Myometrial Cell Line Research Articles

CD38/cADPR-mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1.

Cyclic ADP-ribose (cADPR) has emerged as a calcium-regulating second messenger in smooth muscle cells. CD38 protein possesses ADP-ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP-ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET-1 elicited [Ca2+]i responses, and 8-Br-cADPR, a cADPR antagonist significantly attenuated agonist-induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.

Bryophyllum pinnatum Inhibits Oxytocin and Vasopressin Signaling in Myometrial Cells.

The medicinal plant Bryophyllum pinnatum was previously shown to block oxytocin (OT)-induced signals in myometrial cells, consistent with its tocolytic effect observed in patients. OT activates not only OT receptors but also V1A receptors, two receptors with high receptor homology that are both expressed in the myometrium and play a crucial role in myometrial contraction signaling. We aimed to study the molecular pharmacology of B. pinnatum herbal preparations using specific receptor ligands, the human myometrial cell line hTERT-C3, and cell lines expressing recombinant human OT and V1A receptors.We found that press juice from B. pinnatum (BPJ) inhibits both OT- and vasopressin (AVP)-induced intracellular calcium increases in hTERT-C3 myometrial cells. In additional assays performed with cells expressing recombinant receptors, BPJ also inhibited OT and V1A receptor-mediated signals with a similar potency (IC50 about 0.5 mg/mL). We further studied endogenous OT- and AVP-sensitive receptors in hTERT-C3 cells and found that OT and AVP stimulated those receptors with similar potency (EC50 of ~ 1 nM), suggesting expression of both receptor subtypes. This interpretation was corroborated by the antagonist potencies of atosiban and relcovaptan that we found. However, using qPCR, we almost exclusively found expression of OT receptors suggesting a pharmacological difference between recombinant OT receptors and native receptors expressed in hTERT-C3 cells.In conclusion, we show that B. pinnatum inhibits both OT and AVP signaling, which may point beyond its tocolytic effects to other indications involving a disbalance in the vasopressinergic system.

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells

Oxytocin acts through the oxytocin receptor (OXTR) to modulate uterine contractility. We previously identified OXTR genetic variants and showed that, in HEK293T cells, two of the OXTR protein variants localized to the cell surface less than wild-type OXTR. Here, we sought to measure OXTR in the more native human myometrial smooth muscle cell (HMSMC) line on both the cell-surface and across the whole cell, and used CRISPR editing to add an HA tag to the endogenous OXTR gene for anti-HA measurement. Quantitative flow cytometry revealed that these cells possessed 55,000 ± 3200 total OXTRs and 4900 ± 390 cell-surface OXTRs per cell. To identify any differential wild-type versus variant localization, we transiently transfected HMSMCs to exogenously express wild-type or variant OXTR with HA and green fluorescent protein tags. Total protein expression of wild-type OXTR and all tested variants were similar. However, the two variants with lower surface localization in HEK293T cells also presented lower surface localization in HMSMCs. Overall, we confirm the differential surface localization of variant OXTR in a more native cell type, and further demonstrate that the quantitative flow cytometry technique is adaptable to whole-cell measurements.

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26nM), AUC 7.30 ± 0.32 (IC50 50.43nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Estradiol Augments Tumor-Induced Neutrophil Production to Promote Tumor Cell Actions in Lymphangioleiomyomatosis Models.

Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease caused by smooth muscle cell-like tumors containing tuberous sclerosis (TSC) gene mutations and found almost exclusively in females. Patient studies suggest LAM progression is estrogen dependent, an observation supported by in vivo mouse models. However, in vitro data using TSC-null cell lines demonstrate modest estradiol (E2) responses, suggesting E2 effects in vivo may involve pathways independent of direct tumor stimulation. We previously reported tumor-dependent neutrophil expansion and promotion of TSC2-null tumor growth in an E2-sensitive LAM mouse model. We therefore hypothesized that E2 stimulates tumor growth in part by promoting neutrophil production. Here we report that E2-enhanced lung colonization of TSC2-null cells is indeed dependent on neutrophils. We demonstrate that E2 induces granulopoiesis via estrogen receptor α in male and female bone marrow cultures. With our novel TSC2-null mouse myometrial cell line, we show that factors released from these cells drive E2-sensitive neutrophil production. Last, we analyzed single-cell RNA sequencing data from LAM patients and demonstrate the presence of tumor-activated neutrophils. Our data suggest a powerful positive feedback loop whereby E2 and tumor factors induce neutrophil expansion, which in turn intensifies tumor growth and production of neutrophil-stimulating factors, resulting in continued TSC2-null tumor growth.

High levels of hypusinated eIF5A in leiomyoma and leiomyosarcoma pathologies: a possible novel therapeutic target

Research questionIs the hypusinated form of the eukaryotic translation initiation factor 5A (EIF5A) present in human myometrium, leiomyoma and leiomyosarcoma, and does it regulate cell proliferation and fibrosis? DesignThe hypusination status of eIF5A in myometrial and leiomyoma patient-matched tissues was evaluated by immunohistochemistry and Western blotting as well as in leiomyosarcoma tissues by immunohistochemistry. Myometrial, leiomyoma and leiomyosarcoma cell lines were treated with N1-guanyl-1,7-diaminoheptane (GC-7), responsible for the inhibition of the first step of eIF5A hypunization, and the proliferation rate was determined by MTT assay; fibronectin expression was analysed by Western blotting. Finally, expression of fibronectin in leiomyosarcoma tissues was detected by immunohistochemistry. ResultsThe hypusinated form of eIF5A was present in all tissues examined, with an increasing trend of hypusinated eIF5A levels from normal myometrium to neoplastic benign leiomyoma up to neoplastic malignant leiomyosarcoma. The higher levels in leiomyoma compared with myometrium were confirmed by Western blotting (P = 0.0046). The inhibition of eIF5A hypusination, with GC-7 treatment at 100 nM, reduced the cell proliferation in myometrium (P = 0.0429), leiomyoma (P = 0.0030) and leiomyosarcoma (P = 0.0044) cell lines and reduced the expression of fibronectin in leiomyoma (P = 0.0077) and leiomyosarcoma (P = 0.0280) cells. The immunohistochemical staining of leiomyosarcoma tissue revealed that fibronectin was highly expressed in the malignant aggressive (central) part of the leiomyosarcoma lesion, where hypusinated eIF5A was also highly represented. ConclusionsThese data support the hypothesis that eIF5A may be involved in the pathogenesis of myometrial benign and malignant pathologies.

CAMP Compartmentalisation in Human Myometrial Cells.

Preterm birth is the leading cause of childhood mortality and morbidity. A better understanding of the processes that drive the onset of human labour is essential to reduce the adverse perinatal outcomes associated with dysfunctional labour. Beta-mimetics, which activate the myometrial cyclic adenosine monophosphate (cAMP) system, successfully delay preterm labour, suggesting a key role for cAMP in the control of myometrial contractility; however, the mechanisms underpinning this regulation are incompletely understood. Here we used genetically encoded cAMP reporters to investigate cAMP signalling in human myometrial smooth muscle cells at the subcellular level. We found significant differences in the dynamics of the cAMP response in the cytosol and at the plasmalemma upon stimulation with catecholamines or prostaglandins, indicating compartment-specific handling of cAMP signals. Our analysis uncovered significant disparities in the amplitude, kinetics, and regulation of cAMP signals in primary myometrial cells obtained from pregnant donors compared with a myometrial cell line and found marked response variability between donors. We also found that in vitro passaging of primary myometrial cells had a profound impact on cAMP signalling. Our findings highlight the importance of cell model choice and culture conditions when studying cAMP signalling in myometrial cells and we provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium.

Phosphoserine-86-HSPB1 (pS86-HSPB1) is cytoplasmic and highly induced in rat myometrium at labour

Uterine myocytes during pregnancy proceed through a series of adaptations and collectively transform into a powerfully contractile tissue by term. Previous work has indicated that members of the heat shock protein (HSP) B family of stress proteins are associated with the process of adaptation and transformation. Utilizing immunoblot analyses, widefield epifluorescence and total internal reflection (TIRF) microscopy, this study investigated the temporal and spatial detection of HSPB1 phosphorylated on serine-86 (pS86-HSPB1) in rat myometrium during pregnancy, the role of uterine distension in regulation of pS86-HSPB1, and the comparative localization with pS15-HSPB1 in rat myometrial tissue as well as in an immortalized human myometrial cell line. Immunoblot detection of pS86-HSPB1 was significantly elevated during late pregnancy and labour. In particular, pS86-HSPB1 was significantly increased at day (d)22 and d23 (labour) compared with all other timepoints assessed. Localization of pS86-HSPB1 in myometrium became prominent at d22 and d23 with cytoplasmic detection around myometrial cell nuclei. Furthermore, pS86-HSPB1 detection was found to be significantly elevated in the gravid rat uterine myometrium compared with the non-gravid tissue at d19 and d23. Both widefield epifluorescence and TIRF microscopy examination of human myometrial cells demonstrated that pS15-HSPB1 was prominently localized to focal adhesions, while pS82-HSPB1 (homologous to rodent pS86-HSPB1) was primarily located in the cell cytoplasm. Our data demonstrate that levels of phosphorylated HSPB1 increase just prior to and during labour, and that uterine distension is a stress-inducing signal for HSPB1 phosphorylation. The exact roles of these phosphorylated forms in myometrial cells remain to be determined.

Dual effect of nifedipine on pregnant human myometrium contractility: Implication of TRPC1.

Nifedipine, an L‐type voltage‐gated Ca2+ channel (L‐VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions, but its efficacy is limited over time, and repeated or maintained nifedipine‐based tocolysis appears to be ineffective in preventing preterm birth. We aimed to understand why nifedipine has short‐lasting efficiency for the inhibition of uterine contractions. We used ex vivo term pregnant human myometrial strips treated with cumulative doses of nifedipine. We observed that nifedipine inhibited spontaneous myometrial contractions in tissues with high and regular spontaneous contractions. By contrast, nifedipine appeared to increase contractions in tissues with low and/or irregular spontaneous contractions. To investigate the molecular mechanisms activated by nifedipine in myometrial cells, we used the pregnant human myometrial cell line PHM1‐41 that does not express L‐VGCC. The in vitro measurement of intracellular Ca2+ showed that high doses of nifedipine induced an important intracellular Ca2+ entry in myometrial cells. The inhibition or downregulation of the genes encoding for store‐operated Ca2+ entry channels from the Orai and transient receptor potential‐canonical (TRPC) families in PHM1‐41 cells highlighted the implication of TRPC1 in nifedipine‐induced Ca2+ entry. In addition, the use of 2‐APB in combination with nifedipine on human myometrial strips tends to confirm that the pro‐contractile effect induced by nifedipine on myometrial tissues may involve the activation of TRPC channels.

The MAP kinase ERK5/MAPK7 is a downstream effector of oxytocin signaling in myometrial cells

The hormone oxytocin (OT) has pleiotropic activities both in the central nervous system as well as in peripheral tissues, including uterotonic effects on the myometrium during parturition. OT effects are mediated by a single transmembrane receptor, belonging to the GPCR (G protein-coupled receptor) superfamily and coupled primarily to Gq- and Gi-containing heterotrimeric G proteins. Upon receptor stimulation, one well-studied downstream effect is activation of the ERK1/2 MAP (mitogen-activated protein) kinase, and studies have shown that induction of COX-2 by OT in the myometrium required ERK1/2 activity. Many studies investigating the role of ERK1/2 in myometrial tissue were based on the use of chemical inhibitors that, to varying degrees, also inhibited ERK5/MAPK7. Here we report that OT activates ERK5 in a human myometrial cell line in a dose- and time-dependent manner through the activation of Gi/o heterotrimers. Using complementary approaches, we demonstrate that OT-induced COX-2 induction and the concomitant release of PGF2α into the media are primarily ERK5-dependent and to a much lesser extent ERK1/2-dependent. Moreover, in contrast to ERK1/2 activation, ERK5 activation is downstream of Gi/o activation. Here, we also found that ERK5 impacted both basal and to a lesser extent, OT-mediated myometrial cell contraction in vitro. Finally, tracking both ERK1/2 and ERK5 activity during different stages of gestation in rat myometrium, we showed that they followed distinct patterns starting at the onset of labor corresponding to the highest COX-2 expression levels. Overall, our results reveal an important, hitherto unrecognized role for ERK5 in myometrial cell contraction involving induction of COX-2. This novel pathway is likely to play an important role in supporting uterine contractions during parturition.

Prolactin is Expressed in Uterine Leiomyomas and Promotes Signaling and Fibrosis in Myometrial Cells.

Uterine leiomyomas are benign, estrogen-sensitive, fibrotic smooth muscle cell tumors occurring in the uterine myometrium. Leiomyomas are a considerable health burden, with a lifetime prevalence of 80% and limited treatment options. Estrogen and progesterone have positive effects on leiomyoma growth, but little is known about the roles of other hormones. One hormone of interest is prolactin, as it has been described to be present and functional in leiomyomas. The current study investigates prolactin production within leiomyomas and its effects on myometrial cells. RNA isolation and quantitative-PCR of human leiomyoma samples relative to matched adjacent myometrium confirms significant expression of prolactin and dopamine receptor D2, a known regulator of prolactin production and release in the pituitary, with no difference in prolactin receptor expression. Immunohistochemistry confirms increased prolactin in leiomyomas compared to adjacent myometrium and uteri from women without leiomyomas. These results suggest that leiomyomas contain cells that produce prolactin, which may then promote signaling in leiomyoma cells to regulate leiomyoma development/growth. Accordingly, we find that prolactin robustly activates STAT5 and MAPK signaling in rat and human myometrial cell lines. Furthermore, prolactin stimulates expression of myofibroblast markers in rat myometrial cells. Our findings suggest that local prolactin production in leiomyomas may stimulate trans-differentiation of myometrial cells to myofibroblasts, which in turn contributes to the fibrotic nature of leiomyomas.

IL-27 Mediates Pro-Inflammatory Effects via the ERK Signaling Pathway During Preterm Labor.

Preterm labor (PTL) is a multifactorial syndrome that results in birth prior to 37 weeks of gestation. However, the specific molecular mechanisms underlying this condition have yet to be elucidated. Previous research demonstrated that the abnormal expression of IL-27, and its receptors, played a role in the pathophysiology of preterm labor. In the present study, we established a Lipopolysaccharide (LPS)-stimulated, infection-induced, preterm mouse model based on wild-type C57BL/6 mice and WSX-1-/-C57BL/6 mice. WSX-1 knockdown led to a significant delay in birth by 11.32 ± 2.157h. In addition, compared with wild-type C57B/6 mice, the expression levels of IFN-γ, IL-1β, IL-6, TNF-α, and CXCL10, in the fetal membrane and myometrium of WSX-1-/-mice were significantly lower, particularly in the myometrium. We also confirmed similar pro-inflammatory effects arising from IL-27 in human amniotic cell line (WISH) and human myometrial smooth muscle cell line (HMSMC). Once stimulated by LPS, the pro-inflammatory action exhibited a synergistic effect and appeared to be time-dependent. Finally, we demonstrated that LY3214996, an inhibitor of the ERK pathway, significantly inhibited the pro-inflammatory effect mediated by IL-27. Overall, our data confirmed that the inflammatory effect mediated by the IL-27/IFN-r/ERK axis is involved in preterm labor. Our findings, therefore, provide an enhancement in our etiological understanding of the mechanisms underlying PTL.

Carcinoembryonic antigen-related cell adhesion molecule 1: a key regulatory protein involved in leiomyoma growth

To assess and characterize the role of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in the development of uterine leiomyoma. Laboratory study. Academic research center. Not applicable. Laboratory investigation. Invitro assessment of human leiomyoma and myometrial tissue specimens as well as immortalized leiomyoma and myometrial cell lines. Western blotting and immunohistochemical analyses were performed to assess differences in CEACAM1 content between leiomyoma and myometrial samples. Small interfering RNA silencing experiments and transient transfection experiments were performed to characterize the regulatory role of CEACAM1 on downstream signaling cascades. Analysis of RNA sequencing data revealed decreased CEACAM1 expression in human uterine leiomyoma specimens compared with that in myometrial samples. This translated to a significant down-regulation in CEACAM1 protein content in human leiomyoma compared with patient-matched myometrial tissue samples (0.236 ± 0.05-fold). A similar decrease in CEACAM1 protein content was observed in matched immortalized leiomyoma cell (ILC) and immortalized myometrial cell lines (0.21 ± 0.07). Immunohistochemical analysis revealed decreased staining intensity in leiomyoma surgical specimens compared with the matched myometrium of placebo patients. Lower CEACAM1 levels in leiomyoma were associated with increased activation of both the mitogen-activated protein kinase (MAPK) and the phosphoinositide 3-kinase/protein kinase B pathways compared with that in myometrial cells. This is significant because activation of these pathways plays an important role in leiomyoma growth. Treatment of myometrial cells with CEACAM1 small interfering RNA resulted in a significant down-regulation of CEACAM1 at the protein level (0.272 ± 0.06-fold) and was associated with increased activation of the MAPK (1.62 ± 0.21-fold) and phosphoinositide 3-kinase/protein kinase B (1.79 ± 0.35-fold) pathways, as well as increased collagen production (2.1 ± 0.49-fold). Rescue of CEACAM1 expression in leiomyoma cells by transient transfection restored regulatory control and resulted in lower activation of the MAPK pathway (0.58 ± 0.37-fold). CEACAM1 is an important protein involved in regulating many signal transduction pathways. Decreased CEACAM1 expression in leiomyoma allows permissive uncontrolled overactivation and up-regulation of downstream pathways that may contribute to leiomyoma growth.

Mechanical stiffness augments ligand-dependent progesterone receptor B activation via MEK 1/2 and Rho/ROCK–dependent signaling pathways in uterine fibroid cells

To test whether mechanical substrate stiffness would influence progesterone receptor B (PRB) signaling in fibroid cells. Uterine fibroids feature an excessive extracellular matrix, increased stiffness, and altered mechanical signaling. Fibroid growth is stimulated by progestins and opposed by anti-progestins, but a functional interaction between progesterone action and mechanical signaling has not been evaluated. Laboratory studies. Translational science laboratory. Human fibroid cell lines and patient-matched fibroid and myometrial cell lines. Progesterone receptor B-dependent reporter assays and messenger RNA quantitation in cells cultured on stiff polystyrene plates (3GPa) or soft silicone plates (930KPa). Pharmacologic inhibitors of extracellular signal-related protein kinase (ERK) kinase 1/2 (MEK 1/2; PD98059), p38 mitogen-activated protein kinase (SB202190), receptor tyrosine kinases (RTKs; nintedanib), RhoA (A13), and Rho-associated coiled-coil kinase (ROCK; Y27632). Progesterone-responsive reporter activation. Fibroid cells exhibited higher PRB-dependent reporter activity with progesterone (P4) in cells cultured on stiff vs. soft plates. Mechanically induced PRB activation with P4 was decreased 62% by PD98059, 78% by nintedanib, 38% by A13, and 50% by Y27632. Overexpression of the Rho-guanine nucleotide exchange factor (Rho-GEF), AKAP13, significantly increased PRB-dependent reporter activity. Collagen 1 messenger RNA levels were higher in fibroid cells grown on stiff vs. soft plates with P4. Cells cultured on mechanically stiff substrates had enhanced PRB activation via a mechanism that required MEK 1/2 and AKAP13/RhoA/ROCK signaling pathways. These studies provide a framework to explore the mechanisms by which mechanical stiffness affects progesterone receptor activation.

395 Dual effect of nifedipine on term pregnant human myometrium contractility

Nifedipine, L-type voltage-gated Ca2+ channels (L-VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions but its efficacy is limited overtime (48h). The aim of this study was to understand why nifedipine has short-lasting efficiency for inhibition of uterine contractions. Term pregnant human myometrial strips with different ex vivo contractile capacities [either regular (n=6) or irregular (n=5) spontaneous contractions] were mounted in an organ bath, and treated with cumulative doses of nifedipine. Tissue contractility in response to nifedipine was analyzed. Then, to investigate the molecular mechanisms activated by nifedipine in myometrial cells with irregular contractile capacity, we used the pregnant human myometrial cell line PHM1-41 that does express L-VGCC. Cells were transfected with siRNA for ORAI1, a non-voltage gated Ca2+ channel, and STIM1, the ORAI1 regulator. Both transfected and non-transfected cells were treated with different doses of nifedipine. Evolution of intracellular Ca2+ level was monitored with a Ca2+ indicator dye. Treatment of myometrial strips with cumulative doses of nifedipine inhibited spontaneous myometrial contraction in tissues with high contractility, even at low doses. On the contrary, nifedipine treatment appeared to increase contractions in tissues with low contractility (Fig1). In vitro, high doses of nifedipine (>10-6M) induced an important intracellular Ca2+ entry in myometrial cells (Fig2). Nifedipine-induced Ca2+ influx was not affected by knock-down of ORAI1 and STIM1 neither individually, nor together This study highlighted existence of a dual action of nifedipine. In addition to inhibit contraction in contracting myometrial tissue, we showed that nifedipine had an opposite pro-contractile effect in weak-contracting muscle. This effect could involve an activation of Ca2+ entry into myometrial cells, but this process does not seem to involve neither STIM1 nor ORAI1 Ca2+ channel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

575 Effect of visfatin on contractility related gene expression in a human myometrial cell line

575 Effect of visfatin on contractility related gene expression in a human myometrial cell line

HMGA2-mediated tumorigenesis through angiogenesis in leiomyoma

To study the role of HMGA2 in promoting angiogenesis in uterine leiomyoma (LM). This study involved evaluation of vessel density and angiogenic factors in leiomyomas with HMGA2 overexpression; examining angiogenic factor expression and AKT signaling in myometrial (MM) and leiomyoma cells by introducing HMGA2 overexpression invitro; and exploring vessel formation induced by HMGA2 overexpression both invitro and invivo. University research laboratory. None. None. The main outcome measures include vessel density in leiomyomas with HMGA2 (HMGA2-LM) or MED12 (MED12-LM) alteration; angiogenic factor expression in primary leiomyoma and invitro cell line model; and vessel formation in leiomyoma cells with HMGA2 overexpression invitro and invivo. Angiogenic factors and receptors were significantly upregulated at mRNA and protein levels in HMGA2-LM. Specifically, HMGA2-LM exhibited increased expression of VEGFA, EGF, bFGF, TGFα, VEGFR1, and VEGFR2 compared to MED12-LM and myometrium. Overexpression of HMGA2 in MM and LM cell lines resulted in increased secretion of angiogenesis-associated factors. Secreted factors promoted human umbilical vein endothelial cell (HUVEC) migration, tube formation, and wound healing. HMGA2 overexpression upregulated IGF2BP2 and pAKT, and silencing the IGF2BP2 gene reduced pAKT levels and reduced HUVEC migration. Myometrial cells with stable HMGA2 overexpression exhibited increased colony formation and cell growth invitro and formed xenografts with increased blood vessels. HMGA2-LM have a high vasculature density, which likely contributes to tumor growth and disease burden of this leiomyoma subtype. HMGA2 plays an important role in angiogenesis and the involvement of IGF2BP2-mediated pAKT activity in angiogenesis, which provides a potential novel target for therapy for this subtype of LM.

Cross-talk between Janus kinase-signal transducer and activator of transcription pathway and transforming growth factor beta pathways and increased collagen1A1 production in uterine leiomyoma cells

To characterize the potential interaction between interleukin-6 (IL6), Janus kinase (JAK)-signal transducer and activator of transcription (STAT)-3 (JAK/STAT3) pathway, and Transforming growth factor beta (TGFβ)-3 , and to determine whether such cross-talk was a contributing factor in the dysregulation of type I collagen production in leiomyomas. Laboratory study. University research laboratory. None. Exposure of leiomyoma and myometrial cell lines to IL6 and STAT3 activators/inhibitors. Western immunoblot analysis and immunohistochemistry. Expression of STAT3, pSTAT3, SOCS3, COL1A1, and TGFb3. We observed that IL6 increased pSTAT3 as well as collagen1A1 in uterine leiomyoma cells. Direct activation of the JAK/STAT3 pathway increased collagen1A1 production in leiomyoma cells, whereas inhibition of the pathway significantly decreased collagen1A1 production. We further observed that modulation of the JAK/STAT3 pathway also increased the expression of TGFβ3 protein. Leiomyoma cells exposed to TGFβ3 demonstrated a significant decrease in pSTAT3 protein. Myometrial cells demonstrated a less sensitive response to STAT3 modulation and collagen production. Cross-talk between the TGFβ pathway and JAK/STAT3 pathway contributes to the fibrotic nature of uterine leiomyomas.

MON-011 NALCN Expression Is Regulated by Progesterone and Estrogen in Human Myometrial Smooth Muscle Cells

During pregnancy, the uterus transitions from a quiescent state to a highly contractile, excitable state. Both ion channels and hormones are essential for this transition. We recently identified that the Na+ leak channel, non-selective (NALCN) contributes to a leak current in human MSMCs and mice lacking NALCN have prolonged and dysfunctional labor. Additionally, NALCN levels change throughout mouse pregnancy suggesting regulation by hormones of pregnancy, specifically estrogen and progesterone. Here, we tested the hypothesis that P4, a pro-quiescent hormone, and E2, a pro-contractile hormone, regulate NALCN expression and current in the myometrium. In a human immortalized myometrial cells (HM6ERMS2), using qPCR we measured a 2.3 fold decrease and a 5.6 fold increase in NALCN mRNA expression in the presence of E2 and P4, respectively. These findings were also confirmed when NALCN protein expression were measured by immunoblot. Conversely, treatment with the ER antagonist, ICI 182,780, significantly increased NALCN mRNA expression, while treatment with the PR antagonist RU486 significantly decreased NALCN mRNA expression suggesting E2 and P4 work through their respective receptors to regulate NALCN. P4 differentially regulates myometrial activity depending on which progesterone receptor is activated: PRA, promotes contractility, whereas PRB promotes quiescence. Thus to study the effect of each PR, we used a human myometrial cell line stably expressing PRA or PRB, and measured similar increases in NALCN mRNA expression in both cell lines treated with P4. To determine the functional consequences of E2 and P4, we measured NALCN-dependent leak current in MSMCs using whole cell patch clamping. We observed that E2 significantly inhibited while P4 significantly enhanced NALCN current. Finally, we identified estrogen response and progesterone response elements (ERE and PRE) in the NALCN promoter and showed that the PREs contributed to P4 regulation while the ERE did not contribute to the regulation of NALCN expression using luciferase based promoter assays. Overall, our findings show that NALCN is upregulated by P4, the pro-quiescent hormone, and downregulated by E2, the pro-contractile hormone. This data reveals a new mechanism by which NALCN is regulated in the myometrium and may suggest a novel role for NALCN during pregnancy. Further investigation into these novel roles can provide an insight into potential targets to modulate uterine quiescence and contractility.

Characterization of the role of Activator Protein 1 signaling pathway on extracellular matrix deposition in uterine leiomyoma

To characterize the role Activator Protein 1 (AP 1) family members play in mediating extracellular matrix deposition in uterine leiomyoma. Laboratory study. University research laboratory. Exposure of leiomyoma and myometrial cell lines to either an AP 1 inhibitor alone, AP 1 inhibitor plus transforming growth factor (TGF)ß3, or TGFß3 alone. Western immunoblot analysis was performed to assess for changes in AP 1 family member protein expression. In patient-matched myometrial and leiomyoma cell lines, the only AP 1 member found to be elevated significantly in leiomyoma compared with myometrium was FOSB (3.47 ± 0.12-fold), whereas others were decreased significantly: FRA1 (0.67 ± 0.02-fold), FRA2 (0.45 ± 0.01-fold), c FOS (0.37 ± 0.01-fold), Phos c FOS (0.19 ± 0.02-fold), Phos c JUN (0.75 ± 0.02-fold), JUNB (0.81 ± 0.04-fold), and JUND (0.65 ± 0.03-fold). c JUN (0.93 ± 0.03-fold) concentration was reduced but at nonsignificant levels. Following stimulation with TGF ß 3, fibronectin (2.16 ± 0.14-fold) and versican (4.71 ± 0.15-fold) protein concentrations were increased at 24 hours. Collagen 1A demonstrated a time-dependent significant increased concentration beginning at 6 hours (1.32 ± 0.01-fold) and increased to (6.49 ± 0.02-fold) at 24 hours. Following treatment with AP 1 inhibitor (SR11302), there were significant reductions in Collagen 1A concentration at 4 hours (0.59 ± 0.03-fold) and 6 hours (0.42 ± 0.05-fold). Activator Protein 1 inhibition did not reduce significantly versican concentration until 6 hours of treatment (0.84 ± 0.04-fold). SR11302 also decreased significantly fibronectin concentration (0.68 ± 0.05-fold) at 8 hours of treatment. Activator Protein 1 signaling is well described in fibrotic diseases, and, herein, we demonstrated that signaling via AP 1 family members promotes extracellular matrix deposition in leiomyoma.