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Proteomics 2016, 16, 1331–1340 DOI 10.1002/pmic.

201500211 1331

RESEARCH ARTICLE

Dp71⌬78-79 dystrophin mutant stimulates neurite


outgrowth in PC12 cells via upregulation and
phosphorylation of HspB1
Candelaria Merino-Jiménez1 , Jorge Aragón1 , Vı́ctor Ceja1 , Griselda Rodrı́guez-Martı́nez1 ,
Febe E. Cázares-Raga2 , Solenne Chardonnet3,4 , Cédric Pionneau3,4 , Alvaro Rendon5
and Cecilia Montañez1
1
Departamento de Genética y Biologı́a Molecular, Centro de Investigación y de Estudios Avanzados del IPN,
México, D.F., México
2
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN,
México, D.F., México
3
UPMC Univ Paris 06, UMS 2 Omique, Sorbonne Universités, Plateforme P3S, Paris, France
4
UMS 29 Omique, INSERM, Plateforme P3S, Paris, France
5
Institut de la Vision, INSERM UMR_S968, CNRS UMR_7210, Université Pierre et Marie Curie Paris 06, Paris, France

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, Received: June 4, 2015
this phenotype is more efficiently achieved when the Dp71⌬78-79 dystrophin mutant is stably Revised: January 24, 2016
expressed in PC12-C11 cells. To investigate the effect of Dp71⌬78-79 overexpression on the Accepted: February 29, 2016
protein profile of PC12-C11 cells, we compared the expression profiles of undifferentiated and
NGF-differentiated PC12-C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein
was downregulated, and five were upregulated. Dp71⌬78-79 overexpression had a greater effect
on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein
with the highest upregulation was HspB1. Changes in HspB1 expression were validated by
Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12-
C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and
HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process.
Our results show that Dp71⌬78-79 affects the expression level of some proteins and that the
stimulated neurite outgrowth produced by this mutant is mainly through upregulation and
phosphorylation of HspB1.

Keywords:
Cell biology / Dp71⌬78-79 / HspB1 / MS / Neurite outgrowth / PC12 cells

 Additional supporting information may be found in the online version of this article at
the publisher’s web-site

1 Introduction [1]. Dp71 transcripts are alternatively spliced in exons 71,


The dystrophin Dp71 protein is encoded by the Duchenne 71–74 and/or 78, generating several isoforms [2, 3]. Although
muscular dystrophy (DMD) gene. It is expressed at high lev- the function of Dp71 has not been fully elucidated, muta-
els in non-muscle tissues and is most abundant in adult brain tions in the DMD gene that disrupt Dp71 expression have
been correlated with the severity of the cognitive impairment
Correspondence: Dr. Cecilia Montañez, Departamento de observed in DMD patients, suggesting that Dp71 might play
Genética y Biologı́a Molecular, Centro de Investigación y de Estu- an important role in the central nervous system [4–6].
dios Avanzados del IPN, Av. IPN 2508, Col. San Pedro Zacatenco, Studies in the PC12 cell line have shown that the ex-
07360 México, D.F., México pression and localization of Dp71 isoforms are differentially
E-mail: cecim@cinvestav.mx regulated during nerve growth factor (NGF)-induced
Fax: +52-55-57473392
differentiation [7] and that the Dp71 isoform expression is
Abbreviations: NGF, neural growth factor; WB, Western blotting essential for neurite outgrowth [8]. Recently, our research


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1332 C. Merino-Jiménez et al. Proteomics 2016, 16, 1331–1340

Significance of the study

Our results show that the expression of the Dp71⌬78-79 dys- growth of PC12-C11 cells. These results provide the basis to
trophin mutant modifies the expression profile of several explore the mechanism by which the Dp71 mutant promotes
proteins. In addition, we demonstrate that the most upregu- neurite outgrowth via enhanced expression and phosphory-
lated protein (HspB1) has an important role in neurite out- lation of HspB1 in PC12 cells.

group showed that a dystrophin Dp71⌬78-79 mutant lacking urea, 30% v/v glycerol, 2% w/v SDS, 75 mM Tris-HCl, pH
exons 78 and 79 (PC12-C11 cells) stimulates NGF-induced 8.8, 0.002% w/v bromophenol blue and 1% w/v DTT) for 15
neuronal differentiation in PC12 cells [9]. In this study, we min, followed by a second treatment in the same solution
investigated the effect of Dp71⌬78-79 overexpression on the where the DTT was replaced by 2.5% w/v iodoacetamide.
protein profiles of undifferentiated and NGF-differentiated Then, equilibrated strips were run in 12% SDS-PAGE using
PC12-C11 cells using 2DE, with the aim of identifying differ- a Mini-Protean Tetra system (Bio-Rad) at 80 V/gel for 3 h.
entially regulated proteins controlled by Dp71⌬78-79 . Gels were stained with Bio-Safe Coomassie Stain (Bio-Rad).
Four 2DE gels were obtained using independent biological
replicates for each condition.
2 Materials and methods

2.1 Cell culture and differentiation 2.4 Image and data analysis

PC12-C11 and PC12 cells were grown and differentiated for 2DE gels were scanned with the Gel DocTM EZ Imager (Bio-
six days as previously described [9]. Rad). To evaluate protein abundance, digitalized gel images
were analyzed using ImageMaster 2D Platinum 6.0 software
(GE Healthcare). The percentage of volume (% volume) was
2.2 Protein sample preparation determined for each spot. Resulting sets of averaged rela-
tive spot volume were analyzed by a non-parametric Mann–
Undifferentiated and differentiated PC12-C11 and PC12 cells Whitney test to detect spots with different expression levels.
were harvested, washed with PBS and homogenized in a Spots with P-values<0.05 were considered statistically signif-
dedicated 2DE lysis buffer (7 M urea, 2 M thiourea, 4% icant. The spots with changes of 1.5-fold or more were excised
CHAPS, 40 mM DTT, 2% IPG buffer, pH 3–10 NL) or in from the 2DE gels and analyzed by MS.
a phosphorylation-preserving lysis buffer (20 mM Tris HCl,
pH 7.8, 1 mM EDTA, 10 mM ␤-glycerophosphate, 1 mM
Na3 CO4 , 11 mM NaF, 10 mM Na pyrophosphate) for specific 2.5 Protein identification
phosphoprotein detection. Both lysis buffers contained 1X of
protease inhibitor cocktail (Roche, Inc.). The proteins were Excised spots were destained in 50% v/v ethanol, 50 mM
precipitated by the addition of ice-cold acetone, and the pellet NH4 HCO3 and dehydrated with ACN. Gel pieces were di-
was dissolved in rehydration buffer (7 M urea, 2 M thiourea, gested overnight with 200 ng of trypsin (G-Biosciences) in
2% CHAPS, 40 mM DTT, 2% IPG buffer, pH 3–10 NL). Pro- 50 mM NH4 HCO3 , 5% ACN. Supernatants were collected,
tein samples were cleaned using the 2-D Clean-Up kit (GE and gel pieces were washed twice with 50% ACN, 0.1% TFA
Healthcare). The protein concentration was determined by in an ultrasonic bath. Peptides were vacuum dried and re-
the Bradford method. suspended in 30% ACN, 0.1% TFA. For MALDI-TOF MS
analysis, 1 ␮L of peptide solution was mixed with 1 ␮L of ma-
trix solution (0.7 mg/mL CHCA in 50% ACN, 0.1% TFA) and
2.3 2DE analyzed using an Autoflex Speed MALDI-TOF/TOF mass
spectrometer (Bruker Daltonics) in reflectron mode or using
For the IEF, IPG strips (pH 3.0–10.0 NL, 7 cm) were rehy- a 4800 Plus MALDI TOF/TOF mass spectrometer (Sciex). LC-
drated for 16 h with rehydration buffer containing 200 ␮g MS/MS analysis was performed with an Ultimate3000 Nano
of protein and 0.05% bromophenol. The strips were run on HPLC (Thermo Fisher Scientific) coupled with an HCT ultra
an Ettan IPGphor 3 Isoelectric Focusing System (GE Health- ion trap mass spectrometer (Bruker Daltonics). LC separation
care), with the following running conditions: step-and-hold at was performed on a PepMap100 column (0.075 mm id, 3-␮m
300 V (200 Vh), gradient to 1000 V (300 Vh), gradient to 5000 V particle size, 15 cm) at a flow rate of 300 nL/min with a linear
(4500 Vh) and finally step-and-hold at 5000 V (2000 Vh). gradient from 0 to 35% of H2 O/ACN/formic acid (5/95/0.1
After IEF, strips were immersed in equilibration buffer (6 M v/v) for 35 min. Proteins were identified using MASCOT


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Proteomics 2016, 16, 1331–1340 1333

version 2.5.1 (MatrixScience) against the Swiss-Prot, TrEMBL group, ten photomicrographs were analyzed, and the percent-
or NCBInr of the Rattus norvegicus data bank, allowing for age of cells bearing neurites at least one cell body length was
two missed cleavages with carbamidomethylation of cysteines enumerated using the ImageJ software (US National Insti-
(fixed modification), oxidation of methionine (variable modi- tutes of Health). Assays were performed in triplicate. The
fication), and MS and MS/MS tolerance of 0.5 Da. Confident expression levels of HspB1 and HspB1Ser86 were measured
matches were defined by a MASCOT score higher than 52 for by WB after treatment. Assays were performed in duplicate.
MALDI-TOF and at least two peptides with a score of 31 for
LC-MS/MS (p-value<0.05).
2.9 Statistical analysis

2.6 Western blot analysis The results obtained from three independent experiments
are expressed as the mean ± SD. Statistical analysis was
Forty ␮g of total proteins was used for Western blot (WB) performed by an unpaired Student’s t-test using GraphPad
assays as previously described [9]. Rabbit polyclonal an- Prism 5 software. P-values<0.05 were considered statistically
tibodies against HspB1, annexin-5, TH, aldose reductase, significant for the quantification of the relative expression of
phospho-specific HspB1Ser86 and mouse monoclonal an- the selected proteins.
tibodies against secretogranin-2 and NF-L were purchased
from Abcam. The mouse monoclonal anti-␤-actin was pro-
vided by Dr. Manuel Hernández (CINVESTAV, México). Im- 3 Results
munoreactivity was detected by the Lightning Plus ECL kit
(PerkinElmer, Inc.). Images were captured using the Kodak 3.1 Protein expression profiles of undifferentiated
Digital Science ID programme. The results of densitometry and differentiated PC12-C11 cells
analyses of WB, obtained using ImageJ software (US National
Institutes of Health), were represented as relative density to To better understand the role of the Dp71⌬78-79 dystrophin
the control (␤-actin). Assays were performed in triplicate. mutant in the stimulation of PC12-C11 neuronal differenti-
ation [9], we compared the expression profiles of undiffer-
entiated and NGF-differentiated PC12-C11 and PC12 cells
2.7 Immunofluorescence assays (Fig. 1). Proteins were considered as differentially expressed
with at least a 1.5-fold change. In undifferentiated PC12-C11
Differentiated PC12-C11 and PC12 cells were grown on cov- cells, 323 protein spots were detected, but only four spots (U1-
erslips precoated with poly-L-lysine and fixed with methanol. U4, where U represents undifferentiated cells) showed differ-
Cells were permeabilized with 0.3% Triton X-100 and blocked ential expression (Fig. 1A and B). In differentiated PC12-C11
with 0.5% gelatin. Primary antibodies (rabbit polyclonal cells, 391 protein spots were detected, and only 15 spots (D1-
anti-HspB1, anti-annexin-5 (Abcam) and mouse monoclonal D15, D for differentiated cells) showed differential expression
anti-SYP (Santa Cruz)) were incubated overnight at 4⬚C. (Fig. 1C and D). Magnified views of all differentially expressed
Immunolabeling was visualized with Alexa-488- or Alexa- proteins in the presence of NGF are presented in Fig. 1E. The
594-conjugated secondary antibodies (Molecular Probes, In- % volume for each differentially expressed spot is shown in
vitrogen). Nuclei were counterstained with DAPI (1 ng/mL, Fig. 1F. Overall, these results indicate that some proteins
Sigma). Mounting was performed in Vectashield mounting are differentially expressed due to the Dp71⌬78-79 dystrophin
medium (Vector Laboratories, Inc.). Immunofluorescence mutant expression in PC12 cells.
was visualized by confocal microscopy (Leica TCS SP8). 15–20
optical Z-sections (0.3–0.5 ␮m thick) were scanned from each
sample. To quantify the fluorescence intensity of each pro- 3.2 Protein identification
tein, thirty photomicrographs of 1014×1014 pixels acquired
at 40X magnification with the Leica TCS SP8 were analyzed To identify the differentially expressed proteins, the selected
using the Leica TCS SP software. spots were excised from 2DE gels and analyzed by MS. A
total of 23 proteins were identified (Table 1), and in some
spots, more than two proteins were found. We selected the
2.8 Immunologic blockade assay proteins with higher numbers of matched peptides. In some
cases, the same protein was identified in two different spots,
PC12-C11 and PC12 cells were grown to 70% confluence on corresponding to distinct isoforms of the protein, which was
collagen-coated culture dishes and treated with 50 ng/mL the case for annexin-2, GAPDH and HspB1. Among the 17
NGF (Invitrogen) for 3 days in presence of 10 ␮M of proteins differentially expressed in differentiated cells, the
anti-HspB1 antibody (sc-1049, Santa Cruz) or the vehicle. most substantially upregulated protein was HspB1, with two
Morphological changes were observed using an Axiovert25 different isoforms that were 5.2-fold (D13) and 3.5-fold (D14)
Zeiss microscope at 40X magnification. For each treatment overexpressed in PC12-C11 cells (Table 1).


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1334 C. Merino-Jiménez et al. Proteomics 2016, 16, 1331–1340

Figure 1. Representative 2DE maps of PC12-C11 and PC12 proteins. (A and B) Protein profiles of undifferentiated (U) PC12-C11 and PC12
cells, respectively. U1-U4 indicates differentially expressed proteins. (C and D) Protein profiles of differentiated (D) PC12-C11 and PC12 cells,
respectively. D1-D15 indicates differentially expressed proteins. (E) Magnified views of the PC12-C11 and PC12 spots that were differentially
expressed in NGF-differentiated cells. (F) The % Volume of spots differentially expressed in PC12-C11 and PC12 NGF-differentiated cells.
The graph shows the average % Volume ± SD for four separate experiments. ** P<0.01 and *** P<0.001. The arrow represents the
nonlinear immobilized pH gradient used for IEF. Positions of standard mass markers (kDa) for the second dimension are shown on the left
side of the images.


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Table 1. Proteins identified by mass spectrometry


Proteomics 2016, 16, 1331–1340


C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Spot ID Variation Fold-change MS Accession Protein Gene MW [kDa] pI MASCOT Match SC (%)b)
in spot no.a) score Pept

U1 – 2.5 LC-MS/MS Q62952 Dihydropyrimidinase-related Dpysl3 61.9 6.0 1359.5 24 45.3


protein 3
U2 + 3.7 LC-MS/MS P85834 Elongation factor Tu, Tufm 49.5 7.9 1321.0 23 60.8
mitochondrial
P41562 Isocitrate dehydrogenase Idh1 46.7 6.6 835.6 16 37.9
[NADP] cytoplasmic
U3 + 2.3 LC-MS/MS O88767 Protein DJ-1 Park7 20.0 6.4 506.8 10 56.6
U4 + n.a. LC-MS/MS P62260 14-3-3 protein epsilon Ywhae 29.2 4.5 1096.1 19 79.6
Q63610 Tropomyosin alpha-3 chain Tpm3 29.0 4.6 699.1 11 40.3
D1 + 2.3 LC-MS/MS Q66HD0 Endoplasmin Hsp90b1 92.7 4.6 1923.8 33 37.1
D2 – 6.1 LC-MS/MS P10362 Secretogranin-2 Scg2 71.0 4.6 1348.9 28 46.4
P20156 Neurosecretory protein VGF Vgf 68.1 4.5 1037.2 16 33.4
D3 + 1.7 LC-MS/MS P06761 78 kDa glucose-regulated Hspa5 72.3 4.9 2563.2 38 48.8
protein
D4 – n.a. LC-MS/MS P19527 Neurofilament light Nefl 61.3 4.5 1648.5 30 47.2
polypeptide
D5 – 2.9 LC-MS/MS Q62952 Dihydropyrimidinase-related Dpysl3 61.9 6.0 1542.9 28 54
protein 3
P04177 Tyrosine 3-monooxygenase Th 55.9 5.7 575.5 9 33.1
D6 + 1.9 LC-MS/MS P18418 Calreticulin Calr 48.0 4.2 679.9 14 30
D7 – 2.2 LC-MS/MS B1WC26 N-acetylneuraminic acid Nans 40.0 6.4 633.1 13 30.9
synthase
Q5XI22 Acetyl-CoA acetyltransferase, Acat2 41.1 7.1 513.1 8 37.3
cytosolic
D8 – 3.4 LC-MS/MS Q5XI22 Acetyl-CoA acetyltransferase, Acat2 41.1 7.1 732.1 10 65
cytosolic
Q64559 Cytosolic acyl coenzyme A Acot7 42.7 9.6 424.3 7 21.5
thioester hydrolase
D9 – 1.6 MALDI P07943 Aldose reductase Akr1b1 35.8 6.3 124.0 13 46.8
D10 + 1.9 LC-MS/MS Q07936 Annexin A2 Anxa2 38.7 8.6 1587.6 25 62.8
P04797 Glyceraldehyde-3-phosphate Gapdh 35.8 9.0 701.2 14 41.7
dehydrogenase
D11 + 2.1 LC-MS/MS Q07936 Annexin A2 Anxa2 38.7 8.6 1507.8 25 64
P04797 Glyceraldehyde-3-phosphate Gapdh 35.8 9.0 561.4 11 28.2
dehydrogenase
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D12 + 2.2 MALDI P14668 Annexin A5 Anxa5 35.7 4.9 926.0 15 66.5
D13 + 3.5 MALDI P42930 Heat shock protein beta-1 Hspb1 22.9 6.1 161.0 13 63.1
D14 + 5.2 MALDI P42930 Heat shock protein beta-1 Hspb1 22.9 6.1 690.0 12 82
D15 – n.a. LC-MS/MS Q00981 Ubiquitin carboxyl-terminal Uchl1 24.8 5.0 541.1 10 53.8
hydrolase isozyme L1
Q5XI73 Rho GDP-dissociation inhibitor Arhgdia 23.4 5.0 448.1 9 30.4
1
a) Accession number according to the Swiss-Prot database of Rattus norvegicus.

1335
b) SC % = % of the sequence identified.
n.a.: not applicable as % volume was only obtained for one of the two PC12-C11 or PC12 cells.
1336 C. Merino-Jiménez et al. Proteomics 2016, 16, 1331–1340

Figure 2. Validation of differen-


tially expressed proteins. (A) Ex-
pression of HspB1, annexin-5,
NF-L, secretogranin-2, TH and
aldose reductase proteins. (B)
Relative expression of HspB1,
annexin-5, NF-L, secretogranin-
2, TH and aldose reductase pro-
teins, respectively, normalized
to ␤-actin expression. The graph
presents the mean ± SD from
three independent experiments.
* P < 0.05, ** P < 0.01 and ***
P < 0.001.

3.3 Validation of differentially expressed proteins tification showed that HspB1 immunoreactivity was 3.3-fold
higher in PC12-C11 compared to PC12 cells, whereas for
To validate the changes in the expression levels observed in annexin-5, it was 2.0-fold higher (Fig. 3M).
the 2DE gels, we focused on HspB1 and annexin-5 because
the first one is implicated in neurite outgrowth [10], and
annexin-5 is a membrane-associated protein with calcium- 3.5 Immunologic blockade assay against HspB1
channel activity [11]. In addition, we also selected some pro-
teins involved in PC12 differentiation induced by NGF that To study the role of HspB1 during NGF-induced differen-
unexpectedly were downregulated by Dp71⌬78-79 dystrophin tiation of PC12-C11 cells, we employed an immunologic
expression (NF-L, secretogranin-2, TH and aldose reductase). blockade assay using an antibody against HspB1. The cell
For all the selected proteins, the relative expression levels differentiation percentage was monitored on the third day
were measured in differentiated PC12-C11 and PC12 cells of treatment. PC12-C11 cells treated with NGF showed
by WB (Fig. 2). Similarly to the results obtained by 2DE 20.71% ± 0.97 differentiation and NGF + control (vehicle
(Fig. 1), HspB1 exhibited a 6.3-fold increase, and annexin- of polyclonal anti-HspB1) showed 21.70% ± 1.36, but in
5 showed a 2.1-fold increase, while NF-L decreased 8.4-fold, the presence of NGF + anti-HspB1, the percentage of cell
secretogranin-2 5.1-fold, TH 2.3-fold and aldose reductase differentiation decreased considerably to 5.38% ± 1.36. In
2.4-fold (Fig. 2). PC12 cells treated with NGF, 15.94% ± 1.44 of cell differen-
tiation was observed, NFG + control was 15.59% ± 0.86, and
NGF + anti-HspB1 was 12.26% ± 1.85. Therefore, our results
3.4 Localization of HspB1 and annexin-5 proteins show that treatment with anti-HspB1 decreased the number
of neurite-bearing cells (Fig. 4C and G). It has been shown
To determine the localization of HspB1 and annexin-5, an that HspB1 is activated upon phosphorylation [10, 12–14].
immunofluorescence assay was performed in differentiated Ser15, Ser78 and Ser82 are the main sites for phosphoryla-
PC12-C11 and PC12 cells using the rabbit polyclonal anti- tion in human, and Ser15 and Ser86 are the main sites in rat
HspB1 and anti-annexin-5 antibodies. In both panels of (Ser82 is the major phosphorylation site and an orthologous
Fig. 3, the neuronal marker synaptophysin is shown in green, site of HspB1Ser86) [15, 16]. To determine the effect of the
HspB1 or annexin-5 are shown in red, and nuclei stained with immunologic blockade assay, we measured the expression
DAPI are represented in blue. The localization of HspB1 levels of HspB1 and HspB1Ser86 by WB. Interestingly, both
in differentiated PC12-C11 cells was mainly cytoplasmic, al- HspB1 and HspB1Ser86 decreased after the treatment with
though it was also observed along the neurites (Fig. 3B). In the antibody (Fig. 4H and I).
contrast, in PC12 cells, immunostaining of HspB1 was barely
observed (Fig. 3E). Annexin-5 was predominantly cytoplasmic
and nuclear and was observed along the length of the neu- 4 Discussion
rites in PC12-C11 cells (Fig. 3H), but in PC12 cells, it was
cytoplasmic, although the immunostaining was lower than In this study, to elucidate how Dp71⌬78-79 stimulates neu-
in PC12-C11 cells (Fig. 3K). The fluorescence intensity quan- ronal differentiation, we compared the proteomic expression


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Proteomics 2016, 16, 1331–1340 1337

Figure 3. Localization of HspB1


and annexin-5 in PC12-C11 and
PC12 differentiated cells. A, D,
G and J depict the localiza-
tion of the synaptophysin pro-
tein (in green) used as a neu-
ronal marker. (B and E) Localiza-
tion of HspB1 (red) in PC12-C11
and PC12 cells, respectively. (H
and K) Localization of annexin-
5 (red) in PC12-C11 and PC12
cells, respectively. C, F, I and
L show the merge of the two
preceding images. Nuclei were
stained with DAPI (blue). Images
are representative of three inde-
pendent experiments. Scale bar
represents 25 ␮m. (M) Quantita-
tion of HspB1 and annexin-5 flu-
orescence intensity. The graph
represents the mean ± SD from
three independent experiments.
** P < 0.01 and *** P < 0.001.


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1338 C. Merino-Jiménez et al. Proteomics 2016, 16, 1331–1340

Figure 4. Anti-HspB1 effect upon neurite outgrowth in PC12-C11 cells. (A–C) PC12-C11 cells, (D–F) PC12 cells. (A and D) Cells treated with
NGF. (B and E) NGF + control (vehicle of polyclonal anti-HspB1), (C and F) NGF + anti-HspB1. (G) Percentage of cells with neurites on
the third day of treatment. The graph represents the mean ± SD of three independent experiments. The scale bar represents 50 ␮m.
(H) Detection of HspB1 and HspBSer86 in cells treated with anti-HspB1. (I) Relative expression of HspB1 normalized to ␤-actin expression
and relative expression of HspB1Ser86 normalized to HspB1 expression. The graph represents the mean ± SD from two independent
experiments. * P < 0.05 and *** P < 0.001.

between PC12-C11 and PC12 cells. We identified several pro- expression of both proteins could explain the morphological
teins in most of the variant spots because the use of 7 cm 2DE changes observed in undifferentiated PC12-C11 cells; PC12
gels resulted in a co-migration in the same spot of proteins cells are round, whereas the PC12-C11 cells are oval and even
having a close pI and molecular weight. We thus considered begin to show neurite outgrowth [9]. DJ-1 has a protective role
that proteins with at least twice as many peptides as others against oxidative stress [20]. Therefore, the observed increase
were responsible for the variation observed; however, we can- of this protein may suggest a neuro-protective effect.
not exclude an effect of some of the less abundant proteins The effect of Dp71⌬78-79 on the protein profile of NGF-
that were observed (Supporting Information Table 1). differentiated PC12-C11 cells was more evident. Annexin-
In undifferentiated cultures, DRP-3 was identified as a 5 and annexin-2 were upregulated proteins. Annexins are
downregulated protein, which has been associated with class calcium-binding proteins involved in membrane traffick-
3 semaphorin signaling, axon guidance [17], neuronal growth ing, membrane-cytoskeleton interactions, calcium channel
cone collapse and cell migration. Interestingly, the expres- activity and signal transduction [21]. Annexin A2 bundles
sion levels of this protein also decreased in differentiated actin filaments and plays a role in their polymerization
cultures, making it important to explore its effect on the [22], whereas annexin A5 modulates the affinity for cal-
growth cone in PC12-C11 differentiated cells. Among the cium ions to create cooperative binding [23]. The increased
upregulated proteins identified, the 14-3-3E protein has been expression of these proteins may suggest a role as ion
reported to enhance catecholamine exocytosis [18] by disas- channels to regulate cytosolic calcium levels in PC12-C11
sembling the actin network to facilitate the movement of cells.
secretory granules [19]. Tropomyosin-3 is implicated in the HspB1 showed the highest increase, and it has been de-
stabilization of cytoskeleton actin filaments. The increased scribed in filament structures within dendrites or axons and


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Proteomics 2016, 16, 1331–1340 1339

in synaptic components [14]. Phosphorylated HspB1 is in- Based on the findings described in this work, our results
volved in cytoskeleton restructuration, actin polymerization showed that the expression of the Dp71⌬78-79 dystrophin mu-
and stress fibre formation, contributing to axonal outgrowth tant altered the protein expression profile of PC12 cells and
[10, 12, 13]. In PC12-C11 cells, the HspB1 protein was located that the induced neuritogenesis takes place via upregulation
in the cytoplasm and along the neurites (Fig. 3B). In addition, and phosphorylation of HspB1.
we determined the biological relevance of HspB1 with an
We gratefully acknowledge Consejo Nacional de Ciencia y Tec-
immunologic blockade assay using a polyclonal anti-HspB1.
nologı́a (Conacyt) for supporting this work (C. Merino Doctoral
In this experiment, the neurite outgrowth of PC12-C11 cells
Fellowship 233893 and C. Montañez Grant CB-2013-222054
was significantly reduced as well as the level of HspB1 and
and ECOS-ANUIES-SEP-Conacyt M11-S02). We thank MSc.
HspB1Ser86, suggesting that the expression and phosphory-
Emmanuel Rı́os Castro at the Unidad de Genómica, Protéomica
lation of HspB1 are required for neurite outgrowth of PC12-
y Metabolómica, LaNSE-CINVESTAV for equipment support.
C11 cells. On the other hand, the neurite outgrowth of NGF-
differentiated PC12 cells was not considerable affected by the The authors have no conflicts of interest to declare.
treatment with anti-HspB1 because the HspB1 expression in
these cells is very low. These results suggest that the neu-
rite outgrowth in PC12 cells is independent of HspB1 in the 5 References
presence of NGF. However, it has been reported that when
PC12 cells are stimulated to induce neurite outgrowth by [1] Tadayoni, R., Rendon, A., Soria-Jasso, L., Cisneros, B., Dys-
pituitary adenylate cyclase-activating polypeptide (PACAP), trophin Dp71: the smallest but multifunctional product of the
HspB1 was upregulated [24]. Furthermore, in a subline of Duchenne muscular dystrophy gene. Mol. Neurobiol. 2012,
PC12 cells (PC6), the neurite outgrowth was dependent on 45, 43–60.
HspB1Ser82 activation through PKA [25]. [2] Aragón, J., Martı́nez-Herrera, A., Romo-Yáñez, J., Ceja, V.
In this work, we found that differentiated PC12-C11 cells et al., Identification of Dp71 isoforms expressed in PC12
show phosphorylation of HspB1Ser86, suggesting an activa- cells: subcellular localization and colocalization with ␤-
tion of this protein, which could be responsible for promoting dystroglycan and ␣1-syntrophin. J. Mol. Neurosci. 2016,
58, 201–209.
neurite outgrowth. Based on all results obtained, HspB1 is a
likely to be a relevant protein for neurite outgrowth in PC12- [3] Austin, R. C., Morris, G. E., Howard, P. L., Klamut, H. J., Ray,
C11 cells. P. N., Expression and synthesis of alternatively spliced vari-
ants of Dp71 in adult human brain. Neuromuscul. Disord.
Surprisingly, some proteins that play important roles
2000, 10, 187–193.
in PC12 neuronal differentiation were downregulated
(secretogranin-2, TH, aldose reductase and NF-L) (Fig. 2). [4] Daoud, F., Angeard, N., Demerre, B., Martie, I. et al., Analysis
of Dp71 contribution in the severity of mental retardation
Secretogranin-2 is involved in secretion and increases during
through comparison of Duchenne and Becker patients differ-
NGF treatment [26]. TH catalyses the conversion of tyrosine
ing by mutation consequences on Dp71 expression. Hum.
to L-DOPA, the first step in biosynthesis of catecholamines Mol. Genet. 2009, 18, 3779–3794.
[27]. Aldose reductase participates in the reduction of 3,4-
[5] Daoud, F., Candelario-Martı́nez, A., Billard, J.-M., Avital, A.
dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite
et al., Role of mental retardation-associated dystrophin-gene
formed by the oxidative deamination of dopamine to 3,4- product Dp71 in excitatory synapse organization, synap-
dihydroxyphenylethanol (DOPET) [28]. The downregulation tic plasticity and behavioral functions. PloS one 2009, 4,
of these proteins in PC12-C11 cells may suggest an alteration e6574.
in the secretion of catecholamines, mainly dopamine. Neuro- [6] Moizard, M.-P., Toutain, A., Fournier, D., Berret, F. et al., Se-
filament proteins contribute to the extension and stabilization vere cognitive impairment in DMD: obvious clinical indica-
of neuronal processes, and it has been demonstrated that the tion for Dp71 isoform point mutation screening. Eur. J.Hum.
degree of PC12 differentiation can be quantified by NF-L ex- Genet. 2000, 8, 552–556.
pression [29]. Rho GDI 1 is also downregulated in PC12-C11 [7] Marquez, F., Cisneros, B., Garcia, F., Ceja, V. et al., Differential
cells and is implicated in the reorganization of the actin cy- expression and subcellular distribution of dystrophin Dp71
toskeleton [30]. The downregulation of proteins relevant to isoforms during differentiation process. Neuroscience 2003,
neuronal differentiation suggests that the observed neurite 118, 957–966.
outgrowth requires lower levels of these proteins compared [8] Acosta, R., Montañez, C., Fuentes-Mera, L., Gonzalez, E. et al.,
to the PC12 cells. Dystrophin Dp71 is required for neurite outgrowth in PC12
Annexins, tropomyosins and heat shock proteins are com- cells. Exp. Cell Res. 2004, 296, 265–275.
monly found as variant proteins in proteomic studies and are [9] Aragón, J., Romo-Yáñez, J., Martı́nez-Herrera, A., Ceja, V.
not considered to be relevant proteins [31]. It is not the case for et al., Characterization of Dp71⌬78-79, a novel dystrophin
this study, where the changes in the expression of annexin-5 mutant that stimulates PC12 cell differentiation. J. Neu-
and HspB1 were validated by WB and immunofluorescence rochem. 2011, 119, 697–707.
assays. Moreover, the biological relevance of the upregulated [10] Williams, K. L., Rahimtula, M., Mearow, K. M., Heat shock
HspB1 in neurite outgrowth was also demonstrated. protein 27 is involved in neurite extension and branching of


C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
1340 C. Merino-Jiménez et al. Proteomics 2016, 16, 1331–1340

dorsal root ganglion neurons in vitro. J. Neurosci. Res. 2006, J., Annexin-phospholipid interactions. Functional implica-
84, 716–723. tions. Int. J. Mol. Sci. 2013, 14, 2652–2683.
[11] Gerke, V., Creutz, C. E., Moss, S. E., Annexins: linking Ca2+ [22] Grieve, A. G., Moss, S. E., Hayes, M. J., Annexin A2 at the
signalling to membrane dynamics. Nat. Rev. Mol. Cell Biol. interface of actin and membrane dynamics: a focus on its
2005, 6, 449–461. roles in endocytosis and cell polarization. Int. J. Cell Bio.
[12] Clarke, J. P., Mearow, K. M., Cell stress promotes the associ- 2012, 2012, 852430.
ation of phosphorylated HspB1 with F-actin. PloS one 2013, [23] Gauer, J. W., Knutson, K. J., Jaworski, S. R., Rice, A. M.
8, e68978. et al., Membrane modulates affinity for calcium ion to create
[13] Guay, J., Lambert, H., Gingras-Breton, G., Lavoie, J. N. et al., an apparent cooperative binding response by annexin A5.
Regulation of actin filament dynamics by p38 map kinase- Biophys. J. 2013, 104, 2437–2447.
mediated phosphorylation of heat shock protein 27. J. Cell [24] Lebon, A., Seyer, D., Cosette, P., Coquet, L. et al., Identifica-
Sci. 1997, 110, 357–368. tion of proteins regulated by PACAP in PC12 cells by 2D gel
[14] Schmidt, T., Bartelt-Kirbach, B., Golenhofen, N., electrophoresis coupled to mass spectrometry. Ann. N. Y.
Phosphorylation-dependent subcellular localization of Acad. Sci. 2006, 1070, 380–387.
the small heat shock proteins HspB1/Hsp25 and HspB5/␣B- [25] Shi, G.-X., Jin, L., Andres, D. A., Pituitary adenylate cyclase-
crystallin in cultured hippocampal neurons. Histochem. Cell activating polypeptide 38-mediated Rin activation requires
Biol. 2012, 138, 407–418. Src and contributes to the regulation of HSP27 signaling dur-
[15] Kostenko, S., Moens, U., Heat shock protein 27 phosphoryla- ing neuronal differentiation. Mol. Cell. Biol. 2008, 28, 4940–
tion: kinases, phosphatases, functions and pathology. Cell. 4951.
Mol. Life Sci. 2009, 66, 3289–3307. [26] Taupenot, L., Harper, K. L., O’Connor, D. T., The
[16] Landry, J., Lambert, H., Zhou, M., Lavoie, J. et al., Hu- chromogranin–secretogranin family. N. Engl. J. Med. 2003,
man HSP27 is phosphorylated at serines 78 and 82 by 348, 1134–1149.
heat shock and mitogen-activated kinases that recognize the [27] Kvetnansky, R., Sabban, E. L., Palkovits, M., Catecholamin-
same amino acid motif as S6 kinase II. J. Biol. Chem. 1992, ergic systems in stress: structural and molecular genetic ap-
267, 794–803. proaches. Physiol. Rev. 2009, 89, 535–606.
[17] Yuasa-Kawada, J., Suzuki, R., Kano, F., Ohkawara, T. et al., Ax- [28] Lamensdorf, I., Eisenhofer, G., Harvey-White, J., Nechustan,
onal morphogenesis controlled by antagonistic roles of two A. et al., 3,4-Dihydroxyphenylacetaldehyde potentiates the
CRMP subtypes in microtubule organization. Eur. J. Neu- toxic effects of metabolic stress in PC12 cells. Brain Res.
rosci. 2003, 17, 2329–2343. 2000, 868, 191–201.
[18] Morgan, A., Burgoyne, R. D., Exol and Exo2 proteins stimu- [29] Schimmelpfeng, J., Weibezahn, K.-F., Dertinger, H., Quantifi-
late calcium-dependent exocytosis in permeabilized adrenal cation of NGF-dependent neuronal differentiation of PC-12
chromaff in cells. Nature 1992, 355, 833–836. cells by means of neurofilament-L mRNA expression and
[19] Roth, D., Burgoyne, R. D., Stimulation of catecholamine se- neuronal outgrowth. J. Neurosci. Methods 2004, 139, 299–
cretion from adrenal chromaffin cells by 14-3-3 proteins is 306.
due to reorganisation of the cortical actin network. FEBS [30] Sasaki, T., Takai, Y., The Rho small G protein family-Rho GDI
Lett. 1995, 374, 77–81. system as a temporal and spatial determinant for cytoskele-
[20] Ariga, H., Takahashi-Niki, K., Kato, I., Maita, H. et al., tal control. Biochem. Biophys. Res. Commun. 1998, 245, 641–
Neuroprotective function of DJ-1 in Parkinson’s dis- 645.
ease. Oxidative Med. Cell. Longevity 2013, 2013, [31] Petrak, J., Ivanek, R., Toman, O., Cmejla, R. et al., Deja vu
683920. in proteomics. A hit parade of repeatedly identified differen-
[21] Lizarbe, M. A., Barrasa, J. I., Olmo, N., Gavilanes, F., Turnay, tially expressed proteins. Proteomics 2008, 8, 1744–1749.


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