Heat Shock Proteins in Fibrosis and Wound Healing Good or Evil

Pharmacology & Therapeutics 143 (2014) 119132
Contents lists available at ScienceDirect
Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera
Associate editor: N. Frossard
Heat shock proteins in brosis and wound healing: Good or evil?

Pierre-Simon Bellaye a,b, Olivier Burgy a,b, Sebastien Causse a,b, Carmen Garrido a,b, Philippe Bonniaud a,b,c,
a
b
c
INSERM U866 quipe labellise Ligue contre le cancer, 21000 Dijon, France
Faculty of Medicine and Pharmacy, University of Burgundy, Dijon 21033, France
Service de Pneumologie, CHU Dijon, 21079 Dijon, France
a r t i c l e
i n f o
Available online 26 February 2014

Keywords:
Fibrogenesis
HSP
TGF-1 signaling
EMT
a b s t r a c t
Heat shock proteins (HSPs) are key regulators of cell homeostasis, and their cytoprotective role has been largely
investigated in the last few decades. However, an increasing amount of evidence highlights their deleterious
effects on several human pathologies, including cancer, in which they promote tumor cell survival, proliferation
and drug resistance. Therefore, HSPs have recently been suggested as therapeutic targets for improving human
disease outcomes. Fibrotic diseases and cancer share several properties; both pathologies are characterized by
genetic alterations, uncontrolled cell proliferation, altered cell interactions and communication and tissue
invasion. The discovery of new HSP inhibitors that have been shown to be efcacious against certain types of
cancers has given rise to a new eld of research that investigates the activity of these compounds in other
incurable human diseases such as brotic disorders. The aim of this review is to discuss new ndings regarding
the involvement of HSPs in the pathogenesis of organ brosis and to note recent discoveries that indicate that
HSPs could be important therapeutic targets to improve the current dismal outcome of brotic diseases.
2014 Elsevier Inc. All rights reserved.
Contents
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.
The brotic processes . . . . . . . . . . . . . . . . . . . . . . . . .
3.
Heat Shock Protein 90 inhibitors: anti-brotic drugs? . . . . . . . . . . .
4.
Heat Shock Protein 70 and brosis: intracellular versus extracellular function
5.
Heat Shock Protein110: a role in cardiac brosis? . . . . . . . . . . . .
6.
Heat Shock Protein47: A therapeutic target to prevent collagen accumulation
7.
Small heat shock proteins (sHSP) . . . . . . . . . . . . . . . . . . . .
8.
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . .
Conict of interest statement . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations: 17-AAG, 17-(Allylamino)-17-demethoxygeldanamycin; AP-1,

Activator protein 1; Apaf-1, Apoptotic protease-activating factor 1; CHIP, Carboxyl terminus of Hsc70-interacting protein; CsA, Cyclosporine A; ECM, Extracellular matrix; EMT,
Epithelial-to-mesenchymal transition; ER, Endoplasmic reticulum; ERK, Extracellular
signal-regulated kinase; GGA, Geranylgeranylacetone; GP96, Glycosylated protein 96;
GR, Glucocorticoid receptor; GRP, Glucose-regulated protein; HSC, Hepatic stellate cell;
HSE, Heat shock element; HSF, Heat shock factor; HSP, Heat shock protein; IL-1,
Interleukin 1 beta; IPF, Idiopathic pulmonary brosis; JNK, c-Jun N-terminal kinase;
MAPK, Mitogen-activated protein kinase; MK2, MAPKAP kinase2; PDB, Peptide-binding
domain; SMAD, Mothers against decapentaplegic homolog; -SMA, -Smooth muscle
actin; SMURF, SMAD-ubiquitin regulatory factor; TRI, TGF- receptor I; TRII, TGF- receptor II; TGF-1, Transforming growth factor 1; TIF1, Transcriptional intermediary factor 1; TRAIL, Tumor necrosis-factor related apoptosis-inducing ligand; TRAP1, Tumor
necrosis-factor receptor-associated protein 1; UUO, Unilateral Ureteral Obstruction.
Corresponding author at: Service de pneumologie et soins intensifs respiratoires, CHU
le bocage, 21079 Dijon, France. Tel.: +33 3 80 29 32 63.
E-mail address: philippe.bonniaud@chu-dijon.fr (P. Bonniaud).
http://dx.doi.org/10.1016/j.pharmthera.2014.02.009
0163-7258/ 2014 Elsevier Inc. All rights reserved.
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1. Introduction
Cells are constantly exposed to damage caused by many environmental factors (heat shock, oxidative stress, UV), exposure to
pharmacologic toxic agents (heavy metals, alcohol, chemotherapy)
or certain pathological conditions. Protective mechanisms are
established in cells to maintain their function. The heat shock
response was rst highlighted in 1964 in Drosophila by Ritossa
(Ritossa, 1964; Tissieres et al., 1974) and is characterized by the
expression of a particular protein superfamily, the heat shock
proteins (HSPs). HSPs are highly conserved cellular proteins present
in many species such as yeast, bacteria, plants, animals and humans.
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P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132
In normal conditions, HSPs are able to interact with mature and immature proteins to assist in the folding of newly synthesized proteins
and their stabilization, helping them to reach organelles, such as the mitochondria or the endoplasmic reticulum, and participating in protein
turnover (Gething & Sambrook, 1992). During stress, the proper folding
of proteins can be affected, leading to a loss of protein function. The accumulation of misfolded proteins enhances the formation of aggregates,
which disrupts the cellular machinery and can lead to cell death. The
presence of stress-inducible proteins ensures the correct folding of
misfolded proteins; if refolding is impaired, they direct misfolded proteins towards the proteasome system where they are degraded (Burel
et al., 1992; Lanneau et al., 2010). While HSPs account for up to 23%
of the total cellular proteins at their baseline level, their expression is
strongly induced upon stress. This induction is mediated by specic
transcription factors, called heat shock factors (HSF). The best characterized of these is HSF-1, which has the ability to bind DNA at specic
sites called Heat Shock Elements (HSE) found in the promoters of HSP
genes (Arrigo, 2005). Under normal conditions, HSF-1 is sequestered
in the cytoplasm by HSP70, HSP90 and several co-chaperones
(Voellmy, 2006; Conde et al., 2009). In stressful conditions, the HSPs release HSF-1 to bind misfolded proteins, allowing the phosphorylation,
activation and trimerization of HSF-1, which migrates into the nucleus,
interacts with HSE and nally induces the transcription of HSP genes.
In mammals, heat shock proteins are grouped into six major families
according to their molecular weight as follows: HSP100, HSP90, HSP70,
HSP60, HSP40 and the small Heat Shock Protein family (sHSP, (Vos et al.,
2008)). Although HSPs share many common properties, each family has
specicities in regard to their cellular localization, their dependence on
ATP, their substrate specicity and the type of diseases in which they
may be involved.
Due to their chaperone activity, HSPs are involved in many essential
cellular mechanisms. For instance, several studies have shown that
HSPs can inhibit apoptosis by interacting with proteins involved in
programmed cell death such as cytochrome c, caspases or Apaf-1 (Apoptotic protease-activating factor 1, Beere et al., 2000; Bruey et al., 2000;
Saleh et al., 2000; Didelot et al., 2006). HSPs can also be involved in
cytokine induction (Asea et al., 2000), inammation (Tamura et al.,
2012) and cytoskeleton-related diseases (Wettstein et al., 2012). The
recruitment of inammatory cells, abundant secretion of probrotic
cytokines (mainly TGF-1) and increase in oxidative stress, apoptosis
and degradation via the proteasome system are all events that occur
during brogenesis and therefore might involve HSPs.
HSPs have been implicated in the pathogenesis of cancer (Jego et al.,
2013). HSPs take part in several molecular mechanisms of cancerous
cells, including proliferation (Ghosh et al., 2008), invasion (Lemieux
et al., 1997) and angiogenesis (Sanderson et al., 2006; Thuringer et al.,
2013). In several reports, HSPs appeared to be benecial for cancerous
cells and thereby deleterious for patients affected by a wide range of
cancer types (Rappa et al., 2012). HSP inhibition thus appears to be of
great interest to improve the efciency of chemotherapy and the disease outcome (Dorard et al., 2011; Rerole et al., 2011; Goloudina et al.,
2012). Fibrogenesis and cancer share several properties; both pathologies are characterized by genetic alterations, uncontrolled cell proliferation, altered cell interaction and communication and tissue invasion
(Vancheri et al., 2010). The current review will discuss the involvement
of HSPs in the process of brogenesis and in established organ brosis.
2. The brotic processes
Fibrosis is an essential process of tissue healing, promoting wound
repair, re-epithelialization and restoration of the normal function of
the affected organs in cases of chronic injury. This protective mechanism can be highly deleterious when it is out of control, leading to
organ failure and diseases such as liver, cardiac, renal or pulmonary
brosis. While the process of brogenesis can be slightly different
depending on the affected organ, the major steps remain the same
(Calabresi et al., 2007; Kisseleva & Brenner, 2008). Epithelial cell injury
and apoptosis appear to be some of the triggering events of the process.
Epithelial cell death results in the recruitment of inammatory cells and
secretion of pro-brotic cytokines such as transforming growth factor1 (TGF-1). This pro-brotic environment leads to the activation of
neighboring cells and contributes to the progression of brosis. One of
the major events during brosis is the abnormal and massive increase
in extracellular matrix (ECM) deposition with notable collagen accumulation. Myobroblasts are considered to be key cells in the brotic
process because they are essentially responsible for ECM synthesis and
are associated with disease progression (Phan, 2002). Several hypotheses have been proposed to explain the origin of these aggressive cells.
Resident broblasts and circulating mesenchymal cells derived from
bone marrow (brocytes) are thought to contribute to the pool of
myobroblasts and ECM deposition (Bucala et al., 1994; Kisseleva &
Brenner, 2008) under TGF-1 stimulation. Epithelial cells are also
major players in brogenesis because they can trans-differentiate and
acquire a myobroblastic phenotype and thus contribute to the
accumulation of ECM in the lung tissue. This transformation, called
the epithelial-to-mesenchymal transition (EMT), is initiated by
transforming growth factor-1 (TGF-1), a growth factor essential to brosis (Carew et al., 2012; Klugman et al., 2012). The brotic process is
thus maintained by both apoptosis of epithelial cells, which leads to the
production of pro-brotic cytokines, mainly TGF-1, and also by the inhibition of apoptosis in myobroblasts, which produce an excess of ECM
thereby causing the disease. This paradox has been widely discussed
in the literature, and the key role of TGF-1 has been conrmed
(Thannickal & Horowitz, 2006; Drakopanagiotakis et al., 2008).
Although TGF-1 inhibits cellular proliferation in several cell types, it
stimulates mesenchymal cell proliferation, induces the secretion of
ECM, inhibits apoptosis in myobroblasts and, therefore, is strongly
pro-brotic (Border & Noble, 1994). In addition, the ability of TGF-1
to induce epithelial cell apoptosis may be another mechanism whereby
brosis is promoted, given that impaired epithelial repair is increasingly
recognized as an important mechanism in brogenesis (Barbas-Filho
et al., 2001). Furthermore, TGF-1 levels are increased in brotic lung
tissue from patients with pulmonary brosis (Coker et al., 2001). The
induction of severe prolonged pulmonary brosis in rodents by overexpression of active TGF-1 in the lung is another conrmation of its
central role in brogenesis (Sime et al., 1997).
TGF- is a 25 kDa protein consisting of two identical 12.5 kDa subunits covalently joined by disulde bonds. There are at least three mammalian isoforms of TGF-, designated 1, 2 and 3, which all mediate
signaling through the same surface receptors. The TGF-1 isoform is the
most commonly secreted isoform and is consistently found to be upregulated in brotic diseases (Khalil et al., 1996). TGF- signaling occurs essentially through the following two main receptors: TGF- receptors
type I and II, known as TRI and TRII, respectively. These receptors
are serine/threonine kinases. TRII is constitutively active, and after
the binding of its ligand, it activates TRI via phosphorylation (Wrana
et al., 1994). In turn, TRI activates a large number of intracellular signaling pathways through kinase activation, including the Smad pathway, which is essential for brogenesis (Massague & Wotton, 2000;
Wells, 2000). Smad proteins are direct effectors of the activated TGF-
receptor complex and mediate signaling from cell surface receptors to
the nucleus (Eickelberg, 2001; Schiller et al., 2004). They can be grouped
into the following three classes: receptor-activated Smad (R-Smad:
Smad1, 2, 3, 5 and 8); common mediator Smad (Co-Smad: Smad4)
and inhibitory Smad (I-Smad: Smad6 and 7). Transphosphorylation of
TRI activates its kinase domain, which can then phosphorylate RSmads. Once activated, R-Smads bind to Smad4 and translocate to the
nucleus to regulate the transcription of a large number of genes. The
regulation of R-Smad activation is mediated in the cytoplasm by ISmads, which prevent the binding and phosphorylation of R-Smad
by activated TGF- receptors. Protein levels of R-Smads are regulated
in part by ubiquitin proteasome-mediated degradation. The Smurf
proteins (Smurf 1 and 2) are E3-ubiquitin ligases that are able to

ubiquitinate R-Smads and direct them to the proteasome system
(Kavsak et al., 2000). Smad3 appears to be a crucial element in the
signal transduction pathways involved in wound healing and brosis
(Roberts et al., 2001; Verrecchia et al., 2001; Roberts, 2002). Indeed,
Smad3-decient mice are protected against radiation-induced skin
brosis (Flanders et al., 2002) and against bleomycin-induced
(Zhao et al., 2002) and TGF-1-induced pulmonary brosis
(Bonniaud et al., 2004).
3. Heat Shock Protein 90 inhibitors: anti-brotic drugs?
HSP90-family proteins are the most abundant proteins in eukaryotic
cells, representing 1% of the total pool of cellular proteins (Csermely
et al., 1998). The HSP90 family is composed of the following 4 main
members: HSP90, HSP90, GP96 and TRAP1. All of the members
belonging to the HSP90 family require ATP for their activity and can
be induced by several stresses.
GP96 (glycosylated protein 96) is a molecular chaperone localized in
the endoplasmic reticulum (ER), which is responsible for protein maturation and the proper folding of newly synthesized proteins. GP96 has
been shown to have an effect on the activation of the immune system.
In this eld, some studies reported that GP96 participates in antigen
presentation, especially during antitumoral antigen chaperoning
(Multhoff, 2006). GP96 has also been shown to have a role in endoplasmic reticulum stress (ER-stress (Rolhion et al., 2011)). Recent articles show the involvement of ER stress in epithelial injuries at the
origin of brosis (Korfei et al., 2008; Mahavadi et al., 2010). However,
the involvement of GP96 in ER stress and brosis has not been investigated yet.
The role of TRAP1, tumor necrosis factor receptor-associated protein
1, is not well understood. It is known to localize in the mitochondria,
and, despite its similarities with HSP90, it does not interact with the
usual client proteins of the HSP90 family.
HSP90 (HSP86) and HSP90 (HSP84) are the two main members
of the HSP90 family. Both isoforms are result of a duplication of the
original gene and share 86% homology. Structurally, the following
three domains make up HSP90: the N-terminal domain, which binds
ATP, the central domain required for ATPase activity and client protein
binding, and the C-terminal domain that allows HSP90 dimerization.
HSP90 acts mainly as a homodimer ( or ), but it can also form
heterodimers () or monomers. As a exible dimer, HSP90 is able
to bind a client protein, and the hydrolysis of an ATP molecule by
HSP90 results in a conformational change that stabilizes the client
protein (Pearl & Prodromou, 2006).
HSP90 is also involved in cytoskeleton stabilization by binding actin
and tubulin. These interactions preserve cellular integrity and the intracytoplasmic localization of proteins after stresses (Czar et al., 1996),
suggesting a role of HSP90 in the cytoskeletal changes often observed
during brogenesis.
The fact that specic inhibitors of HSP90 already exist renewed
the interest in these chaperones at the clinical level. 17-AAG (17(allylamino)-17-demethoxygeldanamycin), a geldanamycin derivative,
is a chemical compound that is able to specically bind the ATP-binding
site of HSP90, thereby inhibiting its activity. 17-AAG is now in phase III
clinical trials for its anti-tumoral properties (for a review see Porter
et al., 2010; Jego et al., 2013). This compound has high therapeutic potential because by inhibiting HSP90, it can sensitize cells to the cytotoxic
agents used in chemotherapy. Furthermore, in cancer cells, the afnity
of HSP90 for ATP is increased 100-fold. Because 17-AAG mimics ATP,
its afnity in tumor cells is enhanced, resulting in anti-tumoral specicity for this inhibitor (Kamal et al., 2003).
Although the number of studies linking HSP90 to brogenesis is
limited, some recent articles report a benecial effect of HSP90 inhibitors on tissue brosis. Noh et al. demonstrated that the HSP90 inhibitor
17-AAG was able to prevent brotic events in vitro and in vivo. Indeed, in
121
kidney cell lines stimulated with rTGF-1 and in a murine model of

renal brosis with obstructed kidney, 17-AAG repressed the myobroblast marker -smooth muscle actin (-SMA) and the ECM components bronectin and collagen I, which are usually upregulated
during brotic processes, while enhancing the epithelial marker Ecadherin, which is usually downregulated during brosis (Noh et al.,
2012). Similar results were found in liver brosis, where the HSP90 inhibitor inhibited the activation and reduced the survival of hepatic stellate cells (HSCs), major participants in hepatic brosis. Indeed, 17-AAG
downregulated collagen I and -SMA expression levels and induced apoptosis of HSCs in vitro (Myung et al., 2009). Furthermore, HSP90 inhibition totally abrogated EMT in lung epithelial cells (A549 and H658)
by inhibiting TGF-1-induced E-cadherin downregulation and therefore
blocking N-cadherin, -SMA and vimentin upregulation. These results
correlated with the inhibition of TGF-1-induced changes in cell
morphology and stress ber formation in lung epithelial A549 cells,
which might be of importance in the context of brosis (Reka et al.,
2011). More recently, it has been demonstrated that skin brosis
was inhibited by HSP90 inhibitors (Tomcik et al., 2013). 17Dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG)
prevented an increase in dermal thickness, collagen deposition and
myobroblast differentiation by inhibiting TGF- signaling in a systemic
sclerosis mouse model (Tomcik et al., 2013).
Several mechanisms have been proposed to explain the role of
HSP90 in brotic processes. Recent articles based on kidney and lung
cell lines showed that 17-AAG was able to block the entire TGF-1 signaling pathway, abrogating Smad2/3, Akt, GSK-3, and ERK phosphorylation (Wrighton et al., 2008; Reka et al., 2011; Noh et al., 2012; Zhang
et al., 2012). Indeed, HSP90 inhibition prevented the transcription of
TGF-1 target genes, thus inhibiting the EMT process (Wrighton et al.,
2008; Reka et al., 2011). HSP90 inhibition by 17-AAG countered the
protective role of HSP90 on TGF-1 receptors (mainly the TGF- type
II receptor, TRII) by decreasing the interaction between HSP90 and
TRII and leading to TRII degradation by the ubiquitin-proteasome
system (Fig. 1). The TRII-E3-ubiquitin ligase, Smurf2, was responsible
for TRII degradation by the proteasome in the absence of HSP90
(Wrighton et al., 2008; Noh et al., 2012; Zhang et al., 2012).
Because activated hepatic stellate cells (HSCs) have been demonstrated to be the main participants in hepatic brosis, their inactivation and the induction of apoptosis of those cells has become an
important anti-brotic therapeutic strategy. In regard to the antiapoptotic functions of HSPs, HSP90 inhibitors (17-AAG, VER-49009
and VER-49009M) have been demonstrated to inhibit proliferation
(Sun et al., 2009) and favor apoptosis (Myung et al., 2009) of HSCs.
During hepatic brosis, HSCs are the most important ECM producers,
and NFB is an essential mediator of HSC activation and survival
(Mann & Smart, 2002). During HSC activation, HSP90 chaperones
the glucocorticoid receptor (GR), which is a binding partner of both
HSP90 and NFB. This sequestration of the GR by HSP90 liberates
NFB and favors its nuclear translocation, resulting in HSC activation.
In this context, the inhibition of HSP90 by 17-AAG enhances GR/
NFB complex formation and prevents NFB-mediated activation of
HSCs (Myung et al., 2009).
In addition, 17-AAG inhibited the activation of the HSP90 client
protein Akt, which also participates in NFB activation (Myung
et al., 2009; Sun et al., 2009). Therefore, HSP90 inhibition could
induce apoptosis in HSCs by disrupting NFB cell survival signals in
two distinct ways, formation of GR/NFB complexes and inactivation
of Akt (Fig. 1). Furthermore, 17-AAG also inhibited HSC activation by
reducing -SMA expression and TGF--induced collagen synthesis
(Myung et al., 2009).
Thus, the chaperone activity of HSP90 on both TGF- and the glucocorticoid receptor participates in the activation of brotic signaling
through the TGF- and NFB pathways. Therefore, inhibition of this
HSP may prevent brotic events and might be of interest in the development of new drugs aimed at limiting brotic disorders.
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Fig. 1. HSP90 inhibition inhibits the TGF-1 pathway. Left: HSP90 forms a complex with TRII, inhibiting its ubiquitination and degradation by the proteasome. In parallel, HSP90 binds the
glucocorticoid receptor (GR), enabling the nuclear translocation and activity of NFB. Right: 17AAG binds the ATP-binding domain of HSP90 and therefore inhibits its activity. The
disruption of the HSP90/TRII complex allows Smurf2 to bind to TRII, thus inducing its ubiquitination and degradation. 17AAG also disrupts GR/HSP90 complexes, thereby enabling
the interaction between GR and NFB. This results in the sequestration of NFB in the cytoplasm and impairs its activity.
4. Heat Shock Protein 70 and brosis: intracellular versus extracellular function

There are eight members of the HSP70 family, all of which are well
conserved in organisms from bacteria to human. The HSP70 family
proteins are the most studied HSPs in regard to immunity, cancer and
brosis. Among the HSP70 family, HSC70 (Heat Shock Cognate 70),
HSP70.2, GRP75 (Glucose-Regulated Protein 75), GRP78 and HSC70
are constitutively expressed and participate in cellular homeostasis,
whereas HSP70 (HSP72) and HSP70-6 are induced by stress conditions
(Daugaard et al., 2007).
HSP70 (HSP72), the primary member of the family, participates in
the prevention of protein aggregation by redirecting unfolded or
misfolded proteins to the proteasomal degradation system. This HSP
also has strong anti-apoptotic properties and participates in ribosome
translocation and assembly under stress conditions (Welch & Suhan,
1986; Goloudina et al., 2012).
HSP70 operates in cooperation with different co-chaperones. One of
these is HSP40, which helps client proteins to bind to the peptidebinding domain of HSP70. The hydrolysis of ATP at the ATP-binding
domain of HSP70 induces a conformational change that allows HSP70
to capture client proteins. The binding of another ATP molecule to
HSP70 releases the properly folded client protein (Hartl & Hayer-Hartl,
2009). If the client protein is still not functional, then it can bind to
HSP70 again or to another HSP and/or be driven to the proteasome
system for degradation.
The main studied function of HSP70 is its anti-apoptotic role, given
that it is able to bind several molecules that are involved in different apoptotic pathways. HSP70 has been shown to inhibit caspase-dependent
and independent apoptotic pathways by inactivating Apaf-1 and AIF
(apoptosis-inducing factor), key factors of caspase-dependent and
caspase-independent apoptosis, respectively (Sabirzhanov et al.,
2012). At the mitochondrial level, HSP70 has been shown to block the
translocation of Bax to the mitochondria and mitochondrial protein
release (Yang et al., 2012) and to increase the stability of Bcl-2 (Jiang
et al., 2011). Moreover, HSP70 is abundantly expressed in cancerous
cells, and thus it is an important factor in cancer cell resistance to anticancer drugs (Goloudina et al., 2012). For these reasons, HSP70 inhibition has become a major interest in improving anti-tumoral therapy.
Unlike the situation for HSP90, only a few HSP70 inhibitors are available. Peptidic aptamers inhibiting HSP70 have been recently developed
(Rerole et al., 2011) and a pharmacologic molecule, getinib, an EGFR
inhibitor utilized in cancer therapy, has been shown to inhibit HSP70
expression (Namba et al., 2011).
Recent studies demonstrate a protective role of HSP70 against
brogenesis. Thus, several methods have been developed to induce
HSP70, using HSP70 transgenic mice, hyperthermia or pharmacologic
agents such as geranylgeranylacetone (GGA).
The protective role of HSP70 against brotic processes is now well
documented. In 2007, Wakisaka et al. demonstrated in vitro and in vivo
that heat shock prevented angiotensin-II-induced cardiac brotic response in the atrium via HSP70 upregulation. Cardiac atrial brosis is
known to be driven by increased activity of the ERK-activating kinases
MEK1/2 leading to increased ERK1/ERK2 phosphorylation (Goette et al.,
2000). Angiotensin-II activates the MEK-ERK cascade by binding to the
AT1 receptor, causing broblast proliferation and activation and favoring
atrial brosis (Booz et al., 1999). HSP70 upregulation induced by heat
shock could lead to the attenuation of angiotensin-II-induced signals,
resulting in negative regulation of ERK1/ERK2 phosphorylation, which
eventually inhibits the differentiation of broblasts into myobroblasts
by preventing -SMA expression, TGF-1 secretion and ECM synthesis
(Wakisaka et al., 2007). HSP upregulation has been proposed as a novel
therapeutic approach to prevent atrial brosis (Takahashi et al., 2012).
GGA is an anti-ulcer drug that has been reported to induce HSP70
expression in vitro and in vivo, improving brosis outcome in several organs such as lung and kidney (Fujibayashi et al., 2009; Tanaka et al.,
2010; Zhou et al., 2010). GGA has been demonstrated to prevent most
important events occurring during brotic processes. In addition, it
reduces organ damage signicantly after the induction of lung and

kidney brosis and leads to limited ECM deposition. In vivo, GGAmediated HSP70 upregulation also prevented myobroblast differentiation and EMT, which decreased the number of -SMA-positive
cells (Fujibayashi et al., 2009; Tanaka et al., 2010). In line with its
cytoprotective role, HSP70 upregulation prevented epithelial apoptosis
in the lung and kidney, one of the major events triggering brogenesis.
In a study performed in 2009 based on HSP70 induction by GGA in a
bleomycin-induced pulmonary brosis model, the authors demonstrated a decrease in the number of recruited inammatory cells. Indeed, the
expression level of MIP-2, the most specic chemokine for neutrophil
recruitment, was dramatically decreased by GGA treatment after the induction of lung brosis. HSP70 upregulation in bleomycin-treated mice
also protects lung epithelial cells from apoptosis and inammation, thus
preventing brosis (Fujibayashi et al., 2009).
In vivo evaluation of the effects of GGA in obstructed kidney models
showed that GGA limited damage and reduced ECM deposition. TGF1-induced myobroblast differentiation via EMT also appeared to be
limited by HSP70 upregulation in vitro and in vivo and correlated with
the inhibition of E-cadherin downregulation and -SMA overexpression. Interestingly, Mao et al. also reported that HSP70 upregulation
via GGA in a kidney brosis model reduced renal tubular TGF-1induced apoptosis (a triggering event in brogenesis), in accordance
with its well-known cytoprotective effect (Mao et al., 2008).
In 2010, Tanaka et al. demonstrated that transgenic mice overexpressing HSP70 (tgHSP70) were protected from lung inammation
and brotic lesions caused by bleomycin. The authors showed that
TGF-1 production and pro-inammatory cytokine expression after
bleomycin administration was lower in tgHSP70 mice compared to
WT mice, explaining the limited brosis observed in mice that overexpress HSP70. In vitro, HSP70 inhibition favored the EMT process induced
123
by TGF-1 but did not activate broblasts (Tanaka et al., 2010). The
same team showed that administration of an HSP70 inhibitor, getinib,
in mice exacerbates pulmonary brosis induced by bleomycin (Namba
et al., 2011).
In a model of kidney brosis, Zhou et al. proposed a mechanism of
action for HSP70 in the TGF- pathway that explained the protective
effect of this HSP in brogenesis. They showed that HSP70 induction
via GGA in vivo and in vitro inhibited phosphorylation and nuclear translocation of Smad3, abrogating EMT and brosis (Fig. 2). Apparently, the
peptide-binding domain (PDB) of HSP70 was necessary for the protective function of HSP70 because the overexpression of a mutant HSP70
lacking the PBD was unable to prevent EMT and brosis in their
model. This result suggests that HSP70 acts as a chaperone of Smad3
via the PBD and sequesters it in the cytoplasm, thus hampering the
TGF-1 contribution to brogenesis (Zhou et al., 2010). Interestingly,
another study revealed a similar effect of HSP70 on the phosphorylation
and nuclear translocation of Smad2 on a kidney cell line (Y. Li et al.,
2011).
An inhibitory effect of HSP70 induction on the TGF-1 pathway was
demonstrated by Yun et al. The authors proposed an alternate mechanism involving a direct role of HSP70 on TGF- receptors I and II (TRI
and TRII). They showed that in hepatic, kidney and lung cell lines the
HSP90 inhibitor geldanamycin induced HSP70 upregulation and
decreased the endogenous expression levels of TRI and TRII in a
time and dose-dependent manner (Yun et al., 2010). Geldanamycin
induced the formation of a complex between HSP70 and TGF- receptors, causing TRI and TRII ubiquitination and proteasomal degradation (Fig. 2). The precise mechanism involved has not been clearly
dened, but the authors suggested a role for the protein CHIP,The research leading to these results was funded by the European Community
7th Framework Programme (FP7/20072013) under grant agreement
Fig. 2. HSP70 induction disrupts the TGF-1 pathway. Left: In the case of low HSP70 levels, the TGF-1 pathway is not affected. Right: HSP70, when overexpressed, binds Smad2 and
Smad3, limiting their phosphorylation and nuclear translocation, thereby hampering TGF-1 signaling. HSP70 is also able to bind TRI and TRII and favors their ubiquitination and
proteasomal degradation. A role for the E3-ubiquitin ligase CHIP in the degradation of TRI and TRII has been suggested.
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HEALTH-F2-2007-202224 eurIPFnet, the FEDER Agence Nationale de la

Recherche Blanc 11-BSV-011-01 meso-IPF (ANR), La Ligue Rgionale
Grand Est Contre le Cancer and l'Institut National du Cancer (INCa).
PS.B. is supported by the EU 7th Frame work Programme (20072013
agreement HEALTH-F2-2007-202224 eurIPFnet.). PSB and O.B. are supported by le Fonds de Dotation Recherche en Sant Respiratoire
(FRSR). The C.G. team is supported by La Ligue National Contre le Cancer, the Association pour la Recherche sur le Cancer and LabEx LipSTIC.
a chaperon-dependent E3 ubiquitin ligase. CHIP is known to regulate
TRI stability and to interact with HSP70 (Connell et al., 2001). Because
all HSP90 inhibitors have been shown to induce HSP70 accumulation,
these results suggest that the anti-brotic effect of these compounds
could be in part mediated indirectly through the upregulation of HSP70.
Kahloon et al. recently highlighted a role of the autoimmune
response in the severity of the idiopathic pulmonary brosis (IPF)
in humans. They demonstrated that an increase in HSP70 autoantibodies in patients affected by IPF correlated with more severe
Forced Vital Capacity (FVC) decline and a signicantly higher mortality
after one year (Kahloon et al., 2013). The authors found that anti-HSP70
autoantibodies in IPF patients interfered with the normal immune
response and caused pro-inammatory reactions in the lungs. Indeed,
anti-HSP70 autoantibodies were able to activate monocytes and
lymphocytes, leading to an increase in IL-8 and IL-4 production, two
cytokines involved in the progression of pulmonary brosis (Kahloon
et al., 2013). For the rst time, an autoimmune response against
HSP70 has been linked to the outcome of brosis in human individuals.
These ndings suggest that the study of the autoimmune response
against HSP70 autoantibodies could provide useful clinical information
in IPF patients and could help understand IPF progression (Kahloon
et al., 2013; Wells & Kelleher, 2013). In contrast to this, Cai et al. demonstrated that extracellular HSP70 had a role in cardiac brosis in a pressure overload model; inhibition of extracellular HSP70 by an antiHSP70 antibody reduced cardiac hypertrophy and brosis. Therefore,
extracellular and intracellular HSP70 appear to have contradictory
functions given that extracellular HSP70 aggravated cardiac brosis

whereas intracellular HSP70 had a protective effect on brogenesis
(Cai et al., 2010). Extracellular HSP70 inhibition limited the binding of
HSP70 to TLR4, thus reducing macrophage inltration, inammation
and TGF-1 production in the heart. The authors suggested that extracellular HSP70 could be a new therapeutic target for cardiac brosis
(Cai et al., 2010). More recently, Gonzlez-Ramos et al. investigated
the mechanisms involved in extracellular HSP70 brogenesis exacerbation (Gonzalez-Ramos et al., 2013). In human aorta smooth muscle cells,
they demonstrated that extracellular HSP70 could bind TLR4, activating
the JNK and ERK protein kinases via MyD88 and TRAF6, causing c-Fos
and c-Jun phosphorylation to active the transcriptional factor, AP-1,
which in turn activates TGF-1 (Fig. 3) (Gonzalez-Ramos et al., 2012).
However, the number of studies revealing a role for intracellular
HSP70 upregulating the TGF- pathway demonstrates the interest of researchers in this HSP in the context of brogenesis. The inhibition of this
major pathway of brogenesis is, indeed, a key strategy for regulating
the myobroblast differentiation and subsequent ECM deposition that
cause brotic diseases. HSP70 seems to be involved at several steps of
the TGF- signaling pathway, including Smad2 and Smad3 phosphorylation and TGF- receptor stability. For this reason, chemical agents
inducing HSP70 expression are interesting molecules for further development of new therapies to treat brotic disorders.
5. Heat Shock Protein110: a role in cardiac brosis?
High molecular-weight HSPs constitute a heterogeneous group of
stress proteins that are conserved from yeast to human (Parsell et al.,
1991). Their structure is close to that of HSP70 and is essentially composed of a peptide-binding domain and a nucleotide-binding domain
that is able to bind ATP. HSP110 chaperones seem to have different
functions. In vitro, HSP110 can prevent protein aggregation (Oh et al.,
1999), and some studies have shown that HSP110 also plays a role in
protein refolding in mammalian cells (Yamagishi et al., 2011).
HSP110 has been shown to interact with HSP70. In this complex,
HSP110 has been shown to be an important factor in the HSP70
refolding machinery by acting as a nucleotide-exchange factor
(Hatayama & Yasuda, 1998; Andreasson et al., 2008).
Proteins in the HSP110 family seem to be involved in many human
diseases, such as colon cancer (Dorard et al., 2011); however, their
role in brosis remains poorly described. Using a murine cardiac
pressure overload model, Mohamed et al. highlighted the role of
HSPA4, a cytosolic member of the HSP110 family, in cardiac hypertrophy and brosis. The expression of HSPA4 has been found to be
upregulated in pressure-overloaded hearts, paralleling the development of cardiac brosis. Cardiac brosis was aggravated in HSPA4/
mice in correlation with an increase in interstitial brosis and enhanced
expression of collagen III and TGF-1 (Mohamed et al., 2012). The lack
of HSPA4 was associated with the activation of several signaling pathways involved in the development of cardiac hypertrophy such as
gp130/STAT3, CaMKII and calcineurin-NFAT (Frey & Olson, 2003). The
authors also showed an increase of misfolded proteins in HSPA4/
cardiac cells, causing an accumulation of polyubiquitinated proteins as
a consequence of the alteration of the chaperone activity normally
driven by HSPA4 (Mohamed et al., 2012).
6. Heat Shock Protein47: A therapeutic target to prevent collagen
accumulation
Fig. 3. Extracellular HSP70 promotes TLR4 signaling leading to TGF-1 activation.

Extracellular HSP70 binds to TLR4, activating JNK and ERK, leading to c-Fos and c-Jun
phosphorylation. This in turn activates TGF-1 signaling through the formation of AP-1.
HSP47 represents the most largely studied heat shock protein in the
context of brogenesis in regard to its role in collagen biosynthesis.
To date, this ATP-independent ER-glycoprotein is the only known
substrate-specic HSP. The C-terminal ER-retention signal RDELsequence ensures that HSP47 remains in the ER (Kambe et al., 1994). Although HSP47 interacts with other proteins (PDI, GRP78 and GRP94 (glucose-regulated HSPs)) in the ER, its major role is in collagen maturation,
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Fig. 4. HSP47 staining in brotic lung correlates with collagen upregulation. HSP47 is upregulated along with collagen during pulmonary brosis. I Lung section from mice 21 days after
intratracheal NaCl (control) administration. II Lung section from mice 21 days after intratracheal bleomycin administration. Bleomycin administration induces pulmonary brosis with
collagen deposition as shown by specic staining for collagen (picrosirius red, lower panel). Hsp47 immunostaining (upper panel) indicates a strong HSP47 overexpression in brotic
lungs.
biosynthesis and secretion. Its expression is always found paralleling collagen biosynthesis, and it is totally absent in non-collagen-secreting cells
(Mala & Rose, 2010). The literature reveals that HSP47 can act in several
steps during collagen maturation, preventing newly formed procollagen
chains from aggregating and being degraded in the ER, promoting the stability of the triple-helical region of procollagen and aiding collagen secretion (Hendershot & Bulleid, 2000; Mala & Rose, 2010). An increase in
HSP47 expression has been shown to correlate with collagen deposition
in brotic disorders affecting the liver (Masuda et al., 1994), kidneys
(Sunamoto et al., 1998) and lungs (Becerril et al., 1999, Fig. 4). HSP47
has also become both a biomarker and a therapeutic target of great interest in brogenesis.
Because collagen accumulation is the hallmark of brotic diseases,
HSP47 has largely been used as a marker of brotic lesions and brosis
progression. In rats, HSP47 upregulation has been shown to be correlated with collagen expression in brotic models of several organs. HSP47
mRNA levels have been demonstrated to be upregulated in parallel with
collagen I and III mRNA levels during liver brosis (Masuda et al., 1994).
Likewise, elevated levels of HSP47 were found in kidney, lung and peritoneal brosis and correlated with collagen deposition and disease progression (Razzaque et al., 1998; Mishima et al., 2003). Another study
showed that HSP47 mRNA was also upregulated in myobroblasts expressing -SMA, type II pneumocytes and macrophages present in
brotic areas (Kakugawa et al., 2010). These results suggest that
myobroblasts may synthesize procollagen during the brotic process
induced in the lung as indicated by an upregulation of HSP47 mRNA,
which indicates the importance of this HSPs' role in brogenesis. This
association between HSP47 and collagen accumulation was conrmed
in patients affected by Oral Submucous Fibrosis (Kaur et al., 2001) and
pulmonary brosis patients (Iwashita et al., 2000).
More recently, researchers tried to obtain a better understanding of
the mechanisms involving HSP47 in brogenesis and to develop new
therapeutic strategies targeting HSP47. The inhibition of HSP47 has
thus become of great interest in limiting or preventing brotic events
in vivo. In 2007, Xia et al. designed a delivery system for siRNA against
HSP47 using cationized gelatin microspheres in a Unilateral Ureteral
Obstruction (UUO) model of kidney brosis. This delivery method
prolonged the inhibitory effect on HSP47 expression compared to classical siRNA administration (14 days instead of 7 days). siRNA was found
in tubular epithelial cells and in tubulointerstitial cells at days 7 and 14
in brotic mice. The authors demonstrated that the inhibition of HSP47
expression reduced type IV, III and I collagen expression and accumulation in mice affected by UUO and considerably improved their renal
interstitial brosis status (Xia et al., 2008). A similar method was recently used with impressive results in peritoneal brosis. The delivery
of HSP47 siRNA suppressed collagen and TGF-1 expression and macrophage inltration, therefore inhibiting the development of peritoneal brosis (Obata et al., 2012).
Similar results were found in intestinal brosis using IL-10 KO mice,
which spontaneously develop colitis over time. IL-10 KO mice develop
characteristic brotic features with collagen deposition associated
with the upregulation of TGF-1 and HSP47 expression. Cells positive
for HSP47 staining localized in collagen-rich areas in the lamina propria,
muscularis mucosa, and submucosal area in colonic tissues. Noting the
correlation between HSP47 and collagen expressions, the authors
targeted HSP47 with an HSP47 siRNA in IL-10 KO mice. As in renal brosis, local inhibition of HSP47 expression drastically reduced collagen
deposition and improved intestinal brosis (Kitamura et al., 2011).
Chen et al. also studied the role of HSP47 in keloid disease, a
broproliferative disorder affecting cutaneous cells. Their rst study in
2007 focused on keloid broblast cells transfected with an HSP47
siRNA. They demonstrated that following HSP47 inhibition, both intracellular and extracellular collagen levels were reduced (Chen et al.,
2007). In 2011, the same authors studied the downregulation of
HSP47 in vivo using a keloid animal model based on subcutaneous implantation of human keloid dermal tissues (Chen et al., 2011). Following
HSP47 downregulation in vivo, the volume of implants was reduced
compared to control mice. This reduction correlated with the reduction
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of collagen expression. From both studies, they concluded that HSP47

overexpression in keloid broblasts and in keloid animal models could
explain the excess of collagen deposition, and therefore they proposed
HSP47 inhibition as a possible therapeutic approach for treating keloid
disease.
Hagiwara et al. reported a protective effect of HSP47 inhibition in
vivo in several lung brosis models (Hagiwara et al., 2007a, 2007b,
2007c). Regardless of the model of pulmonary brosis used (induced
by paraquat, bleomycin or LPS), HSP47 downregulation reduced lung
injury and collagen deposition and improved lung morphology and
function in rats. Although in those studies HSP47 siRNA was administered after the induction of brosis, the authors proposed that HSP47
inhibition might be of great interest to stop or even reverse the progress
of brogenesis and the resulting diseases.
Surprisingly, among the many publications highlighting a role for
HSP47 in the process of brosis, only a few propose a mechanism of action. A study performed in 2007 showed a role for neutrophils in HSP47
upregulation. The authors suggest that -defensines secreted by neutrophils during their recruitment to the site of tissue injury are capable
of activating the MAPK pathway in neighboring epithelial cells, thus inducing an increase in HSP47 expression (Yoshioka et al., 2007). Furthermore, in a renal brosis model using human proximal tubular epithelial
cells stimulated with TGF-1, Xiao et al. demonstrated that ERK1/2, JNK
and MAPK inhibitors were able to block HSP47 expression induced by
TGF-1. In their model, the inhibition of those pathways suppressed
the overexpression of ECM proteins and PAI-1, thus limiting brogenesis. The authors suggested that during renal brosis TGF-1 could
enhance HSP47 expression through the ERK1/2 and JNK MAPK signaling
pathways, thereby enhancing collagen synthesis (Xiao et al., 2012).
TGF-1 is not the only cytokine that is able to induce HSP47. IL1, an inammatory cytokine, has also been shown to upregulate HSP47 at the
protein and mRNA levels by itself or in combination with TGF-1

(Sasaki et al., 2002). TGF-1 and IL1 were able to favor nuclear localization of HSF1 and enhance HSF1 trimerization, leading to an increase in
HSF1 binding to its' specic gene response element such as the one
found in the HSP47 promoter (Fig. 5). Thus, this work suggests that
HSP47 could be a good target to prevent brogenesis given that TGF1 and IL1, two crucial molecules in the development of brogenesis,
are able to induce its expression via the trimerization of HSF1 (Sasaki
et al., 2002).
7. Small heat shock proteins (sHSP)
7.1. Heat Shock Protein 27: favors pulmonary brosis
HSP27 belongs to a family of low molecular weight heat shock proteins, the small heat shock proteins (sHSP). It is the most studied and
most expressed HSP at specic stages of development and cellular differentiation. Its basal expression is relatively low but is largely increased
during heat shock and various cellular stresses. HSP27 is constitutively
expressed in the cytoplasm (Lindquist & Craig, 1988), but in case of
heat shock it rapidly translocates into the nucleus (Arrigo et al., 1988).
The N-terminal domain contains a WDPF motif required for the formation of oligomers, whereas the C-terminal domain is involved in the
stabilization of those oligomers. In the cell, there is an equilibrium
between dimeric HSP27 and larger oligomeric forms. Oligomerization
is a dynamic process driven by the phosphorylation status of HSP27
(Kato et al., 1994). Phosphorylated HSP27 forms dimers, and dephosphorylated HSP27 oligomerizes (Mehlen & Arrigo, 1994; Mehlen et al.,
1997). HSP27 can be phosphorylated mainly on three serines located
at positions 15, 78 and 82. This phosphorylation is regulated by MK2
(MAPKAP Kinase 2) and p38MAPK (Stokoe et al., 1992).
Fig. 5. HSP47 favors brotic response through its role in collagen biosynthesis. TGF-1 and/or IL-1 signaling triggers HSF1 nuclear translocation and trimerization. Active HSF1 trimers
bind to a response element in the HSP47 promoter region and enhance HSP47 expression. HSP47 stabilizes procollagen and facilitates the formation of procollagen triple helices. As a
consequence, HSP47 favors collagen secretion by activated cells, promoting brosis.
HSP27 is known to be involved in cytoskeletal dynamics. Indeed, the

dephosphorylated form of HSP27 is able to bind to the terminal end of
actin laments and inhibit their polymerization, whereas its phosphorylated form separates from actin and allows polymerization (Kostenko
et al., 2009). In addition to its effect on actin, HSP27 has been shown
to have anti-apoptotic properties (Bruey et al., 2000) and to promote
cell survival. For instance, HSP27 induces the ubiquitination and degradation of the NF-B inhibitor, I-B, thus allowing nuclear translocation
of NF-B and activation of target genes promoting cell survival such as
Bcl-2, BcL-XL and IAP (inhibitor of Apoptosis Protein, (Parcellier et al.,
2003)).
HSP27 has also been demonstrated to be overexpressed in cancerous
cells, protecting them from apoptosis and favoring their proliferation
(Garrido et al., 2003). In addition, HSP27 seems to promote metastasis
through its effect on cytoskeletal components and favors resistance to
chemotherapeutic agents (Hino et al., 2000; Katoh et al., 2000; G.P. Li
et al., 2011). Choi et al. showed that HSP27 expression was directly
responsible for colorectal cancer cell resistance to the chemotherapeutic
agent irinotecan. By inhibiting HSP27, the authors could decrease the
resistance of those cells to the treatment (Choi et al., 2007). Moreover,
Zhuang et al. demonstrated that HSP27 inhibition sensitized A549
cancerous lung cells to apoptosis induced by TRAIL (Tumor necrosis
factor-alpha-Related Apoptosis-Inducing Ligand, (Zhuang et al., 2010)).
The number of studies investigating the role of HSP27 in brogenesis
is quite limited. However, upregulation of HSP27 in the lungs of patients
affected by pulmonary brosis has been reported in cells located near broblastic foci, more specically in regions where ECM-producing cells
are found during the disease (Korfei et al., 2011). Another study showed
that HSP27 was upregulated in epithelial cells located around broblastic foci. The authors suggested that epithelial cell proliferation and migration could play a role in the morphological changes occurring in
the lungs during pulmonary brosis (Chilosi et al., 2006). Two other articles showed that HSP27 was involved in the process of EMT. Vargha
et al. showed in 2008 that HSP27 was upregulated in vitro during EMT
in mesothelial cells from peritoneum whereas it was diminished in
vivo during chronic EMT in dialysis-induced peritoneal brosis
(Vargha et al., 2008). The authors suggested that the discordant
HSP27 expression between in vivo and in vitro EMT might be due to
morphological changes that are more important during in vitro TGF1-induced EMT. Indeed, in their model, during in vivo EMT induced
by peritoneal dialysis uids, cells underwent EMT, but only a subpopulation of cells presented morphological broblast-like changes. In vitro,
TGF-1 was associated with strong morphological changes and SMA upregulation. The correlation between -SMA and HSP27 upregulation might reect cytoskeletal alterations induced by TGF-1 during
myobroblastic transformation of mesothelial cells. In vitro, HSP27 upregulation protected cells from apoptosis induced by peritoneal dialysis
uids (Vargha et al., 2008). Vidyasagar et al. postulated the involvement
of HSP27 in renal brosis via its role on EMT. The authors showed that
HSP27 was overexpressed during kidney brosis and that it colocalized with TGF-1, -SMA and E-cadherin. Overexpression of
HSP27 in renal epithelial cells induced E-cadherin expression and repressed the expression of Snail, the major transcription factor promoting EMT. The authors concluded that HSP27 had a protective role
against brosis and that its upregulation could slow down brosis and
EMT (Vidyasagar et al., 2008). The same authors recently demonstrated
that HSP27 upregulation in tubular epithelial cells lead to decreased
brogenesis associated with a decline in phosphorylated p38MAPK, collagen III, and -SMA, following induction of renal brosis in transgenic
mice. In their model, E-cadherin levels remained unchanged in tubular
cells after the induction of brosis (Vidyasagar et al., 2013).
Another recent study highlighted the effect of HSP27 overexpression
during EMT in epithelial and mesothelial cells during pulmonary and
pleural brosis. In this study, HSP27 was upregulated within broblastic
foci in vivo in patients affected by pulmonary brosis (Wettstein et al.,
2013). HSP27 induced phenotypic changes in cells, which acquired a
127
myobroblast-like shape along with -SMA upregulation and Ecadherin downregulation. Furthermore, the inhibition of HSP27 in vivo
using OGX-427, an antisense oligonucleotide already in clinical trials
as a chemo-sensitizing agent in cancer therapy (Zoubeidi & Gleave,
2012), protected rats from pleural brosis induced by TGF-1 overexpression. In addition to the inhibition of brosis, repression of HSP27
also inhibited the migration of pleural myobroblasts towards the
lung parenchyma, an essential event in the in vivo model of TGF-1induced pleural brosis (Wettstein et al., 2013). The therapeutic use of
OGX-427 against pleuro-pulmonary brosis has been patented
(Oncogenics).
HSP27 chaperones and stabilizes the essential transcription factor in
the induction of EMT, Snail. HSP27 protects Snail from degradation by
the proteasome, leading to its nuclear accumulation and activation of
its EMT-inducing target genes (Fig. 6). By extension, inhibition of
HSP27 in vivo induced degradation of Snail and stalled EMT, conrming
that HSP27 may be an interesting therapeutic target against pulmonary
brosis (Wettstein et al., 2013).
HSP27 is a well-documented pro-brotic agent in pulmonary brosis. However, this function has yet to be conrmed in other organs although HSP27 has been reported to protect from renal brosis in vitro
and in vivo (Vidyasagar et al., 2008; Vidyasagar et al., 2013).
7.2. B-crystallin: a pro-brotic factor
Like HSP27, B-crystallin is another ubiquitous sHSP, one that is
highly inducible under stress conditions in many organs such as the
brain, heart, smooth muscles and lungs (Bhat & Nagineni, 1989).
HSP27 and B-crystallin are two closely related proteins, and it has
been shown that these proteins co-localized in many organs in normal
and pathological conditions. Some publications even report a synergistic role of these two sHSP, which are able to interact with each other
(MacIntyre et al., 2008). In 2001, a study showed that HSP27 prevented
structural changes and aggregation of B-crystallin induced by heat
shock and thus may have a role in its stabilization (Fu & Liang, 2003).
Like HSP27, B-crystallin has a chaperone activity and is able to bind
hydrophobic areas on the surface of misfolded proteins, preventing
their aggregation and protecting cellular integrity (Markov et al.,
2008). The function of B-crystallin is modulated by its oligomerization
and its phosphorylation at three serine residues, serines 19, 45 and 59.
B-crystallin has a role in many physiological and pathological processes such as cell growth and cell differentiation, apoptosis, tumorigenesis,
signal transduction and the modulation of cytoskeletal proteins such as
intermediate laments, the latter being one of the most important functions of B-crystallin (Nicholl & Quinlan, 1994; Wisniewski & Goldman,
1998; Launay et al., 2006). Indeed, B-crystallin is known to interact
with intermediate laments and contribute to their homeostasis. The
damage to the cytoskeleton architecture caused by mutations of Bcrystallin, which sometimes leads to severe diseases in humans,
shows the importance of these interactions (Wettstein et al., 2012).
Brady et al. established the role of B-crystallin in skeletal and cardiac muscle through the study of mice decient for the gene encoding Bcrystallin (Brady et al., 2001). Surprisingly, the eye-lens structure of Bcrystallin-decient mice was normal, suggesting that the development
and maintenance of lens transparency do not strictly require the presence of this chaperone. Although the lens does not seem to be affected
by the absence of B-crystallin, this mutation causes a severe phenotype in aged mice characterized by hunched posture, a signicant loss
of body weight after 40 weeks, and muscle cell degeneration. Bcrystallin is also expressed at high levels during early embryonic development of the heart and also in the fully formed heart. However, the
heart structure of B-crystallin decient mice appeared normal, even
in older mice (Brady et al., 2001).
B-crystallin is involved in many cellular processes, including
apoptosis. Several studies have demonstrated that B-crystallin
overexpression had a protective effect against a wide range of
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Fig. 6. HSP27 stabilizes the transcription factor Snail and favors EMT. Left: HSP27 is able to bind Snail and protect it against proteasomal degradation. After activation of the TGF- signaling
pathway, Snail translocates to the nucleus and induces the transcription of EMT genes. Right: When HSP27 is inhibited, Snail degradation by the proteasome is enhanced, thus inhibiting
the EMT process and preventing brosis.
apoptotic stimuli, whereas its inhibition signicantly sensitizes cells

to programmed cell death (Kamradt et al., 2001; Kamradt et al.,
2002; Kamradt et al., 2005; Garrido et al., 2012). The antiapoptotic effect of B-crystallin has been shown to involve the inhibition of caspase-3, a key apoptosis effector protein (Kamradt et al.,
2001; Mao et al., 2001; Kamradt et al., 2002; Morrison et al., 2003). It
has been demonstrated that B-crystallin interacts with procapase3 and thereby inhibits its cleavage and activation. This effect of Bcrystallin is critical to its anti-apoptotic function. Morrison et al.
demonstrated that phosphorylation of B-crystallin on serine 59
appeared to be necessary and sufcient to ensure maximal protection against apoptosis in cardiomyocytes (Morrison et al., 2003).
B-crystallin was also able to bind to pro-apoptotic proteins such
as Bax, Bcl-Xs (Mao et al., 2004) and p53 (Liu et al., 2007) and prevent their translocation into the mitochondria, thus inhibiting
apoptosis.
Another recently identied function of B-crystallin is its ability to
facilitate the degradation of specic proteins by the ubiquitin/proteasome system (Garrido, 2002). B-crystallin phosphorylated on serine
19 and 45 is able to interact with FBX4, a component of the SCF
ubiquitin-ligase (SKP1/CUL1/F-box) that ubiquitinates client proteins
to target them to the proteasome (den Engelsman et al., 2003). The
B-crystallin/FBX4 complex facilitates the degradation of various
substrates.
B-crystallin has been shown to be upregulated in several human
diseases (Aoyama et al., 1993; Pinder et al., 1994; Lo et al., 2007;
Cherneva et al., 2010, 2012), but its role in brogenesis remains elusive. In a model of liver brosis, it was shown in vitro and in vivo that
B-crystallin was quickly upregulated after HSC (hepatic stellate
cell) activation and acquired nuclear localization in heat-shock conditions. B-crystallin was totally absent in non-activated HSCs, suggesting a role for it in the activation of these cells, a key step in liver
brosis (Lang et al., 2000; van de Bovenkamp et al., 2008). Rezzani
et al. also showed that overexpression of B-crystallin is induced
by cyclosporine A (CsA) in a model of vascular brosis. CsA has a direct effect on the structure and spatial arrangement of the cytoskeleton and especially on the expression of vimentin and desmin. The
authors suggested that B-crystallin expression is induced to protect
cells from the toxic effects of cytoskeletal remodeling, thereby offering protection from vascular brosis (Rezzani et al., 2005). Interestingly, other studies have shown that B-crystallin expression can
be induced by TGF-1 (Welge-Lussen et al., 1999; Yu et al., 2007),
and a recently published article by Huang et al. showed a role for
B-crystallin in the EMT process in a model of hepatocellular carcinoma. The authors showed that B-crystallin formed a complex
with the protein 14-3-3, protecting it from degradation by the proteasome. This results in an increase in the pool of 14-3-3 in the
cell, leading to the activation of the ERK signaling pathway (Fig. 7).
Therefore, B-crystallin favors the activation of the ERK phosphorylation cascade, leading to the activation of the transcription factor
Slug, an inducer of EMT (Huang et al., 2013). This process, highlighted in a model of hepatic carcinoma, may be particularly important in
brogenesis because, as already mentioned, EMT is a process involved in brosis.
A study recently demonstrated a role for B-crystallin in pulmonary brosis. For the rst time, B-crystallin has been shown to be
overexpressed in hyperplastic alveolar epithelial cells and also
within broblastic foci in lungs from patients affected by pulmonary
brosis. Furthermore, B-crystallin was upregulated during brogenesis in mouse models of pulmonary brosis induced by several
agents including bleomycin, TGF-1 and IL-1. Mouse lacking Bcrystallin were not able to produce TGF-1 in an efcient way, and
we identied a direct role for B-crystallin in the TGF-1 pathway.
B-crystallin was able to modulate the localization of the protein
Smad4, which is responsible for the translocation of Smad2 and
Smad3 into the nucleus, where they can activate TGF-- responsive
genes. The lack of B-crystallin abrogated the nuclear localization
of Smad4s, thereby blocking the TGF-1 pathway and subsequent
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Fig. 7. B-crystallin inhibition favors Smad4 nuclear export. Left: In the absence of B-crystallin, Smad4 is monoubiquitinated by TIF1, enhancing the nuclear export of Smad4. Right: Bcrystallin reduces the interaction between Smad4 and TIF1, thus limiting Smad4 monoubiquitination and nuclear export. The TGF-1 pathway is therefore inhibited. In parallel, Bcrystallin interacts with the protein 14-3-3, allowing Raf dimerization and activation of the ERK phosphorylation cascade.
brosis (Fig. 7, Bellaye et al., 2014). The nuclear localization of

Smad4 is driven by its monoubiquitination by TIF1. In vitro, the
overexpression of B-crystallin disrupted the interaction between
TIF1 and Smad4, mono-ubiquitination of Smad4 and its nuclear export. As a consequence, Smad4 was sequestered in the nucleus, favoring the TGF--induced brosis (Fig. 7, Bellaye et al., 2014).
These studies demonstrate that B-crystallin is a new potential
therapeutic target in brosis and the urgent need for specic Bcrystallin inhibitors.
be of interest because they prevent brogenesis in several animal

models of tissue brosis. Their efciency has yet to be evaluated in clinical trials. Furthermore, we believe that, although it has yet to be investigated, the now well-known role of extracellular HSPs in inammation
and immunomodulation may also contribute to their overall function in
the process of brogenesis.
Conict of interest statement
The authors declare that there are no conicts of interest.
8. Concluding remarks
Heat shock proteins are involved in several cellular processes and
display a wide range of functions. Further investigation is needed to
clearly determine their roles in brogenesis, but recent publications
have demonstrated their potential use in therapeutic approaches
against brotic diseases. Interestingly, depending on the particular
HSP, HSPs can have opposing effects on brotic processes. HSP110 and
HSP70 have been demonstrated to be anti-brotic factors and therefore
are benecial in the context of brosis, whereas HSP90, HSP47, HSP27
and B-crystallin have been demonstrated to favor brogenesis. Except
for HSP47, which acts directly on collagen stability and synthesis, the
most relevant role for HSPs is affecting the TGF-1 pathway. HSP90,
HSP27 and B-crystallin are able to enhance the TGF-1 pathway by
stabilizing TGF- receptors (HSP90), Snail (HSP27) or by favoring the
nuclear localization of Smads (B-crystallin). In contrast, HSP70 and
HSP110 proteins have been demonstrated to induce TGF- receptor
degradation, thus limiting Smad phosphorylation and inhibiting the
TGF- pathway. Because the major role of the TGF- pathway in brosis
is now well documented, modulation of the expression and activity of
HSPs might represent a new therapeutic approach for brotic disorders
such as pulmonary brosis for which no curative treatment currently
exists. Indeed, inhibitors of HSP90, HSP27 or B-crystallin appear to
Acknowledgment
The research leading to these results was funded by the European
Community 7th Framework Programme (FP7/20072013) under
grant agreement HEALTH-F2-2007-202224 eurIPFnet, the FEDER
Agence Nationale de la Recherche Blanc 11-BSV-011-01 meso-IPF
(ANR), La Ligue Rgionale Grand Est Contre le Cancer and l'Institut National du Cancer (INCa). PS.B. is supported by the EU 7th Frame work
Programme (20072013 agreement HEALTH-F2-2007-202224
eurIPFnet.). PSB and O.B. are supported by le Fonds de Dotation
Recherche en Sant Respiratoire (FRSR). The C.G. team is supported
by La Ligue National Contre le Cancer, the Association pour la Recherche
sur le Cancer and LabEx LipSTIC.
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Heat Shock Proteins in Fibrosis and Wound Healing Good or Evil

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Pharmacology & Therapeutics 143 (2014) 119132

Contents lists available at ScienceDirect

Pharmacology & Therapeutics

Associate editor: N. Frossard

Heat shock proteins in brosis and wound healing: Good or evil?

Available online 26 February 2014

Abbreviations: 17-AAG, 17-(Allylamino)-17-demethoxygeldanamycin; AP-1,

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

proteins (Smurf 1 and 2) are E3-ubiquitin ligases that are able to

kidney cell lines stimulated with rTGF-1 and in a murine model of

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

4. Heat Shock Protein 70 and brosis: intracellular versus extracellular function

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

reduces organ damage signicantly after the induction of lung and

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

HEALTH-F2-2007-202224 eurIPFnet, the FEDER Agence Nationale de la

functions given that extracellular HSP70 aggravated cardiac brosis

Fig. 3. Extracellular HSP70 promotes TLR4 signaling leading to TGF-1 activation.

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

of collagen expression. From both studies, they concluded that HSP47

protein and mRNA levels by itself or in combination with TGF-1

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

HSP27 is known to be involved in cytoskeletal dynamics. Indeed, the

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

apoptotic stimuli, whereas its inhibition signicantly sensitizes cells

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

brosis (Fig. 7, Bellaye et al., 2014). The nuclear localization of

be of interest because they prevent brogenesis in several animal

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

P.-S. Bellaye et al. / Pharmacology & Therapeutics 143 (2014) 119132

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