Primary Myometrial Cells Research Articles

Engineered uterine primary myometrial cells with high-mobility group AT-hook 2 overexpression display a leiomyoma-like transcriptional and epigenomic phenotype

To determine if engineered HMGA2 overexpressing uterine primary myometrial cells recapitulate the transcriptional and epigenomic features of HMGA2-subtype leiomyomas. Isolated primary, "normal" myometrial cells from 3 patients were engineered to overexpress HMGA2 to determine how HMGA2 establishes transcriptomic and epigenomic features of HMGA2-overexpressing leiomyoma. Academic research laboratory PATIENTS: Primary myometrial cells were isolated from normal myometrium obtained from three patients undergoing hysterectomy. Not applicable MAIN OUTCOME MEASURES: Determined genome-wide transcriptomic and epigenomic features of engineered HMGA2-overexpressing uterine primary myometrial cells. Engineered HMGA2-V5-overexpressing primary myometrial cells approximated the HMGA2 expression level observed in HMGA2-overexpression subtype leiomyoma. HMGA2-V5 expression resulted in differential expression of 1612 genes (FDR < 0.05) which were found to be enriched in pathways associated with leiomyoma formation, including extracellular matrix organization. Comparative gene expression analysis between HMGA2-V5 engineered primary cells and HMGA2-overexpression subtype leiomyoma revealed significant overlap of differentially expressed genes. Mechanistically, HMGA2-V5 overexpression resulted in 41,323 regions with differential H3K27ac deposition (FDR < 0.05) and 205,605 regions of altered chromatin accessibility (FDR < 0.05). Transcription factor binding site analysis implicated the AP-1 family of transcription factors. • What clinical problem is addressed by these studies? About 10-15% of the uterine leiomyoma cases exhibit increased expression of HMGA2; our study addresses the primary role of HMGA2 overexpression in the pathogenesis of leiomyoma. • What are the key findings? Ectopic expression of HMGA2 in primary myometrial cells transforms transcriptional and epigenomic machinery in these cells which to some extent resemble features of leiomyoma. • How do these findings apply to human fertility or the reproductive process? Our study provides a comprehensive analysis into the changes occurring in epigenome of myometrial cells when HMGA2 is overexpressed. Because epigenome can be targeted with drugs, therefore understanding alterations in epigenome provides new avenues for treating leiomyoma which affects reproductive age women.

Prostaglandin F2α requires activation of calcium-dependent signalling to trigger inflammation in human myometrium.

Preterm birth is one of the major causes of neonatal morbidity and mortality across the world. Both term and preterm labour are preceded by inflammatory activation in uterine tissues. This includes increased leukocyte infiltration, and subsequent increase in chemokine and cytokine levels, activation of pro-inflammatory transcription factors as NF-κB and increased prostaglandin synthesis. Prostaglandin F2α (PGF2α) is one of the myometrial activators and stimulators. Here we investigated the role of PGF2α in pro-inflammatory signalling pathways in human myometrial cells isolated from term non-labouring uterine tissue. Primary myometrial cells were treated with G protein inhibitors, calcium chelators and/or PGF2α. Nuclear extracts were analysed by TranSignal cAMP/Calcium Protein/DNA Array. Whole cell protein lysates were analysed by Western blotting. mRNA levels of target genes were analysed by RT-PCR. The results show that PGF2α increases inflammation in myometrial cells through increased activation of NF-κB and MAP kinases and increased expression of COX-2. PGF2α was found to activate several calcium/cAMP-dependent transcription factors, such as CREB and C/EBP-β. mRNA levels of NF-κB-regulated cytokines and chemokines were also elevated with PGF2α stimulation. We have shown that the increase in PGF2α-mediated COX-2 expression in myometrial cells requires coupling of the FP receptor to both Gαq and Gαi proteins. Additionally, PGF2α-induced calcium response was also mediated through Gαq and Gαi coupling. In summary, our findings suggest that PGF2α-induced inflammation in myometrial cells involves activation of several transcription factors - NF-κB, MAP kinases, CREB and C/EBP-β. Our results indicate that the FP receptor signals via Gαq and Gαi coupling in myometrium. This work provides insight into PGF2α pro-inflammatory signalling in term myometrium prior to the onset of labour and suggests that PGF2α signalling pathways could be a potential target for management of preterm labour.

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.

Myometrial oxidative stress drives MED12 mutations in leiomyoma

BackgroundMore than 70% of leiomyomas (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131(GG). The cause of MED12 mutations in myometrial cells remains largely unknown. We hypothesized that increased ROS promotes MED12 mutations in myometrial cells through the oxidation of guanine nucleotides followed by misrepair.MethodsGenomic oxidative burden (8-OHdG) was evaluated in vitro and in vivo by immunohistochemistry. MED12 mutations were examined by Sanger sequencing and deep sequencing. Transcriptome examined by RNA-seq was performed in myometrium with and without LM, in primary myometrial cells treated with ROS. 8-OHdG mediated misrepair was analyzed by CRISPR/Cas9.ResultsUteri with high LM burden had a significantly higher rate of MED12 mutations than uteri with low LM burden. Compelling data suggest that the uterus normally produces reactive oxidative species (ROS) in response to stress, and ROS levels in LM are elevated due to metabolic defects. We demonstrated that genomic oxidized guanine (8-OHdG) was found at a significantly higher level in the myometrium of uteri that had multiple LM compared to myometrium without LM. Transcriptome and pathway analyses detected ROS stress in myometrium with LM. Targeted replacement of guanine with 8-OHdG at MED12 c.130 by CRISPR/Cas9 significantly increased the misrepair of G>T. Exposure of primary myometrial cells to oxidative stress in vitro increased misrepair/mutations as detected by duplex sequencing.ConclusionsTogether, our data identified a clear connection between increased myometrial oxidative stress and a high rate of MED12 mutations that may underlie the risk of LM development and severity in women of reproductive age.Graphical

Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin.

Tryptophan 2,3-Dioxygenase-2 in Uterine Leiomyoma: Dysregulation by MED12 Mutation Status

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.

Hop and artichoke extracts inhibit expression of extracellular matrix components in uterine leiomyoma cells

To screen 14 different plant extracts for their antifibrotic effect on human primary leiomyoma and healthy myometrial cells. Preclinical study. University research laboratory. Human uterine leiomyoma and matched myometrial tissues were obtained from Caucasian premenopausal women with symptomatic uterine fibroids at the time of hysterectomy. Primary human leiomyoma and myometrial cells were cultured in the absence or presence of the plant extracts. Quantification of the expression of extracellular matrix components, such as fibronectin 1 (FN1), collagen type I alpha 1 (COL1A1), and versican (VCAN), and the profibrotic growth factor activin A or inhibin beta A subunit (INHBA). The cells were treated with the 14 extracts for 48 hours, and we measured FN1 messenger RNA (mRNA) expression. Of the 14 extracts, about (ABO) ABO-2 (hop) and ABO-9 (artichoke) significantly reduced FN1 expression in both the cell types. Next, we evaluated the effect of fractions of these 2 extracts on the mRNA expression of FN1 and other extracellular matrix components, such as COL1A1, VCAN, and INHBA, in leiomyoma and myometrial cells. We found that ABO-2 (hop) and ABO-9 (artichoke) as well as their fractions, ABO-AR-2016-015 (fraction of ABO-2) and ABO-AR-2014-168 (fraction of ABO-9), reduced the mRNA expression of FN1, COL1A1, VCAN, and INHBA in primary leiomyoma cells. In primary myometrial cells, the mRNA expression of FN1, COL1A1, VCAN, and INHBA was not greatly affected. These results suggest that the hop and artichoke extracts possess antifibrotic properties and support additional evaluation using invivo models.

PKA and AKIP1 interact to mediate cAMP-driven COX-2 expression: A potentially pivotal interaction in preterm and term labour.

Previously, we showed that cAMP increased COX-2 expression in myometrial cells via MAPK. Here, we have extended these observations, using primary myometrial cell cultures to show that the cAMP agonist, forskolin, enhances IL-1β-driven COX-2 expression. We then explored the role of A-kinase interacting protein (AKIP1), which modulates the effect of PKA on p65 activation. AKIP1 knockdown reversed the effect of forskolin, such that its addition inhibited IL-1β-induced COX-2 mRNA expression and reduced the IL-1β-induced increase in nuclear levels of p65 and c-jun. Forskolin alone and with IL-1β increased IκBα mRNA expression suggesting that in the context of inflammation and in the presence of AKIP1, cAMP enhances p65 activation. AKIP1 knockdown reversed these changes. Interestingly, AKIP1 knockdown had minimal effect on the ability of forskolin to repress either basal OTR expression or IL-1β-stimulated OTR mRNA expression. AKIP1 was up-regulated by IL-1β, but not stretch and was repressed by cAMP. The mRNA expression of AKIP1 increased in early labour in tandem with an increase in COX-2 mRNA and protein. AKIP1 protein levels were also increased with inflammation and stretch-induced preterm labour. Our results identify a second important cAMP effector-switch occurring at term in human myometrium and suggest that a hitherto unrecognized interaction may exist between AKIP1, NFκB and AP-1. These data add to the proposition that cAMP acts as a key regulator of human myometrial contractility.

SAA1 regulates pro-labour mediators in term labour by activating YAP pathway.

Term labour is associated with activation of inflammation which results in myometrial contractility, cervical ripening and decidual/membrane rupture. Serum amyloid A1 (SAA1) is an acute response protein, whose role and underlying regulatory mechanisms in human labour remain unknown. In this study, we found that the mRNA and protein expression of SAA1 in human myometrium at term was increased in labouring tissues compared to non-labouring tissues. In addition, the expression of SAA1 was significantly increased in human primary myometrial cells treated with the pro-inflammatory cytokines interleukin-1 beta (IL-1β) or tumour necrosis factor-alpha (TNF-α). Knockdown of SAA1 using siRNA (siSAA1) resulted in a significant reduction in the expression and secretion of pro-inflammatory cytokines (IL8, IL6), chemokines (CXCL5, CCL2), adhesion molecules (ICAM1, ICAM5) and contraction-associated factors (COX2, PGE2). Mechanistically, the effects of SAA1 were mediated through activation of the Yes-associated protein (YAP) pathway. There was a decrease in the protein expression of phosphorylated YAP (pYAP) after treatment of siSAA1-transfected human primary myometrial cells with IL-1β or TNF-α. Moreover, enhanced expression of YAP reversed the effect of siSAA1 on pro-labour mediators. In conclusion, these experiments demonstrated that SAA1 accelerates the inflammatory response associated with parturition by activating YAP pathway, which may be a novel understanding of the molecular mechanism of labour onset.

Neuromedin B receptor mediates neuromedin B-induced COX-2 and IL-6 expression in human primary myometrial cells

The precise mechanisms that lead to parturition remain unclear. In our initial complementary DNA (cDNA) microarray experiment, we found that the neuromedin B receptor (NMBR) was differentially expressed in the...

Anti-inflammatory effects of gallic acid in human gestational tissues in vitro.

Spontaneous preterm birth is the leading cause of neonatal mortality and morbidity globally. Activation of the maternal immune system leads to a downstream cascade of proinflammatory events that culminate in the activation of spontaneous uterine contractions and the rupture of the foetal membranes. Anti-inflammatory agents may be a novel therapeutic approach to prevent inflammation-induced myometrial contractions and premature rupture of foetal membranes. The polyphenol gallic acid has been previously shown to exert potent anti-inflammatory effects. Thus, this study aimed to determine the effect of gallic acid on proinflammatory and pro-labour mediators in cytokine-stimulated gestational tissues in vitro. In primary human cells isolated from myometrium and foetal membranes (decidua, and amnion mesenchymal and epithelial cells), gallic acid treatment suppressed inflammation-induced expression of proinflammatory cytokines and chemokines and extracellular matrix-degrading and matrix-remodelling enzymes. Gallic acid also significantly inhibited inflammation-induced myometrial activation as evidenced by decreased expression of contraction-associated proteins, the uterotonic PGF2α and collagen cell contractility. Using a global proteomic approach, gallic acid may differentially regulate proteins associated with collagen synthesis, cell contractility and protein synthesis in primary myometrial and decidual cells. In summary, gallic acid inhibited inflammation-induced mediators involved in active labour in primary cells isolated from myometrium and foetal membranes. These in vitro studies suggest that the polyphenol gallic acid may be able to suppress the production of proinflammatory and pro-labour mediators involved in myometrial contractions and rupture of foetal membranes. Future preclinical studies may elucidate the efficacy of gallic acid in preventing inflammation-driven preterm birth.

Inhibition of GPR91 Reduces Inflammatory Mediators Involved in Active Labor in Myometrium.

Results GPR91 mRNA expression was significantly higher in myometrium from women during term spontaneous labor compared to no labor. Likewise, in mice, GPR91 mRNA expression was significantly upregulated in myometrium during inflammation-induced preterm labor compared to preterm no labor. In myometrial cells, IL1B and TNF significantly increased GPR91 mRNA expression. Knockdown of GPR91 by siRNA in myometrial cells significantly suppressed the secretion and/or expression of IL1B- and TNF-induced proinflammatory cytokines (GM-CSF, IL1A, IL1B, and IL6) and chemokines (CXCL8 and CCL2), myometrial contractility (expression of the contraction-associated proteins PTGFR and CX43, secretion of the uterotonic PGF2α, and in situ collagen gel contraction), and the transcription factor NF-κB. Conclusion Our findings demonstrate that GPR91 is involved in the genesis of proinflammatory and prolabor mediators induced by IL1B or TNF and collectively suggest that GPR91 may contribute to augmentation of the labor processes.

Macrophage-induced reactive oxygen species promote myometrial contraction and labor-associated mechanisms†.

At labor, the myometrium is infiltrated by a massive influx of macrophages that secrete high levels of pro-inflammatory cytokines inducing the expression of specific labor-associated markers. However, the interactions between myocytes and macrophages and the role of macrophages in the myometrium at labor remain to be elucidated. In this work, we studied the role of myometrium-infiltrated macrophages and their interaction with myocytes in lipopolysaccharide-induced preterm labor. A co-culture model of human primary myometrial cells and macrophages was developed and validated. Collagen lattices were used to evaluate myocyte contraction. Differentiation steps were assessed by (i) phalloidin and vinculin staining for cytoskeleton reorganization, (ii) gap junction protein alpha 1 expression and scrape loading/dye transfer with Lucifer Yellow for gap junction intercellular communication, and (iii) calcium imaging for cell excitability. We demonstrated that macrophages favored lipopolysaccharide-induced contraction and early differentiation of myometrial cells. Transwell assays showed that previous activation of macrophages by lipopolysaccharide was essential for this differentiation and that macrophage/myocyte interactions involved macrophage release of reactive oxygen species (ROS). The effects of macrophage-released ROS in myometrial cell transactivation were mimicked by H2O2, suggesting that superoxide anion is a major intermediate messenger in macrophage/myocyte crosstalk during labor. These novel findings provide the foundation for innovative approaches to managing preterm labor, specifically the use of antioxidants to inhibit the initial stages of labor before the contractile phenotype has been acquired. In addition, the co-culture model developed by our team could be used in future research to decipher pathophysiological signaling pathways or screen/develop new tocolytics.

Variable Responsiveness to Agonists for TLR2 and TLR7 in Myometrial Cells from Different Sources: Correlation with Receptor Expression.

The myometrium plays a vital role in maintenance of pregnancy. Disruption of myometrial sensitivity to pro-contractile stimuli might lead to preterm labor. Inflammation and/or infection are common precursors to preterm birth, in part by initiating pro-contractile stimuli through toll-like receptor (TLRs) activation. In this study, we investigated the responses specific to inflammatory stimuli for both human primary myometrial cells (HPMCs) and PHM1-41 cells, a human immortalized myometrial cell line. Both these types of cells are commonly used to study labor and pregnancy. Both cell lines were treated with lipopolysaccharide (LPS), peptidoglycan (PGN), or imiquimod (IQ) (ligands for TLRs 2, 4, and 7, respectively). We demonstrate that inflammatory cytokines increase significantly with LPS treatment; however, no change occurs with PGN and IQ, suggesting lack of TLR2- and TLR7-specific signaling in both HPMCs and in the PHM1-41 cell line. Absence of TLR2- and TLR7-specific protein bands on western blots confirmed the lack of these receptors in both HPMCs maintained in long-term culture and PHM1-41 cells. However, TLR2 expression was present in freshly collected matched human myometrial tissue (i.e., the tissues used to create the HPMC cultures), showing loss of TLR2 receptors by HPMCs during the cell culturing process. TLR7 protein expression was lacking both in myometrial tissue and in cultured cells. These results demonstrate the limited applicability and reliability of cellular models to investigate the role of the myometrium during pregnancy and labor.

Role of IRG1 in Regulating Pro-inflammatory and Pro-labor Mediators in Human Myometrium.

Preterm birth is a major contributor to neonatal deaths and associated long-term morbidities for the survivors, yet therapies remain elusive, given our incomplete understanding of the mechanisms driving human labor and delivery. Human labor is an inflammatory process, and we investigated whether IRG1 (immunoresponsive gene-1) plays a role in these processes. We demonstrate that IRG1 mRNA and protein expression is significantly increased in myometrium with human term labor, compared to no labor samples, and with preterm (LPS) labor in a mouse model. Pro-labor mediators such as pro-inflammatory cytokines TNF and IL1B, and TLR ligands fsl-1, flagellin, LPS, and poly(I:C) also increased IRG1 mRNA expression in myometrial explants. IRG1 silencing, using siRNA in primary myometrial cells, displayed a decrease in the expression of inflammation-induced pro-inflammatory cytokines (IL1A, IL6), chemokines (CCL2, CXCL1, CXCL8), adhesion molecules (ICAM1, VCAM1), and contractility (PTGFR mRNA expression, prostaglandin F2α release, and in situ gel contraction assay). Our results suggest that IRG1 is involved when pro-labor mediators activate the inflammatory processes of human labor, warranting further investigation.

The impact of progesterone and RU-486 on classic pro-labour proteins & contractility in human myometrial tissues during 24-hour exposure to tension & interleukin-1β

Increased expression of pro-labour genes that encode cyclooxygenase-2 (COX-2), oxytocin receptor (OTR) and connexin-43 (Cx43) at parturition is often attributed to P4 functional withdrawal, based on findings from animal models and human primary myometrial cells. However, the cause of reduced myometrial P4 responsiveness that promotes contractions at labour is not fully determined. Uterine stretch occurs with advancing gestation but most in vitro experimental models do not take this into consideration. We aimed to examine whether tissue-level myometrial stretch influences the ability of P4 to regulate pro-labour protein abundance by using myometrial biopsies from term gestation pregnant women to assess the impact of 24 h exposure to combinations of (i) stretch-mediated tension, (ii) P4 (100 nM) and (iii) an anti-progestin, RU-486 (1 μM). Firstly, we observed baseline COX-2 and Cx43 protein levels increased, whereas P4 content along with calponin-1 and progesterone receptor (PR) protein abundance decreased, in vehicle-treated tissues. P4 supplementation subtly reduced COX-2 levels in un-stretched tissues. Spontaneous and oxytocin-augmented contractility were unchanged by tissue culture exposure to P4 and/or RU-486. Interleukin-1β (IL-1β; 1 ng/ml) enhanced COX-2 protein and PGE2 content in un-stretched tissues. Overall, tissue stretch may, in part, regulate P4-sensitive pro-labour protein levels, but this is likely to be reliant on interaction with other in utero factors that were absent in our tissue cultures. More complex culture conditions should be evaluated in future to aid further development of a physiologically relevant model to improve our understanding of in utero myometrial P4 responsiveness.

Neuromedin B mediates IL-6 and COX-2 expression through NF-κB/P65 and AP-1/C-JUN activation in human primary myometrial cells.

Neuromedin B (NMB) and its receptor regulate labor onset by mediating inflammatory factors; however the underlying mechanisms remain poorly understood. The present study is aimed to investigate the mechanisms of NMB-induced cyclo-oxygenase 2 (COX-2) expression and interleukin (IL)-6 generation in human primary myometrial cells. The results indicated that NMB could increase phosphorylation of nuclear factor κB (NF-κB) transcription factor p65 (p65) and Jun proto-oncogene, activator protein 1 (AP-1) transcription factor subunit (c-Jun), and in turn, markedly up-regulated the expression levels of COX-2 and IL-6. This up-regulation was significantly attenuated by knockdown of p65 or c-Jun, and enhanced by overexpression of p65 or c-Jun. Furthermore, we identified a potential interaction between p65 and c-Jun following NMB stimulation. In addition, a significant positive correlation was observed between the amount of phosphorylated p65 and the levels of COX-2 and IL-6, and between the amount of phosphorylated c-Jun and COX-2 and IL-6 levels. These data suggested that NMB-induced COX-2 and IL-6 expression were mediated via p65 and c-Jun activation.

Myometrial progesterone hyper-responsiveness associated with increased risk of human uterine fibroids

BackgroundUterine Fibroids (UFs) growth is ovarian steroid-dependent. Previous studies have shown that estrogen and progesterone play an important role in UF development. However, the mechanism underlying progesterone induced UF pathogenesis is largely unknown. In this study, we determined the expression of progesterone receptor and compared the expression level of progesterone-regulated genes (PRGs) in human myometrial cells from normal uteri (MyoN) versus uteri with UFs (MyoF) in response to progesterone.MethodsPrimary human myometrial cells were isolated from premenopausal patients with structurally normal uteri (PrMyoN). Primary human myometrial cells were also isolated from uterus with UFs (PrMyoF). Isolated tissues were excised at least 2 cm from the closest UFs lesion(s). Progesterone receptor (PR) expression was assessed using Western blot (WB). Expression levels of 15 PRGs were measured by qRT-PCR in PrMyoN and PrMyoF cells in the presence or absence of progesterone.ResultsWB analysis revealed higher expression levels of PR in PrMyoF cells as compared to PrMyoN cells. Furthermore, we compared the expression patterns of 15 UF-related PRGs in PrMyoN and PrMyoF primary cells in response to progesterone hormone treatment. Our studies demonstrated that five PRGs including Bcl2, FOXO1A, SCGB2A2, CYP26a1 and MMP11 exhibited significant progesterone-hyper-responsiveness in human PrMyoF cells as compared to PrMyoN cells (P < 0.05). Another seven PRGs, including CIDEC, CANP6, ADHL5, ALDHA1, MT1E, KIK6, HHI showed gain in repression in response to progesterone treatment (P > 0.05). Importantly, these genes play crucial roles in cell proliferation, apoptosis, cell cycle, tissue remodeling and tumorigenesis in the development of UFs.ConclusionThese data support the idea that progesterone acts as contributing mechanism in the origin of UFs. Identification and analysis of these PRGs will help to further understand the role of progesterone in UF development.

Comparative analysis of myometrial and vascular smooth muscle cells to determine optimal cells for use in drug discovery

Novel therapeutic regulators of uterine contractility are needed to manage preterm labor, induce labor and control postpartum hemorrhage. Therefore, we previously developed a high-throughput assay for large-scale screening of small molecular compounds to regulate calcium-mobilization in primary mouse uterine myometrial cells. The goal of this study was to select the optimal myometrial cells for our high-throughput drug discovery assay, as well as determine the similarity or differences of myometrial cells to vascular smooth muscle cells (VSMCs)-the most common off-target of current myometrial therapeutics. Molecular and pharmacological assays were used to compare myometrial cells from four sources: primary cells isolated from term pregnant human and murine myometrium, immortalized pregnant human myometrial (PHM-1) cells and immortalized non-pregnant human myometrial (hTERT-HM) cells. In addition, myometrial cells were compared to vascular SMCs. We found that the transcriptome profiles of hTERT-HM and PHM1 cells were most similar (r = 0.93 and 0.90, respectively) to human primary myometrial cells. Comparative transcriptome profiling of primary human myometrial transcriptome and VSMCs revealed 498 upregulated (p ≤ 0.01, log2FC≥1) genes, of which 142 can serve as uterine-selective druggable targets. In the high-throughput Ca2+-assay, PHM1 cells had the most similar response to primary human myometrial cells in OT-induced Ca2+-release (Emax = 195% and 143%, EC50 = 30 nM and 120 nM, respectively), while all sources of myometrial cells showed excellent and similar robustness and reproducibility (Z’ = 0.52 to 0.77). After testing a panel of 61 compounds, we found that the stimulatory and inhibitory responses of hTERT-HM cells were highly-correlated (r = 0.94 and 0.95, respectively) to human primary cells. Moreover, ten compounds were identified that displayed uterine-selectivity (≥5-fold Emax or EC50 compared to VSMCs). Collectively, this study found that hTERT-HM cells exhibited the most similarity to primary human myometrial cells and, therefore, is an optimal substitute for large-scale screening to identify novel therapeutic regulators of myometrial contractility. Moreover, VSMCs can serve as an important counter-screening tool to assess uterine-selectivity of targets and drugs given the similarity observed in the transcriptome and response to compounds.

GIT2 deficiency attenuates inflammation-induced expression of pro-labor mediators in human amnion and myometrial cells†.

Untimely activation of the inflammatory response by sterile or infective insults in uterine tissues can result in preterm birth. Pro-inflammatory cytokines and pathogenic activation of toll-like receptors (TLRs) initiate a biochemical cascade of events leading to myometrial activation and contractility, cervical dilatation, and rupture of the chorioamniotic membranes. GIT2 is a signaling protein known to play a role in innate and adaptive immunity; however, its role in the inflammatory pathways of human labor is not known. In this article, we report that GIT2 expression is lower in human myometrium and fetal membranes with term labor, and in preterm amnion with histological chorioamnionitis. GIT2 knockdown by siRNA in primary myometrial and amnion cells exhibited reduced expression of pro-inflammatory cytokines and chemokines in response to inflammatory challenge by cytokines or TLR ligands. In addition, the pro-inflammatory cytokines IL1B and TNF could not induce the expression of extracellular matrix degrading enzymes in GIT2-deficient amnion cells. Myometrial activation in response to pro-inflammatory cytokines was also significantly suppressed in GIT2-deficient cells as evidenced by decreased prostaglandin release and expression of contraction-associated proteins. Further to this, collagen gel assays demonstrated that TNF had a reduced ability to induce myometrial contractility in situ in GIT2-deficient myometrial cells compared to control-transfected cells. In summary, the loss of GIT2 diminishes the effects inflammatory mediators have in promoting myometrial contraction and fetal membrane rupture in vitro, suggesting that GIT2 could be a possible target for preterm birth therapies.