Induction of expression and phosphorylation of heat shock protein B5 (CRYAB) in rat myometrium during pregnancy and labour

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J G NicolettiDepartment of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
One Reproductive Health Research Group, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

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B G WhiteOkanagan College, Salmon Arm Campus, Salmon Arm, British Columbia, Canada

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E I MiskiewiczDepartment of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
One Reproductive Health Research Group, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

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D J MacPheeDepartment of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
One Reproductive Health Research Group, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Division of Biomedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John’s, Newfoundland, Canada

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During pregnancy the myometrium undergoes a programme of differentiation induced by endocrine, cellular, and biophysical inputs. Small heat shock proteins (HSPs) are a family of ten (B1–B10) small-molecular-weight proteins that not only act as chaperones, but also assist in processes such as cytoskeleton rearrangements and immune system activation. Thus, it was hypothesized that HSPB5 (CRYAB) would be highly expressed in the rat myometrium during the contractile and labour phases of myometrial differentiation when such processes are prominent. Immunoblot analysis revealed that myometrial CRYAB protein expression significantly increased from day (D) 15 to D23 (labour; P<0.05). In correlation with these findings, serine 59-phosphorylated (pSer59) CRYAB protein expression significantly increased from D15 to D23, and was also elevated 1-day post-partum (P<0.05). pSer59-CRYAB was detected in the cytoplasm of myocytes within both uterine muscle layers mid- to late-pregnancy. In unilaterally pregnant rats, pSer59-CRYAB protein expression was significantly elevated in the gravid uterine horns at both D19 and D23 of gestation compared with non-gravid horns. Co-immunolocalization experiments using the hTERT-human myometrial cell line and confocal microscopy demonstrated that pSer59-CRYAB co-localized with the focal adhesion protein FERMT2 at the ends of actin filaments as well as with the exosomal marker CD63. Overall, pSer59-CRYAB is highly expressed in myometrium during late pregnancy and labour and its expression appears to be regulated by uterine distension. CRYAB may be involved in the regulation of actin filament dynamics at focal adhesions and could be secreted by exosomes as a prelude to involvement in immune activation in the myometrium.

Abstract

During pregnancy the myometrium undergoes a programme of differentiation induced by endocrine, cellular, and biophysical inputs. Small heat shock proteins (HSPs) are a family of ten (B1–B10) small-molecular-weight proteins that not only act as chaperones, but also assist in processes such as cytoskeleton rearrangements and immune system activation. Thus, it was hypothesized that HSPB5 (CRYAB) would be highly expressed in the rat myometrium during the contractile and labour phases of myometrial differentiation when such processes are prominent. Immunoblot analysis revealed that myometrial CRYAB protein expression significantly increased from day (D) 15 to D23 (labour; P<0.05). In correlation with these findings, serine 59-phosphorylated (pSer59) CRYAB protein expression significantly increased from D15 to D23, and was also elevated 1-day post-partum (P<0.05). pSer59-CRYAB was detected in the cytoplasm of myocytes within both uterine muscle layers mid- to late-pregnancy. In unilaterally pregnant rats, pSer59-CRYAB protein expression was significantly elevated in the gravid uterine horns at both D19 and D23 of gestation compared with non-gravid horns. Co-immunolocalization experiments using the hTERT-human myometrial cell line and confocal microscopy demonstrated that pSer59-CRYAB co-localized with the focal adhesion protein FERMT2 at the ends of actin filaments as well as with the exosomal marker CD63. Overall, pSer59-CRYAB is highly expressed in myometrium during late pregnancy and labour and its expression appears to be regulated by uterine distension. CRYAB may be involved in the regulation of actin filament dynamics at focal adhesions and could be secreted by exosomes as a prelude to involvement in immune activation in the myometrium.

Introduction

The uterine smooth muscle or myometrium undergoes many structural and physiological changes throughout pregnancy to become a powerful, contractile tissue at term. These changes occur in a series of four phases during pregnancy and one phase post-partum. Each phase is marked by unique characteristics that have been well defined in the pregnant rat model (Shynlova et al. 2013). At the beginning of rat pregnancy until day (D) 14, the myometrium enters the proliferation phase. Myometrial cells increase in number and the expression of anti-apoptotic factors, including BCL2, is increased within these cells contributing to an overall increase in proliferation (Shynlova et al. 2006). The next phase is the synthetic phase from D15 to D21, where the protein:DNA ratio in the myometrium is increased, cells hypertrophy and increase their production of interstitial extracellular matrix proteins (ECM) and remodel focal ­adhesions (MacPhee & Lye 2000, Shynlova et al. 2004). In the contractile phase, there is an increased expression of basement membrane matrix proteins such as fibronectin and associated integrin receptors in myometrial cells (Shynlova et al. 2004, Williams et al. 2005, 2010). During the final phase of pregnancy, the labour phase, the expression of contractile-associated proteins (CAPs) is elevated, such as the gap junction protein GJA1 and OXTR (oxytocin receptor; Tabb et al. 1992, Ou et al. 1998).

Immune system activation and immune cell infiltration of the myometrium is also thought to play a role in the initiation of labour (Thomson et al. 1999, Osman et al. 2003, Shynlova et al. 2013). Foetal growth-induced uterine stretch may positively regulate both the production of chemokines and cytokines and chemokine-mediated infiltration of immune cells into the myometrium during term and pre-term labour aiding cytokine-mediated myometrial contractility (Shynlova et al. 2008, 2013).

The small heat shock protein B (HSPB) family comprises ten small-molecular-weight proteins (15–40 kDa; B1–B10) that are key for cellular homeostasis and are also induced by physiological stressors such as mechanical forces (Kampinga & Garrido 2012). HSPB members not only act as ATP-independent molecular chaperones, but also assist in cell death regulation, cytoskeletal rearrangements, and immune system activation (Acunzo et al. 2012, van Noort et al. 2012, ­Wettstein et al. 2012). The HSPB family is characterized by a conserved C-terminal region named the α-crystallin domain, a more variable N-terminal sequence, and in most cases, a short variable C-terminal tail (Garrido et al. 2012). Phosphorylation of these proteins, particularly on serine residues, is critical for the regulation of structure and function. For example, HSPB1 phosphorylation induces the dissociation of large oligomers of HSPB1 and marked loss of chaperoning activity (Kato et al. 1994, Garrido et al. 2012).

CRYAB, previously known as αB-crystallin, was discovered as a highly abundant protein in the eye lens, where it maintains the transparency of the structure (Bloemendal 1982, Clark et al. 2012). To this end, it acts as a molecular chaperone to aid cytoprotection and prevent aggregation of denatured proteins (­Horwitz 1992). CRYAB is expressed in a multitude of other ­tissues, and mutations in CRYAB can lead to congenital cataracts, cardiac myopathies, and neurodegenerative diseases (Acunzo et al. 2012, ­Boncoraglio et al. 2012). CRYAB phosphorylation on serine 59 (pSer59) residues also regulates CRYAB–actin ­interaction (Singh et al. 2007).

From an immune system perspective, CRYAB has also been shown, in vitro, to induce an interleukin-10 (IL10) regulatory macrophage immune response at low concentrations; whereas, at higher concentrations, it can increase the T-cell production of interferon-γ (IFN-γ), which activates macrophages (van Noort et al. 2010, 2012). CRYAB can also alter the immune system by increasing the expression of endothelial cell adhesion molecules, such as ICAM1 and SELE (E-selectin), that are responsible for slowing leukocyte rolling and facilitating leukocyte entry into tissues (Dieterich et al. 2013).

Despite current knowledge, the expression of CRYAB in the myometrium has never been fully characterized. Thus, we examined the spatiotemporal expression of CRYAB in the rat myometrium during pregnancy and in a human myometrium-derived cell line, as well as the potential regulation of CRYAB expression by uterine distension. Due to the potential role of CRYAB in aiding actin filament dynamics and immune cell activation, it was hypothesized that CRYAB would be highly expressed in the myometrium during the contractile and labour phases of myometrial differentiation.

Materials and methods

Animals

Sprague–Dawley rats were acquired from the Mount Scio Vivarium (Memorial University of Newfoundland, St John’s, NL, Canada) and used for all experiments. Animals were individually housed and cared for under standard environmental conditions (12h light and 12h darkness) in the Animal Care Unit at the Health Sciences Centre, Memorial University of Newfoundland. The rats had access to water ad libitum and were maintained on LabDiet Prolab RMH 3000 (PMI ­Nutrition International, Brentwood, MO, USA). For all experiments, ­virgin female rats weighing approximately 220–250 g were mated with stud males. Day 1 of the gestational period was designated following the observation of a vaginal plug the morning after mating. The time of delivery under these standard conditions was D23 of the gestational period. All experiments were granted ethical approval by the institutional animal care committee under protocols 08-02-DM to 11-02-DM.

Experimental design

Cell culture

The hTERT-HM myometrium-derived cell line was a gracious gift from Dr Ann Word (University of Texas Southwestern Medical Center, Dallas, TX, USA) and established via stable transfection of human myometrial cells with the expression vectors containing the human telomerase reverse transcriptase (hTERT), which maintains telomere length and immortalizes cells (­Condon et al. 2002). These cells retain myometrial cell characteristics such as the expression of CNN1 (calponin) and OXTR proteins (Condon et al. 2002). hTERT-HM cells were cultivated in DMEM/F12 media with l-glutamine and 15mM HEPES (catalogue number (Cat #): 11330-032; Life Technologies) plus 10% foetal bovine serum and 1% penicillin–­streptomycin (Cat #: 15140-122; Life Technologies). Cell cultures were maintained at 37°C and 5% CO2 in air. The medium was refreshed every 24h and cells were passaged when they reached ∼90% confluence using trypsin-EDTA solution (0.05% v/v trypsin in EDTA, Cat #: 15400-054; Life Technologies).

Tissue collection

Carbon dioxide-induced asphyxiation was used for killing all animals before sample collection. For normal gestation, samples were collected from animals at ten time points throughout gestation including non-pregnant (NP), D6, D12, D15, D17, D19, D21, D22, D23 (labour), and 1-day post-partum (PP). For immunofluorescence detection, a portion of the rat uterine horn was fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.4) while shaking overnight at room temperature and then washed in PBS for 24h. Tissues were processed, paraffin embedded, sectioned, and mounted on microscope slides by the Histology Unit of Memorial University of Newfoundland School of Medicine. Cross sections of the uterine horn were used for experiments and both the longitudinal and the circular muscle layers of the myometrium were included in all sections. All sections were treated under identical conditions at the same time for each experiment.

For immunoblot analysis, uterine horns were removed, excised, and opened longitudinally, after which foetuses and placentae were discarded. Uterine tissue was then placed in ice-cold PBS and a scalpel blade was used to gently scrape away the endometrial layer, as described previously (White et al. 2005). All myometrial samples were flash-frozen in liquid nitrogen and stored at −80°C.

Unilaterally pregnant rat model

Virgin female rats (∼220g) were administered an intramuscular injection of anaesthesia (100mg/kg ketamine, 20mg/kg xylazine; Ketaset, Wyeth Animal Health, Guelph, ON, Canada; Rompun, Bayer) and then received a unilateral tubal ligation as described previously (White & MacPhee 2011). Animals were monitored post-operatively and subsequently allowed to recover for at least 5 days before mating was attempted. Samples of gravid and non-gravid horns were collected on ­gestational D19 and D23.

Immunoblot analysis

Immunoblot analysis was performed on at least three independent sets of myometrium samples from normal rat gestation and unilaterally pregnant rat models (n≥3 for each timepoint analyzed). Tissue samples were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (50mM Tris–HCl [pH 7.5], 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)) plus protease and phosphatase inhibitors (Completemini, Cat # 04693124001; PhosSTOP, Cat # 04906845001; Roche Diagnostics). Protein concentration was determined using the Bio-Rad Protein Assay Kit II according to the manufacturer’s instructions (Cat #: 500-0002; Bio-Rad). Protein samples (20μg/lane of protein for the rat gestational profile; 15μg/lane of protein for the unilaterally pregnant model) were electrophoretically separated under reducing conditions in 12% SDS–polyacrylamide gels and subsequently electroblotted onto 0.2μm nitrocellulose membranes (Cat #: 162-0097; Bio-Rad).

The membranes were stained with a Memcode Reversible Protein Stain kit to verify protein transfer (Cat #: 24580; Thermo Fisher Scientific) as per the manufacturer’s instructions. Blots were then incubated for 1h with antisera that recognized both unphosphorylated and phosphorylated CRYAB (total-CRYAB) or CRYAB specifically phosphorylated on serine-59 (pSer59-CRYAB; given in Table 1 for all antisera used for experiments). Antisera were diluted in blocking solution consisting of 5% milk in Tris-buffered saline containing Tween 20 (TBST; 20mM Tris–HCl [pH 7.6], 150mM NaCl, 0.1% Tween 20) or 5% bovine serum albumin (BSA) in TBST for phospho-specific antibodies. An appropriate horseradish peroxidase (HRP)-conjugated secondary antibody diluted in blocking solution was used for immunoblot development. Protein detection on immunoblots was accomplished using a SuperSignal West Pico chemiluminescence substrate detection system (Cat # 34080; Thermo Fisher Scientific) and multiple exposures were acquired using a Bio-Rad ChemiDoc MP digital imaging system. Membranes were subsequently stripped with Restore Western Blot Stripping Buffer according to the manufacturer’s instructions (Cat #: 21059; Thermo Fisher Scientific) and re-probed for ­glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, which served as a loading control, using a rabbit polyclonal GAPDH-specific antibody (Table 1).

Table 1

Antibodies used for immunoblot (IB) or immunofluorescence assays with myometrial tissue sections (IF-T) or hTERT-HM cells (IF-C).

Antibody Supplier Catalogue number Method and dilution
Rabbit anti-total CRYAB Enzo Life Sciences, Farmingdale, NY, USA ADI-SPA-223 IB: 1: 10,000
Rabbit anti-total CRYAB Sigma-Aldrich HPA-028724 IF-T: 1:100
Mouse anti-total CRYAB Stressmarq Biosciences, Victoria, BC, CA SMC-159 IF-C: 1:100
Rabbit anti-pSer59-CRYAB Enzo Life Sciences, Farmingdale, NY, USA ADI-SPA-227 IB: 1: 10,000

IF-T, IF-C: 1:100
Mouse anti-FERMT2 Millipore MAB2617 IF-C: 1:100
Rabbit anti-ACTA1 Abcam ab5694 IF-C: 1:20
Mouse anti-ACTA1 Sigma-Aldrich A2547 IF-C: 1:1000
Mouse anti-CD63 Thermo Fisher Scientific MA5 11501 IF-C: 1:20
Rabbit anti-GAPDH Abcam ab9485 IB: 1:10,000
ChromePure rabbit IgG Jackson ImmunoResearch 011-000-003 IF-T, IF-C: N/A*
ChromePure mouse IgG Jackson ImmunoResearch 015-000-003 IF-C: N/A*
HRP goat anti-rabbit IgG Promega W4011 IB: 1:10,000
FITC sheep anti-rabbit IgG Sigma-Aldrich F7512 IF-T, IF-C: 1:250
RRX goat anti-mouse IgG Jackson ImmunoResearch 715-295-150 IF-C: 1:150

*Depending on the concentration of the primary antisera used. FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; RRX, rhodamine red-X.

Immunofluorescence analysis

Rat myometrium during pregnancy

Tissue sections from three independent sets of rat uterine tissue samples from normal rat gestation and unilaterally pregnant rat models were used for experiments (n=3 for each timepoint analyzed). Tissue sections were deparaffinized in xylene three times for 5min each and then rehydrated in a descending series of ethanol (100, 95, 90, 80, 70, and 50%). Heat-induced epitope retrieval was conducted with boiling 10mM sodium citrate buffer (pH 6.0) for 10min followed by enzyme-induced epitope retrieval with 1mg/mL trypsin solution (Cat # T7168; Sigma Chemical Co, Oakville, ON, Canada) at room temperature for 10min. Sections were then incubated for 1h in a blocking solution consisting of 5% normal goat serum, 1% normal horse serum, and 1% foetal bovine serum in PBS.

Tissue sections were incubated overnight at 4°C with either total-CRYAB or pSer59-CRYAB-specific antisera diluted in blocking solution. Non-specific isotype-matched rabbit IgG (Table 1), used at the same concentration as primary antisera, was used as a negative control in all experiments. Sections were washed with PBS and then incubated with fluorescein isothiocyanate (FITC)-conjugated sheep anti-rabbit IgG as a secondary antibody. Following two washes in ice-cold 0.02% Tween 20 in PBS, sections were then mounted in ProLong Gold Anti-Fade Reagent with DAPI (Cat #: P36931; Life Technologies). Images were collected using an Olympus BX51 microscope with widefield epifluorescence capabilities and a DP70 Olympus digital camera (Olympus).

hTERT-HM cells

Following cell collection by trypsinization, cells were counted with a Bio-Rad TC20 cell counter, and 1×105 cells were seeded onto 22mm×22mm sterile glass coverslips placed within six-well tissue culture plates. After cell cultivation for 24h, cells were fixed with 4% PFA in PBS for 5min at room temperature and then washed with PBS. Cells were then treated with PBS containing 0.1% Triton X-100 for 15min at room temperature and subsequently with blocking solution for 30min at room temperature. Combinations of primary antisera specific for total CRYAB, pSer59-CRYAB, the focal adhesion protein Kindlin-2 (FERMT2), α-smooth muscle actin (ACTA1), and the tetraspanin CD63 were used for experiments (Table 1). For negative controls, affinity-purified mouse or rabbit IgG was used in place of primary antisera and at the same concentration. FITC-conjugated anti-rabbit IgG and a rhodamine red-X (RRX)-conjugated anti-mouse IgG were used as secondary antibodies. Cells were then mounted to glass slides using ProLong Gold Anti-Fade Reagent with DAPI. Independent experiments were conducted three times and the images were collected using an Olympus IX83 microscope equipped with widefield epifluorescence capabilities, an Andor Zyla sCMOS camera, and CellSens software (Olympus) or a Leica TCS-SP5 laser scanning confocal microscope equipped with Leica LAS AF imaging software (Leica). For confocal imaging, a z-series of optical slices were collected for each antibody pair and analyzed with the Leica LAS AF imaging software. A plan apochromat 63X oil/NA 1.40 objective was used for analysis (for additional parameters, Supplementary Fig. 1, see section on supplementary data given at the end of this article) and appropriate lookup tables used to ensure pixel saturation were prevented. Further analyses were conducted using orthogonal views (XZ and YZ axes) using the Stacks function within the Fiji open-source platform for biological image analysis (Schindelin et al. 2012). The z-series of optical slices also underwent image deconvolution with AutoQuant X3 (Bitplane USA, Concord, MA, USA), for calculation of Pearson’s correlation coefficient using the JACoP plug-in within ImageJ (Bolte & Cordelieres 2006, Adler & Parmryd 2010).

Statistical analysis

Densitometric analysis was performed on immunoblot data by using Image Lab software (Bio-Rad). Densitometric measurements of total-CRYAB protein or pSer59-CRYAB protein on immunoblots were normalized to the densitometric measurements of GAPDH protein expression. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software). Data from normal gestational profiles were subjected to a one-way Analysis of Variance and Tukey–Kramer multiple comparisons tests. Data sets from unilateral pregnancies were assessed by performing a two-tailed t-test. Values with a P<0.05 were considered to be significantly different.

Results

Expression of CRYAB in the myometrium throughout gestation

The temporal expression of total and pSer59-CRYAB was determined via immunoblot analysis. Total CRYAB protein expression significantly increased from D15 to D23 (labour) compared with NP and D6 (Fig. 1; *P<0.05). Expression was also significantly elevated from D15 to D22 compared with d12 (**) and at D17, D19, and D22 compared with D23 and PP (#). Lastly, expression on D21 was significantly elevated vs PP (+). pSer59-CRYAB expression was significantly increased from D15 to PP (*) or from D17 to PP (**) compared with expression at NP-D6 and D12–D15, respectively (Fig. 1; P<0.05). The highest expression was observed at D22 compared with D17–D21 and D23-PP (***). pSer59-CRYAB expression was also significantly higher at D23 compared with D17 and PP (#). Notably, expression of pSer59-CRYAB displayed a slower increase at D15 compared with total CRYAB and a less rapid decrease at D23 and PP. This likely reflects the time required for addition and loss of post-translational modification (serine phosphorylation) of CRYAB, respectively.

Figure 1
Figure 1

Immunoblot analysis of total and serine-59-phosphorylated (pSer59) CRYAB protein expression during pregnancy, parturition, and post-partum. Representative immunoblots and densitometric analyses of total CRYAB, pSer59-CRYAB, and GAPDH expression during gestation are shown. Total CRYAB protein expression significantly increased from D15 to D23 (labour) compared with NP and D6 (*). Expression was also significantly elevated from D15 to D22 compared with d12 (**) and at D17, D19, and D22 compared with D23 and PP (#). Lastly, expression on D21 was significantly elevated vs PP (+). pSer59-CRYAB expression was significantly increased from D15 to PP (*) or from D17 to PP (**) compared with expression at NP-D6 and D12–D15, respectively. The highest expression was observed at D22 compared with D17–D21 and D23–PP (***). pSer59-CRYAB expression was also significantly higher at D23 compared with D17 and PP (#). Data presented are from four independent experiments (n=4) and error bars represent the standard error of the mean. Values were considered significantly different when P<0.05. NP, non-pregnant; PP, 1-day post-partum; CRYAB, total CRYAB; pSer59-CRYAB, serine-59-phosphorylated CRYAB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Citation: Reproduction 152, 1; 10.1530/REP-16-0092

The spatial expression of pSer59-CRYAB and total CRYAB was examined by immunofluorescence analysis. pSer59-CRYAB was virtually undetectable from NP to D12 of gestation in myometrial cells from longitudinal and circular muscle layers and subsequently increased in detection with prominent immunolocalization from D21 to D23 (Figs 2 and 3). Expression was restricted to the entire cytoplasm of myocytes in both muscle ­layers with minimal immunodetection in the tissue stroma. Total CRYAB was similarly localized throughout the cytoplasm of myometrial cells at the same timepoints (data not shown).

Figure 2
Figure 2

Immunofluorescence analysis of serine-59-phosphorylated (pSer59) CRYAB protein expression in the rat longitudinal smooth muscle layer of the myometrium. The representative spatiotemporal expression of pSer59-CRYAB assessed in longitudinal muscle layers of non-pregnant (NP) and pregnant rat myometrium on gestational day (D) 6, D15, D21, D23, and 1-day post partum (PP) using rabbit polyclonal anti-human pSer59-CRYAB-specific antisera is shown. pSer59-CRYAB was virtually undetectable from NP to D12 of gestation in myometrial cells and subsequently increased in detection with prominent immunolocalization from D21 to D23. Expression was restricted to the entire cytoplasm of myocytes with minimal immunodetection in the tissue stroma. IgG; isotype-matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm.

Citation: Reproduction 152, 1; 10.1530/REP-16-0092

Figure 3
Figure 3

Immunofluorescence analysis of serine-59-phosphorylated (pSer59) CRYAB protein expression in the rat circular smooth muscle layer of the myometrium. The representative spatiotemporal expression of pSer59-CRYAB assessed in circular muscle layers of non-pregnant (NP) and pregnant rat myometrium on gestational day (D) 6, D15, D21, D23, and 1 day post-partum (PP) using rabbit polyclonal anti-human pSer59-CRYAB-specific antisera is shown. pSer59-CRYAB was virtually undetectable from NP to D12 of gestation in the myometrium and then increased in detection with prominent immunolocalization from D21 to D23. Expression was restricted to the entire cytoplasm of myocytes. IgG; isotype-matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm.

Citation: Reproduction 152, 1; 10.1530/REP-16-0092

Uterine stretch induces myometrial CRYAB expression

Since foetal growth-induced uterine distension is a powerful stimulator of protein expression, uterine ­tissue and myometrial protein extracts were collected from unilaterally pregnant rats at D19 and D23 for immunofluorescence and immunoblot analyses, respectively. Immunoblot analysis demonstrated that pSer59-CRYAB protein expression was markedly increased in myometrial lysates from gravid horns compared with non-gravid horns at D19 and D23 of gestation (Figs 4A and 5A). Immunofluorescence analysis supported these results as immunolocalization of pSer59-CRYAB was also markedly increased in the cytoplasm of myometrial cells from gravid horns at D19 and D23, in both muscle layers, compared with contralateral non-gravid uterine horns (Figs 4B and 5B). Similar results were obtained for total CRYAB expression at the same timepoints (data not shown).

Figure 4
Figure 4

Expression of serine-59-phosphorylated (pSer59) CRYAB protein in the rat myometrium at day 19 of pregnancy is significantly induced by uterine stretch. (A) Representative immunoblots of pSer59-CRYAB and GAPDH protein expression in myometrium from non-gravid (NG) and gravid (G) uterine horns at day (D) 19 of rat gestation are shown. Densitometric analyses illustrated that pSer59-CRYAB protein expression significantly increased in myometrium from gravid uterine horns compared with non-gravid horns (*P<0.05). Values are from three independent experiments (n=3) and error bars represent the standard error of the mean. (B) Immunofluorescence analysis of pSer59-CRYAB protein expression in longitudinal (Long) and circular (Circ) muscle layers of non-gravid and gravid uterine horns at D19 of rat gestation. pSer59-CRYAB expression was highly detectable in the cytosol of myocytes in both muscle layers of gravid horns when compared with non-gravid horns. Representative micrographs are shown. IgG; isotype-matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm. pSer59-CRYAB, serine-59-phosphorylated CRYAB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Citation: Reproduction 152, 1; 10.1530/REP-16-0092

Figure 5
Figure 5

Expression of serine-59-phosphorylated (pSer59) CRYAB protein in the rat myometrium at day 23 (labour) is significantly induced by uterine stretch. (A) Representative immunoblots of pSer59-CRYAB and GAPDH protein expression in myometrium from non-gravid (NG) and gravid (G) uterine horns at day (D) 23 of rat gestation are shown. Densitometric analyses illustrated that pSer59-CRYAB protein expression significantly increased in myometrium from gravid uterine horns compared with non-gravid horns (*P<0.05). Values are from three independent experiments (n=3) and error bars represent the standard error of the mean. (B) Immunofluorescence analysis of pSer59-CRYAB protein expression in longitudinal (Long) and circular (Circ) muscle layers of non-gravid and gravid uterine horns at day 23 (labour). pSer59-CRYAB expression was highly detectable in the cytosol of myocytes in both muscle layers of gravid horns when compared with non-gravid horns. Representative micrographs are shown. IgG; isotype matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm. pSer59-CRYAB, serine-59-phosphorylated CRYAB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Citation: Reproduction 152, 1; 10.1530/REP-16-0092

Immunolocalization of pSer59-CRYAB within hTERT-human myometrial cells

To better understand the potential pSer59-CRYAB interactome within the myometrium, the spatial localization of this protein with potential partners was examined in hTERT-HM cells. The cytoskeletal protein ACTA1 showed characteristic immunodetection in actin stress fibres within hTERT-HM cells and CRYAB co-immunolocalized along stretches of these filaments, particularly when observed in orthogonal views (e.g. YZ and XZ axes) of optical slices from confocal microscopy (Fig. 6 and Supplementary Fig. 1A). By contrast, within all cells examined, pSer59-CRYAB did not appreciably co-localize with long stretches of ACTA1 filaments, but instead co-localized with the focal adhesion protein FERMT2 at the ends of ACTA1 filaments in close proximity to myocyte membranes (Figs 6, 7 and Supplementary Fig. 1B and C).

Figure 6
Figure 6

Immunofluorescence analysis of total CRYAB or serine-59-phosphorylated (pSer59) CRYAB protein localization with alpha (α)-smooth muscle actin (ACTA1) in hTERT-HM myometrial cells. Upper panels: representative images of ACTA1 (green) and total CRYAB (red) protein localization. Middle panels: representative images of ACTA1 (red) and pSer59-CRYAB (green) protein localization. Lower panels: Representative mouse and rabbit IgG controls. Mounting media containing DAPI was used to visualize nuclei. ACTA1, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; CRYAB, total CRYAB; pSer59-CRYAB, serine-59-phosphorylated CRYAB; Merge, ACTA1 and CRYAB or ACTA1 and pSer59-CRYAB co-localization. Scale bar=50μm.

Citation: Reproduction 152, 1; 10.1530/REP-16-0092

Figure 7
Figure 7

Immunofluorescence analysis of Kindlin-2 (FERMT2) or CD63 protein localization with serine-59-phosphorylated (pSer59) CRYAB in hTERT-HM myometrial cells. Upper panels: representative images of FERMT2 (red) and pSer59-CRYAB (green) protein localization. Middle panels: representative images of CD63 (red) and pSer59-CRYAB (green) protein localization. Lower panels: representative mouse and rabbit IgG controls. Mounting media containing DAPI was used to visualize nuclei. FERMT2, Kindlin-2; DAPI, 4′,6-diamidino-2-phenylindole; pSer59-CRYAB, serine-59-phosphorylated CRYAB; Merge, FERMT2 and pSer59-CRYAB or CD63 and pSer59-CRYAB co-localization. Scale bar=50μm.

Citation: Reproduction 152, 1; 10.1530/REP-16-0092

sHSPs have recently been shown to be extracellular signalling molecules that can be secreted via exosomes (reviewed by Van Noort et al. 2012); thus, we examined the co-immunolocalization of pSer59-CRYAB with the exosomal marker and tetraspanin CD63. In all hTERT-HM cells examined, CD63 showed characteristic immunofluorescence detection not only near the nuclei of cells, but also in proximity to plasma membrane regions. pSer59-CRYAB also co-localized with CD63 at these areas, particularly when examined in orthogonal views of optical slices from confocal microscopy (Fig. 7 and Supplementary Fig. 1D). Importantly, in all confocal image collections, the co-localization was confirmed following image deconvolution and calculation of the mean Pearson correlation coefficients for image data sets (Supplementary Fig. 1A, B, C and D).

Discussion

The HSPB family of small stress proteins is a group of ATP-independent chaperones that also have important roles in processes such as cell death regulation, cell growth, and cytoskeletal organization (Acunzo et al. 2012, ­Wettstein et al. 2012). Several family members are dynamically expressed in the myometrium during pregnancy (White et al. 2005, Cross et al. 2007, MacIntyre et al. 2008, White & MacPhee 2011, Marsh et al. 2015), but CRYAB, originally discovered in the eye lens, has never been thoroughly examined in this tissue. Since CRYAB appears to aid actin filament dynamics and immune cell activation, it was hypothesized that CRYAB would be highly expressed in the myometrium during the contractile and labour phases of myometrial differentiation.

Myometrial CRYAB expression during pregnancy and the influence of uterine distension

The increased detection of the phosphorylated form of CRYAB by immunoblot analysis, slightly after significant increases in total CRYAB levels, suggested that CRYAB was produced in large amounts in the synthetic phase of myometrial programming and then quickly serine phosphorylated for subsequent phases. CRYAB is phosphorylated on Ser59 by the p38 MAPK (MAPK14) pathway (Kato et al. 1998) and Oldenhof and coworkers (2002) reported that activated MAPK14 is readily expressed in rat myometrium during late pregnancy. Thus, this pathway may be responsible, in part, for the increased detection of pSer59-CRYAB during late pregnancy and labour. The significant increase in the expression of total CRYAB observed beginning at D15 and subsequent significant decrease at D23 and PP compared with D17, D19, and D22 is also notable as MacIntyre and coworkers (2008) reported that CRYAB protein expression decreased by 71% in the myometrium of labouring women compared with non-­labouring women. However, these authors did not examine the serine phosphorylated form of CRYAB, and our results indicate the importance of assessing the expression of phosphorylated forms of sHSPs. pSer59-CRYAB was significantly elevated at D23 compared with D17 and PP and not significantly different from expression at D19 and D21. Phosphorylation of these proteins on serine residues is critical for the regulation of structure and function (Kato et al. 1994, Garrido et al. 2012).

Both total (data not shown) and pSer59-CRYAB were detected from D15 to PP throughout the cytosol of myocytes in both circular and longitudinal muscle ­layers. By contrast, MacIntyre and coworkers (2008) only detected total CRYAB in perinuclear regions of myocytes within the myometrium of non-labouring patients from 37 to 40weeks of gestation. Detection of total CRYAB was barely above background levels in the myometrium of ­labouring patients within the same timeframe. This may reflect ­species-specific differences in total CRYAB expression and localization in the myometrium at labour.

Both total (data not shown) and pSer59-CRYAB protein expression and immunolocalization were markedly increased upon uterine distension at D19 and D23 of pregnancy representing the synthetic and labour phases of myometrial differentiation. To our knowledge, this is the first demonstration of mechanical forces inducing CRYAB expression in smooth muscle. In terms of pSer59-CRYAB expression, Oldenhof and coworkers (2002) demonstrated that activated MAPK14 expression was significantly increased in myometrium from gravid rat uterine horns compared with myometrium from non-gravid horns. Again, this implicates the MAPK14 pathway in the serine phosphorylation of CRYAB. It is also well reported that CRYAB can readily interact and form a functional complex with HSPB1 (reviewed by Arrigo 2013). Both total and pSer15-HSPB1 expression are readily induced in the rat myometrium by uterine distension at d19 and d23 of pregnancy (White & MacPhee 2011). Thus, both of these sHSPs may be working together during this period of gestation in response to uterine distension.

Potential roles for CRYAB in the myometrium

The serine phosphorylation of CRYAB, like serine phosphorylation of HSPB1, leads to the production of small oligomers from very large complexes (Ito et al. 2001). As a result, both HSPB1 and CRYAB can interact with ACTA1 fibres (Wettstein et al. 2012, Arrigo & Gilbert 2013). Singh and coworkers (2007) showed that CRYAB can interact directly with ACTA1 in rat H9C2 cardiomyoblasts, regulate actin filament dynamics, and that the association is dependent on serine phosphorylation of CRYAB. We detected pSer59-CRYAB with the focal adhesion protein FERMT2 at the ends of ACTA1 filaments. By contrast, total CRYAB largely co-localized with filamentous ACTA1. Thus, CRYAB may help in the maintenance of filamentous ACTA1 (Singh et al. 2007), and localization of total CRYAB to these filaments may also reflect localization of other phosphorylated CRYAB species such as pSer19 and/or pSer45-CRYAB.

The dynamic modulation of ACTA1 microfilament formation plays a large role in smooth muscle contraction (Taggart & Morgan 2007). For example, Shaw et al. (2003) reported that agonist-induced contraction of non-pregnant rat myometrium was reduced by inhibition of ACTA1 polymerization with cytochalasin D. Focal adhesions or smooth muscle dense plaques are sites on the plasma membrane where clusters of integrins and integrin activators such as FERMT2 help to initiate a structural link between the ECM and the ACTA1 cytoskeleton. ACTA1 filaments are very active at focal adhesion locations, remodelling and anchoring the plasma membrane to the ECM and to other cells, and focal adhesion signalling is important for promoting myometrial cell contraction in late pregnancy (Launay et al. 2006, Li et al. 2007). Due to the increase in pSer59-CRYAB expression during late pregnancy and labour and induction by uterine distension, we speculate that pSer59-CRYAB may be part of a mechano-adaptive response, perhaps in partnership with pSer15-HSPB1, to modulate ACTA1 polymerization dynamics at focal adhesions in the myometrium during late pregnancy and to facilitate phasic labour contractions.

In contrast to a role with the cytoskeleton, sHSPs have also been shown to be extracellular signalling molecules that can be secreted via exosomes (Clayton et al. 2005, Rayner et al. 2008, Sreekumar et al. 2010, Gangalum et al. 2011, reviewed by van Noort et al. 2012). Results of this study demonstrate that pSer59-CRYAB readily co-immunolocalized with the exosome marker CD63. This suggests that pSer59-CRYAB may be localized in vesicles destined to be released as exosomes from hTERT-HM cells; however, late endosomes can also contain CD63 because exosomal vesicles have an endocytic origin (Thery et al. 2002). Thus, we cannot rule out localization of pSer59-CRYAB to these late endosomes as well in myometrial cells. Exosome production has been demonstrated in vascular smooth muscle and recently implicated in myometrium (Liao et al. 2000, Martin-Ventura et al. 2004, Cretoiu et al. 2013). The uptake of exosomes by macrophages through phagocytosis and the release of contents has also been demonstrated (Feng et al. 2010). In this manner, CRYAB and other HSPB family members can signal to macrophages or macrophage-like cells and induce innate immune responses (Bhat & Sharma 1999, van Noort et al. 2010). Interestingly, van Noort et al. (2010) demonstrated that HSPB family members appear to promote the activation of macrophages into an immune regulatory state that stimulates tissue repair and attenuates inflammation. We speculate that the detection of CRYAB in myometrium post-partum may be indicative of such a role in tissue repair and signalling to immune cells at this time as the involution process is similar to wound healing (Shynlova et al. 2013). The immune response, however, likely depends on the local concentration of CRYAB because high local concentrations can stimulate a pro-inflammatory immune response (van Noort et al. 2010, 2012). The maternal immune system during pregnancy and labour undergoes an immunological transformation from initiation to tolerance and then activation in concert with myometrial programming (­Shynlova et al. 2013). Thus, CRYAB may have chronologically specific anti-inflammatory and pro-inflammatory roles in the myometrium during pregnancy and post-partum to aid this immunological transformation.

Overall, the spatial and temporal expressions of CRYAB changes dynamically in the myometrium during late pregnancy and labour and is regulated, in part, by uterine distension. CRYAB, and particularly pSer59-CRYAB, could play an important role in facilitating the contractility of myometrial smooth muscle cells by regulating actin filament dynamics at focal adhesions and/or help to regulate immune responses within the myometrium via exosome-mediated delivery during late pregnancy and labour. Further investigations at the ­cellular and molecular levels are required to understand the exact role(s) and underlying mechanism(s) of CRYAB signalling in myometrial function.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-16-0092.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The research was funded by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (Grant #250218) and a Canada Foundation for Innovation John R Evans Leaders Fund (Project # 32512) to Daniel J MacPhee. Jessica Nicoletti was funded, in part, by a devolved scholarship from the College of Graduate Studies and Research, University of Saskatchewan.

Acknowledgements

The authors wish to thank Ms Shanna Banman, Dr Eiko Kawamura, and the Western College of Veterinary Medicine Imaging Centre for excellent technical assistance with laser scanning confocal microscopy and image analysis.

References

  • Acunzo J , Katsogiannou M & Rocchi P 2012 Small heat shock proteins HSP27 (HSPB1), alphaB-crystallin (HSPB5) and HSP22 (HSPB8) as regulators of cell death. International Journal of Biochemistry & Cell Biology 44 16221631. (doi:10.1016/j.biocel.2012.04.002)

    • Search Google Scholar
    • Export Citation
  • Adler J & Parmryd I 2010 Quantifying colocalization by correlation: The Pearson correlation coefficient is superior to the Mander’s overlap coefficient. Cytometry Part A 77A 733742. (doi:10.1002/cyto.a.v77a:8)

    • Search Google Scholar
    • Export Citation
  • Arrigo AP 2013 Human small heat shock proteins: protein interactomes of homo- and hetero-oligomeric complexes: an update. FEBS Letters 587 19591969. (doi:10.1016/j.febslet.2013.05.011)

    • Search Google Scholar
    • Export Citation
  • Arrigo AP & Gilbert B 2013 Protein interactomes of three stress inducible small heat shock proteins: HSPB1, HSPB5, HSPB8. International Journal of Hyperthermia 29 409422. (doi:10.3109/02656736.2013.792956)

    • Search Google Scholar
    • Export Citation
  • Bhat NR & Sharma KK 1999 Microglial activation by the small heat shock protein, alpha-crystallin. NeuroReport 10 28692873. (doi:10.1097/00001756-199909090-00031)

    • Search Google Scholar
    • Export Citation
  • Bloemendal H 1982 Lens protein. Critical Reviews in Biochemistry and Molecular Biology 12 138. (doi:10.3109/10409238209105849)

  • Bolte S & Cordelieres FP 2006 A guided tour into subcellular colocalization analysis in light microscopy. Journal of Microscopy 224 213232. (doi:10.1111/jmi.2006.224.issue-3)

    • Search Google Scholar
    • Export Citation
  • Boncoraglio A , oia M & Carra S 2012 The family of mammalian small heat shock proteins (HSPBs): implications in protein deposit diseases and motor neuropathies. International Journal of Biochemistry & Cell Biology 44 16571669. (doi:10.1016/j.biocel.2012.03.011)

    • Search Google Scholar
    • Export Citation
  • Clark AR , Lubsen NH & Slingsby C 2012 sHSP in the eye lens: crystallin mutations, cataract and proteostasis. International Journal of Biochemistry & Cell Biology 44 16871697. (doi:10.1016/j.biocel.2012.02.015)

    • Search Google Scholar
    • Export Citation
  • Clayton A , Turkes A , Navabi H , Mason MD & Tabi Z 2005 Induction of heat shock proteins in B-cell exosomes. Journal of Cell Science 118 36313638. (doi:10.1242/jcs.02494)

    • Search Google Scholar
    • Export Citation
  • Condon J , Yin S , Mayhew B , Word RA , Wright WE , Shay JW & Rainey WE 2002 Telomerase immortalization of human myometrial cells. Biology of Reproduction 67 506514. (doi:10.1095/biolreprod67.2.506)

    • Search Google Scholar
    • Export Citation
  • Cretoiu SM , Cretoiu D , Marin A , Radu BM & Popescu LM 2013 Telocytes: ultrastructural, immunohistochemical and electrophysiological characteristics in human myometrium. Reproduction 145 357370. (doi:10.1530/REP-12-0369)

    • Search Google Scholar
    • Export Citation
  • Cross BE , O’Dea HM & MacPhee DJ 2007 Expression of small heat shock-related protein 20 (HSP20) in rat myometrium is markedly decreased during late pregnancy and labour. Reproduction 113 807817. (doi:10.1530/REP-06-0291)

    • Search Google Scholar
    • Export Citation
  • Dieterich LC , Huang H , Massena S , Golenhofen N , Phillipson M & Dimberg A 2013 alphaB-crystallin/HSPB5 regulates endothelial-leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin. Angiogenesis 16 975983. (doi:10.1007/s10456-013-9367-4)

    • Search Google Scholar
    • Export Citation
  • Feng D , Zhao WL , Ye YY , Bai XC , Liu RQ , Chang LF , Zhou Q & Sui SF 2010 Cellular internalization of exosomes occurs through phagocytosis. Traffic 11 675687. (doi:10.1111/j.1600-0854.2010.01041.x)

    • Search Google Scholar
    • Export Citation
  • Gangalum RK , Atanasov IC , Zhou ZH & Bhat SP 2011 AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells. Journal of Biological Chemistry 286 32613269. (doi:10.1074/jbc.M110.160135)

    • Search Google Scholar
    • Export Citation
  • Garrido C , Paul C , Seigneuric R & Kampinga HH 2012 The small heat shock proteins family: the long forgotten chaperones. International Journal of Biochemistry & Cell Biology 44 15881592. (doi:10.1016/j.biocel.2012.02.022)

    • Search Google Scholar
    • Export Citation
  • Horwitz J 1992 Alpha-crystallin can function as a molecular chaperone. PNAS 89 1044910453. (doi:10.1073/pnas.89.21.10449)

  • Ito H , KameiK , IwamotoI , Inaguma Y , Nohara D & Kato K 2001 Phosphorylation-induced change of the oligomerization state of alpha B-crystallin. Journal of Biological Chemistry 276 53465352. (doi:10.1074/jbc.M009004200)

    • Search Google Scholar
    • Export Citation
  • Kampinga HH & Garrido C 2012 HSPBs: small proteins with big implications in human disease. International Journal of Biochemistry & Cell Biology 44 17061710. (doi:10.1016/j.biocel.2012.06.005)

    • Search Google Scholar
    • Export Citation
  • Kato K , Hasegawa K , Goto S & Inaguma Y 1994 Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27. Journal of Biological Chemistry 269 1127411278.

    • Search Google Scholar
    • Export Citation
  • Kato K , Ito H , Kamei K , Inaguma Y , Iwamoto I & Saga S 1998 Phosphorylation of alphaB-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation. Journal of Biological Chemistry 273 2834628354. (doi:10.1074/jbc.273.43.28346)

    • Search Google Scholar
    • Export Citation
  • Launay N , Goudeau B , Kato K , Vicart P & Lilienbaum A 2006 Cell signaling pathways to alphaB-crystallin following stresses of the cytoskeleton. Experimental Cell Research 312 35703584. (doi:10.1016/j.yexcr.2006.07.025)

    • Search Google Scholar
    • Export Citation
  • Li Y , Gallant C , Malek S & Morgan KG 2007 Focal adhesion signaling is required for myometrial ERK activation and contractile phenotype switch before labour. Journal of Cellular Biochemistry 100 129140. (doi:10.1002/jcb.v100:1)

    • Search Google Scholar
    • Export Citation
  • Liao DF , Jin ZG , Baas AS , Daum G , Gygi SP , Aebersold R & Berk BC 2000 Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells. Journal of Biological Chemistry 275 189196. (doi:10.1074/jbc.275.1.189)

    • Search Google Scholar
    • Export Citation
  • MacIntyre DA , Tyson EK , Read M , Smith R , Yeo G , Kwek K & Chan EC 2008 Contraction in human myometrium is associated with change in small heat shock proteins. Endocrinology 149 245252. (doi:10.1210/en.2007-0662)

    • Search Google Scholar
    • Export Citation
  • MacPhee DJ & Lye SJ 2000 Focal adhesion signaling in the rat myometrium is abruptly terminated with the onset of labour. Endocrinology 141 274283. (doi:10.1210/endo.141.1.7264)

    • Search Google Scholar
    • Export Citation
  • Marsh NM , Wareham A , White BG , Miskiewicz EI , Landry J & MacPhee DJ 2015 HSPB8 and the cochaperone BAG3 are highly expressed during the synthetic phase of rat myometrium programming during pregnancy. Biology of Reproduction 92 131. (doi:10.1095/biolreprod.114.125401)

    • Search Google Scholar
    • Export Citation
  • Martin-Ventura JL , Duran MC , Blanco-Colio LM , Meihac O , Leclercq A , Michel JB , Jensen ON , Hernandez-Merida S , Tunon J & Vivanco F et al. 2004 Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis. Circulation 110 22162219. (doi:10.1161/01.CIR.0000136814.87170.B1)

    • Search Google Scholar
    • Export Citation
  • Oldenhof AD , Shynlova OP , Liu M , Langille BL & Lye SJ 2002 Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells. American Journal of Physiology 283 C1530C1539. (doi:10.1152/ajpcell.00607.2001)

    • Search Google Scholar
    • Export Citation
  • Osman I , Young A , Ledingham MA , Thomson AJ , Jordan F , Greer IA & Norman JE 2003 Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term. Molecular Human Reproduction 9 4145. (doi:10.1093/molehr/gag001)

    • Search Google Scholar
    • Export Citation
  • Ou CW , Chen ZQ , Qi S & Lye SJ 1998 Increased expression of the rat myometrial oxytocin receptor messenger ribonucleic acid during labour requires both mechanical and hormonal signals. Biology of Reproduction 59 10551061. (doi:10.1095/biolreprod59.5.1055)

    • Search Google Scholar
    • Export Citation
  • Rayner K , Chen YX , McNulty M , Simard T , Zhao X , Wells DJ , de Belleroche J & O’Brien ER 2008 Extracellular release of the atheroprotective heat shock protein 27 is mediated by estrogen and competitively inhibits acLDL binding to scavenger receptor-A. Circulation Research 103 133141. (doi:10.1161/CIRCRESAHA.108.172155)

    • Search Google Scholar
    • Export Citation
  • Schindelin J , Arganda-Carreras I , Frise E , Kaynig V , Longair M , Pietzsch T , Preibisch S , Rueden C , Saalfeld S & Schmid B et al. 2012. Fiji: an open-source platform for biological-image analysis. Nature Methods 9 676682. (doi:10.1038/nmeth.2019)

    • Search Google Scholar
    • Export Citation
  • Shaw L , Ahmed S , Austin C & Taggart MJ 2003 Inhibitors of actin filament polymerisation attenuate force but not global intracellular calcium in isolated pressurised resistance arteries. Journal of Vascular Research 40 110. (doi:10.1159/000068940)

    • Search Google Scholar
    • Export Citation
  • Shynlova O , Mitchell JA , Tsampalieros A , Langille BL & Lye SJ 2004 Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium. Biology of Reproduction 70 986992. (doi:10.1095/biolreprod.103.023648)

    • Search Google Scholar
    • Export Citation
  • Shynlova O , Oldenhof A , Dorogin A , Xu Q , Mu J , Nashman N & Lye SJ 2006 Myometrial apoptosis: activation of the caspase cascade in the pregnant rat myometrium at midgestation. Biology of Reproduction 74 839849. (doi:10.1095/biolreprod.105.048124)

    • Search Google Scholar
    • Export Citation
  • Shynlova O , Tsui P , Dorogin A & Lye SJ 2008 Monocyte chemoattractant protein-1 (CCL-2) integrates mechanical and endocrine signals that mediate term and preterm labour. Journal of Immunology 181 14701479. (doi:10.4049/jimmunol.181.2.1470)

    • Search Google Scholar
    • Export Citation
  • Shynlova O , Lee YH , Srikhajon K & Lye SJ 2013 Physiologic uterine inflammation and labour integration of endocrine and mechanical signals. Reproductive Sciences 20 154167. (doi:10.1177/1933719112446084)

    • Search Google Scholar
    • Export Citation
  • Singh BN , Rao KS , Ramakrishna T , Rangaraj N & Rao ChM 2007 Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo. Journal of Molecular Biology 366 756767. (doi:10.1016/j.jmb.2006.12.012)

    • Search Google Scholar
    • Export Citation
  • Sreekumar PG , Kannan R , Kitamura M , Spee C , Barron E , Ryan SJ & Hinton DR 2010 alphaB crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells. PLoS ONE 5 e12578. (doi:10.1371/journal.pone.0012578)

    • Search Google Scholar
    • Export Citation
  • Tabb T , Thilander G , Grover A , Hertzberg E & Garfield R 1992 An immunochemical and immunocytologic study of the increase in myometrial gap junctions (and connexin 43) in rats and humans during pregnancy. American Journal of Obstetrics and Gynecology 167 559567. (doi:10.1016/S0002-9378(11)91453-7)

    • Search Google Scholar
    • Export Citation
  • Taggart MJ & Morgan KG 2007 Regulation of the uterine contractile apparatus and cytoskeleton. Seminars in Cell & Developmental Biology 18 296304. (doi:10.1016/j.semcdb.2007.05.006)

    • Search Google Scholar
    • Export Citation
  • Thery C , Zitvogel L & Amigorena S 2002 Exosomes: composition, biogenesis and function. Nature Reviews Immunology 2 569579. (doi:10.1038/nri855)

    • Search Google Scholar
    • Export Citation
  • Thomson AJ , Telfer JF , Young A , Campbell S , Stewart CJ , Cameron IT , Greer IA & Norman JE 1999 Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process. Human Reproduction 14 229236. (doi:10.1093/humrep/14.1.229)

    • Search Google Scholar
    • Export Citation
  • van Noort JM , Bsibsi M , Gerritsen WH , van der Valk P , Bajramovic JJ , Steinman L & Amor S 2010 alphaB-crystallin is a target for adaptive immune responses and a trigger of innate responses in preactive multiple sclerosis lesions. Journal of Neuropathology & Experimental Neurology 69 694703. (doi:10.1097/NEN.0b013e3181e4939c)

    • Search Google Scholar
    • Export Citation
  • van Noort JM , Bsibsi M , Nacken P , Gerritsen WH & Amor S 2012 The link between small heat shock proteins and the immune system. International Journal of Biochemistry & Cell Biology 44 16701679. (doi:10.1016/j.biocel.2011.12.010)

    • Search Google Scholar
    • Export Citation
  • Wettstein G , Bellaye PS , Micheau O & Bonniaud P 2012 Small heat shock proteins and the cytoskeleton: an essential interplay for cell integrity? International Journal of Biochemistry & Cell Biology 44 16801686. (doi:10.1016/j/biocel.2012.05.024)

    • Search Google Scholar
    • Export Citation
  • White BG & MacPhee DJ 2011 Distension of the uterus induces HspB1 expression in rat uterine smooth muscle. American Journal of Physiology 301 R1418R1426. (doi:10.1152/ajpregu.00272.2011)

    • Search Google Scholar
    • Export Citation
  • White BG , Williams SJ , Highmore K & MacPhee DJ 2005 Small heat shock protein 27 (Hsp27) expression is highly induced in rat myometrium during late pregnancy and labour. Reproduction 129 115126. (doi:10.1530/rep.1.00426)

    • Search Google Scholar
    • Export Citation
  • Williams SJ , White BG & MacPhee DJ 2005 Expression of alpha5 Integrin (Itga5) is elevated in the rat myometrium during late pregnancy and labour: implications for development of a mechanical syncytium. Biology of Reproduction 72 11141124. (doi:10.1095/biolreprod.104.035626)

    • Search Google Scholar
    • Export Citation
  • Williams SJ , Shynlova O , Lye SJ & MacPhee DJ 2010 Spatiotemporal expression of alpha1, alpha3 and beta1 integrin subunits is altered in rat myometrium during pregnancy and labour. Reproduction, Fertility and Development 22 718732. (doi:10.1071/RD09163)

    • Search Google Scholar
    • Export Citation

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    Immunoblot analysis of total and serine-59-phosphorylated (pSer59) CRYAB protein expression during pregnancy, parturition, and post-partum. Representative immunoblots and densitometric analyses of total CRYAB, pSer59-CRYAB, and GAPDH expression during gestation are shown. Total CRYAB protein expression significantly increased from D15 to D23 (labour) compared with NP and D6 (*). Expression was also significantly elevated from D15 to D22 compared with d12 (**) and at D17, D19, and D22 compared with D23 and PP (#). Lastly, expression on D21 was significantly elevated vs PP (+). pSer59-CRYAB expression was significantly increased from D15 to PP (*) or from D17 to PP (**) compared with expression at NP-D6 and D12–D15, respectively. The highest expression was observed at D22 compared with D17–D21 and D23–PP (***). pSer59-CRYAB expression was also significantly higher at D23 compared with D17 and PP (#). Data presented are from four independent experiments (n=4) and error bars represent the standard error of the mean. Values were considered significantly different when P<0.05. NP, non-pregnant; PP, 1-day post-partum; CRYAB, total CRYAB; pSer59-CRYAB, serine-59-phosphorylated CRYAB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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    Immunofluorescence analysis of serine-59-phosphorylated (pSer59) CRYAB protein expression in the rat longitudinal smooth muscle layer of the myometrium. The representative spatiotemporal expression of pSer59-CRYAB assessed in longitudinal muscle layers of non-pregnant (NP) and pregnant rat myometrium on gestational day (D) 6, D15, D21, D23, and 1-day post partum (PP) using rabbit polyclonal anti-human pSer59-CRYAB-specific antisera is shown. pSer59-CRYAB was virtually undetectable from NP to D12 of gestation in myometrial cells and subsequently increased in detection with prominent immunolocalization from D21 to D23. Expression was restricted to the entire cytoplasm of myocytes with minimal immunodetection in the tissue stroma. IgG; isotype-matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm.

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    Immunofluorescence analysis of serine-59-phosphorylated (pSer59) CRYAB protein expression in the rat circular smooth muscle layer of the myometrium. The representative spatiotemporal expression of pSer59-CRYAB assessed in circular muscle layers of non-pregnant (NP) and pregnant rat myometrium on gestational day (D) 6, D15, D21, D23, and 1 day post-partum (PP) using rabbit polyclonal anti-human pSer59-CRYAB-specific antisera is shown. pSer59-CRYAB was virtually undetectable from NP to D12 of gestation in the myometrium and then increased in detection with prominent immunolocalization from D21 to D23. Expression was restricted to the entire cytoplasm of myocytes. IgG; isotype-matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm.

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    Expression of serine-59-phosphorylated (pSer59) CRYAB protein in the rat myometrium at day 19 of pregnancy is significantly induced by uterine stretch. (A) Representative immunoblots of pSer59-CRYAB and GAPDH protein expression in myometrium from non-gravid (NG) and gravid (G) uterine horns at day (D) 19 of rat gestation are shown. Densitometric analyses illustrated that pSer59-CRYAB protein expression significantly increased in myometrium from gravid uterine horns compared with non-gravid horns (*P<0.05). Values are from three independent experiments (n=3) and error bars represent the standard error of the mean. (B) Immunofluorescence analysis of pSer59-CRYAB protein expression in longitudinal (Long) and circular (Circ) muscle layers of non-gravid and gravid uterine horns at D19 of rat gestation. pSer59-CRYAB expression was highly detectable in the cytosol of myocytes in both muscle layers of gravid horns when compared with non-gravid horns. Representative micrographs are shown. IgG; isotype-matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm. pSer59-CRYAB, serine-59-phosphorylated CRYAB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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    Expression of serine-59-phosphorylated (pSer59) CRYAB protein in the rat myometrium at day 23 (labour) is significantly induced by uterine stretch. (A) Representative immunoblots of pSer59-CRYAB and GAPDH protein expression in myometrium from non-gravid (NG) and gravid (G) uterine horns at day (D) 23 of rat gestation are shown. Densitometric analyses illustrated that pSer59-CRYAB protein expression significantly increased in myometrium from gravid uterine horns compared with non-gravid horns (*P<0.05). Values are from three independent experiments (n=3) and error bars represent the standard error of the mean. (B) Immunofluorescence analysis of pSer59-CRYAB protein expression in longitudinal (Long) and circular (Circ) muscle layers of non-gravid and gravid uterine horns at day 23 (labour). pSer59-CRYAB expression was highly detectable in the cytosol of myocytes in both muscle layers of gravid horns when compared with non-gravid horns. Representative micrographs are shown. IgG; isotype matched rabbit IgG used in place of primary antisera to serve as a negative control. Scale bar=50μm. pSer59-CRYAB, serine-59-phosphorylated CRYAB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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    Immunofluorescence analysis of total CRYAB or serine-59-phosphorylated (pSer59) CRYAB protein localization with alpha (α)-smooth muscle actin (ACTA1) in hTERT-HM myometrial cells. Upper panels: representative images of ACTA1 (green) and total CRYAB (red) protein localization. Middle panels: representative images of ACTA1 (red) and pSer59-CRYAB (green) protein localization. Lower panels: Representative mouse and rabbit IgG controls. Mounting media containing DAPI was used to visualize nuclei. ACTA1, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; CRYAB, total CRYAB; pSer59-CRYAB, serine-59-phosphorylated CRYAB; Merge, ACTA1 and CRYAB or ACTA1 and pSer59-CRYAB co-localization. Scale bar=50μm.

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    Immunofluorescence analysis of Kindlin-2 (FERMT2) or CD63 protein localization with serine-59-phosphorylated (pSer59) CRYAB in hTERT-HM myometrial cells. Upper panels: representative images of FERMT2 (red) and pSer59-CRYAB (green) protein localization. Middle panels: representative images of CD63 (red) and pSer59-CRYAB (green) protein localization. Lower panels: representative mouse and rabbit IgG controls. Mounting media containing DAPI was used to visualize nuclei. FERMT2, Kindlin-2; DAPI, 4′,6-diamidino-2-phenylindole; pSer59-CRYAB, serine-59-phosphorylated CRYAB; Merge, FERMT2 and pSer59-CRYAB or CD63 and pSer59-CRYAB co-localization. Scale bar=50μm.