as
a negative control. For Re-ChIP assays, a second immunoprcipitation was conducted with
5 pg o f rabbit anti-p300 polyclonal antibody, after eluting the immunoprecipitated NF-kB
p65-DNA complex from the G protein agarose beads.
before
immunoprcipitation)
was
used to
verify
amount o f DNA
in
each
In supershift
experiments, 3 pg o f mouse monoclonal anti-NF-xB p65 antibody was used per sample and
added 20 min prior to the addition o f the radiolabeled probes. In competition experiments,
1OX or 100X o f the unlabeled probe NBM2 was added to the sample at the same tim e o f
labeled probe. Gels were dried and autoradiographed on an Imaging Screen-K (Kodak) for
18 h and imaged using a Bio-Rad M olecular Imager FX apparatus and data were acquired
using Quantity One software from Bio-Rad.
W estern immunoblotting
After nucleotide stimulation, Caco-2 cells were lysed and processed as previously
described (10).
49
human COX-2 was performed using a 1:750 dilution o f mouse monoclonal anti-COX-2.
Specific protein band on membranes were detected using a 1:10,000 dilution o f HRPconjugated anti-mouse as the secondary antibody and visualized on autoradiographic film
using the Millipore chemiluminescences system. Normalization o f the signal was realized
as previously described (10).
Statistical analysis
Results are expressed as the mean standard error o f the mean (SEM).
The statistical
legends.
13. Results
P 2 Y 2R expression is increased in Caco-2 and IEC-6 intestinal epithelial cells under
inflammatory-like conditions.
Recently, we showed that P 2 Y 2R mRNA expression was increased in colonic tissues o f
patients suffering from Crohns disease and ulcerative colitis (10). Sim ilar observations
were made in colonic epithelium o f mice in which intestinal inflammation was induced by
adding DSS to the drinking water (10). Using real-time PCR, we found that addition o f
DSS for 3 to 6 h to Caco-2 intestinal epithelial cells causes more than a 2-fold increase in
level o f P2Y2R mRNA (Fig. 1A). In addition, treatment o f Caco-2 cells with LPS 12.5
pg/ml resulted in a 8-fold increase in level o f P 2 Y 2R mRNA after 24 h (Fig. IB ) which was
also confirmed in a normal intestinal epithelial cell line, IEC-6, which resulted in more than
50
7-fold increase in level o f P 2 Y2R mRNA after 12 h (Fig. ID). Moreover, treatm ent o f IEC6 cells with IL-6 (10 ng/ml) causes an increase o f more than 2.5-fold in level o f P 2 Y2
mRNA after 18 h (Fig. 1C).
05h
Ik
3h
DSS (0.5% w *)
<h
IS
2
s?
I ! 10
II
E.
> 1T
1
se
in
24k
IL -6 (IO a s rn f)
LPS <12-5
51
PCR in confluent IEC-6 and 3-days post-confluent Caco-2 cells. A, B) Increases in mRNA
expression were significant after a 3 and 6 h incubation with 0.5% (w/v) dextran sulfate
sodium (DSS) and 24 h incubation with 12.5 pg/m l LPS in Caco-2 cells. C, D) Increases in
mRNA expression were significant after 12 h incubation with 12.5 pg/m l LPS and 18 h
treatment with 10 ng/ml IL-6 in IEC-6 cells. Data are expressed as P 2 Y 2R mRNA
expression induced by stimuli relative to the untreated control (ctrl). Results were
normalized to the expression o f TBP mRNA. Values are the means SEM o f results from
three separate experiments done in duplicate. Statistical significance was determined by
unpaired t test, where *p < 0.05 and **p < 0.01, as compared to crtl.
RLM-
RACE is a more sensitive method than prim er extension analysis since it removes
uncapped mRNA, DNA and other non-m RN A molecules by treatment o f the RNA sample
with calf intestine phosphatase (34, 35).
amplification products revealed that Caco-2 cells express only variant 2 o f the human
P 2 Y 2R mRNA and a single TSS, as shown by the presence o f a single band for both clones
analyzed from the nested PCR assays with hY 2R l and hY2R3 oligonucleotide primers
(supplemental data, Fig. IB). Sequence alignment o f representative clones obtained from
the nested PCR with the 5 -UTR sequence o f human P 2 Y 2R (variant 2; NM 002564)
allowed us to localize the TSS (+1) 72 nucleotides downstream from the first nucleotide o f
the published P 2 Y 2R gene sequence, as indicated in supplemental - Fig. 1C. We also found
52
a mutation at +32 where a thymine (T) was replaced by a cytosine (C) residue, as indicated
by the arrowhead (supplemental data, Fig. 1C). Finally, the translation start point (ATG)
was located at position +262, as indicated by the bold letters (supplemental data, Fig. 1C).
We aligned the sequence o f the promoter from the two different cell types and identified by
computational analysis potential binding sites for Spl and NF-kB (supplem ental data, Fig.
2). The P 2 Y 2R promoter o f these cell types is 99.2% identical.
53
promoter (Fig. 2B, p < 0.01) but increased to more than 7-fold in response to DSS (Fig. 2B,
p < 0.05).
To
luciferase activity o f the minimal P2Y2A-350-Luc promoter construct by more than 60%,
whereas mutation o f NBM1 and NBM3 had no effect on luciferase activity (Fig. 2E),
suggesting a role for NBM2 in the transcriptional regulation o f P 2 Y 2R expression by NFkB. We also have identified other potential binding sites for transcription factors in the
P 2 Y 2R promoter region, such as SP1 (supplemental data, Fig. 2) that rem ain to be
characterized.
54
-II* *
-1572*
-7 1 4 *
-1200*
'''Nun!
-U S *
NBM2
NBM)
-271 Is -200
KO*
200 *
-100*
I*
- J*
10
, *
i*
;
p fK Y A -ll* )
I *
1
mA
DSS O SS
NF-dtyS
ptWY.-fat
pOM.IO
prfjYjA-TM
ptf2Yj-330
"I............
0.5
|.0
Lactfcnac actmty
(FU vanatMa over M l le a f* promoter)
K)
pRY-A-UBSM
"T
oi
1.0
ij
L a c tfe ra a c a c t m t y
/////
t///
FIGURE 2. N F-kB p65 transactivates the P 2 Y2R promoter region under basal conditions
and this transactivation is enhanced following DSS-induced stress in Caco-2 cells. A)
Schematic representation o f the P 2 Y2R promoter constructs. B) Subconfluent Caco-2 cells
were transiently cotransfected with the P 2 Y2R promoter-luciferase construct (prP2Y2-luc)
and the N F-kB p65-expressing vector or the empty pG L4.10 vector (control). Cells were
incubated with or without 0.5% (w/v) DSS for 6 h and luciferase activity was assayed after
48 h. C) Subconfluent Caco-2 cells were cotransfected with the P 2 Y2R promoter-deletion
luciferase constructs prP2Y2A-350bp, prP2Y2A-784bp or prP2Y2A-l 165bp and the NF-kB
p65-expressing or control vector. D) Subconfluent Caco-2 cells were cotransfected with
the P 2 Y2R promoter-deletion luciferase constructs prP2Y2A-350/PmlI or prP2Y2A-350/StuI
and the NF-kB p65-expressing or control vector. E) prP2Y2ANBM l, prP2Y2ANBM2,
prP2Y2ANBM3, P 2 Y2R full-length promoter constructs or prP2Y2A-350bp promoter
construct were transiently cotransfected with the N F-kB p65-expressing or control vector.
55
Luciferase activity is expressed as the fold increase relative to the activity o f the empty
vector cotransfected with the NF-kB p65-expressing vector. Data are the means SEM o f
results from at least 4 separate experiments done in triplicate. Statistical analysis was
performed by an ANOVA test, where *p < 0.05 and **p < 0.01, as com pared to respective
controls and indicated on the figures. In panel E, s s p < 0 .0 1 , as compared to empty vector
control (EV).
We verified by electrophoretic mobility and supershift assays, the ability o f labeled NBM 1,
NBM2 and NMB3 double-stranded oligonucleotides to bind NF-kB p65 in Caco-2 nuclear
extracts (Fig. 3A).
O f the three NBM sites, the NBM 2-protein complex was the major
complex supershifted by a NF-kB p65 antibody (Fig. 3A). We also did a com petition assay
using unlabeled NBM 2 probe with NBM2 labeled probe and observed alm ost complete
diminution o f the intensity o f the labeled protein-DNA complex in the presence o f 100X o f
unlabeled probe (Fig. 3B).
PGE 2, can modulate the inflammatory response o f the intestinal m ucosa (43-45).
56
57
c >
2-5 - ,
' ...""I...
N F - B p A 5
pJOO
FIGURE 3. N F- k B p65 binds to the P 2 Y2R prom oter sequence. A) N uclear extracts from
Caco-2 cells were incubated with the putative [y- P]ATP-labeled N F - k B p65 DNAbinding site probes NBM1, NBM 2 or NBM 3 and anti-NF-KB p65 antibodies for
electrophoretic mobility and supershift assays. DNA-protein complexes were separated
from the free probe on a native polyacrylam ide gel. N F - k B p65 D NA-binding and
supershifted complexes are indicated by the arrow. B) Nuclear extracts from Caco-2 cells
were incubated with the putative [y-32P]ATP-labeled N F - k B p65 DNA-binding site probe
NBM2 and 10X or 100X o f unlabeled NBM2 for electrophoretic mobility and competition
assays.
DNA-protein complexes were separated from the free probe on a native
polyacrylamide gel. Results are representative o f three independent experiments. N F - k B
p65 DNA-binding complex is indicated by the arrow.
C) Chromatin was
immunoprecipitated with or without rabbit IgG antibody or anti-NF-KB p65 antibody. The
re-ChIP assay was performed with anti-p300 antibody following the first
58
immunoprcipitation with anti-p65 antibody. Samples were verified by quantitative RTPCR analysis with oligonucleotides amplifying the -221 bp to -155 bp region o f the P 2 Y2R
promoter and expressed as fold increase over normal rabbit IgG normalized to input.
Representative results from 3 independent experiments are shown.
14. Discussion
We recently reported the upregulation o f P 2 Y 2R mRNA in the colonic tissues
isolated from patients suffering from IBDs (10). Similar observations were also made in
colon isolated from mice suffering from chemically induced colitis (10).
It is well
neutrophils
and
using the adenocarcinoma-derived Caco-2 cells and the non-cancerous IEC - 6 cells that the
increased expression o f the P 2 Y 2R transcript could be attributable in part to the
upregulation o f receptor expression by IECs (Fig. 1).
expression and function has been associated to enhanced tissue dam ages as reported in
Sjogrens syndrome-like phenotype in mice (14, 50), chronic pancreatitis (13) and the
induction o f intimai hyperplasia in an animal model o f human atherosclerosis (15, 51).
More recently, P 2 Y 2R increased expression and function in rat prim ary cortical neurons
have been associated to the a-secretase-dependent processing o f amyloid precursor protein,
a well described molecule involved in neurodegenerative disorders (52).
In addition,
59
potential,
we
also
looked
at
p300,
coactivator
with
histone
By computational analysis
(Supplemental Data, Fig. 2), we identified three potential Spl binding sites 50 bp upstream
o f the P 2 Y 2R TSS. Further studies will be necessary to define the role o f these potential
binding sites, since multiple Spl elements can act independently or synergistically to
promote gene transcription (55).
60
J t
$.<*>
3
4
UTP(100nM)
0)
ATP(IOO|iM)
ctrt
0.3
72k0>
if BM*
t rig fl
FIGURE 4. ATP and UTP stimulate COX-2 expression and PGE 2 released by Caco-2.
Addition o f 100 pM ATP (A) or UTP (B) rapidly stimulated the mRNA expression o f
COX-2 in Caco-2 cells. C, Caco-2 cells were stimulated w ith 100 pM ATP or UTP for 0
(control; Ctrl), 3, 6 , 18 and 24 h. COX-2 protein expression was detected by W estern
analysis. D, PGE 2 released in the cell culture m edia was determined after a 24 h
stimulation o f Caco-2 cells with lOOpM o f ATP or UTP. For panels A, B and D, data are
the means SEM o f results from 3 separate experiments done in duplicate. Statistical
analysis was performed by an unpaired t test, where *p < 0.05; **p < 0.01 and *** p <
0.001, is comparison to unstimulated cells (ctrl). Panel C presented blot is typical o f three
separate sets o f experiments.
Truncation o f the 5 -region o f the P 2 Y 2R promoter has allowed us to identify the
region between -273 and -33 nt as important for transactivation o f P 2 Y 2R gene expression
by the transcription factor NF-kB p65. We then conducted computational analysis o f this
61
promoter region and indeed found three potential NF-kB binding sites (NBM 1-3).
specific
mutations
o f these
NBMs
in the
P2Y2A-350bp promoter
Site-
followed
by
cotransfection with NF-kB p65 in Caco-2 cells further supported the postulate that NBM2
is a binding site for NF-kB. Actually, mutation o f the NBM 2 site resulted in a decreased
luciferase activity o f more than 50%. This result was confirmed by EM SA experiments
which demonstrated that p65 interacts strongly in vitro with NBM2, as compared to NBM1
and NBM3.
ChIP assays revealed that p65 binds to the region (-221bp to -105bp) that
contains the NBM2 motif. Interestingly, transactivation o f the P 2 Y 2R prom oter by NF-GB
p65 occurs under both basal and pro-inflammatory conditions. Our results suggest that the
induction o f inflammation in intestinal epithelial cells allows nuclear translocation o f NFkB p65 and leads to an increased transactivation o f the P2Y2R promoter and subsequently
the upregulation o f P 2 Y 2R mRNA. These results are in agreement with recently published
data demonstrating that a 24h stimulation o f rat prim ary cortical neurons w ith IL -ip
induced P 2 Y 2R mRNA expression possibly through a NF-KB-dependent mechanism (52).
To circumvent the cancerous nature o f Caco-2 cells, we used the non-cancerous intestinal
epithelial cell line IEC - 6 and measured an increased in P 2 Y 2R mRNA expression following
IL - 6 or LPS stimulation.
62
the inflammatory response o f the intestinal m ucosa thus resulting in increasing tissue
damages but also at limiting entry o f foreign molecules to the systemic circulation, as well
as facilitating the repair o f damage tissues.
inflammation, these same molecules could enhance epithelium repair by stimulating IEC
proliferation and increasing the IECs barrier integrity (44, 56).
S)
_
hY2R1 hY2&
bp -
cDNA
soo300-
C)
*
*Y261
n t 0 3 2 5 6 4 & ^ ^ O ^ T ^ S A C M I ^ C C r r C T O C A C A G C ^ T k t:c r a C C C * l3 A A M A T r ;
' 4Y 263 K A K 9 -M n t3 :6 M 3 M X 0 C C C C T t< :r^ A e c ^ ^ A C T W C T Q C C rjtG A M A T Y ;
MJKI Oi^OC*C7WC(yuU(WJWCCCCTTSrG*C*CCA:AC'TCCTlX'CCG*AA*Ar'5
k* 00356 rtc^T^'Tcwc^CTi^cTirtnfcrwTncciiiS
hfj*3 S*0CKa3GCCrt5M:CtXIW!X5rtOCGC*CCTQrTriTO.'WTrTOCCCCAilkG
h3l ncsAQSCToeoccrosci.'cciiGScrrosacACrfoTTrtTccKrrTcei.'cAAi
n t 053 56.4 rTcccTCcjuxccoiT^auwTcnjujseaTO'roakTrcATatcrcywiiAAcnccToo*
iliii.i rrc.ccwi5!xorrcc*ffrcc*awTCTcc*TTCMfi*cT4S3Juu:ocaYc
&rl rrcCCtTJkCCCi3TCakOCTCC*l5CCCTCr5C*TTCTCCTfJM3rJ5*CCCOTOCA
m 055564 cecocrcjuiCATccrGjurcT&oAOjuxAQi^recTCjooGxaaocxiicaijOJicci
Y63 03C3CTGlbEC3kTCrrClkCC-r0S*ajUIC3MiCG!iCTSCTataCSCG>Maau3CAakCC-t
i y 5 6 i c ^ o :Y ^ J v c c 6 r 7 0 J u ;c 7 G C * M ;
6Y56) (SCKTCG*TI*t6CCAKJk*TaC^.CTO^tSOKA7
M 5RI
................
........
64
C om am u
prfYJ Caco-2
pfS2Y2HCAC
C o n ta n t*
ptS2T2Ca-2
ph tch ca k
caccgoEoo
aaxK^i^aaaxKM aaasmsos(aaasaxmaacaaakt*osK.
GCAGGGGCEGGACAGGGtnAGGGIGGCGCGGIGCCIIjGGCIjCAAAGGIC
CCg^TTf^<TA(riCy/K/^/Clt^CKXXAA*ACmGOCGTCIt<iAAA
CCGCAGTOCGCCACOCAGGCACCGGGCTtiACCTGGCAAAACTITBGCGilCICtGAAA
OCEC<6IOO6COCSOCGC<COGCCCIC<CCTGCCAAAACTTTGCOGIOOGAAA
NF k B (N BM I)
ContanM
faSTT] Caco-2
PMT2HCAK
ACIQGGIMCCAOCICOnTCtAGCtnGAMGAGCC^^
ACCTCHiCIAAOOGClOOCnCTACCGIGAGGGAGOCEGGAGGCCTCCTICIOGCC
ConMmw
aiCogiGACAGCxnqaxccccgAcitr iccicsncoccAcccc^^
lf2Y2 Caco-2
pif7Y2HCAfiC
CGCOGnMGAGOncXXXGCCiaWKiriCCOCTTGOCCXCGCnAOGAOCGa
COGCAGTGACAOCSTOOCGCCCGACCCICCPin OCACCACCOGTAOGACACGCC
ACCTCTOGTAACCAOCTCCCTICTAGCGIOAGGGACCCGOOiGOCCTCCTTCTCGCC
NfkB(NBM2)
r^rrrrr^*srT rr,vrj^ J & r f ^ ^ r s jsjrj-j7Trj-srjr*m rTu-rr
Consens*
p tO m Caco-2
prfZY2HCAK
GACCCCGCGOCCTTGICGCIOCOCOGCXiICKGGAGCGGGIOGOSCGGTGICIACCC
GACCirGCGGCCTTGTCGCTCOCSGCGTCCCGGAGiCOGiGTGGCSCGGTCrCTAOCC
C om ns*
prF2Y2 Caco-2
pf2Y2 HCAEC
OCuCCCTTGACCCCGOlOCOGGCICAGICAAAAGilOOGCCCCCiCCCOCIXaOC-GO
CXUCG0GT1GAC(XICXTCCC<CGGTCMnCAAA<G1GCGCCCGC(ZXC10CC10C
COGCCGGTTGAGGOCGCTGCCAGGCTCAGTCAAAAGTCCGCCCCGCCCCCTOCCSGO
SOI
prf2Y2 Caco-2
prWYJHCAK
Gomma*
prMM Caco-2
prP2Y2 HCAtC
NFkB(NBM3)
CCtECCTTCGGGGTTCGGGAAiCAGCGCA(jG<l*OCTGGOT%CCCOOCTCCCAG<jCAC
CCCGGCTICGGCCnGGOGAACAGCOCAOOGAOO'CiGGTAOCCGCKiCTCCCAGCAe
aX00CTTOWWTT0GGGAACAGC<XAOMi<iiaiG'AOCCGG0CKCCAG*f
! '.bp rss3bp
G!GCCK1CTOCG0CTOCO(jCC<CiC>ACCCG<iC>C1OGCACCCCi(i(iAGCGGCCCiCGAC
GTGGGTCTCTCiC<jGCIGCGCGG6<CCG&G< 'CTGGCaCCCGGGAGCG&CGGCGAC
GIWXiTnCTWGaiGCGCiCGGGACCrGOGr'CCrACrCrGCJUKGGCGGCWC
Contant*
pif JYJ Caco-2
p if JY2 HCAiC
CjGCACCCrGAGA.C<iMi*ACCCiCMjCGCACiTG<jCCAf>ACGrG<jijGTC<iGGCG'rCTGcjA
GGCACCCCGACAOGAGAAGCClCACiCGCAG'GGCCiACiMiGrGGGGICiGCiGCGTOGGA
CGCACCCTGA&aGGACiAAGCGCWiCGC *r,rf<<W>(^3C.Crfiin5CiCGTCTC^
Cornant*
pif2V2 Caco-2
piP2Y2HCAiC
GGAACAAGGCG f U O b p
6G&WCAAGGCG
C-GCAACXCGCG
65
A e -H 4 <ly t )
Ctrl -
66
Sequence (5 > 3 )
P 2 Y 2R U pstream a
P 2 Y 2R D ow nstream a
GCCTCC AG ATGGGTCT AT GA
P 2 Y2R Upstream b
ATGTCGCTTCAACGAGGACT
P2 Y 2R Downstream b
T GCCAGGTGAAACATGTAGG
COX-2 U pstream 3
TGAAACCCACTCCAAACAC
COX-2 Downstream 3
TBP U pstream 3
T G AGGATAAGAGAGCCACGAA
TBP D ow nstream 3
TBP Upstream b
T AT A AT CCC AAGCGGTTT GC
TBP D ow nstream b
CCCACCATGTTCTGGATCTT
Sequence (5 > 3 )
CACCAACCTTTACTGCAGCA
Sequence (5 > 3 )
GCTAGCCAGGAATGCAGTTTCCTCAGCTG
AGATCTCGCCTTGTTCCCTCCAGA
67
Table 4.
mutants
Oligonucleotide
Sequence (5 - 3 )
AAGCTGGCTAGCCACTATGGGCTGTGTGTCCA
Sequence (5
3 )
Sequence (5 3 )
ACCGGTACAGACACGCTGAC
Sequence (5-> 3 ) e
CCGGGA GG CCTCCTT
GGGCGGCGTCCCGGA
GAGGGCGGTGCCAGG
16. Acknowledgements
The authors thank Naomie Turgeon and Isabelle Frechette for their technical help w ith the
ChIP and EMSA studies as well as Dr. Cheikh I. Seye and Sophie Tousignant for critical
and careful reading o f the manuscript.
68
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72
Footnotes
1. Correspondence: Dr. Femand-Pierre Gendron, Dpartement d anatom ie et de biologie
cellulaire, Facult de mdecine et des sciences de la sant, Universit de Sherbrooke, 3001
12e Avenue Nord, Sherbrooke, QC, Canada, J1H 5N4; Tel. (819) 564-5272; Fax. (819)
564-5320; E-mail: Femand-P.Gendron@ USherbrooke.ca
2. This research was supported by the C rohns and Colitis Foundation o f Canada Grant in
Aid o f Research, the Canadian Institutes o f Health Research (CIHR, N M D -94729) and an
establishment grant from the Fonds de la recherche en Sant du Qubec (FRSQ) to F.P.G.,
and by grants from the Canadian Institutes o f Health Research (CIHR) and The Arthritis
Society (TAS to J.S. andN IH grants AGI 8357, DE07389, and D E I7591 to G.A.W . F.P.G.
is a scholar from the FRSQ and he is mem ber o f the FRSQ-funded Centre de Recherche
Clinique tienne-Le Bel. D.M.G. is a recipient o f scholarship from the Natural Sciences
and Engineering Research Council o f Canada. J.S. was a recipient o f a new investigator
award from the CIHR and E.G. Lavoie o f a scholarship from the FRSQ.
3. Abbreviations: AMV, avian myeloblastosis virus; DSS, dextran sulfate sodium; HCAEC,
human coronary artery endothelial cells; IBD, inflammatory bowel diseases; IECs,
intestinal epithelial cells; NBM, NF-DB binding motif; TBP, TATA binding protein; TSS,
transcription
starting
site
CHAPITRE 2
P2Y2 receptor promotes intestinal epithelial cell migration through a Go protein and
integrin a v pathway allowing microtubule stabilization and mucosal re-epithelization
in experimental colitis.
Auteurs de larticle: Emilie Degagn, Jade Degrandmaison, Djordje M. Grbic, Valrie
Vinette, Guillaume Arguin et Fem and-Pierre Gendron.
Statut de larticle : Sous presse dans le Journal o f Cellular Physiology, 2012.
Avant-propos : Ma contribution pour cet article a t d crire le manuscrit et de m ettre en
forme les figures pour la premire version de la publication. J ai effectu la m ajorit du
travail exprimental des 8 figures rgulires et des 2 figures supplmentaires de ce papier
lexception de la figure 5 A-G, des figures 6 A et 7A et de la figure supplm entaire 1 A.
74
chambres de Boyden ou en utilisant un modle de blessure par lame de rasoir. Nous avons
activ le rcepteur avec ses agonistes endognes soit lATP et lUTP ou en utilisant un
agoniste de synthse soit le 2-thioUTP.
75
Department o f Anatomy and Cell Biology, Canadian Institutes o f Health Research Team
J1H
5N4.
Tel.
819-564-5272,
Fax:
819-564-5320;
P.Gendron@ USherbrooke.ca
E-mail:
Fernand-
76
19. Abstract
P 2 Y 2 receptor expression is increased in intestinal epithelial cells (IECs) during
inflammatory bowel diseases (IBDs).
IECs, suggesting a role in wound healing. For this study, we have used the non-cancerous
IEC -6 cell line.
IEC -6 cell migration was determined using Boyden chambers and the
single-edged razor blade model o f wounding. The receptor was activated using ATP, UTP
or 2-thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and
interfering RNAs were used to characterize the signaling events.
Following P 2 Y 2 activation, we
demonstrated that GSK3P activity was inhibited in response to Akt activation. This leads
to MT stabilization and increased number o f focal adhesions. In vivo, P 2 Y 2 activation
stimulates remission, as illustrated by a reduction in the disease activity index values and
histological scores as compared to control mice. These findings highlight a novel function
for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the
development o f innovative therapies for the treatm ent o f IBDs.
77
20. Introduction
Crohn's disease and ulcerative colitis are chronic and relapsing inflammatory bowel
diseases (IBDs) that can affect the entire length o f the intestine (Abraham and Cho, 2009).
IBDs are the consequence o f intestinal epithelial cells (IECs) and immune cell dysfunction
resulting in impaired or abnormal mucosal immune responses (Laukoetter et al., 2008).
The regenerative capacity o f the mucosal surface epithelium is essential to the maintenance
o f the intestinal mucosal barrier integrity even after extensive damages (Sturm and Dignass,
2008). However, the inflammatory burst associated with the relapsing episodes observed in
IBDs, impaired the regenerative capacity o f the IEC monolayer. As a consequence, it is
possible
to
observed
crypt
abscess
formation,
uncontrolled
bacterial
infiltration,
exacerbated inflammatory responses and loss o f tissue architecture and functions (Sartor,
2004; Sturm and Dignass, 2008).
migrate into the wound depolarize, form pseudopodia-like structures, reorganize their
cytoskeleton, and repolarize after wound closure. This restitution process is independent o f
cell proliferation (Sturm and Dignass, 2008). Intestinal epithelial restitution occurs within
minutes to hours both in vivo and in vitro. Second, epithelial cell proliferation is necessary
to replace and maintain the IEC monolayer.
78
2009). Nucleotides are released in the extracellular environment o f IECs under normal
physiological conditions, but are massively liberated following inflammation and injury (Di
Virgilio et al., 2001; Grbic et al., 2008; Luttikhuizen et al., 2004).
P 2 Y 2 agonists can also negatively regulate immunity and inflammation (Di Virgilio et al.,
2009), and could thus affect both mucosal inflammation and the healing process (Boucher
et al., 2010; Braun et al., 2006; Crooke et al., 2009; Dignass et al., 1998).
In fact, the
involved and the signaling events associated with intestinal wound healing have never been
investigated.
Signaling
through
the
Gq-coupled
P 2 Y2
involves
the
hydrolysis
of
79
intracellular calcium and triggers calcium-dependent cell responses, such as the activation
o f protein kinase C (PKC) (Erb et al., 2006). PKC stimulation o f M APK activity regulates
various cellular processes, notably migration and proliferation. In addition to MAPK, P 2 Y2
can stimulate PI3K/Akt activity through a PLC/IP 3/Ca2+ and PKC pathway (Bilbao et al.,
2010).
domain found in its first extracellular loop (Bagchi et al., 2005; Erb et al., 2001).
Association o f P 2 Y 2 to the a v integrin subunit is necessary for the recruitm ent o f Go and
the initiation o f Go-mediated signaling events leading to cell migration (Bagchi et al.,
2005).
The participation o f the actin cytoskeleton in cell migration has been well described.
It provides a protrusion force at the cell front and a contractile force in the cell body
(Gardel et al., 2010). In contrast, the role o f microtubules (M T) is far more diverse and the
tight regulation o f MT dynamics is required for proper directed cell m igration (EtienneManneville, 2010; Wen et al., 2004). Hence, the regulation o f MT dynamics is essential to
cytoskeleton and focal adhesion organization (Blikslager et al., 2007; Gardel et al., 2010).
One key component o f MT is a-tubulin (a-T ub), which is stabilized through protein
actylation (Creppe et al., 2009). Consequently, detection o f the acetylated form o f a-T ub
is an indication o f MT stabilization (Creppe et al., 2009; Etienne-M anneville, 2010). MT
dynamics is regulated by numerous effectors including, but not limited to, G SK3p and
PI3K/Akt. The PI3K/Akt pathway is important for MT dynamics as well as being a key
element for cell migration and proliferation (Sun et al., 2009), whereas GSK3P regulates
MT dynamics by inhibiting the activation o f M T-associated proteins like CLIP, EB1 and
APC that are responsible for MT elongation and stabilization (Hur et al., 2011; Wen et al.,
2004).
In this study, we have investigated the role o f P 2 Y 2 in intestinal epithelial wound
healing processes.
monolayer o f IECs.
80
LY294002, H-Arg-Gly-Asp-Ser-OFl (RGDS) peptide and control were acquired from EMD
chemicals (Mississauga, ON, Canada). The rabbit anti-phospho-Akt (Ser473), anti-phosphoGSK-3J3 (Ser9), anti-Ac-a-tubulin (Lys40), anti-integrin a v, anti-Akt antibodies and rabbit
monoclonal anti-GSK3p antibody were purchased from Cell Signaling Technology
(Pickering, ON, Canada). Integrin a v neutralizing antibody and normal mouse IgG were
purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).
receptor antibody, mouse monoclonal anti-actin (clone C4), DAPI and -ECL reagent were
purchased from Millipore (M ississauga, ON, Canada).
conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG were from GE
Health Care Bio-Sciences Corp (Piscataway, NJ). Dextran sulfate sodium (DSS; Mx
36,000-50,000) was obtained from MP Biomedicals (Solon, OH). Pertussis toxin (PTX)
was purchased from Enzo Life Sciences (Burlington, ON, Canada).
Cell culture
The non-cancerous rat intestinal epithelial cell line IEC -6 (ATCC #CRL-1592) was
grown as previously described (Degagne et al., 2009; Langlois and Gendron, 2009). Cells
were incubated in serum free medium at 37C, 24 h prior to experiments.
81
'j 1
and Mg
"yA-
and incubated
for 25 min at 37C to ensure complete hydrolysis o f the Fluo-4/AM. Cells were then
centrifuged again and re-suspended in 16 ml o f HBSS with Ca2+ and M g2+ and 2 ml o f cells
suspension was gently stirred in a quartz cuvette while [Ca2+]j was m onitored on a RF-5301
PC Shimadzu spectrofluorometer (M an-Tech, Guelph, ON) with excitation and emission
wavelengths o f 488 and 520 nm, respectively. Change in intracellular Fluo-4 fluorescence
intensity (F) was acquired using the Panorama fluorescence 1.1 software. At the end o f
each recording, maximal (Fmax) and minimal (Fmin) fluorescence were determined by
adding successively 0.1% Triton X-100 and 50 mM EDTA to cell suspensions. The
2+
9+
following equation was used to relate the fluorescence intensity to Ca levels: [Ca ] = Kd
x (7r-Fm in)/(Fm ax-/:r). Kd is the Ca2+ dissociation constant o f Fluo-4 (345 nM)
(G rynkiew iczetal., 1985).
M igration assay
IEC - 6 cells or IEC -6 cell populations for shP 2 Y 2 (IEC- 6 /shY 2), shNT (IEC - 6 /shNT)
or EV (IEC- 6 /EV) were grown to confluence and serum-deprived 24 h before assays.
Briefly, cells were trypsinized and resuspended in 600 pi o f migrating buffer (0.05%
gelatine and 25 mM HEPES in DMEM). The upper cham ber o f an 8 pm Transwell was
loaded with 175,000 cells. The lower chamber was filled with 600 pi o f migrating buffer
supplemented or not with P 2 Y 2R agonists, with or without P2 receptor antagonists suramin
82
or PPADS. Antagonists were added 30 min prior to cell stimulation with 100 pM A TP or
UTP, or 10 pM 2-thioUTP. After a 6 h incubation period, the upper and lower chambers
were washed using PBS, The remaining cells in the upper cham ber were removed with a
cotton swab. Cells present beneath the membrane were fixed with ice-cold methanol. The
filter was then removed and placed on a microscope slide. M igrating epithelial cells were
counted in 4 random fields using a Leica DM2500 microscope equipped with a Hamamatsu
ORCA-R 2 digital cam era under bright field illumination.
Wounding assay
Cells were grown to confluence in 100-mm dishes. They were incubated in serumfree medium for 24 hours, wounded with a single-edged razor blade and incubated in the
presence or absence of 100 pM ATP or UTP, or 10 pM 2-thioUTP for 24 hours. To assess
the signaling pathways involved, IEC - 6 cell m onolayers were pre-treated for 30 min with
10 pg/ml anti-integrin a v neutralizing antibody, 10 pg/ml normal m ouse IgG or 10 pM o f
LY294002 wounded and stimulated in the presence or absence o f ATP or UTP.
In the
assays in which PTX was used, PTX was added at a concentration o f 250 ng/ml for 16 h
prior to wounding and nucleotide stimulation. Photom icrographs were taken at 4 locations
per wound, and the area o f migrating cells was measured using ImageJ software.
Western blotting
After treatments, IEC - 6 cells were lysed and samples processed for protein
immunoblotting, as previously described (Grbic et al., 2008).
performed using a 1/1,000 dilution o f rabbit anti-phospho-Akt (Ser 473), anti-phosphoGSK-3(3 (Ser 9), anti-acetyl-a-tubulin K40 and anti-P 2 Y 2 anti-integrin a v. Specific protein
bands on membranes were detected using a 1:10,000 dilution o f HRP-conjugated anti
rabbit as the secondary antibody and visualized on autoradiographic films using the
Millipore chemiluminescence system.
stripped o f antibodies (Grbic et al., 2008) and reprobed w ith a 1/1,500 dilution o f rabbit
anti-Akt, a 1/5,000 dilution o f mouse anti-GSK3p or a 1/10,000 dilution o f mouse
monoclonal anti-P-actin followed by a 1/10,000 dilution o f HRP-conjugated anti-rabbit IgG
or HRP-conjugated anti-mouse IgG as the secondary Ab, respectively.
Indirect immunofluorescence
83
IEC -6 cells grown on sterile glass coverslips were washed twice with ice-cold PBS
after treatments and fixed with 3% paraformaldehyde for 30 min at 4C. Cells were washed
with PBS, quenched with 100 mM glycine for 10 min and permeabilized with PBS
containing 0.1% Triton X-100 for 10 min at RT. Cells were washed and then blocked with
PBS containing 2% BSA for 20 min at RT. They were then immunostained for 2 h with the
rabbit anti-acetyl-a-tubulin as the primary antibody, followed by the Alexa Fluor 488 goat
anti-rabbit IgG (H+L) as the secondary antibody for 1 h. Nuclei were stained with 4', 6 diamidino-2-phenylindole (DAPI). Staining for the focal adhesion proteins vinculin and Factin using phalloidin was realized using the Actin Cytoskeleton and Focal Adhesion
Staining Kit (M illipore) as recommended by the manufacturer. Negative controls (normal
IgG antibody) were included in all experiments. Slides were washed in PBS, mounted, and
images were captured on Leica DM 2500 microscope using a Hamamatsu O RCA -R 2 digital
camera.
Mouse model o f IBD and 2-thioUTP injections
Adult CD -I mice (30-35 g) were purchased from Charles River Laboratories (St.
Constant, QC, Canada). Colitis-like disease was induced by adding 4% (w/v) DSS to the
drinking water for 7 days as previously described (Grbic et al., 2011; Grbic et al., 2008).
After a 24 h recovery period, mice received daily rectal enemas o f a 2-thioUTP solution at
a dose o f 2 pg per gram o f body weight versus saline. The dose o f 2-thioUTP used was
based on a recent paper by Grbic et al. (Grbic et al., 2011) in which UDP was given by
enemas at a dose o f 100 pg per gram o f body weight (pg/g) to stimulate, in vivo, the P2Y 6
receptor. The reported EC 50 for 2-thioUTP toward P 2 Y 2 receptor is ranging from 35 to 50
nM (El-Tayeb et al., 2006) as compared to previously reported average EC 50 values ranging
from 1.5 to 5.8 pM for ATP and UTP (Velazquez B. et al. 2000). Thus, 2-thioUTP is more
potent for the P 2 Y 2 receptor by about a 90 fold factor as compared to ATP or UTP.
Adenosine 5'-triphosphate (ATP) has been previously used in clinical trial as a cancer
treatment at doses ranging from 24 pg/g (Beijer et al., 2010) to 135 pg/g (Agteresch et al.,
2000) and even up to 228 pg/g (Haskell et al., 1998) for an average dose o f about 129 pg/g.
Given the potency and selectivity o f 2-thioUTP as compared to ATP we decided to use a
dose o f 2 pg/g, which reflect the difference in potency between P 2 Y 2 receptor natural
agonists and 2-thioUTP and also similar to the recently reported level o f nucleotide
84
secreted by colonic mucosa in vitro (Patel et al., 2011). The 2-thioUTP solution was
prepared in normal saline and sterilized by filtration (0.22 pm filter pores). The 2-thioUTP
solution or saline was injected in the distal colon up to the first curvature leading to the
transverse colon as we previously described (Grbic et al., 2011). Colitis was assessed by
clinical and histological scoring, as previously described (Cooper et al., 1993; Dielem an et
al., 1998; Grbic et al., 2011; Grbic et al., 2008). Spleen was harvested to measure bacterial
translocation. All procedures were approved by the Universit de Sherbrooke Animal Care
Committee and performed according to the Canadian Guidelines for Care and Use o f
Experimental Animals.
Bacterial translocation
Enteric bacterial translocation to spleen was assessed in C D -I mice that were
treated with 2-thioUTP after receiving 4% (w/v) DSS in their drinking water for 7 days.
The harvested tissues were removed under strictly aseptic conditions and were
homogenized in sterile PBS. Homogenates were plated on tryptic soy agar under aerobic
conditions and were allowed to grow for 24 h at 37C. CFUs were counted and expressed
as the LogioCFU/organ.
Histological analysis
Mice colon were fixed in 4% paraformaldehyde overnight at 4C, embedded in
paraffin, cut into 5-pm sections, and applied to Superfrost/Plus slides (Fisher Scientific), as
previously described (Grbic et al., 2008). Hematoxylin and eosin (H&E) staining was
performed by the QTRN/CToDE intestinal phenotyping platform from the Universit de
Sherbrooke. Images were captured on a Leica DMLB2 microscope using a Leica DC300
camera.
Statistical analysis
Results are expressed as the means standard error o f the mean (SE).
Statistical
significances were determined using ANOVA tests or as indicated in the figure legends.
The numbers o f replicates for each experiment is presented in the figure legends.
22. Results
P 2 Y 2 agonists stimulate IECs migration in vitro
85
As shown in
generated with the empty vector (EV), a non-targeting scrambled shRNA (shNT), or a
shRNA targeting the receptor. Neither the EV nor the shNT constructs affected the
expression o f P 2 Y 2 (Fig. ID). However, the expression o f shP2Y2-08 showed a 60%
decrease in receptor expression and was used in subsequent experiments (Fig. ID). The
migration o f IEC- 6 /shP 2 Y 2 cells in response to agonists was brought back to non
stimulated control levels (Fig. IE), thus clearly suggesting a role for P 2 Y 2 in IECs
migration.
86
B 1.6
S
1.6
il
11
S
r*
1.4
14
I1
fl 10 a . a
l . l
5 S 1.2
1.0
3 ? 0.8 t
S S 1.2
3 I 0.8
I 0.4
0.4
0.0
+ ATP
- Suramin
+ PPADS
0.0
UTP
- Suramin
+ PPADS
I S-actin
+ 2-thioUTP g
- Suramin
PPADS
ni
ATP
UTP
EV shNT 05
shP2Y,
EV
H shNT
sh P 2 Y 2
2-thioUTP
87
Ctrt
ATP
UTP 2-thioUTP
EV
shNT
shP2Y
ATP
UTP
2-thioUTP
88
89
~ 20
]5 1.8
**
g 16
S o 1.4
1.2
il
II
1.0
0.8
P E
?
it
0.4
00
Ctrl ATP UTP
"ST
Integrin a
(Vactin
> t 0.5
anti-ITAV
i A*
fi li
2 3
v co
O E 0.8
04
qxfxp
0.0
Ctrl ATP UTP
+
+ anti-a,,
- IgG
90
measured 16 hours after wounding. Results represent fold variation o f the means SE of
three separate experiments normalized to control. Statistical significance was determined
by one-way ANOVA in which *, p < 0.05 and **, p < 0.01 and ***, p < 0.001 as compared
to unstimulated cells.
91
(C). The acetylated form o f a-tubulin (a-T ub) was detected with an anti-acetyl-a-Tub
(Lys40) antibody (green filaments). Nuclei (blue) were counterstained with 4',6-diamidino2-phenylindole (DAPI). Presented results are typical immunofluorescence micrographs o f
two to three independent experiments. The original magnification was set to 40x and scale
bars = 50 pm.
92
LV294002
FIGURE 5. P2 Y2 promotes cell migration by activating the PI3-K signaling pathway and
the stabilization of microtubules.
IEC-6 cells were stimulated with 100 pM ATP (A) or UTP (B) for 0 (control), 5 ,1 5 , 30, 60
and 120 min. Akt phosphorylation was detected by Western blot analysis. Presented blots
are typical of three separate sets o f experiments. Indirect immunofluorescence o f acetyl-atubulin in IEC-6 cells stimulated with 100 pM ATP () or UTP (D) for 15 min in the
presence o f 20 pM LY294002 or in the absence of inhibitor (panels E and F). Unstimulated
control IEC-6 cells are presented on panel G. Original magnification o f 63x with scale bars
= 30 pm. (H) IEC-6 cell monolayers were pre-treated with 20 pM o f LY294002 for 30
min, wounded and either left unstimulated (ctrl) or stimulated with 100 pM o f ATP or
UTP, and the wound area was measured 16 hours after wounding. Results represent fold
variation of the means SE of three separate experiments normalized to control. Statistical
significance was determined by one-way ANOVA in which *, p < 0.05 and **, p < 0.01
and ***, p < 0 .001 as compared to unstimulated cells, and <(), p < 0.05 and <()<j>, p < 0.01 vs.
nucleotide stimulated cells.
93
phoipt-GSK3p
15
30
60
ATP (100 mM)
| GSK-3P
120 Time |mm)
phopfto-GSK3(S
15
30
60
UTP (100 |M )
| GSK-3P
120 Tin (min(
phoipxHiSK3p
GSK-30
Cttl
ATP
UTP
CW
ATP UTP
LV204002
94
to 100 pM ATP (Fig. 7C) or UTP (Fig. 7D) when compared to IEC-6 cells stimulated with
ATP (Fig. 7E) or UTP (Fig. 7F) alone.
pnospho-Akt
PTX
*
ii.
CM
ATP UTP
Ctrl
ATP UTP
PTX
95
2-thioUTP administrations during the remission phase o f mice suffering from colitis-like
disease improve recovery
Non-inflamed mice treated for three days with PBS enemas displayed typical colon
architecture with well define crypts and undisturbed surface epithelium (Fig. 8A-A'), as
shown by H&E staining. On the contrary, m ice suffering from D SS-induced colitis had
regions where the epithelium was completely absent (Fig. 8B) and where the crypt
architecture was lost (Fig. 8B'). As for the non-inflam ed mice treated with PBS enemas,
non-inflamed mice treated with 2-thioUTP also displayed normal colon architecture (Fig. 8
C -C ). Finally, inflamed mice treated with 2-thioUTP had regained part o f their epithelium
although crypts and surface epithelium were not as well defined (Fig. 8D-D').
Hence,
massive infiltrates o f immune cells were still visible in the colon o f inflamed mice treated
with the P2Y2 agonist (Fig. 8D, white arrow).
recovery significantly reduced the disease activity index (DAI) value as com pared to mice
receiving PBS enemas (Fig. 8E).
histological damages following treatment with 4% DSS prior to daily injections o f 2thioUTP for three days using the scoring chart described by Jeffers et al. (Jeffers et al.,
2002). In Fig. 8F, we showed that 2-thioUTP treated mice had a score significantly lower
than control PBS mice. As an indication o f IECs barrier function, we determined bacterial
translocation in spleen. We observed that bacterial translocation to the spleen (Fig. 8G)
was significantly reduced in mice treated with daily injections o f 2-thioU TP during
recovery as compared with non-treated mice.
96
pS^^W oUTP
P8S
P#
2-thoUTP
97
23. Discussion
IECs constitute a physical barrier to bacteria and other luminal contents that could
trigger an uncontrolled immune response (Blikslager et al., 2007). Following wounding,
cells located at the wound margin rapidly migrate to seal the epithelial defect and
reconstitute the barrier (Sturm and Dignass, 2008). In a recent study, it was shown that
inhibition o f P 2 Y 2 expression in vivo using siRNA applied to a model o f com eal wounding
delayed the restitution response to receptor agonists (Crooke et al., 2009). IEC -6 cell is an
accepted and validated in vitro model to study epithelial restitution as it gives reproducible
wounds.
proliferation at the wound edge even 24 h after wounding (M cCormack et al., 1992). Using
this cell model, it was previously reported that ATP stimulation increased the cell migration
rate when compared to control (Dignass, 2001). However, no mechanisms or any receptors
were identified as being involved in this process. The reported EC 50 values for the
recombinant P 2 Y 2 receptor range from 1.5 to 5.8 pM (Velazquez B. et al. 2000) and
although, 300 pM o f ATP or UTP elicited the maximal variation in intracellular calcium,
we chose the 100 pM concentration as the working concentration based on our previous
publication (Degagne et al., 2009; Langlois and Gendron, 2009). Our data clearly showed
that the most efficient 2-thioUTP-concentration stimulating cell migration was 10 pM.
Hence, we showed that P 2 Y 2 is involved in wound closure o f an IEC-6 monolayer. In fact,
the selective inhibition o f P 2 Y 2 expression by shRNA highlighted the important role o f this
receptor in cell migration and the wound healing processes triggered by ATP and UTP.
P 2 Y 2 cooperates with integrin a v through a RGD m otif found in the receptor's first
extracellular loop to promote cell migration (Erb et al., 2001). This cooperation results in
the recruitment o f Go (Bagchi et al., 2005), the clustering o f a v integrin and the activation
o f Mek/Erk pathway dependent on PI3K activity (Chm a et al., 2007). Integrin clustering
can be seen as focal adhesions in cells and these cellular entities act as a platform for
different signaling pathways involved in cell m igration (W elf et al., 2011). Using a RGD
blocking peptide and an anti-integrin a v neutralizing antibody, we demonstrated that P 2 Y 2induced cell migration required the coordinate action o f integrin a v and P 2 Y 2 .
The
reduction o f a v integrin subunit expression following treatm ent with the integrin blocking
98
antibody is in accordance with the literature (Chm a et al., 2007). Using U-937 cell line,
Chm a et al. demonstrated that incubation o f these cells for 24 h with a v integrin blocking
antibody leads to the internalized o f this integrin, resulting in a decrease o f integrin
expression at the cell surface by 35% (C hm a et al., 2007).
We report here a novel mechanism by which P 2 Y 2 enhances IECs m igration that
involves the stabilization o f MT through the stimulation o f a-Tub actylation.
In fact,
recent studies have showed that the stabilization o f MT requires the coordinate actions o f
microtubule-associated proteins to catalyze the actylation o f a-T ub and thus promote cell
migration (Creppe et al., 2009; Etienne-M anneville, 2010; Gundersen and Bulinski, 1988).
MT stabilization is regulated by numerous factors, but GSK3(3 seems central to this
process. Active GSK3P was reported to inhibit MT stabilization by dow n-regulating MTassociated protein CRMP2 activity and by negatively regulating Tau (Sun et al., 2009).
GSK3P activity is inhibited by phosphorylation o f serine 9 residue found in its N-terminal
domain. Consequently, inhibition o f GSK3P results in Tau and CRMP2 activation, as well
as the subsequent elongation and stabilization o f MT. We showed that P 2 Y 2 activation
leads to an Akt-dependent GSK3P phosphorylation, ensuing actylation o f a-tubulin on
Lys40 in IEC -6 cells. These findings are not only in accordance with the role o f Akt and
MT stabilization in cell migration (Creppe et al., 2009; Etienne-M anneville, 2010), but
most interestingly propose that P 2 Y 2 is involved in the control o f MT dynamics.
Furthermore, indirect immunofluorescence for Ac-a-Tub in IEC - 6 cells pretreated with
PTX showed a reduction in the actylation o f a-Tub induced by ATP and UTP (Fig. 7). On
the other hand, the presence of PTX prior to ATP and UTP stimulation did not influence
the number o f vinculin positive FA (data not shown). These results are thus suggesting that
activation o f a PTX-sensitive Go protein is responsible for the increase in the acetylated
form o f a-Tub.
The maintenance o f the intestinal epithelium integrity is a necessity to prevent the
translocation o f bacteria or entry o f luminal molecules that could trigger an excessive and
uncontrolled immune response and lost o f tissue architecture and function.
Here, we
reported that following P 2 Y 2 activation there is an increase in the acetylated form o f a-T u b
and in the number o f FA. Both a-T ub actylation and FA are essential to IECs migration.
99
FA are composed in part o f integrin a vP3 and other signaling molecules, like vinculin, FAK
and Src, that are involved in signaling cascades responsible for the attachm ent o f migrating
cells to the extracellular matrix (Hamadi et al., 2009).
contribution o f MT to IECs migration is not well defined. In the course o f cell migration,
there is a reorientation o f the microtubule organizing center towards the leading edge that
allows the asymmetrical polarization and elongation o f microtubule to the m igrating front
(Kaverina et al., 1998; Wehrle-Haller and Imhof, 2003). M T are then pulled at their plus
ends by plus-end tracking proteins and stabilized by protein like CLIP (Schober et al.,
2007). MT at the leading edge can thus deliver important signaling m olecules necessary to
cell migration. In fact, disruption o f a-T ub actylation results in a significant decreased in
the binding and motility o f kinesin-1 (Reed et al., 2006).
transport o f cargo towards the MT plus-end. M oreover, it was showed that Necl-5 interacts
with PDGF receptor and intregrin a vP3 at the leading edge o f NIH3T3 to promote
directional cell migration (Minami et al., 2010; Minami et al., 2007). They have showed
that activation o f Necl-5 allows the growth and attraction o f microtubules at the leading
edge in an intregrin a vP3-dependant manner.
increasing the dose o f 2-thioUTP or the length o f treatment we could favor the healing
process.
indicators that 2-thioUTP treatments favored the reepithelization process, since bacterial
100
translocation can be the result o f increased barrier permeability, as observed in IBDs (Berg,
1999).
In this study, we provided evidence that activation o f P2Y 2 during the remission
phase following acute inflammation could facilitate wound healing. Therefore, not only
have we described a novel function for this receptor in IECs, we are also the first to show
that P2Y 2 could modulate M T dynamics. Regulation o f M T dynamics is essential for
wound closure (Etienne-M anneville, 2010), but deregulation o f this phenom enon is also
linked to cancer progression (Etienne-M anneville, 2010). In fact, as proposed by our in
vivo experiments, our findings could lead to the development o f novel therapies aimed at
stimulating intestinal wound healing, which could represent a step forward in the treatm ent
o f IBDs (Ardizzone et al., 2011; Pineton de Chambrun et al., 2010).
101
UTP
(nucleolideal (pM)
0.1
(2-ttiioUTP)
102
25. Acknowledgments
We thank Gabriel Mitchell from the department o f Biology from the Universit de
Sherbrooke for his technical help with the bacterial translocation studies. This research was
supported by the Crohns and Colitis Foundation o f Canada Grant in Aid o f Research
(2009-2012) to F.P.G. F.P.G. is member o f the FRSQ-funded Centre de Recherche
Clinique Etienne-Le Bel. D.M.G. is a recipient o f an Alexander Graham Bell scholarship
from the Natural Sciences and Engineering Research Council o f Canada.
Disclosure: The authors have no conflict of interest to disclose.
103
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CHAPITRE 3
108
109
Abbreviations:
C/EBPp,
CCAAT/enhancer-binding
protein
P;
ChIP,
chromatin
Key Words: C/EBP, colitis, gene regulation, intestinal mucosa, P 2 Y 2 receptor, intestinal
epithelial cells.
110
28. Summary
Inflammatory bowel diseases (IBD) are characterized by relapses and rem ission periods
during which numerous factors, including stress factors such as nucleotides, are mobilized
to reestablish intestinal mucosal homeostasis.
We thus
Ill
29. Introduction
Crohn's disease and ulcerative colitis are chronic and relapsing inflammatory bowel
diseases (IBD) [1] caused, among others, by intestinal epithelial cell (IECs) and immune
cell dysfunction. These impaired or abnormal mucosal immune responses lead to epithelial
breaches potentially harmful [2]. Usually, the epithelial regenerative capacity m aintains the
integrity o f the intestinal mucosal barrier even after extensive damages [3].
However,
during inflammation associated with IBD, repair o f the epithelial m onolayer is impaired,
leading to crypt abscess formation, uncontrolled bacterial infiltration, exacerbated
inflammatory responses and loss o f tissue architecture and functions [3, 4],
As a
epithelial barrier breach may result in exacerbated and uncontrolled immune responses,
leading to IBD [1]. In contrast, a limited and controlled immune response will regulate
foreign pathogen and noxious agent entry, and will favor wound healing and rem ission [ 1 ,
4, 8 ].
namely ATP, UTP and diphosphate derivatives, have recently been described as dangerassociated molecular pattern molecules [7, 9, 10] and as immunodepressants [11]. These
labile molecules can either stimulate the inflammatory response or act as anti-inflamm atory
and pro-healing mediators [11-14].
nucleotides depends not only on the microenvironment, but also on the P2 receptor subtype
activated [7, 9, 11-13].
P2 receptor subtypes include P2X ATP-gated ion channels and P2Y G protein-coupled
receptors [15, 16].
equipotently by extracellular ATP and UTP [17]. P 2 Y 2 activation regulates among others,
cell proliferation, inflammation and angiogenesis [17-19], Signaling through Gq-coupled
P 2 Y 2 involves phosphatidylinositol-4, 5-bisphosphate hydrolysis by phospholipase C
(PLC), thus generating inositol 1, 4, 5-trisphosphate (IP 3) and diacylglycerol [20].
IP 3
increases the concentration o f intracellular calcium and triggers calcium -dependent cell
responses, such as the activation o f protein kinase C (PKC) [20].
MAPK activity regulates various cellular processes, notably migration and proliferation. In
112
addition to MAPK, P 2 Y 2 stimulates PI3K/Akt activity through a PLC/IP 3/Ca2+ and PKCdependent pathway [2 1 ]. P2 Y 2 is also coupled to integrins a vp3 and a vp5 via binding to a
RGD integrin-binding domain in the first extracellular loop [22, 23]. P 2 Y 2 and a v integrin
subunit interaction is necessary for Go recruitment and the initiation o f Go-mediated
signaling events leading to cell migration [22].
activation increases the rate o f IEC migration in an integrin a v-dependent m anner that
translated into an improvement in the rem ission phase o f mice suffering from chemically
induced colitis [14],
Furthermore, P 2 Y2 expression is increased in the colonic tissue o f IBD patients and in a
mouse model o f chemically induced colitis [7].
demonstrate that NF-kB p65 cooperates with C/EBPP to transactivate the P 2Y i promoter.
30. Results
C/EBPp regulates
2 Y 2 expression
Following our studies on the regulation o f P 2 Y 2 by NF-kB [12], we have identified putative
C/EBP DNA-binding sites by TRANSFAC analysis.
113
expression more than 7-fold as compared to control, as assessed by luciferase assays using
the cloned proximal human P 2 Y2 promoter in the luciferase-containing pGL4.10 vector
[12] (Fig. IB). To determine the C/EBP DNA-binding site involved, we replaced the three
motifs by inserting scrambled CEBP1, CEBP2, and CEBP3 sequences (Fig. 1C). While
mutation o f the CEBP2 and CEBP3 sites did not affect luciferase activity, m utation o f the
CEBP1 site significantly reduced luciferase activity o f the minimal P2Y2A350-Luc
promoter construct by more than 50% (Fig. 1C). We confirmed by electrophoretic mobility
and supershift assays with HEK 293T C/EBPP overexpressing nuclear extracts, C/EBP
binding to the CEBP1 site, and the presence o f the C/EBPP isoform (Fig. ID). This result
was verified by the absence o f C/EBPp binding to a mutated CEBP1 double-stranded
oligonucleotide. These results indicate that CEBP1 ~229GTT GCA GCA C 220 is a potential
binding site required for the transcriptional regulation o f P 2 Y2 expression by C/EBPp.
114
P2Y..V50
_350bf>
-l?72 bp
-lioO bp
-sltO
-400 bp
CKBP2
CEBPl
-7* bp 1 0 - 6 9 bp
-229 bp to - 720 bp
bp O ^Obp
-200 bp
-UK) bp
t T Hp\
bp to - 48 bp
11 bp
*93 bp
~ 40-,
+
+
350
D
antbC/EBPfl
Nuclear extract
pcDNA 3 1
pGL4 10
pcONA31-C/EBPp
pGL4 10-prP2Y.
CBP2
C/EBP3 mutCBPI
+ . . +
115
experiments done in triplicate. Statistical analysis was performed by a one way ANOVA,
where *p < 0.05 and **p < 0.01, as compared to respective controls and as indicated on the
figure. (D) Nuclear extracts from HEK 293T cells were incubated with the putative [y32P]ATP-labeled C/EBP(3 DNA-binding site probes CEBP1, CEBP2, CEBP3 or C E B Plm ut
and anti-C/EBPp antibodies for electrophoretic mobility and supershift assays. DNAprotein complexes were separated from the free probe on a native polyacrylam ide gel.
C/EBPP DNA-binding and supershifted complexes are indicated by the bracket and the
supershifted complex by the star.
Results are representative o f three independent
experiments.
C/EBPp regulates
P 2Y2
to mechanical stress
Caco-2 cells were treated with pro-inflammatory cytokines IFN-y, lipopolysaccharides
(LPS) (Fig. 2A) and dextran sulfate (DSS) (Fig. 2B).
subjected to mechanical stress by using a scratch wound model (Fig. 2C). It is known that
IFN-y and LPS are inflammatory mediators activating C/EBPp [24], Stimulation for 6 h
with IFN-y or LPS, o f Caco-2 cells transfected with the C/EBPp expression-vector and the
P 2 Y2-Luc promoter constructs, led to significantly increased transcription o f P 2 T? when
compared to unstimulated controls, respectivelyl.5 0.07 fold (p < 0.01) for IFN- y, and
1.4 0.03-fold (p < 0.001) for LPS (Fig. 2A). Again, treatment with DSS, a chemical
agent used in vivo to induce colitis in mice, increased P2Y2 expression by a 3.0 0.22-fold
(p < 0.001) (Fig. 2B). Interestingly, Caco-2 cell m onolayer wounding led to a significant
increase o f C/EBPP binding to the P2 Y2 promoter. Indeed, as showed in Fig. 2C, C/EBPP
was associated with the P 2 Y2 prom oter region in unwounded Caco-2 cells. However, 6 h
after wounding, there was a significant increase in the level o f C/EBPP associated with the
P 2 Y2 promoter region when compared to unwounded control (Fig. 2C).
2 Y 2 transcription
We have previously identified a NF-kB p65 binding site in the P 2 Y2 prom oter [12]. This
binding m otif was located between base pairs -181 to -172 [12], proximal to the CEBP1
site (-229 bp to -220 bp) identified in this study.
116
cells with expression vectors for both transcription factors and performed luciferase assays.
As shown in Fig. 3A, N F-kB p65 and C/EBPp upregulated P 22 expression by 10- and 25fold, respectively. However, when both transcription factors were co-expressed, luciferase
activity was increased more than 45-fold (Fig. 3A).
cooperation between N F-kB and C/EBPp in the regulation o f P22 expression. Indeed, reChlP assays o f Caco-2 chromatin extracts, in which the first immunoprcipitation was
performed with an NF-kB p65 antibody followed by a second immunoprcipitation o f the
eluted NF-kB p65 complex with an antibody against C/EBPp, show that both transcription
factors occupied the P 2 Y2 promoter region between -221 bp to -105 bp (Fig. 3B).
A
.A
_______
pGL4 10
P2Y:-Luc
C/EBPp
anti-C/EBPp
Wound
117
incubated with (A) 500 ng/ml IFN-y or 12.5 ng/m l LPS or (B) with 0.5% DSS for 6 h and
luciferase activity determined. Luciferase activity is expressed as the fold-increase relative
to the activity o f the empty vectors. Data are the means SEM o f results from at least 4
separate experiments done in triplicate. Statistical analysis was perform ed by one-way
ANOVA, where **p < 0.01 and ***p < 0.001, as compared to respective controls, and
where <t><[><|) p< 0.001 vs. non-DSS treated cells. (C) Caco-2 cell monolayers were wounded
with a pipet tip and ChIP assays were perform ed 6 h after wounding using an anti-C/EBPp
antibody or normal rabbit IgG antibody.
Quantification o f sam ples was done by
quantitative real-time RT-PCR analysis with oligonucleotides amplifying the -221 bp to 155 bp region o f the P2Y2 promoter, and was expressed as the fold-increase over negative
control normalized to input. Results are presented as the mean SEM o f 3 independent
experiments. Statistical significance was determined by an unpaired t-test, where * p <
0.05 and*** p < 0.001.
C/EBPp regulates
P 2Y 2
expression
in v iv o
CD-I mice were treated for 7 days with 4% DSS in drinking water. First, we showed that
C/EBPP protein expression was absent in C/EBP'7' murine colon (Supplem ental Fig. SI).
In response to DSS treatment, C/EBPp protein levels were increased in wild-type colonic
epithelial
cells,
as
assessed
by
W estern
blot
analysis
(Supplemental
Fig.
SI).
Staining was
specific since the non-immune controls were negative (Fig. 4E, 4F). To determine whether
P 2 Y 2 expression, previously found to be induced in IBD [7, 12], was regulated in part by
C/EBPs, we isolated total RNAs from colonic epithelial cells o f w ild-type and C /EB Pp7'
mice treated with or without DSS. Real time quantitative PCR analysis showed similar
levels o f P2Y2 expression in non-treated wild-type and C/EBPp7' mice.
However, P2Y2
expression was significantly reduced in C /EB Pp7' mice treated with DSS, as opposed to
control mice (Fig. 4G), suggesting that C/EBPp indeed regulates P2Y2 receptor expression
in inflamed IECs.
118
+
B
NF-*B pp65
NF-B
+ C/EBPp
0
Ctrl
w-ChlP
Fig. 3. C/EBPP and NF-kB p65 cooperate to induce P 2 Y2 transcription. (A) HEK 293T
cells were transiently transfected with the P 2 Y2 full-length prom oter-luciferase construct
(pGL 4 . 1 0 -prP 2 Y 2) with or without the C/EBPP and NF-kB p65-expressing vectors.
Luciferase activity was assayed 48 h after transfection. Luciferase activity is expressed as
the fold-increase relative to the activity o f the empty vectors. Data are the means SEM o f
results from at least 4 separate experiments done in triplicate. Statistical analysis was
performed by one-way ANOVA, where **p < 0.01 and ***p < 0.001, as compared to
controls. (B) Re-ChIP assay was perform ed with anti-C/EBPp antibody following the first
immunoprcipitation with anti-NF-xB p65 antibody. Samples were verified by quantitative
RT-PCR analysis with oligonucleotides amplifying the -221 bp to -105 bp region o f the
P 2 Y2 promoter and expressed as fold-increase over negative control normalized to input.
Representative results from 3 independent experiments are shown.
31. Discussion
We have previously shown that P 2 Y 2 receptor expression is increased in IBD [7,
12]. We have recently reported that NF-kB p65 is an important regulator o f P2Y:
transcription in IECs, in response to inflammation [12]. Beside NF-kB p65, C/EBPp is also
an important modulator o f intestinal inflammation, by regulating among others acute phase
protein expression [25-27], We have found that P 2 Y 2 is a novel C/EBPP target. Indeed, we
have identified a functional C/EBP DNA-binding site in the P 2 Y2 promoter, and we have
shown that C/EBPp transmits inflammatory signals to increase P 2 Y2 expression, from
cytokines such as IFN-y, from bacterial products such as LPS, from epithelial injury
increased C/EBPP and P2Y2 expression in colonic cells from DSS-treated mice. Indeed, the
absence of C/EBPp in C/EBPP'A mice reduces dramatically P2Y2 induction during DSSinduced colitis. Interestingly, both C/EBPp and NF-kB p65 synergize to transactivate P2Y'2,
and both bind simultaneously the P2Y2 promoter, with a close proximity o f the NF-kB p65
consensus motif [12] and the C/EBPp DNA-binding domain identified in this study. It is
possible that the synergy also depends on direct interactions between C/EBPp and NF-kB
p65, as proposed in other cell systems [29-31], as well as close recruitment. The exact
molecular interactions between C/EBPp and NF-kB p65 that allow the synergistic
transactivation of the P2 Y2 promoter remain to be determined.
wr
CEBPfP
WT
CEBP0'
OSS
120
C/EBPP is located in epithelial cell nuclei o f the colonic gland. (B) N uclear DAPI staining
o f colonic tissue. (C) CD-I mice were treated with 4% DSS in their drinking water for 7
days and C/EBPP expression was determined by indirect immunofluorescence. C/EBPP
expression is increased both in the nuclear as well as in the cytoplasmic compartments. (D)
N uclear DAPI staining o f DSS treated colonic tissue. (E-F) Non-immune control for
C/EBP staining and normal nuclear DAPI staining. M icrographs show typical results for at
least 4 different animals. (G) P22 mRNA expression was determined by quantitative RTPCR o f mRNAs obtained from DSS-treated wild-type or C/EBPP knock-out (C /EB Pp'7')
enriched colonic epithelial cells. Data are the means SEM o f results from at least 4
animals per group. Statistical analysis was perform ed by a One-way ANOVA, where *p <
0.05; **p < 0.01 and *** p < 0.001 as compared to controls.
In addition to the regulation o f pro-inflammatory gene expression, C/EBPP is also
involved in cell migration, as showed by his reported role in the mediation o f keratinocyte
growth factor-induced epidermis migration during wound healing [32] and in the promotion
o f melanocyte motility [33], C/EBPp also regulates the expression o f anti-inflamm atory
cytokines such as IL-24, which suppresses mucosal inflammation in IBD [28].
It thus
recently showed that P 2 Y 2 activation increases intestinal epithelial cell migration in vitro,
and the stimulation o f the rate o f remission o f mice suffering from DSS-induced colitis
[14]. In fact, in IBD, there are increasing evidences suggesting that anti-inflamm atory and
pro-healing responses are activated even in the presence o f acute inflammation, in order to
control the invasion o f pathogenic components and to rapidly reseal the mucosal breach [ 1,
34], In this context, C/EBPP may be a key player in the establishment o f an appropriate
intestinal mucosal response by regulating the expression o f pro-inflammatory genes
necessary to the recruitment o f immune cells at the site o f inflammation [24], and by
stimulating the expression anti-inflammatory and pro-healing components, such as IL-24
[28] and P 2 Y 2 receptor.
Reagents
Dulbeccos modified Eagle medium (DM EM ), penicillin-streptomycin, HEPES, and FBS
were purchased from W isent (St. Bruno, QC, Canada).
121
inactivated by heating at 50C for 60 min. Opti-M EM , GlutaMax, LipofectAM INE 2000
and Alexa Fluor 488 goat anti-rabbit IgG (H+L) were from Invitrogen Life Technologies
(Burlington, ON, Canada). Dextran sulfate sodium (DSS; Mr 36,000-50,000) was obtained
from MP Biochemicals (Solon, OH). The pGL4.10 and pcDNA3.1 vectors were purchased
from Promega (Madison, WI) and Invitrogen Life Technologies, respectively.
LPS
( Escherichia coli 0 5 5 :B5) and IFN-y were purchased from EMD (M ississauga, ON,
Canada)
and
BioShop
Canada
(Burlington,
ON,
Canada),
respectively.
The
kindly provided by Dr. Marc Servant (Universit de M ontral, Facult de Pharmacie, QC,
Canada).
The rabbit anti-CEBPp and the mouse anti-NF-KB p65 were purchased from
(clone C4), DAPI and ECL reagent were purchased from M illipore (M ississauga, ON,
Canada).
Cell culture
The human colon carcinoma cell line Caco-2 (ATCC, HTB-37) and the human embryonic
kidney cell line HEK 293T (ATCC, C R L -11268) were grown as previously described [12].
Cells were incubated in serum free medium for 24 h at 37C before experiments.
122
buffered saline (PBS) and used either for paraffin embedding and immunofluorescence
studies or epithelial cell enrichment and RNA isolation. All mice were maintained on a
12:12-h light-dark cycle at 23C, were given standard lab chow and had food and w ater ad
libitum.
Committee and performed according to the Canadian Guidelines for Care and Use o f
Experimental Animals.
Indirect immunofluorescence
Deparaffmized and rehydrated slides were subjected to microwave antigen retrieval as
previously described [7]. Slides were washed in PBS, and then blocked with 2% BSA in
PBS for 20 min at RT. The rabbit anti-C/EBPP (2 pg/pl) antibody was diluted 1:100 in
PBS containing 0.1% BSA and 0.2% Triton X-100 (PBT) and incubated with the sections
overnight at 4C. Slides were washed in PBS and incubated with Alexa Fluor 488 donkey
anti-rabbit IgG in PBT for 2 h at room temperature. Slides were washed in PBS, mounted
and images were captured on a Leica DMLB2 microscope using a Leica DC300 camera.
Western blots
Colons were excised from C/EBP' ' and wild-type control m ice and washed with ice-cold
PBS. Matrisperse-isolated intestinal epithelial cells [36] were homogenized in Triton buffer
(40 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.2 mM sodium
orthovanadate, 40 mM P-glycerophosphate, 0.1 mM PMSF, and protease inhibitor mixture
from
Sigma-Aldrich)
and
samples
were
processed
as
previously
described
[7].
Immunoblotting for C/EBPP expression was performed with a 1:1,000 dilution o f rabbit
anti-C/EBPp antibody. Specific protein bands were detected using a 1:10,000 dilution o f
HRP-conjugated anti-rabbit IgG and visualized on autoradiographic film using the
Millipore chemiluminescence system. Signal normalization was realized as described with
an anti-P-actin antibody [7],
123
Life
Technologies, Burlington, ON, Canada) from murine colonic epithelial cells enriched with
BD cell recovery solution (BD Biosciences, M ississauga, ON, Canada) or from Caco-2
cells stimulated with 100 pM ATP or UTP. Complementary DNA was then synthesized
from 2 pg o f purified RNA by reverse transcription using the Superscript II system
(Invitrogen Life Technologies, Burlington, ON, Canada). Five percent o f the synthesized
cDNA was used as a template for real-time PCR using the Brilliant III Ultra-fast SYBR
Green QPCR M aster Mix (Agilent Technologies, Mississauga, ON, Canada).
The
sequence-specific primers for mouse P2Y2 were 5- GGC A AC AGC ACG TAC TTG AA3 and 5-CAG GCC TGT GCA TAT GTG A G -3.
The
upstream amplification was performed with the prP 2 Y 2/N h eI oligonucleotide prim er (5GCT AGC CAG GAA TGC AGT TTC CTC AGC TG -3) and the prP 2 Y 2A C E B P lR (5GTC TGT ACC GTC ATA ACG CGT GGA GGG TC G -3), prP2Y2ACEBP2R (5-TCC
CTG CGC TTA CCT ACG CGT CCG AAG C CG -3), or prP 2 Y 2ACEBP3R (5-CTG GGA
GCC CCC TAG GCT GGA TCC CTG C G C-3) primers. The downstream amplification
was performed with the prP 2Y2A C E B P lF (5 -CGA CCC TCC ACG CGT TAT GAC GGT
ACA GAC-3), prP 2Y2ACEBP2F (5 -CGG CTT CGG ACG CGT AGG TAA GCG CAG
GGA-3), or prP 2Y2ACEBP3F (5-GCG CAG GGA TCC AGC CTA GGG GGC TCC
CAG-3) and the prP2Y2/B glII (5-AGA TCT CGC CTT GTT CCC TCC A G A -3)
primers. Products resulting from these two PCRs were used as DNA tem plates for the final
PCR using the prP 2Y2/N hel and prP 2Y2/B glII oligonucleotides. The PCR fragments were
then cloned into the pGL4.10 luciferase vector. The presence o f the mutations was verified
124
125
to -1 0 5 bp (5-ACC GGT ACA GAC ACG CTG A C -3 and 5- GGC GGA CTT TTG
ACT GAC C-3). Data are expressed as fold increase over background (negative control)
normalized to input, as previously described [ 1 2 ].
Statistical analysis
Results are expressed as the mean SEM.
performing unpaired t or ANOVA tests. The num ber o f replicates for each experiment is
presented in figure legends.
126
33. Acknowledgements
This research was supported by a Crohns and Colitis Foundation o f Canada Grant in Aid
o f Research (2009-2012) to F.P.G. and a C rohns and Colitis Foundation o f Canada Grant
in Aid o f Research (2011-2014) to C.A. and F.P.G. F.P.G. and C.A. are m em bers o f the
FRQ-S-funded Centre de Recherche Clinique tienne-Le Bel.
127
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to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP1-dependent manner in epithelial cells, Inflammatory bowel diseases.
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stimulating the epithelial cell P2Y(2) receptor, European jo u rn a l o f immunology. 39, 2895905.
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increased by NF-kappa B and stimulates cyclooxygenase-2 expression and PGE2 released
by intestinal epithelial cells, J Immunol. 183, 4521-9.
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pathways paves the way for drug discovery, Curr Opin Pharmacol. 9, 445-6.
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P. (2012) P2Y2 receptor promotes intestinal epithelial cell migration through a Go protein
and integrin av pathway allowing microtubule stabilization and mucosal re-epithelization in
experimental colitis, J Cell Physiol, (in press).
15. Abbracchio, M. P. & Bumstock, G. (1994) Purinoceptors: are there families o f P2X
and P2Y purinoceptors?, Pharmacol Ther. 64, 445-75.
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Naunyn Schmiedebergs Arch Pharmacol. 362, 310-23.
17. Abbracchio, M. P., Bumstock, G., Boeynaems, J. M., Barnard, E. A., Boyer, J. L.,
Kennedy, C., Knight, G. E., Fumagalli, M., Gachet, C., Jacobson, K. A. & W eisman, G. A.
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on CD44v6 regulated via NF-kappa B, Egr-1, and C/EBP-beta, J Invest Dermatol. 130,
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34. Mammen, J. M. & Matthews, J. B. (2003) Mucosal repair in the gastrointestinal tract,
Crit Care Med. 31, S532-7.
35. Stemeck, E., Tessarollo, L. & Johnson, P. F. (1997) An essential role for C/EBPbeta in
female reproduction, Genes Dev. 11, 2153-62.
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human intestinal epithelial cells, Exp Cell Res. 245, 34-42.
C/EBPp (LIP)
p-acPn
CBPp - CBPP"
D ISC U SSIO N
132
analyse bioinformatique, nous avons identifi trois sites de liaison potentiels pour le facteur
de transcription S p l. La mutagense dirige de chacun de ces sites seul ou en combinaison
pourrait nous indiquer limportance de ce facteur de transcription dans la rgulation de
lexpression du rcepteur P 2 Y 2. Ainsi nous pourrions nous attendre que la m utation des
sites de liaison pour Spl abolisse la transactivation basale du promoteur du rcepteur P2Y2.
De plus, Spl peut interagir directement avec NF-icB/p65 et entraner la transactivation des
promoteurs (Pazin et al., 1996; Perkins et al., 1994; Perkins et al., 1993). Puisque nous
avons montr que NF-icB/p65 peut transactiver le prom oteur du rcepteur P2Y2, nous
pourrions dterminer leffet de la cotransfection de ces deux facteurs de transcription sur la
transactivation du promoteur ainsi nous pourions peut-tre observer une synergie de la
transactivation du promoteur du rcepteur P2Y2.
Chez les patients atteints de maladies inflammatoires intestinales, linteraction entre
les cellules prsentatrices d antignes et la microflore locale contribue lactivation
incontrle des lymphocytes T CD4+ de la muqueuse entranant la relche de cytokines
pro-inflammatoires comme lIL- 6 , lIL-12, lIL-23, lIL-27 et lIL-17 (Derer et al., 2012;
M udter et Neurath, 2007; Perrier et Rutgeerts, 2011). Une tude prcdente de notre
laboratoire a montr que lexpression du rcepteur P2Y 2 tait augmente en condition
d inflammation intestinale (Grbic et al., 2008). Nous avons donc valid, laide de modles
cellulaires, ces observations en stimulant les cellules IEC - 6 et Caco-2 laide de la
cytokine pro-inflammatoires IL- 6 , le lipopolysaccharide (LPS) qui est une composante
antignique des parois bactriennes, et le DSS, une molcule utilise in vivo pour induire
des colites chimiques murines. Par cette approche, nous avons montr q u un stress
inflammatoire amenait la transactivation du prom oteur du rcepteur P2Y 2 et que cette
rgulation tait ralise par les facteurs de transcription C/EBPP et NF-icB/p65. Ces
rsultats taient les premiers, non seulement identifier et caractriser le prom oteur de ce
rcepteur mais aussi les premiers identifier formellement un mcanisme m olculaire et
cellulaire responsable de la rgulation de lexpression du rcepteur P2Y 2 et des rcepteurs
P2 en gnral. Bien que linteraction C\EBPp-NF-icB tait connu dans plusieurs types
cellulaires (Stein et al., 1993), mes travaux de recherche ont mis en lumire que ces deux
facteurs de transcription agissaient en synergie pour induire la transcription du rcepteur
P2Y 2 dans les CEI. En fait, les sites de liaison que nous avons identifis pour chacun de ces
133
facteurs de transcription sont situs moins de 40 bp lun de lautre. Il est donc possible,
que leffet synergique observ rsulte de la formation d un dimre N F-kB-C/EBPP au
niveau du promoteur comme il a t propos dans certaines tudes (Cha-M olstad et al.,
2000; M ukeijee et al., 2007). Les domaines protiques impliqus dans la dim risation de
ces deux facteurs pourraient comprendre le dom aine d homologie Rel de NF-icB/p65 et la
rgion bZIP de C/EBPj3 (M ukeijee et al., 2007). Cependant, une autre tude propose que la
formation de dimre NF-kB-C/EBPP
domaine bZIP de C/EBPP (Khanjani et al., 2011). Il serait donc intressant de faire des
essais lucifrase en cotransfectant des constructions de ces deux facteurs de transcription
dont certains domaines auront t enlevs afin caractriser linteraction entre les deux
protines ncessaires la transactivation en synergie du promoteur du rcepteur P2Y i- De
plus, deux tudes ont rapport quun seul site de liaison pour C/EBP tait suffisant pour sa
coopration avec NF-kB et quun dimre de C/EBPP une fois li son site de liaison
lADN pouvait favoriser le recrutement de NF-kB son propre site de liaison (Cha-M olstad
et al., 2000; Mukerjee et al., 2007). Ainsi, il est possible que la synergie observe dans la
transactivation du promoteur du rcepteur P 2 Y 2 ne soit pas d la formation d un dimre
entre NF-kB et C/EBPP mais bien par un recrutement facilit, par exem ple de NF-kB,
suivant la liaison du dimre de C/EBPp.
Les facteurs de transcription de la famille des C/EBP reconnaissent tous la mme
squence concensus sur lADN (Ramji et Foka, 2002). Ainsi, on pourrait supposer que le
site de liaison de C/EBPp identifi sur le promoteur du rcepteur P2 Y 2 pourrait lier d autres
membres de cette famille. Parmi ceux-ci les facteurs de transcription C /EB Pa et C/EBP 8
seraient de bons candidats puisquils rgulent lexpression de gnes lis linflammation
dont des protines de la phase aigu (Boudreau et al., 1998; Dinic et al., 2005). Des
rsultats non publis ont dmontr autant en condition basale que suivant un traitement
avec le DSS que le facteur de transcription C /EB Pa transactive le prom oteur du rcepteur
P2Y 2 tel que dtermin par essais lucifrase (Voir annexe 3). Alors que le facteur de
transcription C/EBP 8 trnsactive la rgion du promoteur du rcepteur P2Y 2 seulement
aprs un traitement avec le DSS. (Voir annexe 3). Turgeon et collaborateurs ont dmontr
quen condition basale le facteur de transcription C/EBP 8 pouvait interargir avec lhistone
dactylase HDAC1 via le domaine bZIP de C/EBP 8 (Turgeon et al., 2008). Ceci
134
135
Bien que nous n ayons pas abord cette hypothse dans les articles publis, il n est
pas exclure que la rgulation de lexpression du rcepteur P 2 Y 2 pourrait tre influence
par les micros ARN (miARN). Il est connu dans la littrature que lexpression d autres
membres de la famille des rcepteurs P2, soient les rcepteurs P2X7 et P 2 Y i 2, peut tre
rgule par des miARN (Landry et al., 2009; Rahman et al., 2010). De plus, de rcentes
tudes ont dmontr quil y a un profil d expression distinct de miRNA dans les cellules
pithliales intestinales de patients atteints de maladie de Crohn et de colite ulcreuse
comparativement aux tissus sains (Wu et al., 2011; Wu et al., 2010; Wu et al., 2008). Par
ailleurs, une analyse de la rgion 3 UTR de P 2 Y 2
136
intgrines et le rcepteur P 2 Y 2 fut dmontre par les travaux de Erb et collaborateurs (Erb
et al., 2001) et ceux de Bagchi et collaborateurs (Bagchi et al., 2005). Cependant, ce type
interaction n a jam ais t dmontre dans un systme dont lexpression du rcepteur P 2 Y 2
tait endogne et encore moins dans les cellules pithliales intestinales. Nous pourrions
donc dterminer par immunoprcipitation linteraction physique du rcepteur P 2 Y 2 avec la
sous-unit a v de lintgrine. Par la suite, nous pourrions tablir plus en dtails le rle de la
stabilisation des microtubules dans la migration cellulaire des cellules pithliales
intestinales. Nous pourrions tout d abord nous intresser la dtyrosination de la tubuline
qui est une modification post-traductionnelle qui augmente la stabilit des m icrotubules et
favorise la migration cellulaire dans diffrents types cellulaires comme les fibroblastes
(Goulimari et al., 2005; Gundersen et Bulinski, 1988; Palazzo et al., 2004). Ainsi, nous
pourrions raliser des immunofluorescences suivant la stimulation du rcepteur P 2 Y 2 avec
ses agonistes en utilisant un anticorps dirig contre la forme dtyrosine de la tubuline.
Finalement, nous pourrions dmontrer limpact rel de la stabilisation des m icrotubules sur
137
138
139
des centrosomes qui servent de site pour la nuclation des microtubules (Alberts et al.,
2008). On compte deux centrosomes dans la cellule en division et ils sont localiss
chacun des ples de la cellule (Alberts et al., 2008). Ainsi, on retrouvera trois classes de
microtubules. Tout d abord les microtubules axiaux qui radient vers le cortex de la cellule,
les microtubules interpolaires qui interagissent avec les microtubules de lautre ple et
finalement les microtubules relis aux kintochores qui leur permettent de sattacher la
chromatide sur et de les aligner sur la plaque mitotique (Alberts et al., 2008). Les
microtubules reprsentent donc des cibles thrapeutiques de choix de lindustrie
pharmaceutique dans le traitement des cancers puisque la dstabilisation des microtubules
aura pour effet d empcher la formation des fuseaux mitotiques et ultimem ent de prvenir
la sgrgation des chromatides surs (Zhou et Giannakakou, 2005). Il en rsultera
lapoptose de la cellule puisque celle-ci ne pourra passer le point de contrle G2/M qui
consiste en la vrification que les chromatides surs sont aligns sur la plaque mitotique
(Alberts et al., 2008; Zhou et Giannakakou, 2005). Ainsi la cellule cancreuse sera donc
cible par des drogues comme le taxol, qui ont pour rle de dstabiliser les m icrotubules et
de les rendre ainsi susceptible lapoptose (Guo et al., 2010; Zhou et Giannakakou, 2005).
Il savre donc que mes travaux de recherche pourraient avoir une porte beaucoup plus
grande que les maladies inflammatoires intestinales, puisquen contrlant lactivation du
rcepteur par des agonistes slectifs nous pourrions, peut tre, agir sur la formation du
fuseau mitotique. Bien sur, cette hypothse demeure tre valide. Par contre, dans ce
contexte, il a t not que lexpression du rcepteur P 2 Y 2 tait augment dans les tumeurs
colorectales (Nylund et al., 2007). Ainsi bien que le rle du rcepteur P 2 Y 2 semble tre
bnfique pour favoriser la rparation de lpithlium intestinal, la stimulation du rcepteur
P 2 Y 2 en contexte tumorale pourrait favoriser la prolifration cellulaire en entranant la
stabilisation des microtubules ncessaires la formation des fuseaux mitotiques.
Comme mes travaux de recherche ne visaient pas seulement comprendre les
mcanismes molculaires rgulant lexpression du rcepteur P 2 Y 2, mais aussi identifier le
rle de ce rcepteur dans les maladies inflammatoires, nous avons dmontr dans un
modle murin de colite chimique induit au DSS que les souris qui ont reu des injections
intra-rectales de 2-thioUTP pendant la phase de rmission ont une diminution significative
de lindex de svrit de la maladie et des comptes histologiques. Le 2-thioUTP est un
140
agoniste de synthse du rcepteur P 2 Y 2 qui possde un E C 50 dix fois plus spcifique pour
ce rcepteur comparativement au rcepteur P 2 Y 4 (El-Tayeb et al., 2006). La concentration
utilise pour les injections intrarectales de 2 pg/g du poids de la souris pourrait donc aussi
activer le rcepteur P 2 Y 4. Par contre, aucune tude ne sest penche sur le rle du rcepteur
P2Y 4 dans la rparation pithliale. Puisquil n existe aucun agoniste spcifique pour ces
rcepteurs P2Y, lutilisation de souris transgnisque dont lexpression du rcepteur P 2 Y 4
ou P 2 Y 2 aurait t invalid serait essentielle pour conclure du rle du rcepteur P2Y 2 dans
la rmission des souris traites au DSS. Cependant, des souris P 2 Y 2_/ atteintes d une colite
chimique ont un index de svrit de la maladie significativement plus lev que les souris
de type sauvage (rsultats non publis, voir Annexe 2). Par ailleurs, la translocation des
bactries
les souris
P 2 Y 2' '
comparativement aux souris de type sauvage suivant un traitement au DSS (rsultats non
publis, voir Annexe 2). Ces rsultats bien que prliminaires indiquent que le rcepteur
P 2 Y 2 possdent un rle important dans le maintien de lintgrit de la barrire pithliale
intestinale.
Bien qu/'n vitro, nous avons dmontr que le rcepteur P2Y 2 favorise la migration
cellulaire suivant une blessure, il n est pas exclure que le rle exact du rcepteur P 2 Y 2 in
vivo implique d autres processus et la participation de cellules autre que les CEI. Le
rcepteur P 2 Y 2 est, en effet, exprim la surface de plusieurs cellules du systme
immunitaire dont les macrophages et les neutrophiles qui ont des rles essentiels dans la
rparation de blessures (Bowler et al., 2003; Chen et al., 2006). Comme mentionn dans
lintroduction, les macrophages activs suivant une blessure vont se retrouver prs de la
niche des cellules souches et favoriser leur prolifration et leur survie (Pull et al., 2005). De
plus, ils vont jouer un rle trs important en limitant lentre de pathognes et en
phagocytant les dbris cellulaires (Reichner et al., 2001). Finalement, les macrophages
permettent de contrler lactivit des neutrophiles en induisant lapoptose de ceux-ci
(M eszaros et al., 2000). Malgr leurs actions destructrices, laction des neutrophiles est
ncessaire au processus de rparation. Les neutrophiles vont scrter des facteurs de
croissance, des lipides mdiateurs de rparation comme les lipoxines et vont participer la
phagocytose des dbris cellulaires accum uls au site de la blessure (Fournier et Parkos,
2012). Il a t dmontr que les macrophages et les neutrophiles vont migrer par
141
un
pisode
aigu
dinflammation
intestinale.
CONCLUSION
Nous avons donc entrepris des tudes visant dterminer les mcanismes
site
P 2 Y 2 et j ai identifi des sites de liaison pour les facteurs de transcription C/EBPp et NFKB/p65. Ces facteurs de transcription rgulent en partie lexpression du rcepteur P 2 Y 2 en
conditions physiologiques mais aussi en conditions inflammatoires tels que celles
retrouves chez les patients atteints de maladies inflammatoires intestinales. Ces rsultats
143
reprsenter l'une
des pierres
angulaires
essentielles au
144
rechutes sont souvent associes une mauvaise rparation (Ardizzone et al., 2011). Ainsi,
la gnration d agonistes de synthse spcifiques et stables pourrait reprsenter une
nouvelle voie de traitement des maladies inflammatoires intestinales.
En conclusion, ltude de la rgulation de lexpression et du rle du rcepteur P 2 Y2
a permis de reconnatre que ce rcepteur est un joueur important dans les MIL
Lidentification du promoteur et des facteurs de transcription rgulant lexpression du
rcepteur P 2 Y 2 auront des impacts dans la comprhension des autres pathologies dans
lesquelles une augmentation de son expression est observe. De plus, les rsultats obtenus
indiquant que le rcepteur P 2 Y 2 favorise la rparation de blessure in vitro et la phase de
rmission in vivo ouvrent la voie toute une nouvelle catgorie de molcules au fort
potentiel thrapeutique pour les patients atteints de MIL
145
REMERCIEM ENTS
Je tiens tout d abord remercier les membres de mon jury soit les Professeurs JeanFranois Ct, Abdelaziz Amrani, Franois Boudreau et Femand-Pierre Gendron pour le
temps et lintrt quils ont investi dans la correction de cette thse.
Un remerciement tout spcial mon directeur de recherche, Pr Fem and-Pierre
Gendron (Patron), qui m a accueillie dans son laboratoire en juin 2006. Au cours de ces
annes passes dans ton laboratoire, j ai eu la chance d apprendre normment. La libert
scientifique que tu m as laisse a t tellement apprcie et m a permis de dcouvrir mon
potentiel scientifique. Tu as aussi fait preuve d une grande patience et confiance envers moi
qui parfois manque un peu dassurance! Je ressors de cette exprience grandie et prte
affronter de nouveaux dfis.
Je voudrais aussi remercier les membres prsents et passs du laboratoire. AndreAnne, Karine, Djordje, Christine, Valrie, Maude, Jean-Franois et Guillaume. On peut
dire quen toutes ces annes, on a eu du plaisir! Que de sorties et party mm orables on a eu
tous ensemble! Je tiens aussi rem ercier les autres membres du dpartement d anatomie et
biologie
cellulaire
particulirement Vronique
Giroux,
Grald
Bem atchez,
Benot
Marchand, Naomie Turgeon et Franois Brial sans qui m on heure de dner aurait t
particulirement moins anim! Je remercie aussi Valrie et Vronique pour les nombreux
desserts quelles ont apports au fil des annes et qui me remplissaient de bonheur chaque
fois! Aussi, je tiens remercier tous les tudiants avec lesquels j ai assist des congrs.
Ce ft un rel plaisir d apprendre vous connatre et de dcouvrir de nouveaux endroits
avec vous.
Je remercie mes parents qui ont su m appuyer dans toutes les tapes de mon
cheminement acadmique. Je veux videmment dire merci ma fille Lily qui a t trs
comprhensive avec sa maman! Finalement, je remercie m on mari Gabriel qui cette
russite appartient aussi puisque sans son support indfectible, mon cheminem ent aurait t
beaucoup plus ardu.
146
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162
ANNEXES
Annexe 1 : Articles publis en tant que deuxime auteur
RT-PCR,
cytokine
164
in
IEC.
METHODS:
Neutrophil
recruitment
was
monitored
by
C xcll
increased
expression
in
IEC
and
the
severity
of
inflammation.
CONCLUSIONS: This study not only describes the P2Y(6) signaling mechanism
regulating CXCL 8 expression in IEC, but it also illustrates the potential o f targeting P2Y(6)
to reduce intestinal inflammation.
165
1510 -
C57/BI6 WT
C57/BI6 P2Y2 KO
Une colite chimique a t induite en ajoutant, durant 7 jours, 3,5% de DSS dans leau de
boisson de souris C57/B16 de type sauvage (C57/B16 WT) ou invalides pour lexpression
du rcepteur P2Y2 (C57/B16 P2Y2 KO). Lindex de svrit de la m aladie est
significativement plus lev chez les souris C57/B16 P2Y2 KO (13.8 units arbitraires)
comparativement aux souris C57/B16 W T (4,7 units arbitraires). Les analyses statistiques
ont t dtermines par le Mann W hitney test (t test), ***, p< 0.002.
Q>
Q.
V)
u.
O)
-J
C57/BI6 WT
C57/BI6 P2Y2 KO
Une colite chimique a t induite en ajoutant, durant 7 jours, 3,5% de DSS dans leau de
boisson de souris C57/B16 de type sauvage (C57/B16 WT) ou invalides pour lexpression
166
167
JS 15
.m
% 10
<0
ai
PCDNA3.1
+
+
pGL4.10
pcDNA 3.1-C /E B P a
p G L 4 .1 0 -p rP 2 Y 2
B)
<2 30-i
AAA
+
+
+
+
pcDNA3.1
pGL.4.10
pcDNA 3 .1 -C/EBPa
pGL4.10 - prP2Y2
DSS
168
C)
AAA
S 15n
+
+
+
+
pcDNA3.1
pGL4.10
pcDNA 3.1 - C/EBP
pGL4.10 - prP2Y2
D)
S 3<h
+
+
+
+
pcDNA3.1
pGL4.10
pcDNA 3 .1 -C/EBP5
pGL4.10 - prP2Y2
DSS
Les cellules Caco-2 ont t transfectes transitoirem ent avec la construction pGL4.10 prP2Y2 et le vecteur d expression de C/EBPa (A et B) ou C/EBP (C et D). Les cellules ont
t incubes en prsence (B et D) ou non (A et C) de 0,5% DSS, 6 h avant d effectuer les
essais lucifrase. Lactivit de la lucifrase est exprime par rapport lactivit de celles
des vecteurs vides. L analyse statistique utilise est le one-way ANOVA o **p < 0.01 et
***p < 0.001 comparativement leur contrle respectif et o AA p < 0.01 et AAA p < 0.001
comparativement leur contrle respectif.