Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells

Alexandra D. Oldenhof1, Oksana P. Shynlova1, Mingyao Liu2,3, B. Lowell Langille2,3, and Stephen J. Lye1,3

1 Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario; 2 Toronto General Research Institute, Toronto, Ontario; and 3 Departments of Obstetrics and Gynecology and Physiology, University of Toronto, Toronto, Ontario, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Evidence indicates that stretch of the uterus imposed by the growing fetus contributes to the onset of labor. Previously we have shown that mechanically stretching rat myometrial smooth muscle cells (SMCs) induces c-fos expression. To investigate this stretch-induced signaling, we examined the involvement of the mitogen-activated protein kinase (MAPK) family. We show that stretching rat myometrial SMCs induces a rapid and transient phosphorylation (activation) of MAPKs: extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. The use of selective inhibitors for the ERK pathway (PD-98059 and U-0126), p38 (SB-203580), and JNK pathway (curcumin) demonstrated that activation of all three MAPK signaling pathways was necessary for optimal stretch-induced c-fos expression. We also demonstrate that upstream tyrosine kinase activity is involved in the mechanotransduction pathway leading to stretch-induced MAPK activation and c-fos mRNA expression. To further examine the role of MAPKs in vivo, we used a unilaterally pregnant rat model. MAPKs (ERK and p38) are expressed in the pregnant rat myometrium with maximal ERK and p38 phosphorylation occurring in the 24 h immediately preceding labor. Importantly, the rise in MAPK phosphorylation was confined to the gravid horn and was absent in the empty uterine horn, suggesting that mechanical strain imposed by the growing fetus controls MAPK activation in the myometrium. Collectively, this data indicate that mechanical stretch modulates MAPK activity in the myometrium leading to c-fos expression.

myometrium; mechanical stretch; tyrosine kinase; mechanotransduction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE INCREASE IN INTRAUTERINE VOLUME during pregnancy, due to fetal growth, is associated with a significant increase in amniotic pressure and myometrial tensile stress (10). This increased load on the myometrium is accentuated in twin pregnancies despite normal amniotic pressure (10, 32) because of the relationship between amniotic pressure, uterine radius, and myometrial tension. Interestingly, the risk of preterm labor is particularly high in pregnancies associated with increased intrauterine volume including multifetal pregnancies (11), polyhydramnios (14), and singleton pregnancies with a larger than average fetus (25). Furthermore, a study showing that human labor can be induced within 24 h of distending the uterus at term (33) provides additional support for a link between uterine stretch and the onset of labor.

We have reported that the onset of labor requires "activation" of the myometrium, which involves an increase in the expression of a cassette of contraction-associated proteins (CAPs), including the gap junction connexin43 (Cx43), the oxytocin receptor (OTR), and the prostaglandin F2alpha receptor (FP) (4). We have also shown that the expression of several activator protein-1 (AP-1) transcription factors including members of the Fos and Jun family are upregulated in the myometrium just before the onset of labor (34). Collectively, the expression of these genes enables the myometrium to produce the coordinated, high-amplitude contractions necessary for the expulsion of the fetus at term. The mechanisms that program activation have yet to be precisely determined; however, we hypothesize that they involve an integration of fetal endocrine and growth signals that provide hormonal and mechanical inputs to the myometrium (36). In rats in which unilateral pregnancy was produced by tubal ligation, increased expression of c-fos (34), Cx43 (36), and OTR (37) mRNA was confined to the gravid uterine horn. Importantly, insertion of a 3-mm tube into the empty uterine horn of these animals to mechanically stretch the uterus restored the myometrial expression of these genes late in pregnancy (36, 37). These studies indicate that stretch induced by the growing fetus is required for the expression of Cx43, OTR, and c-fos before the onset of labor.

Studying the direct effects of mechanical forces in vivo is complex. Recently, we developed an in vitro stretch system for myometrial smooth muscle cells (SMCs) and demonstrated that they respond to stretch by increasing mRNA levels of c-fos (43). The c-fos gene encodes a protein (Fos) belonging to the AP-1 family of transcription factors. Fos proteins heterodimerize with Jun proteins and subsequently translocate to the nucleus where they bind to specific AP-1 control elements present in the promoters of various genes to activate transcription (45). We speculate that c-fos plays an important role in the onset of labor, because c-fos mRNA is elevated in the myometrium before the rise in CAP expression with the onset of both term and preterm labor (39). Interestingly, Cx43 and OTR both contain consensus AP-1 sites in their promoters (18, 27), raising the possibility that c-fos may mediate stretch-induced CAP expression in the myometrium.

The mechanism(s) by which myometrial SMCs sense mechanical stimuli and convert them to changes in gene expression has not been characterized. Studies in other cell types that employed in vitro stretch systems have demonstrated that cellular mechanotransduction activates multiple signaling molecules (41), including phospholipase C (PLC)-protein kinase C (PKC) (41), cAMP-protein kinase A (PKA) (8), the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathways (38), phosphatidylinositol 3-kinase (12), G proteins (13), small GTPases (1), receptor and nonreceptor tyrosine kinases (5, 15, 20, 21, 30), and, most notably, members of the mitogen-activated protein kinase (MAPK) family (24, 28, 42, 44).

Three MAPKs have been well characterized: extracellular-regulated protein kinase (ERK1/2), c-Jun NH2-terminal protein kinase (JNK), and p38 (45). All three are serine-threonine kinases that transmit extracellular signals to the nucleus to control gene expression and regulate cellular process such as growth, differentiation, and apoptosis. Mechanical stimulation activates ERK, JNK, and p38 in a number of cell types including vascular SMCs (28), endothelial cells (24), cardiac myocytes (42), and mesangial cells (19).

A well-recognized role of all three MAPK members is the phosphorylation of the transcription factor Elk-1 to enhance complex formation with the serum response factor (SRF) at the serum response element (SRE) of the c-fos promoter to induce transcription (45). Because the SRE is also the stretch-responsive element of the c-fos promoter (41), we hypothesized that strain-induced c-fos expression in myometrial SMCs depends on MAPK activation. Involvement of ERK is supported by the findings that shear-induced c-fos mRNA expression in endothelial cells (2) and pressure-induced c-fos protein expression in mesangial cells (23) are blocked by the ERK pathway-specific inhibitor PD-98059. However, roles for JNK and p38 in strain-induced c-fos mRNA expression have not been investigated.

In this study, we examined the involvement of tyrosine kinases and the MAPK members (ERK1/2, JNK, and p38) in stretch-induced c-fos expression in myometrial SMCs. These in vitro data were complemented by in vivo studies using a unilaterally pregnant rat model.


    MATERIAL AND METHODS
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ABSTRACT
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MATERIAL AND METHODS
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Myometrial smooth muscle cell isolation and culture. Primary rat myometrial SMC isolation was performed as previously described (35, 43). Briefly, cells were harvested from rat uteri by collagenase treatment and subjected to a differential attachment technique to select for SMCs. Freshly isolated myometrial SMCs were directly seeded onto six-well flexible-bottomed culture plates coated with collagen I (Flexcell International, McKeesport, PA) at a plating density of 3 × 106 cells/well. The cells were grown to confluence within 3-4 days in phenol red-free DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS (Cansera, Rexdale, Ontario, Canada), 25 mM HEPES, 100 U/ml penicillin-streptomycin (GIBCO), and 2.5 µg/ml amphotericin B (Sigma, St Louis, MO). At the time of cell confluence, there were ~100-120 µg total protein/well.

Application of static stretch. Static stretch was applied utilizing a Flexercell strain unit (Flexcell FX-2000, Flexercell International). The stretch unit consists of a computer-controlled vacuum unit and a base plate to hold the six-well culture dish, which is placed in a humidified incubator with 5% CO2 at 37°C. Adjustment of the vacuum pump allows the magnitude and duration of the applied stretch to be precisely varied in this system. For the experiments described herein, static strain was applied by deforming the membrane with 150 mmHg of vacuum, which produces a maximal 25% elongation. Force analysis of the strain on the Flexcell plate by Flexcell International shows that deformation of the flexible membrane is not uniform but rather generates a gradient stretch, with the greatest deformation occurring at the periphery of the well. Therefore, the results of the present studies represent an average of cells exposed to different degrees of stretch. For 25% stretch, the average elongation is ~10% calculated for cells over the entire culture plate surface. Control cells were cultured under identical conditions but remained stationary. Before exposure to mechanical stretch, the confluent cells were incubated for 12 h in serum-free DMEM. For the tyrosine kinase inhibitor studies, cell monolayers were preincubated with genistein (50 µM; Sigma) for 1 h and with herbimycin A (1 µM; Sigma) for 16 h before the application of stretch. For the MAPK inhibitor studies, cell monolayers were preincubated with PD-98059 (25 µM; New England Biolabs, Beverly, MA), U-0126 (5 µM; New England Biolabs), SB-203580 (25 µM; Sigma), or curcumin (25 µM; Sigma) for 1 h before application of stretch. All inhibitors were reconstituted in DMSO, and control cells were preincubated with equal amounts of DMSO in the absence of the inhibitor.

Unilaterally pregnant rats. Virgin female rats were subjected to unilateral tubal ligation as described in Ou et al. (36). Animals were allowed to recover from surgery for at least five days. Female virgin Wistar rats (Charles River, St. Constance, Canada) were mated with male Wistar rats. Day 1 of gestation was designated as the day a vaginal plug was observed. Female rats were then housed individually under standard environmental conditions (12:12-h light-dark cycle) and fed Purina rat chow (Ralston Purina, St. Louis, MO) and water ad libitum. Under these conditions, the time of delivery was during the morning of day 23. Animals were designated as "in labor" when at least one pup was delivered. After mating, myometrial samples were collected on days 15, 17, 19, 21, 22, 23, or 1 day postpartum (1PP; n = 3). At the time of tissue collection, the ligated empty horn was separated from the gravid horn. All animal experiments were approved by the institutional animal care committee.

Tissue collection. Animals were killed by asphyxiation in carbon dioxide gas. Uterine horns were removed, bisected longitudinally, dissected away from both pups and placentas, and placed in ice-cold PBS. The endometrium was separated from the myometrial tissue by mechanical scraping on ice using a scalpel blade. The two uterine horns were collected separately. The tissue was then flash frozen in liquid nitrogen and stored at -70°C.

RNA isolation and Northern blotting. SMC monolayers were washed twice with ice-cold PBS, and total RNA was isolated using TRIzol (GIBCO). Ten micrograms of total RNA from each sample was separated in 1% agarose-3.7% formaldehyde denaturing gel and transferred overnight by capillary blotting onto nylon GeneScreen membranes (DuPont NEN Research Products, Boston, MA) in 0.1 M sodium phosphate (NaP). RNA was cross linked to the membrane by ultraviolet irradiation. Blots were prehybridized for 1 h at 55°C in a solution containing 1% (wt/vol) BSA, 0.35 M NaP, 7% SDS, and 30% formamide. The membrane was hybridized with 32P-labeled cDNA probe for rat c-fos (provided by Dr. Tom Curran, Roche Research Center, Nutley, NJ) and 18S rRNA (provided by Dr. Denhardt, Rutgers University, Piscataway, NJ). Probes were radiolabeled with [alpha 32P]dCTP using random priming (multiprime DNA labeling system; Amersham, Oakville, Canada) to a specific activity of 108 cpm/µl or a final concentration of ~106 cpm/ml according to manufacturer's instructions. Hybridization was carried out in a fresh hybridization solution at 55°C for 20 h, followed by washes to a final stringency of 30 mM NaP/0.1% SDS at 55°C. The membrane was exposed to X-ray film (Reflection; DuPont NEN Research Products) with the aid of an intensifying screen at -70°C. Autoradiographs were quantified by densitometry and data, normalized to 18S rRNA levels, and expressed as relative optical density (ROD) units.

Protein isolation and Western blotting. SMC monolayers were washed twice with ice-cold PBS and scraped in radioimmunoprotective assay (RIPA) lysis buffer containing 50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1% (vol/vol) sodium deoxycholate, and 0.1% (wt/vol) SDS, supplemented with 100 µM sodium orthovanadate and protease inhibitor cocktail tablets (Complete Mini; Roche, Quebec, Canada). Cell extracts were pipetted up and down and centrifuged (10 min at 15,000 g) at 4°C, and supernatants were collected. Protein content was determined by the Bradford technique (3), using Bio-Rad protein assay system (Bio-Rad, Hercules, CA). Laemmli sample buffer was added to equal protein amounts of each sample and boiled for 5 min. Protein samples were separated on 10% SDS-PAGE according to the method of Laemmli (26) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) in 48 mM Tris, 39 mM glycine, and 0.037% (wt/vol) SDS, pH 8.3, for 1.5 h at 350 mA at 4°C. Membranes were blocked in Tris-buffered saline (50 mM Tris and 150 mM NaCl, pH 7.4) with 0.1% (vol/vol) Tween-20 (TBST) supplemented with 0.2% I-block (Tropix, Bedford, MA) for 1 h at 37°C and then incubated for 2 h at room temperature with primary antibodies including the phosphospecific ERK1/2 (1:5,000), JNK (1:5,000), and p38 (1:2,000) antibodies (Promega, Madison, WI). After primary antibody incubation, membranes were washed several times with TBST buffer, followed by incubation with a secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000; Amersham, Bucks, UK), in blocking solution for 1 h. Membranes were repeatedly washed with TBST buffer and exposed to enhanced chemiluminescence reagent (ECL; Amersham). To confirm that equal amounts of ERK1/2, JNK, and p38 protein were electrophoresed in each reaction, the blot was stripped and reprobed with anti-ERK1/2, anti-JNK1, and anti-p38 antibodies at a 1:1,000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA) using the probing conditions described above. The intensity of phosphorylation at the 44/42-kDa bands of ERK, 46-kDa band of JNK, and 38-kDa bands of p38 was quantified by densitometry, and phospho-ERK, -JNK, and -p38 levels were normalized to their respective protein levels and expressed as ROD units.

Statistics. The results are presented as means ± SE (n = at least three independent experiments). Data from time course experiments were analyzed by one-way ANOVA, followed by pairwise multiple comparison procedures (Student-Newman-Keuls method) to determine differences between groups. Data from inhibitor studies and tubal ligation studies were analyzed by two-way ANOVA, followed by pairwise multiple comparison procedures as described above. For all these tests, a value of P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunodetection of phosphorylated MAPK members. The phosphospecific antibodies used for Western analysis recognized proteins of the appropriate sizes for all MAPK family members. Major signals for ERK were detected at 42 and 44 kDa. The JNK antibody detected a strong signal at 46 kDa, a weaker signal at 49 kDa, and a very weak signal at 54 kDa. The p38 antibody recognizes the alpha , gamma , and delta  isoforms of p38, all of which show slight differences in mobility during SDS-PAGE (29). In some blots, depending on the duration of exposure, a single p38 band was observed, whereas in other blots two or three closely associated bands were observed.

Static stretch stimulates ERK1/2, JNK, and p38 phosphorylation. To investigate whether static stretch activates MAPK family members in myometrial SMCs, we examined whether stretch phosphorylates ERK1/2, JNK, and p38. Stretch resulted in a rapid and significant phosphorylation of ERK1/2, indicating the activation of this kinase (Fig. 1A). The mean values presented in the bar graph represent the phosphorylation level of both ERK1 and ERK2 (normalized to total ERK1/2). After a 5-min stretch, ERK1/2 phosphorylation was significantly increased (2.1-fold) compared with the nonstretch control. ERK1/2 phosphorylation peaked after 15 min of stretch (2.6-fold) and returned to nonstretch levels by 120 min.


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Fig. 1.   Time course of mitogen-activated protein kinase (MAPK) phosphorylation by static stretch. Myometrial smooth muscle cells (SMCs) were subjected to 25% static stretch for the indicated time periods. MAPK phosphorylation was analyzed by Western blot using phosphospecific MAPK antibodies. Shown are representative immunoblots for p-ERK1 and p-ERK2, phosphorylated ERK1 and ERK2, respectively (A); p-c-Jun NH2-terminal kinase (JNK) (p49) and p-JNK (p46), phosphorylated JNK (p49) and JNK (p46) respectively (B); and p-p38, phosphorylated p38 (top panels) or a control anti-ERK1/2, -JNK (p46), -p38 antibody (bottom panels) (C). The intensity of phosphorylation at the 44/42-kDa bands of ERK, 46-kDa band of JNK, and 38-kDa bands of p38 was quantified by densitometry, and phospho-ERK, -JNK and -p38 levels were normalized to their respective protein levels and represented in the corresponding bar graph as means ± SE. Data from 4 independent experiments were subjected to one-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). The control activity at 0 min is designated as 1.0. *P < 0.05, **P < 0.01, ***P < 0.001 indicates significant difference vs. time 0 (nonstretch control).

In parallel studies, the effect of mechanical stretch on JNK phosphorylation was determined. The phosphorylation levels of JNK (p49) and JNK (p54) were very low. For this reason, we focused our attention on JNK (p46). Stretch caused an increase in JNK (p46) phosphorylation by 3 min (Fig. 1B). JNK (p46) phosphorylation peaked after 5 min of stretch. At this time, there was a 12-fold increase in phosphorylation levels compared with the nonstretch control. JNK (p46) phosphorylation levels declined after 10 min of stretch and returned to nonstretch levels by 30 min. Therefore, stretch-induced JNK (p46) phosphorylation precedes, and is greater in magnitude than, stretch-induced ERK1/2 phosphorylation.

Stretch also caused a significant (13-fold) increase in p38 phosphorylation by 5 min compared with the nonstretch control (Fig. 1C). Stretch-induced phosphorylation of p38 remained elevated up to 30 min and returned to nonstretch levels by 120 min. We confirmed that equal amounts of ERK, JNK, and p38 protein were electrophoresed in each reaction by Western analysis using antibodies against these kinases (Fig. 1, A-C).

Specificity of MAPK inhibitors. To investigate whether MAPK members are involved in stretch-induced c-fos expression, we pretreated myometrial SMCs with PD-98059, U-0126, SB-20385, or curcumin. PD-98059 binds to the inactive form of MEK1, preventing its ability to activate ERK1/2 (9). U-0126 blocks the ability of both MEK1 and MEK2 to activate ERK1/2 and is therefore a more potent inhibitor than PD-98059. SB-203580 binds p38alpha and p38beta directly at the ATP-binding site, thereby blocking its catalytic activity but not its ability to be phosphorylated by upstream MAPKKs (47). Curcumin, a dietary pigment found in curry, has been shown to inhibit JNK activation by various agonists via an unknown mechanism (6). To first confirm inhibitor specificity to their respective MAPK pathways, we measured ERK, JNK, and p38 phosphorylation in the presence of these inhibitors after 5 min of stretch (Fig. 2). Five minutes of stretch was selected because all three MAPKs were shown to be significantly phosphorylated at this time point (Fig. 1). Mechanical stretch-induced ERK1/2 phosphorylation was attenuated by 25 µM PD-98059 pretreatment and completely abolished in the presence of 5 µM U-0126. Both PD-98059 and U-0126 were specific to the ERK pathway because neither inhibitor affected stretch-induced JNK or p38 phosphorylation (Fig. 2). As expected, SB-203580 (25 µM) failed to inhibit stretch-induced p38 phosphorylation (Fig. 2), which is consistent with the reported mode of action of this inhibitor. Importantly, SB-203580 had no impact on stretch-induced ERK or JNK phosphorylation (Fig. 2). A 1-h incubation with 25 µM curcumin completely abolished stretch-induced JNK phosphorylation while not impinging on the ERK and p38 pathways. Collectively, these results confirm that the inhibitors used in the present study were specific to their designated MAPK pathways.


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Fig. 2.   Specificity of MAPK inhibitors to their designated MAPK pathways. Myometrial SMCs were pretreated with 0.04% DMSO, 25 µM PD-98059, 5 µM U-0126, 25 µM SB-203580, or 25 µM curcumin for 60 min and subjected to 25% static stretch for 5 min. ERK, JNK, and p38 phosphorylation was assessed by Western blot using phosphospecific MAPK antibodies. "S" represents cells subjected to stretch and "NS" represents nonstretch control cells. Shown are representative Western blots probed with a phosphospecific ERK (p-ERK, top), JNK (p-JNK, middle), and p38 (p-p38, bottom) antibody. Data represent similar results from 3 independent experiments.

ERK pathway inhibitors attenuate stretch-induced c-fos expression. Myometrial SMCs were pretreated with 25 µM PD98059, 5 µM U0126, or 0.04% DMSO (control vehicle) for 1 h and then stretched (25% elongation) for 30 min to analyze c-fos mRNA expression. In the absence of PD-98059 or U-0126, stretch resulted in a 12- to 16-fold increase in c-fos expression compared with the nonstretch control cells (Fig. 3, A and B). When myometrial SMCs were pretreated with 25 µM PD-98059, stretch-induced c-fos expression was significantly attenuated, compared with the untreated stretch group (Fig. 3A). The more potent inhibitor of ERK1/2 activation, U0126, was also only able to partially inhibit c-fos expression in response to stretch (Fig. 3B). Importantly, neither drug was able to completely eliminate stretch-induced c-fos mRNA expression even when ERK activation was entirely abolished (see Fig. 2). These results indicate that ERK1/2 activation only in part mediates stretch-induced c-fos expression in myometrial SMCs.


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Fig. 3.   Effect of MAPK inhibitors on stretch-induced c-fos expression. Myometrial SMCs were pretreated with 25 µM PD-98059 (A), 5 µM U-0126 (B), 25 µM SB-203580 (C), and 25 µM curcumin (D)for 60 min and subjected to 25% static stretch for 30 min to assess c-fos mRNA expression. Shown are representative Northern blots for c-fos (top) and the same membrane stripped and reprobed with 18S (bottom) and the corresponding bar graph showing the means ± SE of c-fos (relative to 18S); n = 3. Data were subjected to two-way ANOVA followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). *P < 0.05, **P < 0.01, ***P < 0.001 indicates significant difference from nonstretch controls, "a" is significantly different from "b" (P < 0.05).

p38 contributes to stretch-induced c-fos expression. Myometrial SMCs were pretreated with 25 µM SB-203580 or 0.04% DMSO (control vehicle) for 1 h and then stretched (25% elongation) for 30 min to analyze c-fos mRNA expression. In the absence of SB-203580, stretch resulted in a 12-fold increase in c-fos expression compared with the nonstretch control cells (Fig. 3C). When SMCs were pretreated with 25 µM SB-203580, stretch-induced c-fos expression was almost completely abolished compared with the untreated stretch group (Fig. 3C). These results indicate that p38 also mediates stretch-induced c-fos expression in myometrial SMCs.

JNK inhibition prevents stretch-induced c-fos expression. To investigate whether JNK is involved in stretch-induced c-fos expression, myometrial SMCs were pretreated with 25 µM curcumin or 0.04% DMSO (control vehicle) for 1 h and then stretched (25% elongation) for 30 min to analyze c-fos mRNA expression. In the absence of curcumin, stretch resulted in a 10-fold increase in c-fos expression compared with the nonstretch control cells (Fig. 3D). In the presence of curcumin, stretch-induced c-fos expression was completely abolished (Fig. 3D). These results suggest that the JNK pathway is also involved in the mechanotransduction pathway leading to c-fos expression.

Effects of tyrosine kinase inhibitors on stretch-induced c-fos expression. To examine whether stretch-induced c-fos expression is dependent on upstream tyrosine kinase activity, myometrial SMCs were pretreated with the tyrosine kinase inhibitors genistein or herbimycin A and then subjected to 25% static stretch for 30 min. In the absence of genistein or herbimycin A, stretch resulted in a 7- to 10-fold increase in c-fos expression compared with the nonstretch control cells (Fig. 4, A and B). When myometrial SMCs were pretreated with 50 µM genistein for 60 min, stretch-induced c-fos expression was abolished (Fig. 4A). Similarly, pretreatment of cells with 1 µM herbimycin A for 16 h also abolished stretch-induced c-fos expression compared with untreated stretched cells (Fig. 4B). These findings indicate that tyrosine kinases are involved in the mechanotransduction pathway leading to c-fos expression.


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Fig. 4.   Effect of tyrosine kinase inhibitors genistein and herbimycin A on stretch-induced c-fos expression. Myometrial SMCs were pretreated with 0.04% DMSO or 50 µM genistein for 60 min or 1 µM herbimycin A for 16 h and subjected to 25% static stretch for 30 min. Shown are representative Northern blots showing stretch-induced c-fos expression in the presence or absence of genistein (A) or herbimycin A (B). Data represent similar results from 3 independent experiments

Effect of tyrosine kinase inhibitor on stretch-induced MAPK phosphorylation. To determine whether tyrosine kinase activity is upstream of the MAPK pathway, myometrial SMCs were pretreated with genistein and subjected to 25% static stretch for 5 min, and ERK, JNK, and p38 phosphorylation was determined by Western blot analysis using phosphospecific MAPK antibodies. In the absence of genistein, stretch increased phosphorylation of all three MAPK family members (Fig. 5). However, when cells were pretreated with genistein stretch-induced ERK, JNK and p38 phosphorylation was completely abolished (Fig. 5). These findings suggest that tyrosine kinases are upstream signaling molecules of stretch-induced MAPK activation leading to c-fos expression in myometrial SMCs.


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Fig. 5.   Effect of tyrosine kinase inhibitor genistein on stretch-induced MAPK phosphorylation. Myometrial SMCs were pretreated with 0.04% DMSO or 50 µM genistein for 60 min and subjected to 25% static stretch for 5 min. Shown are representative Western blots showing stretch-induced ERK, JNK, and p38 phosphorylation (p-ERK, p-JNK, and p-p38) in the presence or absence of genistein. Data represent similar results from 3 independent experiments.

MAPK activation in pregnant rat myometrium. We have previously shown that increased expression of the CAP genes Cx43 and OTR and the transcription factor c-fos during labor occurs only in the gravid horn of unilaterally pregnant rats (34, 36, 37). In conjunction with our in vitro stretch experiments, we have interpreted these data as demonstrating a role for mechanical stretch in inducing CAP and c-fos gene expression. To determine whether MAPK activity exhibits a similar regulation, we measured phosphorylation of MAPKs in empty and gravid horns of unilaterally pregnant rats. Western analysis indicated a dramatic increase in ERK phosphorylation in the gravid horn across gestation, peaking within the 24 h before the onset of labor (day 22; Fig. 6A). The levels of ERK phosphorylation remained relatively unchanged in the empty horn across gestation (Fig. 6A). ERK phosphorylation was significantly higher in the gravid horn compared with the empty horn on day 21, day 22, and during labor (day 23; Fig. 6A), suggesting that strain imposed on the uterine wall by the growing fetus during late pregnancy accounts for the increased ERK phosphorylation. Interestingly, ERK phosphorylation dropped precipitously one day postpartum (1PP) when the uterus was no longer exposed to the strain imparted by the presence of the fetuses (Fig. 6A). Statistical analysis revealed a significant induction of p38 phosphorylation in the gravid horn compared with the empty horn only on day 22 (Fig. 6B). We were unable to detect JNK phosphorylation in either empty or gravid horns throughout gestation with the phosphospecific antibody used in this study (data not shown).


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Fig. 6.   MAPK phosphorylation in empty and gravid horns of unilaterally pregnant rats during gestation. MAPK phosphorylation was analyzed in gravid (G) and empty (E) horns of unilaterally pregnant rats on the indicated days of gestation by Western blot using phosphospecific MAPK antibodies. Shown are immunoblots for p-ERK1 and p-ERK2, phosphorylated ERK1 and ERK2, respectively (A), and p-p38, phosphorylated p38 (top panels) or a control anti-ERK1/2, -p38 antibody (bottom panels) (B) for 1 of 3 sets of Western analysis. The intensity of phosphorylation at the 44/42-kDa bands of ERK and 38-kDa bands of p38 was quantified by densitometry, and phospho-ERK, and -p38 levels were normalized to their respective protein levels and represented in the corresponding bar graph as means ± SE. Data from 3 independent experiments were subjected to two-way ANOVA, followed by all pairwise multiple comparison procedures (Student-Newman-Keuls method). Data labeled with different letters are significantly different from each other (P < 0.05). *P < 0.05, **P < 0.01, ***P < 0.001 indicates significant difference vs. empty horn of the same gestational day.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study provides several novel insights into the mechanotransduction pathways leading to c-fos gene expression in myometrial SMCs. We show that static stretch induces a transient phosphorylation (activation) of ERK, JNK, and p38 and that all three MAPKs are involved in the mechanical signaling pathway leading to c-fos expression. We also show that stretch-induced c-fos expression and MAPK activation are dependent on upstream tyrosine kinase activity. Furthermore, MAPKs (ERK and p38) are expressed in the pregnant rat myometrium, and their phosphorylation levels increase only in the gravid horn (not in the empty horn) late in pregnancy, suggesting that strain imposed on the uterine wall by the growing fetus stimulates myometrial MAPK activity.

Our finding of stretch-induced phosphorylation of ERK1/2, JNK, and p38 in myometrial SMCs in vitro is consistent with data from endothelial cells (24), vascular SMCs (28), mesangial cells (17), and cardiac myocytes (42). Importantly, our finding that ERK1/2 and p38 phosphorylation are increased in gravid (but not nongravid) myometrium before the onset of labor indicate that our in vitro data are informative of physiological events in vivo. The magnitude of JNK and p38 phosphorylation in response to in vitro stretch was greater than that for ERK. This is consistent with other cell types subjected to various types of stress in which JNK and p38 are activated strongly whereas ERK is only weakly activated (45). In contrast, in cells treated with mitogens, ERK is more strongly activated than JNK and p38 (45). The temporal profile of stretch-induced phosphorylation was different for each MAPK, suggesting their activation occurs via distinct signal transduction pathways. Stretching endothelial cells increases the association of PKC-alpha and PKC-epsilon with Raf-1 (MAPKKK of ERK1/2 pathway) (7), thereby providing a mechanism by which PKC can selectively regulate the ERK pathway. The rapid activation of distinct G proteins subunits could account for temporal differences in stretch-induced ERK, JNK, and p38 activation. In endothelial cells, Galpha i2 mediates shear-induced Ras-ERK activation and Gbeta /gamma mediates shear-induced Ras-JNK activation (22). The activation of receptor tyrosine kinase signaling may represent another mechanism by which mechanical inputs are converted into differential MAPK activation. Platelet-derived growth factor receptor-alpha is activated in vascular SMCs in response to mechanical stretch in a ligand-independent fashion, leading to ERK2 activation and enhanced AP-1 binding activity (15). Using a general growth factor receptor inhibitor, Li et al. (28) showed that ERK activation, but not p38 or JNK activation, was dependent on growth factor receptor activation in vascular SMCs.

The phosphorylation of all three MAPKs was transient despite continuous exposure to in vitro stretch. A highly coordinated mechanism of MAPK deactivation may be responsible for this transient activation. In vascular SMCs, mechanical stress induces MAPK phosphatase 1 (MKP-1) protein expression, and a decline in ERK1/2, JNK, and p38 activity occurred when high levels of MKP-1 protein persisted (28), suggesting a role for MKP-1 in the rapid inactivation of MAPKs after their stretch-induced activation. The temporal nature of MAPK activation is critically important in determining cell fate. JNK activation in endothelial achieved with colchicine treatment induces apoptosis, whereas transient shear stress-induced JNK activation did not lead to apoptosis (16).

All three MAPK signaling pathways control the transcriptional activation of the c-fos gene (45). This effect is mediated by phosphorylation and thereby activation of the transcription factor Elk-1, which complexes with SRF at the SRE of the c-fos promoter (45). We found that stretch-induced c-fos mRNA expression was attenuated when myometrial SMCs were pretreated with inhibitors for the ERK pathway (PD-98059 and U-0126), p38 (SB-203580), or the JNK pathway (curcumin). These findings provide evidence that all three MAPKs are involved in the c-fos response in stretched myometrial SMCs. A mediatory role for ERK in shear-induced c-fos mRNA expression has also been demonstrated in endothelials cells (2). To our knowledge, the present study is the first to directly link p38 and JNK activation to stretch-induced c-fos mRNA expression. Studies have implicated p38 and JNK pathways in increased AP-1 activity in response to mechanical forces. Nuclear extracts from stretched mesangial cells (19) and endothelial cells (24) have increased DNA binding activity to the AP-1 consensus sequence, which can be attenuated by blocking the ERK or p38 pathway. Furthermore, transfection experiments using an AP-1 binding site-containing reporter gene reveal that stretch-induced AP-1 activity in endothelial cells is dependent on the ERK, JNK, and p38 pathways (24).

Our finding that all three MAPKs pathways are required for optimal stretch-induced c-fos expression suggests functional interaction among these pathways. Similarly, Kito et al. (24) showed that specific inhibitors and dominant negative mutants that target any one MAPK pathway completely abolished stretch-induced AP-1 binding and activity. It is conceivable that activation of all three MAPKs is necessary to properly activate the downstream target, Elk-1, which requires phosphorylation of several residues in the carboxy terminus. ERK, JNK, and p38 are targeted to distinct amino acid residues in Elk-1 (46), raising the possibility that each MAPK may phosphorylate a unique subset of sites on Elk-1 to achieve optimal activation. In addition to activating Elk-1, JNK and p38 can phosphorylate ATF-2, which can promote transcription of the c-fos gene at the cAMP response element (CRE) (45). ERK is also able to phosphorylate and activate STAT for transcriptional activation of the c-fos promoter at the sis-inducible element (SIE). Therefore, blocking any one of the MAPK pathways could have an impact on their effect on other elements of the c-fos promoter and thereby severely reduce c-fos transcription. Transgenic mice carrying c-fos-lacZ fusion genes with mutations in any one of the control elements in the c-fos promoter (SIE, SRE, AP-1, and CRE) exhibit severally reduced expression of the transgene, indicating that cooperation between elements in the c-fos promoter is a strict requirement for c-fos transcription in vivo (40).

We have also demonstrated a requirement for protein tyrosine kinase activity in the signal transduction pathway leading to MAPK activation and c-fos expression in stretched myometrial SMCs. Because genistein is a general tyrosine kinase inhibitor, the identities of the tyrosine kinase(s) influencing MAPK activation and downstream c-fos expression remain unclear. However, herbimycin A, an inhibitor which has some specificity towards the Src tyrosine kinases, also blocked stretch-induced c-fos expression; therefore, the tyrosine kinase dependence may be associated with Src activity. Src is rapidly activated in endothelial cells in response to shear stress, and it is a common upstream mediator of shear-induced ERK and JNK activation, transcriptional activity of Elk-1, and subsequent induction of the c-fos promoter (21).

In previous studies, we have shown that the increase in c-fos expression during labor is associated with an increase in the plasma estrogen: progesterone ratio (39). Furthermore, estrogen administration significantly increases c-fos expression in the nonpregnant rat myometrium, and this is followed by an increase in CAP (Cx43) expression (39). We speculate that c-fos may mediate both endocrine (estrogen) and stretch-induced CAP expression, leading to myometrial "activation" and the onset of labor. This possibility is attractive because the increase expression of CAPs including the gap junction connexin43 (Cx43) and the oxytocin receptor (OTR), the induction of which requires both hormonal and mechanical signals during term and preterm labor, is paralleled by a similar increase in c-fos mRNA (39). Furthermore, the Cx43 and OTR promoters contain several putative AP-1 sites, which bind Fos proteins (18, 27).

The present data indicates that MAPKs are upstream mediators of stretch-induced c-fos expression in myometrial SMCs. Our in vivo data show that during late pregnancy MAPK phosphorylation (ERK and p38) increases in the myometrium of the gravid horn in unilaterally pregnant rats (31). This increase in ERK and p38 phosphorylation occurs before the maximal induction that has been observed for c-fos mRNA, suggesting a regulatory role for MAPK pathways in myometrial c-fos expression leading to CAP expression and the initiation of labor. Furthermore, phosphorylation levels of ERK and p38 were significantly higher in the gravid uterine horn compared with the empty horn near the end of gestation, implying a role for mechanical factors (due to growth of fetuses) in the induction of MAPK activity in the pregnant myometrium. Importantly, our in vivo data supports the validity and physiological relevance of our in vitro stretch system.

In conclusion, our data suggest that tyrosine kinases and all three MAPK members contribute to the mechanical signaling pathway, leading to c-fos mRNA expression in the myometrium. Understanding the process of mechanotransduction in myometrial SMCs is critical because increased uterine tension or stretch may contribute to preterm labor, especially in those pregnancies associated with increased intrauterine volume (e.g., multifetal pregnancies).


    ACKNOWLEDGEMENTS

A. Oldenhof and O. Shynlova contributed equally to the work presented in this manuscript.


    FOOTNOTES

This work was supported by a grant from National Institute of Child Health and Human Development, Grant Number HD-37942-01.

Address for reprint requests and other correspondence: S. J. Lye, Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, 600 Univ. Ave, ON, Canada, M5G 1X5 (Email: Lye{at}mshri.on.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 3, 2002;10.1152/ajpcell.00607.2001

Received 19 December 2001; accepted in final form 25 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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