Uterine Myometrial Cells Research Articles

Blockade of the endothelinA receptor prevents hypoxia induced smooth muscle cell proliferation and endothelin secretion in patients with uterine leiomyomas

Hypoxia, low oxygen conditions, has been implicated as a possible mechanism by which uterine myometrial cells undergo aberrant cell growth and differentiation into leiomyoma (fibroid) cells. Endothelin-1 (ET-1), a vasoconstrive peptide, is increased in fibroid explants in response to hypoxia and inhibits apoptosis of leiomyoma cells in vitro. The objective of this study was to determine if hypoxia induces ET-1 secretion from myometrial smooth muscle cells (mSMCs) to levels comparable to ET-1 secretion fibroid smooth muscle cells (fSMCs). Basic science research study using human tissue samples. Fibroid and myometrium tissue were excised from the uterus of women undergoing hysterectomies for uterine fibroids (n=10). Tissue was washed in phosphate-buffered saline and digested in collagenase solution for 6hrs. The resultant smooth muscle cells were grown until confluent, trypsinized and seeded at 50,000 cells/well and cultured under normoxia (6% oxygen) or hypoxia (1% oxygen) for a period of 24hrs with/without 1μM of endothelin A (ETA) receptor antagonist. ET-1 was measured from cell culture supernatant using a Quantikine ET-1 ELISA. Results were analyzed with paired student's t-test. Under normoxia fSMCs secreted more ET-1(840.9+234ng/mg/mL) compared to mSMCs (265.2+65ng/mg/mL;p=.03). Hypoxia increased ET-1 secretion from mSMCs (903.1+186ng/mg/mL) to levels comparable with normoxic fSMCs (p=.854) and levels significantly higher than mSMCs cultured at normoxia (p<.05). Blockade of the ETA receptor for 24hrs significantly decreased hypoxia induced ET-1 secretion from mSMCs (148.3+18) compared to untreated hypoxic fSMCs (p=.05). Hypoxia significantly increased mSMC proliferation (p<.05) compared to normoxic mSMCs. Blockade of the ETA receptor decreased hypoxia induced increase in mSMC proliferation (p=.05). These results indicate that hypoxia may increase mSMC proliferation through activation of the ETA receptor.

BKca Channel Alpha Subunit Variants are Differentially Modulated by the Beta-1 Subunit

Uterine myometrial cells are maintained in a quiescent state during pregnancy, in part due to hyperpolarization of the membrane by the large-conductance Ca2+-activated K+ (BKCa) channel. During labor, uterine contractility is increased and BKCa current is suppressed, however, expression of BKCa is increased, suggesting that multiple mechanisms regulate BKCa channel function. We have focused on the roles of an auxiliary β1-subunit and two N-terminal α-subunit BKCa variants, whose peptide sequences start at different methionines, MANG and MDAL. We found that whereas MDAL expression was equivalent in all samples, MANG was expressed at a higher level in myometrium from term laboring women than from term non-laboring or non-pregnant women. To examine the possibility that differential expression of these variants reduces BKCa channel activity at the onset of labor, we expressed the MANG and MDAL variants, with or without the β1-subunit, in a heterologous system and recorded currents using patch-clamp. Although whole-cell voltage-gated currents were similar between variants, the β1-subunit enhanced Ca2+ activation of MDAL but not MANG. Inside-out single-channel recordings confirmed that MANG currents were not modulated by β1. Our finding that β1 co-immunoprecipitated with both MDAL and MANG variants suggests that the longer N-terminus of MANG does not affect the interaction between BKCa α- and β1-subunits, but instead inhibits the functional modulation of the channel by β1. These observations suggest that BKCa variants are differentially regulated by auxiliary subunits to regulate myometrial contractility during pregnancy and labor.

Proliferative Action of the Androgen Receptor in Human Uterine Myometrial Cells—A Key Regulator for Myometrium Phenotype Programming

During pregnancy, the myometrium undergoes a phenotype programming starting from an early proliferative stage, to an intermediate synthetic stage, to a late contractile stage, after which the cells commit to labor. Steroid receptors play important roles in regulating myometrial cell phenotype during pregnancy, although detailed mechanisms are not fully defined. The aim of the study was to investigate the expression and function of the androgen receptor (AR) in myometrial cells during pregnancy. Human primary myometrial cells, immortalized myometrial cells, rat pregnant and tubal ligation models were used. Immunohistochemistry, Western blot and real-time PCR, cell proliferation, and flow cytometry assays were applied. The AR is highly expressed in the proliferative stage of pregnancy, starts to decrease in the synthetic stage, and reaches the lowest levels in the contractile stage. Both the mechanical stretch by the growing fetus and the decreased ratio of progestin:estrogen are responsible for AR protein reduction. AR regulates myometrial cell proliferation ligand-independently. Decreased AR expression delays the G(1)-S phase transition of human myometrial cell cycling and reduces expression of several cyclins. These AR actions are mediated through reducing IGF-I receptor protein stability, thus weakening PI3K/Akt signal cascade downstream of IGF-I. AR is required for IGF-I receptor protein stability by preventing the IGF-I receptor from ubiquitylation and protein degradation through both proteosomal and lysosomal pathways. AR is a key regulator for myometrial cell proliferation, suggesting its critical role in myometrium phenotype programming during pregnancy.

Uterine-Specific Knockout of TSC2: A Mouse Model for Leiomyoma and LAM.

Lymphangioleiomyomatosis (LAM) is a rare disease characterized by proliferation of abnormal smooth muscle (SM) cells in the lungs. With time, LAM cells proliferate and lead to progressive loss of lung function. No effective treatment for this life-threatening disease is available, and patients often require lung transplantation. LAM has two unique characteristics: 1) LAM demonstrates strong gender specificity, affecting almost exclusively reproductive-age women; and 2) LAM cells contain mutations in genes (TSC1 or TSC2) that encode the tuberous sclerosis complex, a signaling module that limits mTOR activity and thereby cell growth. The origin of LAM cells is not known; however, data support a metastatic model of LAM pathogenesis. Interestingly, LAM shares many features with uterine leiomyomas, the leading reason for hysterectomy among women. Like LAM, leiomyomas are characterized by abnormal SM cell proliferation and are most prevalent during reproductive years. Furthermore, both LAM and leiomyomas express steroid hormone receptors and respond to estrogen and possibly progesterone. We propose that mutations in TSC2, in addition to estrogen and/or progesterone stimuli, trigger myometrial cells to proliferate and metastasize to the lungs similar to LAM cells. To test our hypothesis, we generated a mouse strain with a uterine-specific loss of TSC2. We show that TSC2 expression is low and mTOR signaling elevated in the uteri of post-pubertal uterine-specific TSC2 knockout (KO) mice. Notably, uteri from TSC2 KO mice develop leiomyomas at 5-6 months of age, and these tumors are prevented by treatment with the mTOR inhibitor rapamycin. Furthermore, these myometrial tumors express high levels of estrogen- and progesterone receptors, and uterine growth can be prevented by eliminating ovarian steroid production through ovariectomy. Importantly, multiple lung lesions were seen in a 34-week old TSC2 KO female. These lung tumors express progesterone receptors and are negative for the lung marker, Nkx2.1, indicating that they might be LAM cells. Together, our studies suggest that LAM may indeed originate from uterine myometrial cells. We hope that our model will improve our understanding of both LAM and leiomyomas, and may lead to the development of novel innovative therapeutic strategies for both tumors.

Uterine contractility of plants used to facilitate childbirth in Nigerian ethnomedicine

Ethnopharmacological relevancePregnant women in Nigeria use plant preparations to facilitate childbirth and to reduce associated pain. The rationale for this is not known and requires pharmacological validation. Aim of studyObtain primary information regarding the traditional use of plants and analyze their uterine contractility at cellular level. Materials and methodsSemi-structured, open interviews using questionnaires of traditional healthcare professionals and other informants triggered the collection and identification of medicinal plant species. The relative traditional importance of each medicinal plant was determined by its use-mention index. Extracts of these plants were analyzed for their uterotonic properties on an in vitro human uterine cell collagen model. ResultThe plants Calotropis procera, Commelina africana, Duranta repens, Hyptis suaveolens, Ocimum gratissimum, Saba comorensis, Sclerocarya birrea, Sida corymbosa and Vernonia amygdalina were documented and characterized. Aqueous extracts from these nine plants induced significant sustained increases in human myometrial smooth muscle cell contractility, with varying efficiencies, depending upon time and dose of exposure. ConclusionThe folkloric use of several plant species during childbirth in Nigeria has been validated. Seven plants were for the first time characterized to have contractile properties on uterine myometrial cells. The results serve as ideal starting points in the search for safe, longer lasting, effective and tolerable uterotonic drug leads.

Hypoxia: The driving force of uterine myometrial stem cells differentiation into leiomyoma cells

We propose that stem cells that display specific phenotypic and functional characteristics exist in human uterine myometrium. Under hypoxic circumstances, estrogen signaling pathways in certain myometrial stem cells are aberrantly activated, which leads to continuous differentiation of this portion of cells into leiomyoma cells. This process also protects the cells from undergoing physiological apoptotic or dedifferentiation. Therefore, we summarized some evidences for our bases and hypothesize that hypoxia may be the key contributor to the pathogenesis of leiomyoma through aberrant estrogen pathway activation of myometrial stem cells.

Cyclic mechanical stretch augments neutrophil chemotactic chemokine and MMP production in cultured human uterine myometrial and decidual cells

Cyclic mechanical stretch augments neutrophil chemotactic chemokine and MMP production in cultured human uterine myometrial and decidual cells

Abstract 676: Induction of apoptosis via control of cell adhesion by peroxisome proliferator activated receptor-gamma agonist in human uterine leiomyoma cells

Abstract Objective : Peroxisome Proliferator-Activated Receptor (PPAR)-gamma ligands constitute important insulin sensitizers that have already been approved for the treatment of human metabolic disorders. It has been previously demonstrated that growth inhibition in human uterine leiomyoma cells mediated by caspase-dependent pathway and death receptor. The present study was designed to further understand the mechanism after uterine leiomyoma and normal myometrial cells were exposed to PPAR gamma agonist, Ciglitizone. Methods : In order to confirm the unknown cellular and biochemical responses to ciglitizone treatment with three uterine leiomyoma and normal myometrial cells, DNA chip analysis were performed. Highly up-regulated genes were confirmed to ascertain effect of PPAR gamma agonist by RT-PCR and Western-blot analysis. To analyze mechanism of invasion and metastasis -associating gene regulation by PPAR-γ ligands, wound healing assay and soft agar assay were performed. Results : DNA chip analysis revealed genes are related with cell adhesion and metastasis genes including plasminogen activating urokinase (PLAU), claudin 1, matrix metallopeptidase 1,2, and 9. Highly down-regulated genes had defense system against cell metastasis in common and was related with adhesion. Treatment with ciglitizone made decrease expression level of above cell adhesion related genes in dose dependent manner by RT-PCR and Western-blot analysis. Furthermore, treatment with ciglitizone in uterine leiomyoma cells decrease the invasion ability of leiomyoma cells and the formation of anchorage-independent colonies in soft agar. Conclusions : These results suggest that PPAR-gamma could be considered as a therapeutic target for diverse disease states in which excessive angiogenesis is implicated, including cancer and uterine leiomyoma. This phenomenon has not been previously reported for PPAR-gamma agonist in uterine leiomyoma. Defining the role of PPARγ in uterine leiomyoma cells will be paramount for a rational design of combinatorial therapy schemes that takes advantage of the potent and well-tolerated PPARγ agonists. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 676.

Xenoestrogen-Induced Regulation of EZH2 and Histone Methylation via Estrogen Receptor Signaling to PI3K/AKT

Although rapid, membrane-activated estrogen receptor (ER) signaling is no longer controversial, the biological function of this nongenomic signaling is not fully characterized. We found that rapid signaling from membrane-associated ER regulates the histone methyltransferase enhancer of Zeste homolog 2 (EZH2). In response to both 17beta-estradiol (E2) and the xenoestrogen diethylstilbestrol, ER signaling via phosphatidylinositol 3-kinase/protein kinase B phosphorylates EZH2 at S21, reducing levels of trimethylation of lysine 27 on histone H3 in hormone-responsive cells. During windows of uterine development that are susceptible to developmental reprogramming, activation of this ER signaling pathway by diethylstilbestrol resulted in phosphorylation of EZH2 and reduced levels of trimethylation of lysine 27 on histone H3 in chromatin of the developing uterus. Furthermore, activation of nongenomic signaling reprogrammed the expression profile of estrogen-responsive genes in uterine myometrial cells, suggesting this as a potential mechanism for developmental reprogramming caused by early-life exposure to xenoestrogens. These data demonstrate that rapid ER signaling provides a direct linkage between xenoestrogen-induced nuclear hormone receptor signaling and modulation of the epigenetic machinery during tissue development.

Effects of chlorinated biphenyls and metabolites on human uterine myocyte proliferation

Uterine myometrial cells are responsive to sex steroids, which could make them susceptible to actions of endocrine disrupting environmental contaminants such as some PCBs. The aim of this investigation was to identify possible effects of some chlorinated biphenyls (CBs) and their metabolites on myometrial cell proliferation. Myometrial cells obtained from women in both phases of the menstrual cycle and from pregnant women were grown in vitro and exposed to CB 101, CB 118, 3' -MeSO2-CB 101, 4'-MeSO2-CB 101, 4-OH-CB 107, 17 beta-estradiol, progesterone, ethinylestradiol or levonorgestrel. The proliferative activity was studied by a BrdU assay. Myometrial cell cultures originating from pregnant women exhibited decreased proliferation in response to 3'-MeSO2-CB 101, 4'-MeSO2-CB 101 and 4-OH-CB 107. Estradiol, a combination of 1 nM 17beta-estradiol and 10 nM progesterone, ethinylestradiol and levonorgestrel also reduced the proliferation of the myometrial cells, regardless of whether the cells were collected from either of the menstrual cycle phases or from pregnant women. To our knowledge this study is the first to demonstrate that some CBs affect the proliferative activity of human uterine myocytes.

The Deoxyribonucleic Acid Repair Protein Flap Endonuclease-1 Modulates Estrogen-Responsive Gene Expression

The ligand-occupied estrogen receptor alpha (ERalpha) initiates changes in gene expression through its interaction with target DNA. The capacity of ERalpha to modulate gene expression is influenced by the association of the receptor with a variety of coregulatory proteins. To further understand the role of these coregulatory proteins in ERalpha-mediated transcription, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus estrogen response element. One of the proteins identified in this complex, flap endonuclease-1 (FEN-1), is required for DNA replication and repair. We show that FEN-1 interacts directly with ERalpha and enhances the interaction of ERalpha with estrogen response element-containing DNA. More importantly, chromatin immunoprecipitation and RNA interference assays demonstrate that endogenously expressed FEN-1 associates with the native pS2 gene in MCF-7 cells and influences estrogen-responsive gene expression. Interestingly, estrogen differentially regulates expression of FEN-1 in mouse uterine epithelial, stromal, and myometrial cells. Together, our studies help to elucidate the functional consequence of the ERalpha-FEN-1 interaction and increase our understanding of the elaborate regulatory mechanisms that drive estrogen-responsive gene expression and DNA repair.

The effect of relaxin on the oxytocin receptor in human uterine smooth muscle cells

Experimental objectives: Activation of the oxytocin receptor (OTR) induces phospholipase C induced PIP 2 turnover in the human uterus. Relaxin (RLX), a polypeptide hormone produced in the corpus luteum of pregnancy as well as in the placenta and decidua inhibits PIP 2 turnover and subsequent signaling in human myometrium. The purpose of this study was to evaluate a possible effect of RLX on OTR regulation in human uterine smooth muscle cells. Primary cultures of myometrium from term pregnant women undergoing elective caesarean section were incubated for different time periods (0–96 h) and with different concentrations of RLX [10 pg/ml–20 μg/ml]. The effects on OTR binding, mRNA and protein expression were evaluated by means of 125I‐OVT binding assay, RT‐PCR and flow cytometry. Results: Prolonged RLX incubation was able to inhibit 30–40% of OTR binding while binding affinity remained unchanged. Oxytocin receptor mRNA and protein expression were down regulated by RLX about 50% and 35% respectively. Conclusion: We report for the first time an effect of RLX on OTR regulation in human uterine myometrial cells. The above results indicate that high local uterine RLX concentrations may be involved in uterine quiescence during human pregnancy by down regulating the OTR.

Myometrial cells induce angiogenesis and salvage damaged myocardium

Characteristically, uterine myometrial cells (MCs) are proliferative, inducing angiogenesis within the female reproductive organ. We evaluated whether MCs implanted into myocardium could also induce angiogenesis and restore heart function after injury. MCs were isolated from the adult rat uterus and cultured for three studies: 1) Intracellular VEGF levels were measured in MCs cultured with progesterone (10(-11), 10(-9), and 10(-7) M) (n = 6 tests per group). 2) Blood vessel density was evaluated 8 days after MCs (3 x 10(6) or 6 x 10(6)), smooth muscle cells (SMCs), or endothelial cells (n = 6 rats per group) were injected with matrigel into the subcutaneous tissue of adult rats. 3) MCs, SMCs (5 x 10(6)/rat), or media were injected into a transmural scar 3 wk after cryoinjury in rat hearts (n = 12 rats per group), and heart function, blood vessel density, and myocardial scar size and thickness were evaluated 5 wk later. In study 1, cultured MCs expressed VEGF, with levels significantly (P < 0.05) upregulated by progesterone at an optimal dose of 10(-11) M. In study 2, MCs injected into the subcutaneous tissue with matrigel induced significantly more blood vessels, especially large-diameter vessels, than did SMCs or endothelial cells (P < 0.01 for all groups). This angiogenic effect was greatest (P < 0.01) at higher doses of MCs and was enhanced by progesterone (10(-11) M). In study 3, MCs implanted into the injured myocardium increased blood vessel density at the implant area, reduced scar size, and improved cardiac function relative to SMCs and media. Overall, MCs induced angiogenesis in vitro and in vivo, prevented cardiac remodeling, and improved heart functional recovery after cardiac injury.

Progesterone upregulates calcitonin gene-related peptide and adrenomedullin receptor components and cyclic adenosine 3'5'-monophosphate generation in Eker rat uterine smooth muscle cell line.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), two potent smooth-muscle relaxants, have been shown to cause uterine relaxation. Both CGRP- and AM-binding sites in the uterus increase during pregnancy and decrease at labor and postpartum. These changes in binding sites appear to be related to the changes in calcitonin receptor-like receptor (CRLR), receptor activity-modified protein 1 (RAMP1), RAMP2, and RAMP3 mRNA levels. It is not clear, however, whether the changes in the receptor components occur in the myometrial cells and whether the steroid hormones can directly alter these receptor components in the muscle cells. In addition, the mechanism of CGRP and AM signaling in the rat myometrium is not well understood. Therefore, we examined the mRNA expression of CGRP- and AM-receptor components, G protein Galphas, CGRP, and AM stimulation of cAMP and cGMP, and the effects of progesterone on these parameters in the Eker rat uterine myometrial smooth-muscle cell line (ELT3). ELT3 cells expressed CGRP- and AM-receptor components CRLR, RAMP1, RAMP2, and RAMP3. Expression of CRLR and RAMP1 mRNA increased with progesterone treatment and decreased with estradiol-17beta treatment. However, RAMP2 and RAMP3 mRNA expressions were unaltered by both progesterone and estradiol. Progesterone increased (P<0.05) Galphas expression and augmented CGRP- and AM-induced increases in cAMP levels. In uterine smooth-muscle cells, the antagonist to Galphas protein NF449 decreased basal as well as CGRP- and AM-stimulated cAMP levels. None of the cell treatments affected cyclic GMP production. Our results suggest that the progesterone-stimulated increases in CGRP and AM receptors, Galphas protein levels, and cAMP generation in the myometrial cells may be responsible for increased uterine relaxation sensitivity to CGRP and AM during pregnancy.

Synergistic effects of androgen and estrogen on the mouse uterus and mammary gland

Many studies have suggested that elevated estrogens and androgens may be etiologically related to the development of breast cancer, endometrial cancer and uterine leiomyomas. We and other investigators have previously shown that estrogen and androgen are synergistic in the induction of mammary carcinogenesis in the Noble rat. However, the mechanisms behind the synergy is unknown, and it is unclear whether such synergy is unique for the Noble rat and for the mammary gland. In this study we treated female FVB mice with 17beta-estradiol (E2) and 5alpha-dihydrotestosterone-bezonate (DHT-B), alone and in combination, using silastic tubing for 2-7 months. The results showed that DHT-B alone induced proliferation of uterine endometrial epithelium and myometrial smooth muscle cells, whereas E2 alone induced much more pronounced growth of endometrial epithelium without affecting smooth muscle cells. Combined treatment with E2+DHT-B caused an even more severe hyperplasia of endometrial epithelium and myometrial muscle cells, compared with the treatment with each hormone alone. Uterine leiomyomas were observed in 2 of 6 mice at 7 months of combined treatment but not in any of 6 or 7 mice receiving each single hormone. DHT-B alone induced growth and secretion of mammary ductal cells, as well as growth of mammary stroma. E2 alone stimulated much more pronounced growth of both ductal cells and alveolar cells and secretion of alveolar cells, but had no effect on mammary stroma. Treatment with both E2 and DHT-B caused more severe hyperplasia of mammary ducts and alveoli, compared to the treatment with each hormone alone. Intraductal hyperplasia occurred early and frequently in the E2+DHT-B- treated mice, but no mammary tumors were observed. These results suggest that E2 and DHT-B have synergistic effects on the growth of uterine endometrial epithelium and myometrial muscle cells, as well as mammary epithelial ducts and alveoli.

Scutellaria barbata D. Don induces c-fos gene expression in human uterine leiomyomal cells by activating beta2-adrenergic receptors

Scutellaria barbata D. Don (Lamiaceae; SB) inhibited the growth of uterine leiomyomal (LM) cells with unknown actions. The expression patterns of beta-adrenergic receptors (beta-ARs) in human uterine LM cells and functional coupling to gene expression have also been investigated. Northern blot analysis showed that beta-AR subtypes are expressed at different levels in the uterine LM cells and myometrial smooth muscle cells (SMCs). beta1-AR expression was to be found approximately at the same level in the two cell types. beta2-ARs were expressed at higher levels in uterine LM cells than that in myometrial SMCs. beta3-AR expression was not found in both the cells. c-fos gene expression was induced by SB in uterine LM cells via increases in adenosine-3',5', cyclic monophosphate (cAMP), which in turn activated the cAMP/protein kinase A (PKA) pathway. The PKA inhibitor, H89, inhibited c-fos gene expression induced by SB. It seems that the mechanism of proto-oncogenes c-fos different leiomyoma from other myometrial cancer. Further studies are necessary to elucidate whether c-fos induction by SB in uterine LM cells influences a regression of leiomyoma or induces other differentiation.

Differential Inhibition of Scutellaria barbata D. Don (Lamiaceae) on HCG‐Promoted Proliferation of Cultured Uterine Leiomyomal and Myometrial Smooth Muscle Cells

Scutellaria barbata D. Don (Lamiaceae)(SB) is a perennial herb which is natively distributed throughout Korea and southern China. This herb is known in traditional Chinese Medicine as Ban‐Zhi‐Lian and traditional Korean medicine as Banjiryun, respectively. SB has been used as an anti‐inflammatory and antitumor agent. We aimed to determine the expressin of cell cycle‐related signal molecules for growth inhibition after HCG treatment by the herb SB in two different human myometrial smooth muscle cells (SMCs) and leiomyomal SMCs. Water‐soluble ingredients of SB, myometrial SMCs and the leiomyomal cell lines were used in vitro. Uterine myomas often enlarge rapidly during pregnancy, implying that human chorionic gonadotrophin (HCG) may influences cell proliferation in uterine leiomyomata. We investigated the effects of SB on the cell proliferation and the expression of cell cycle‐related proteins in these cells. Although HCG/LH receptor was present in both cultured myometrial and leiomyomal cells, as assayed by reverse transcription polymerase chain reaction analysis, treatment with HCG significantly increased cell proliferation in both myometrial and leiomyomal cells. However, SB reduced the proliferative effect of HCG in leiomyoma and myometrial cells, respectively. In HCG‐treated leiomyomal cells, the expression of proliferating cell nuclear antigen, cyclin E and cdc2 was significantly reduced by SB treatment. These results suggest that SB reduced the HCG‐promoted proliferation of myometrial and leiomyomal cells.

Cyclic mechanical stretching and interleukin-1α synergistically up-regulate prostacyclin secretion in cultured human uterine myometrial cells

Prostacyclin (PGI2), a potent uterine smooth muscle relaxant, is postulated to be a major prostaglandin (PG) secreted from the human myometrium. PGI2metabolite concentrations in the maternal plasma were reported to be elevated during pregnancy, especially during labor. Recently, we developed cultured human myometrial cells from pregnant women and reported that cyclic mechanical stretching mimicking labor increased PGI2secretion from these cells by up-regulating PGI2synthase promoter activities. Since elevation of cervical/vaginal interleukin-1α (IL-1α) concentrations is also a characteristic feature of delivery, and IL-1α is a known stimulator of PGsynthesis, we investigated a possible synergistic effect of cyclic mechanical stretching and IL-1α on PGI2production in cultured human myometrial cells. Treatment with IL-1α (10 ng/ml) significantly augmented (4- to 60-fold) the secretion of PGI2, prostaglandin E2(PGE2), prostaglandin F2α(PGF2α) and thromboxane A2(TXA2) from cultured human myometrial cells obtained from non-pregnant and pregnant women as well as in cultured human umbilical artery and cultured human coronary artery smooth muscle cells (p<0.05 for all comparisons). However, labor-like cyclic mechanical stretching up-regulated IL-1α-augmented PGI2secretion from myometrial cells obtained from nonpregnant and pregnant women 2.1- to 2.8-fold (p<0.05 for all comparisons), but not PGE2, PG F2αnor TXA2. Moreover, such an augumentation of PGI2secretion by cyclic mechanical stretching was not observed in cultured human umbilical artery nor in cultured human coronary artery smooth muscle cells. These results suggest that cyclic mechanical stretching by labor, in concert with IL-1α stimulation, contributes to the increase in myometrial PGI2secretion during delivery.

Cyclic mechanical stretch augments prostacyclin production in cultured human uterine myometrial cells from pregnant women: possible involvement of up-regulation of prostacyclin synthase expression.

Prostacyclin (PGI(2)), a potent smooth muscle relaxant, is a major prostaglandin secreted from human myometrium. The concentrations of PGI(2) metabolites in the maternal plasma were reported to be elevated during pregnancy, especially in labor. To clarify the mechanism in PGI(2) secretion from the myometrium, we first investigated the protein expression of cytosolic phospholipase A(2), cyclooxygenase (COX)-1, COX-2, and prostacyclin synthase (PGIS) in the human uterine myometrium at various gestational ages before labor. To elucidate the involvement of labor in the increase in PGI(2) production during labor, we next examined the effect of labor-like cyclic mechanical stretch on PGI(2) production by cultured human myometrial cells. Pregnancy specifically increased COX-1 and PGIS protein expression in the myometrial tissues before labor (P < 0.01 for both). Cyclic mechanical stretch augmented PGIS promoter activity, via activation of activator protein-1 site, and PGIS mRNA and protein expression in cultured human myometrial cells and resulted in a 3.5-fold increase in the concentration of 6-keto-prostaglandin F(1alpha), the stable metabolite of PGI(2), in the culture medium (P < 0.05). However, stretch did not affect the levels of prostaglandin E(2), prostaglandin F(2alpha), or thromboxane A(2) secreted into the same culture media. These results suggest that cyclic mechanical stretch during labor may contribute to the increase in the PGI(2) concentration in the maternal plasma during parturition.

Platelet-activating factor-induced intracellular calcium waves in human uterine myometrial cells

Objective: We visualized and investigated the intracellular calcium waves propagated by platelet-activating factor (PAF) in cultured human myometrial cells. Study design: Myometrial cells were stimulated with PAF ranging between 10 −8 and 10 −15 M. For the observation of calcium waves, calcium green-1 and a confocal laser microscopy were used. Cells were also stimulated with 10 −9 M of PAF in a calcium-free solution. Results: In physiological solution, PAF at concentrations ranging between 10 −9 and 10 −15 M induced intracellular calcium waves. Mean wave speed was 16.1±5.6 μm/s. Wave speeds were independent of the PAF concentration. Similar results were observed in the absence of added calcium, with the exception that the wave speeds were significantly slower (7.3±3.3 μm/s). Conclusion: This is the first study to demonstrate the calcium waves propagated by PAF stimulation in human myometrial cells. These observations further support the proposed role of PAF in parturition.