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. 2012 Nov 7;87(6):142. doi: 10.1095/biolreprod.112.104448

Chronic Hypoxia Differentially Up-Regulates Protein Kinase C-Mediated Ovine Uterine Arterial Contraction via Actin Polymerization Signaling in Pregnancy1

DaLiao Xiao 1,2, Xiaohui Huang 1, Lubo Zhang 1
PMCID: PMC4435429  PMID: 23136295

ABSTRACT

Chronic hypoxia (CH) during pregnancy is associated with increased uterine vascular tone. The present study tested the hypothesis that CH up-regulates protein kinase C (PKC)-mediated actin polymerization, resulting in enhanced uterine vascular contraction in pregnancy. Uterine arteries were isolated from nonpregnant (NPUA) and near-term (∼140 days of gestation) pregnant (PUA) sheep that had been maintained at sea level (∼300 m) or exposed to high altitude (3801 m) hypoxia for 110 days. In normoxic animals, the induced contractions by the PKC activator phorbol 12,13-dibutyrate (PDBu) were greater in NPUA than in PUA, which was abrogated by an actin polymerization inhibitor cytochalasin B (Cyto B). In hypoxic animals, PDBu-induced contractions were significantly increased in PUA but not in NPUA, which was inhibited by Cyto B. In contrast, neither pregnancy nor hypoxia affected Cyto B-mediated inhibition of norepinephrine (NE)-induced contractions. Prolonged ex vivo treatment of NPUA with 17beta-estradiol and progesterone significantly attenuated PDBu-induced actin polymerization and contractions, and the hormonal treatment did not alter the inhibitory effect of Cyto B on PDBu- or NE-induced contractions in either normoxic or hypoxic animals. 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one potentiated PDBu-mediated actin polymerization and enhanced PDBu-induced contractions of PUA in normoxic but not hypoxic animals, which was abrogated by Cyto B. The results suggest that chronic hypoxia during pregnancy causes attenuation of steroid hormone-mediated ERK1/2 signaling and results in increased actin polymerization and uterine vascular tone, linking gestational hypoxia to aberrant uteroplacental circulation.

Keywords: actin polymerization, chronic hypoxia, pregnancy, protein kinase C (PKC), uterine vascular contraction

Chronic hypoxia increases protein kinase C-mediated uterine vascular contraction via actin polymerization signaling, resulting in decreased uterine blood flow.

INTRODUCTION

Pregnancy is associated with a significant increase in uterine blood flow that optimizes the delivery of oxygen and substrates to the developing fetus via the placenta. The increase in uterine vascular relaxation and decrease in uterine arterial constriction and vascular tone play an important role in keeping the uterine circulation functions as a low-resistance shunt to accommodate the large increase of uterine blood flow required for the normal fetal development [1–6]. Chronic hypoxia during pregnancy is a common stress to the mother and the developing fetus. Hypoxia during gestation alters uterine vascular reactivity and impairs the adaptation of uterine blood flow, which is associated with an increased risk of preeclampsia and fetal intrauterine growth restriction [3, 7–10].

Among other mechanisms, previous studies suggested that protein kinase C (PKC) played a pivotal role in the regulation of vascular contractility both in physiologic conditions and in pathologic conditions, including hypoxia exposure [11–15]. Activation of PKC by pressure or agonists has been proposed to induce vasoconstriction and increased vascular tone without increases in intracellular Ca2+ concentrations or myosin light chain phosphorylation [13, 16]. Our recent studies have also demonstrated in ovine uterine arteries that activation of PKC by agonist causes sustained contractions without changes in 20-kDa myosin light chain (MLC20) phosphorylation levels [6]. These findings suggest that PKC-mediated vascular tone of the uterine arteries is mainly regulated through a thin filament regulatory mechanism, that is, Ca2+ or MLC20 phosphorylation-independent pathway. Actin is the key component in thin filament regulatory signaling pathways. Previous studies have demonstrated that increased actin polymerization plays an important role in actin-myosin interactions, force production, and development of vascular tone [17–19]. Furthermore, our recent studies have demonstrated that activation of PKC causes actin polymerization and contractions of ovine uterine arteries and that inhibition of actin polymerization blocks PKC-mediated contractions [15]. In addition, uterine vascular tone attenuated by pregnancy is associated with a decrease in PKC activity and PKC-induced actin polymerization [15, 20–22].

However, the effect of chronic hypoxia on PKC-mediated actin polymerization in the regulation of uterine artery contractility remains undetermined. Given the findings that chronic hypoxia up-regulates uterine vascular tone through its direct effect in suppressing extracellular signal-regulated kinase 1/2 (ERK1/2) activity and increasing the PKC signal pathway during pregnancy [11, 23], the present study further tested the hypothesis that chronic hypoxia during gestation up-regulates PKC-mediated actin polymerization and increases uterine arterial vascular tone. We first determined the role of actin polymerization in PKC- and norepinephrine (NE)-induced contractions of uterine arteries in nonpregnant and pregnant sheep maintained at sea level as normoxic control or exposed to high altitude hypoxia for 110 days. We then determined whether inhibition of actin polymerization affected chronic hypoxia-mediated up-regulation of PKC-induced contractions. Additionally, because steroid hormones play an important role in pregnancy-mediated down-regulation of PKC-induced actin polymerization in uterine artery [15], we also determined whether chronic hypoxia attenuated the role of steroids hormones in the down-regulation of PKC-induced actin polymerization.

MATERIALS AND METHODS

Tissue Preparation

All the procedures and protocols used in the present study were approved by the Animal Research Committee of Loma Linda University and followed the guidelines by the National Institutes of Health's Guide for the Care and Use of Laboratory Animals. As previously described [24], nonpregnant and time-dated pregnant sheep were obtained from the Nebeker Ranch in Lancaster, CA (altitude: ∼300 m; arterial PaO2: 102 ± 2 mm Hg). Uterine arteries were obtained from nonpregnant and near-term (∼140 days of gestation) pregnant sheep. Normoxic animals were studied between December and July. For chronic hypoxic treatment, nonpregnant and pregnant (30 days of gestation) animals bred during the time period of March to June were transported to the Barcroft Laboratory, White Mountain Research Station, Bishop, CA (altitude, 3,801 m; maternal PaO2, 60 ± 2 mm Hg) and maintained there for ∼110 days. Starting from August to October, the animals were transported to the laboratory immediately before the studies. Ewes were anesthetized with thiamylal (10 mg/kg) administered via the external left jugular vein. The animals were then intubated and anesthesia was maintained on 1.5% to 2.0% halothane in oxygen throughout surgery. An incision in the abdomen was made and the uterus exposed. The uterine arteries were isolated and removed without stretching, and placed into a cold physiological salt solution containing (in mM): 130 NaCl, 10.0 HEPES, 6.0 glucose, 4.0 KCl, 4.0 NaHCO3, 1.80 CaCl2, 1.2 MgSO4, 1.18 KH2PO4, and 0.025 ethylenediaminetetraacetic acid, pH 7.4. After removal of the tissues, animals were killed with an euthanasia solution (T-61; Hoechst-Roussel). For nonpregnant animals, uterine arteries were only obtained from animals during the luteal phase of the ovarian cycle. The uterine arteries were isolated bilaterally in nonpregnant animals but only isolated from the gravid uterine horn in pregnant animals.

Contraction Studies

The fourth generation branches of main uterine arteries from both pregnant and nonpregnant sheep were isolated and cut into 2-mm ring segments and mounted in 10-ml tissue baths containing modified Krebs solution equilibrated with a mixture of 95% O2 and 5% CO2. Phorbol 12,13-dibutyrate (PDBu; Sigma)- or NE-induced isometric tensions in the absence or presence of an actin polymerization inhibitor, 10 μM cytochalasin B (Cyto B) (Sigma) were measured in tissue baths at 37°C as described previously [15, 25]. PDBu-induced contractions were also determined in the absence or presence of 30 μm 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059; Sigma) or 30 μm PD098059 plus 10 Cyto B.

Steroid Hormones Treatment

As previously described [15, 25, 26], freshly isolated uterine arteries were incubated in phenol red-free Dulbecco modified Eagle medium (Mediated Cellgro) with 1% charcoal-stripped fetal bovine serum for 48 h at 37°C in a humidified incubator with 5% CO2/95% air in the absence or presence of 0.3 nM estradiol-17β (E2) plus 100 nM progesterone (P4). After the treatment, the vessels were mounted in 10-ml tissue baths for functional contractile studies or used to determine the status of actin polymerization.

Measurement of Actin Polymerization

Actin polymerization from nonomeric globular actin (G-actin) to filamentous actin (F-actin) was determined in the uterine arteries. The concentrations of F-actin and G-actin in the tissues were measured by the method described previously [4, 15, 27]. Briefly, each of the artery rings after agonist stimulation was homogenized in 200 μl F-actin stabilization buffer (50 mM Pipes, pH 6.9, 50 mM NaCl, 5 mM MgCl2, 5 mM ethylene glycol tetraacetic acid, 5% glycerol, 0.1% Triton X-100, 0.1% Nonidet P40, 0.1% Tween 20, 0.1% β-mercaptoethanol, 0.0011% antifoam, 1 mM ATP, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 10 μg/ml benzamidine, 500 μg/ml tosyl arginine methyl ester). The supernatants of protein extracts were collected after centrifugation at 100 000 × g for 60 min at 30°C. The pellets were then resuspended in ice-cold distilled H2O plus 1 μM cytochalasin D and then incubated on ice for 1 h to dissociate F-actin. The supernatant of the resuspended pellets was collected after centrifugation at 2300 × g for 2 min at 4°C. The proteins from the first supernatant (G-actin) and second supernatant (F-actin) were subjected to analysis by immunoblot using anti-actin antibody (1:2000, catalog #A2547; Sigma). The total amounts of G-actin and F-actin from the original soluble and insoluble fractions were calculated based on the total protein in each fraction.

Data Analysis

Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad Software). The results were expressed as means ± SEM obtained from the number of experimental animals given. Differences were evaluated for statistical significance (P < 0.05) by ANOVA or Student t-test, where appropriate.

RESULTS

Effect of Actin Polymerization Inhibition on PKC-Mediated Contractions of Uterine Arteries

In normoxic sheep, PKC activator PDBu-induced maximum contraction (percent KCl response) was significantly greater in nonpregnant (Fig. 1A) than in pregnant (Fig. 1B) uterine arteries (100.4% ± 8.9% vs. 34.9% ± 2.3%, P < 0.05). Inhibition of actin polymerization by Cyto B significantly decreased PDBu-induced contractions in both nonpregnant (100.4% ± 8.9% vs. 29.0% ± 5.7%, P < 0.05) and pregnant (34.9% ± 2.3% vs. 19.9% ± 1.1%, P < 0.05) uterine arteries. Chronic hypoxic had no significant effect on PDBu-induced maximum contraction of uterine arteries in nonpregnant animals (100.4% ± 8.9% vs. 101.8% ± 6.4%, P > 0.05) (Fig. 1, A and C). Similar to the findings in normoxic animals, Cyto B significantly decreased PDBu-induced contractions in hypoxic nonpregnant sheep (101.8% ± 6.4% vs. 36.9% ± 3.1%, P < 0.05). In contrast to the findings in nonpregnant animals, chronic hypoxia significantly increased PDBu-induced uterine artery contractions in pregnant sheep from 34.9% ± 2.3% in normoxic animals (Fig. 1B) to 61.5% ± 4.8% (P < 0.05) in hypoxic animals (Fig. 1D); Cyto B abrogated the increase, reducing it to 27.5% ± 2.7% in hypoxic animals (Fig. 1D).

FIG. 1.

FIG. 1

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Effect of cytochalasin B on phorbol 12,13-dibutyrate (PDBu)-induced contractions in uterine arteries. Cumulative concentration-response curves to PDBu were determined in uterine arteries obtained from nonpregnant and pregnant ewes with normoxia (A, B) and long-term high altitude hypoxia (C, D) treatment in the absence or presence of 10 μM cytochalasin B (Cyto B) pretreatment for 20 min. Data are expressed as percentages of contraction generated by 120 mM KCl, and each point represents the means ± SEM of tissues from five to seven animals.

Effect of Actin Polymerization Inhibition on NE-Mediated Contractions of Uterine Arteries

As shown in Figure 2, Cyto B also significantly inhibited NE-induced contractions of uterine arteries. However, neither pregnancy nor hypoxia altered Cyto B-mediated inhibition of NE-induced contractions.

FIG. 2.

FIG. 2

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Effect of cytochalasin B on norepinephrine (NE)-induced contractions in uterine arteries. Cumulative concentration-response curves to NE were determined in uterine arteries obtained from nonpregnant and pregnant ewes with normoxia (A, B) and long-term high altitude hypoxia (C, D) treatment in the absence or presence of 10 μM Cyto B pretreatment for 20 min. Data are expressed as percentages of contraction generated by 120 mM KCl, and each point represents the means ± SEM of tissues from five to seven animals.

Effect of Steroid Hormones on Actin Polymerization Inhibitor-Mediated Vascular Contractility

Our recent studies have demonstrated that steroid hormones inhibit PKC-mediated contractions of uterine arteries [26]. In order to test whether the pregnancy-mediated changes in actin polymerization was regulated through steroid hormones, nonpregnant uterine arteries isolated from normoxic and hypoxic animals were treated in the absence or presence of 0.3 nM E2 and 100 nM P4 for 48 h as described previously [26]. As shown in Figure 3, the Cyto B-mediated inhibition of PDBu-induced contractions after chronic pretreatment with E2 and P4 in normoxic (Fig. 3A) or hypoxic (Fig. 3B) nonpregnant animals were similar to those without treatment by E2 and P4 in normoxic (Fig. 1A) or hypoxic (Fig. 1C) nonpregnant animals. The percent inhibition of PDBu-induced maximal response by Cyto B after pretreatment with E2 and P4 in normoxic (68.5% ± 5.8%) or hypoxic (55.0% ± 5.8%) nonpregnant animals were not different as compared with those in normoxic (68.5% ± 4.9%) and hypoxic (60.3% ± 6.4%) nonpregnant animals (Fig. 4A). Similar to the effect of Cyto B on PDBu-induced contractions, the inhibitory effect of Cyto B on NE-induced contraction in both normoxic and hypoxic nonpregnant animals was also not affected by pretreatment with E2 and P4 in the uterine arteries (Figs. 3, C and D, and 4B).

FIG. 3.

FIG. 3

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Effect of steroid hormones on Cyto B-mediated contractions in uterine arteries. Nonpregnant uterine arteries obtained from normoxic (A, C) and hypoxic (B, D) ewes were treated with 0.3 nM 17β-estradiol (E2) and 100 nM progesterone (P4) or vehicle control for 48 h. Then, the hormone-treated vessel rings were submitted to the cumulative additions of PDBu (A, B) or NE (C, D) in the presence of 10 μM Cyto B pretreatment for 20 min. Data are expressed as percentages of contraction generated by 120 mM KCl, and each point represents the means ± SEM of tissues from six to seven animals.

FIG. 4.

FIG. 4

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The percent inhibition of Cyto B on maximal responses of PDBu- and NE-induced contractions in uterine arteries. PDBu- or NE-induced maximal responses of contractions in uterine arteries of nonpregnant sheep were obtained from the cumulative concentration-response curves indicated in Figures 1–3. The percent inhibition of Cyto B on PDBu-induced (A) and NE-induced (B) maximal responses were calculated from the difference between with and without Cyto B treatment divided by without Cyto B treatment. Data are means ± SEM of tissues from five to seven animals.

To test whether steroid hormones affect PKC-mediated actin polymerization, uterine arteries of nonpregnant sheep were treated in the absence or presence of 0.3 nM E2 and 100 nM P4 for 48 h. Then the actin polymerization was determined by Western blot analyses of G-actin and F-actin in the uterine artery. As shown in Figure 5, pretreatment with E2 and P4 significantly decreased the density level of basal F-actin/G-actin ratio in nonpregnant uterine arteries (16.0 ± 0.4 vs. 11.3 ± 1.1, P < 0.05). In addition, pretreatment with E2 and P4 also significantly inhibited PDBu-induced increased F-actin/G-actin ratio in the uterine arteries (36.8 ± 2.3 vs. 20.2 ± 1.9, P < 0.05).

FIG. 5.

FIG. 5

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Effect of steroid hormones on PDBu-induced changes of F-actin/G-actin ratio. Uterine arteries of nonpregnant sheep were treated with 0.3 nM 17β-estradiol (E2) and 100 nM progesterone (P4) or vehicle control for 48 h, then the artery rings were stimulated with 1 μM PDBu or without PDBu (control) for 5 min. F-actin and G-actin were separated by differential sedimentation and determined by Western blot analyses. Data are means ± SEM of tissues from five animals. a(P < 0.05) +E2P4 versus −E2P4; b(P < 0.05) PDBu versus control.

Effect of PD098059 on PKC-Mediated Contractions of Uterine Arteries

In normoxic pregnant sheep (Fig. 6, upper panel), pretreatment with extracellular signal-regulated kinase (ERK) inhibitor, PD098059, significantly potentiated PDBu-induced maximal contractions of uterine arteries compared with the control (29.3% ± 3.1% vs. 41.7% ± 4.4% KCL response, P < 0.05). Pretreatment with both PD098059 and Cyto B significantly inhibited PDBu-induced maximal responses of uterine artery contractions (29.3% ± 3.1% vs. 15.6% ± 2.2% KCL response, P < 0.05), which was not different compared with Cyto B alone (15.6% ± 2.2% vs. 19.6% ± 1.8% KCL response, P > 0.05). In hypoxic pregnant sheep (Fig. 6, lower panel), PD098059 had no effect on PDBu-induced maximal responses of uterine artery contractions compared with the control (55.1% ± 6.9% vs. 41.6% ± 4.4% KCL response, P > 0.05). In addition, pretreatment with both PD098059 and Cyto B significantly inhibited PDBu-induced maximal responses of uterine artery contractions (55.1% ± 6.9% vs. 20.4% ± 2.2% KCL response, P < 0.05) but was not different compared with Cyto B alone (20.4% ± 2.2% vs. 22.2% ± 1.9% KCL response, P > 0.05).

FIG. 6.

FIG. 6

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Effect of ERK1/2 inhibitor PD098059 on PDBu-induced contractions in uterine arteries. Uterine arteries were isolated from normoxic (upper panel) and hypoxic (lower panel) pregnant ewes. Cumulative concentration response curves to PDBu were determined in the uterine artery rings in the absence or presence of 30 μM PD098059, 10 μM Cyto B, or 30 μM PD098059 plus 10 μM Cyto B for 20 min. Data are expressed as percentages of contraction generated by 120 mM KCl, and each point represents the means ± SEM of tissues from eight to nine animals.

The effect of PD098059 on PKC-induced actin polymerization was determined by Western blot analyses of G-actin and F-actin in the uterine artery. As shown in Figure 7, the PKC activator (PDBu) induced an increase in the F-actin/G-actin ratio from 10.72 ± 0.64 to 16.34 ± 0.66 (P < 0.05) in pregnant uterine artery, which was further potentiated treatment with PD098059 (16.34 ± 0.66 vs. 22.54 ± 1.13, P < 0.05).

FIG. 7.

FIG. 7

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Effect of PD098059 on PDBu-induced changes of the F-actin/G-actin ratio. Uterine arteries isolated from pregnant ewes were stimulated without PDBu (control), with 1 μM PDBu, or with 1 μM PDBu plus 30 μM PD098058 (pretreatment for 20 min). F-actin and G-actin were separated by differential sedimentation and determined by Western blot analyses. Data are means ± SEM of tissues from five animals. a(P < 0.05) PDBu versus control; b(P < 0.05) PDBu + PD098059 versus PDBu.

DISCUSSION

The current study offers the following key observations: 1) the PKC activator (PDBu)-induced contractions of uterine arteries were attenuated by pregnancy, and the actin polymerization inhibitor (Cyto B) significantly attenuated the difference of PDBu-induced contractions between pregnant and nonpregnant animals; 2) PDBu-induced contractions were enhanced by chronic hypoxia only in pregnant but not in nonpregnant animals, and chronic hypoxia significantly enhanced the inhibitory effect of Cyto B on PDBu-induced contractions in pregnant but not nonpregnant animals; 3) Cyto B significantly inhibited α1-adrenoreceptor agonist (NE)-induced contractions of uterine arteries, which was not altered either by pregnancy or hypoxia; 4) chronic treatment of steroid hormones (estrogen and progesterone) in nonpregnant uterine arteries did not alter the inhibitory effect of Cyto B on PDBu- or NE-induced contractions, but the steroid hormones significantly attenuated the basal level F-actin/G-actin ratio and PDBu-induced F-actin/G-actin ratio in nonpregnant uterine arteries; 5) the ERK inhibitor (PD098059) potentiated PDBu-induced contractions of pregnant uterine arteries in normoxic animals but not in hypoxic ones, and PD098059 did not alter the inhibitory effect of Cyto B on PDBu-induced contractions; and 6) pretreatment of PD098059 enhanced PDBu-induced increase in the F-actin/G-actin ratio in pregnant uterine arteries.

In the present study, the activation of PKC by PDBu or the activation of the α1-adrenoreceptor by NE produced a dose-dependent development of uterine artery contractions that were inhibited by the actin filament polymerization inhibitor Cyto B. These observations suggest that alteration of actin polymerization dynamics is one of the key mechanisms in PKC- and α1-adrenoreceptor-mediated uterine vascular contractility. The findings that both pregnancy and chronic hypoxia significantly altered the inhibitory effect of Cyto B on PDBu- but not NE-induced uterine vascular contraction suggest that PKC-mediated actin polymerization signaling plays an important role in uterine vascular contractile adaptation to pregnancy and high altitude hypoxia. Previous studies have demonstrated that PKC activities in the uterine arteries are significantly decreased during pregnancy [15, 20–22], resulting in a decrease in uterine artery vascular tone [5, 6, 26], which may play a key role in increased uterine blood flow in pregnancy. Consistent with the previous studies, our current findings that PDBu-induced contractions were significantly decreased in pregnant uterine arteries compared with nonpregnant uterine arteries further confirmed that pregnancy attenuates the role of PKC in mediated uterine vascular function. In certain vasculatures, including uterine artery smooth muscle cells, PKC-induced contraction is independent of changes in intracellular Ca2+ concentration or myosin light chain phosphorylation levels. It suggests that PKC-mediated contraction is regulated predominantly through thin filament regulatory pathway [28–31]. In agreement with previous study [15], our present observations that Cyto B inhibited PDBu-induced contraction and attenuated the difference of PKC-mediated contractions between nonpregnant and pregnant uterine arteries suggest that the attenuated PKC-induced contraction of uterine artery in pregnancy may be due to down-regulation of actin polymerization signaling.

In contrast to the inhibitory effect of pregnancy on PKC-mediated signaling, our current data indicate that high altitude hypoxia significantly enhanced PDBu-induced contractions of the uterine arteries in pregnant sheep. This observation further supports the idea that chronic hypoxia-enhanced uterine vascular tone is due to its direct effect on the up-regulation of PKC signaling pathway in the uterine arteries during pregnancy [11]. In agreement with our current findings, several previous studies from others have demonstrated that the activation of PKC plays a major role in regulation of hypoxia-induced pulmonary vasoconstriction and hypertension [32–35]. The mechanisms underlying the hypoxia-mediated increase in PKC-induced uterine artery contractions are not fully understood. However, the present findings that the inhibitory effect of Cyto B on PDBu-induced contractions in pregnant uterine arteries was higher in hypoxic than normoxic animals suggest that chronic hypoxia up-regulates actin polymerization signaling, which, at least partly, plays a key role in hypoxia-enhanced PKC-induced contraction in pregnancy.

In our recent studies, we have demonstrated that steroid hormones (estrogen and progesterone) play an important role in pregnancy-induced down-regulation of PKC-mediated uterine vascular tone [15, 26]. Direct steroid hormones treatment attenuates basal level of actin polymerization in nonpregnant uterine arteries and steroid hormone receptor blockers enhance basal value of actin polymerization in pregnant uterine arteries [15], which suggests a direct effect of the steroid hormones in the down-regulation of actin polymerization signaling in pregnancy. This is further supported by the present observation that chronic steroid hormones treatment significantly attenuated the PDBu-induced F-actin/G-actin ratio in the uterine arteries. Because steroid hormones directly inhibit PKC-mediated uterine artery contraction [26], the present findings that Cyto B produced a similar inhibitory effect on PDBu-induced contractions both in the absence and in the presence of steroid hormones suggest that Cyto B and steroid hormones may act on a common mechanism, that is, inhibition of actin polymerization [36, 37]. In the present study, chronic hypoxia selectively enhanced PKC-mediated actin polymerization signaling only in pregnant animals but not in nonpregnant animals. This suggests that chronic hypoxia during pregnancy may attenuate the effect of sex steroid hormones on PKC-mediated actin polymerization signaling in uterine arteries. Indeed, in the same animal model, our previous studies have demonstrated that high altitude hypoxia during pregnancy suppresses estrogen receptor expression in the uterine artery [23], which may provide a mechanism for the chronic hypoxia-induced inhibition of steroid hormone-mediated responses in uterine arteries.

Although the pathophysiological mechanisms underlying how chronic hypoxia during pregnancy alters steroid hormones-mediated down-regulation of PKC-actin polymerization signaling pathway are unclear, ERK1/2 signaling may be one of the direct links. ERK1/2 has been demonstrated to regulate smooth muscle contraction in different animal models [38–40]. Our previous studies have demonstrated that pregnancy significantly enhances ERK1/2 gene expression and its activity in ovine uterine artery smooth muscle cells [5, 11, 41]. Direct treatment with steroid hormones (E2 and P4) can mimic the effect of pregnancy on ERK1/2 gene expression and its effect on uterine vascular tone [26]. The steroid hormone-mediated increases in ERK1/2 gene expression act as upstream signals in suppressing PKC-mediated contractions and myogenic tone of uterine arteries in pregnant animals [5, 26, 41]. However, chronic hypoxia during pregnancy attenuates ERK/12 gene expression and its activity, resulting in increased PKC activity and PKC-mediated uterine vascular tone via attenuation of sex steroid hormone-mediated signaling [11, 23]. In agreement with previous studies, the current findings that the ERK1/2 inhibitor PD098059 enhanced PDBu-induced contractions only in normoxic but not in hypoxic animals further confirm that ERK1/2 had an inhibitory effect on PKC-mediated contraction of uterine artery and chronic hypoxia could abolish the inhibitory effect of ERK1/2 in pregnancy. The findings that treatment with Cyto B in the absence or presence of PD098059 produced a similar inhibitory effect on PDBu-induced contractions of pregnant uterine arteries suggest that Cyto B and PD098059 may act on the common signaling pathway, that is, inhibition of actin polymerization signaling. Furthermore, the findings that PDBu produced an increase in the levels of the F-actin/G-actin ratio in pregnant uterine arteries, which was enhanced by PD098059, further suggest that ERK1/2 acts as an upstream signal that inhibits PKC-mediated actin polymerization, resulting in suppressing PKC-mediated contractions and myogenic tone of uterine arteries in pregnant animals. The relation between PKC and MEK/ERK1/2 signaling is debatable and controversial. It has been proposed in several studies that ERK1/2 is one of the downstream signals of PKC-mediated pathway in the vascular smooth muscle [42–44], but in cardiac fibroblasts and rat vascular smooth muscle cells, PKC failed to regulate ERK1/2 signaling [45, 46]. Our previous studies in ovine nonpregnant uterine arteries have demonstrated that PKC-mediated contraction is not affected by activation of ERK1/2 [5, 6, 30, 31], suggesting that PKC does not interact with ERK1/2 signaling. However, our current findings, which agree with previous studies in ovine pregnant uterine [5, 6, 30, 31] and cerebral [47] arteries, are that inhibition of ERK1/2-enhanced PKC-mediated contraction suggest that ERK/12 serves as an upstream signal that has an inhibitory effect on PKC-mediated signaling and contraction in pregnant uterine artery.

In summary, the present study has demonstrated an important role of actin polymerization in regulation of PKC-mediated contraction in the uterine artery and its maladaptation to high altitude hypoxia during pregnancy. As shown in Figure 8, in nonpregnant animals, activation of PKC directly causes actin polymerization, resulting in vasoconstriction. However, in pregnant animals, the increase in sex steroid hormones and their receptors during pregnancy enhance ERK1/2 gene expression and its activity in uterine artery smooth muscles. ERK1/2, acting upstream, inhibits PKC activity and PKC-mediated actin polymerization. Pregnancy attenuates the F-actin/G-actin dynamics of uterine artery, resulting in a reduced vascular tone. Chronic hypoxia enhances PKC/actin polymerization via inhibiting the steroid hormone-mediated adaptation of ERK1/2 signaling, leading to an enhanced uterine vascular tone during pregnancy. Thus, the maladaptation of steroid hormone-mediated exaggerated PKC/actin polymerization-induced uterine vascular tone to chronic hypoxia may contribute to the increased risk of uteroplacental circulation dysfunction during pregnancy and fetal growth restriction.

FIG. 8.

FIG. 8

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Proposed mechanism underlying chronic hypoxia enhances uterine vascular tone during pregnancy. In nonpregnant animals, activation of PKC directly causes actin polymerization, resulting in vasoconstriction. In pregnant animals, the increased sex steroid hormones and their receptors during pregnancy up-regulate ERK1/2 gene expression and its activity in uterine artery smooth muscle cells. MEK/ERK1/2 serves as an upstream inhibitor of PKC signaling and inhibits F-actin/G-actin dynamics in the uterine artery. The attenuated actin polymerization results in decreased uterine vascular tone in pregnancy. However, chronic hypoxia during pregnancy enhances PKC-induced actin polymerization via down-regulation of steroid hormone-mediated signaling, resulting in increased uterine vascular tone.

Footnotes

1

Supported by National Institutes of Health grants HL089012 (L.Z.), HD031226 (L.Z.), HL110125 (L.Z.), and DA032510 (D.X.).

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