Oksana Shynlova

p54nrb Is a Transcriptional Corepressor of the Progesterone Receptor that Modulates Transcription of the Labor-Associated Gene, Connexin 43 (Gja1)

The progesterone receptor (PR) plays important roles in the establishment and maintenance of pregnancy. By dynamic interactions with coregulators, PR represses the expression of genes that increase the contractile activity of myometrium and contribute to the initiation of labor. We have previously shown that PTB-associated RNA splicing factor (PSF) can function as a PR corepressor. In this report, we demonstrated that the PSF heterodimer partner, p54nrb (non-POU-domain-containing, octamer binding protein), can also function as a transcription corepressor, independent of PSF. p54nrb Interacts directly with PR independent of progesterone. In contrast to PSF, p54nrb neither enhances PR protein degradation nor blocks PR binding to DNA. Rather, p54nrb recruits mSin3A through its N terminus to the PR-DNA complex, resulting in an inhibition of PR-mediated transactivation of the progesterone-response element-luciferase reporter gene. PR also repressed transcription of the connexin 43 gene (Gja1), an effect dependent on the presence of an activator protein 1 site within the proximal Gja1 promoter. Mutation of this site abolished PR-mediated repression and decreased the recruitment of PR and p54nrb onto the Gja1 promoter. Furthermore, knockdown p54nrb expression by small interfering RNA alleviated PR-mediated repression on Gja1 transcription, whereas overexpression of p54nrb enhanced it. In the physiological context of pregnancy, p54nrb protein levels decrease with the approach of labor in the rat myometrium. We conclude that p54nrb is a transcriptional corepressor of PR. Decreased expression of p54nrb at the time of labor may act to derepress PR-mediated inhibition on connexin 43 expression and contribute to the initiation of labor.

The expression of transforming growth factor β in pregnant rat myometrium is hormone and stretch dependent

From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor beta (TGFbeta) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFbetas. The expression of TGFbeta1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfbeta1-3 genes were expressed differentially in pregnant myometrium. Tgfbeta2 gene was not affected by pregnancy, whereas the Tgfbeta1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfbeta3 gene expression throughout pregnancy. Tgfbeta3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfbeta3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20-23) caused a significant decrease in the expression of Tgfbeta3 gene. In addition, Tgfbeta3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFbeta family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.

Inhibition of infection‐mediated preterm birth by administration of broad spectrum chemokine inhibitor in mice

Preterm birth (PTB) is the single most important cause of perinatal and infant mortality worldwide. Maternal infection can result in PTB. We investigated the ability of a Broad Spectrum Chemokine Inhibitor (BSCI) to prevent infection‐induced PTB in mice. PTB was initiated in pregnant mice by intraperitoneal injection of lipopolysaccharide (LPS; 50 μg). Half the mice received BSCI (10 mg/kg) 24 hrs prior to and immediately before LPS administration. The impact of LPS alone or LPS plus BSCI was assessed on (i) injection‐to‐delivery interval, foetal survival rate, placental and neonates weight; (ii) amniotic fluid and maternal plasma cytokine levels (by Luminex assay); foetal and maternal tissue cytokine gene expression levels (by Real‐Time RT‐PCR); (iii) immune cells infiltration into the uterine tissue (by stereological immunohistochemistry). Pre‐treatment with BSCI (i) decreased LPS‐induced PTB (64% versus 100%, P < 0.05); (ii) significantly attenuated cytokine/chemokine expression in maternal tissues (plasma, liver, myometrium, decidua); (iii) significantly decreased neutrophil infiltration in the mouse myometrium. BSCI‐treated mice in which PTB was delayed till term had live foetuses with normal placental and foetal weight. BSCI represents a promising new class of therapeutics for PTB. In a mouse model of preterm labour, BCSI suppresses systemic inflammation in maternal tissues which resulted in the reduced incidence of LPS‐mediated PTB. These data provide support for efforts to target inflammatory responses as a means of preventing PTB.

A New Role for Monocytes in Modulating Myometrial Inflammation During Human Labor

ABSTRACT Here we fully characterize the cytokine profile of laboring human myometrium using Luminex analysis of 48 cytokine proteins, and stereologically quantified infiltration of monocytes and neutrophils into the myometrium. We hypothesized that monocytes can regulate their accumulation in the myometrium by disruption of proinflammatory cytokines to prevent an uncontrolled inflammatory response after labor onset. We isolated primary human myometrial cells (HMCs) from term, nonlaboring myometrial biopsies. Confluent HMCs were cocultured directly with human monocytic (THP-1) or lymphocytic (U937) cells, and with the same cells spatially separated by a membrane insert. After 72 h, HMCs and THP-1 were harvested separately, and RNA was extracted and analyzed by quantitative PCR. Coculture supernatants were collected and analyzed by Luminex assay and ELISA. We found that the laboring human myometrium produces significantly higher amounts of interleukin (IL) 6, IL9, IL18, IL1RA, CCL2, CCL7, CXCL8, CSF3, and tumor necrosis factor alpha, which coincides with the influx of immune cells. The direct contact or presence of THP-1 monocytes (but not U937 cells) significantly decreased CCL2 protein levels and increased IL1RA protein levels secreted by HMCs. This time-dependent decrease of CCL2 was greater with increasing number of monocytes being in direct contact with HMCs. We suggest a novel mechanism by which monocytes are first recruited to the myometrium by multiple cytokines and contribute to the physiologic inflammation of labor. After completing transmigration, activated monocytes disrupt locally established CCL2 gradients (possible by CCR2-mediated consumption) to limit their accumulation in the uterus. This mechanism may serve as a negative feedback loop to control the local inflammation and promote a return to homeostasis.

Expression and Function of Myometrial PSF Suggest a Role in Progesterone Withdrawal and the Initiation of Labor

Progesterone (P4), acting through its receptor (PR), is essential for the maintenance of pregnancy. P4 acts by suppressing uterine contractility and the expression of contraction-associated proteins (CAP) such as connexin 43 (Cx43). P4 levels must be reduced or its actions blocked to allow the increased expression of CAP genes and the initiation of labor. Although the importance of progesterone in pregnancy has been known for about 80 yr, the fundamental mechanisms by which P4/PR maintains myometrial quiescence and by which this signaling is blocked at term labor remain to be determined. In this manuscript, we demonstrate that ligand-bound PR interacts with the Cx43 gene promoter through activator protein-1 transcription factors. We show that the ability of PR to repress Cx43 transcription is conferred through the recruitment of the PR coregulator, polypyrimidine tract binding protein-associated splicing factor (PSF), and the further recruitment of the yeast switch independent 3 homolog A/histone deacetylase corepressor complex. PSF expression is elevated during pregnancy but falls toward term as a result of increased mechanical stretch of the myometrium and a rise in the concentrations of circulating estrogen. These data together indicate that PSF is a critical regulator of P4/PR signaling and labor. We suggest that decreased PSF at term may result in a de-repression of PR transcriptional control of CAP genes and thereby contributes to a functional withdrawal of progesterone at term labor.

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

CONTEXTnDuring 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.nnnOBJECTIVEnThe aim of the study was to investigate the expression and function of the androgen receptor (AR) in myometrial cells during pregnancy.nnnDESIGN AND SETTINGnHuman 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.nnnRESULTSnThe 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.nnnCONCLUSIONnAR is a key regulator for myometrial cell proliferation, suggesting its critical role in myometrium phenotype programming during pregnancy.

Expression of Extracellular Matrix-Remodeling Proteins Is Altered in Vaginal Tissue of Premenopausal Women With Severe Pelvic Organ Prolapse

Aim: The molecular etiology of pelvic organ prolapse (POP) is complex and not well understood. We compared the expression/activity of extracellular matrix (ECM)-processing (procollagen I N-proteinase/ a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]-2,-3,-14) and ECM-degrading (matrix metalloproteinase [MMP]-1, -2, -7, -8, -9, -12) enzymes and their natural tissue inhibitors (tissue inhibitors of metalloproteinase [TIMP]-1,-2,-3,-4) in vaginal tissues from premenopausal women with advanced POP (POP-Q stage ≥ 3) and asymptomatic controls (POP-Q = 0). Study Design: We sampled the anterior vaginal wall of 36 premenopausal women (17 patients with POP and 19 controls) undergoing total hysterectomy. Exclusion criteria include steroid therapy, malignancy, previous pelvic surgery, and connective tissue diseases. Total RNAs and proteins were quantified by real-time polymerase chain reaction, immunoblotting, and Luminex assay; MMPs activity was analyzed by zymography and tissue localization by immunohistochemistry. Results: The MMP-2 gelatinase activity as well as expression of 58-kDa isoform of ADAMTS-2 was upregulated in patients with POP, irrespective of menstrual phase status, secretory or proliferative, when compared to controls (P < .05). The TIMP-1-4 gene and TIMP-1 protein expression were significantly (P < .05) reduced, whereas protein expression of MMP-12 (pro and active forms) was significantly increased in vaginal biopsies of patients with POP in the proliferative phase of the menstrual cycle compared to corresponding controls. Analyses of MMP-12, TIMP-1, and ADAMTS-2 tissue immunostaining indicate similar localization in the vaginal specimens from control and patients with POP. Conclusion: Expression of ECM-remodeling proteins is altered in the vagina of premenopausal patients with severe POP. We speculate that dysregulation of MMP/TIMP complexes and ADAMTS-2 proteins may cause connective tissue defects, which result in weakened vaginal wall support and POP development.

Matrix Metalloproteinase Expression in the Rat Myometrium During Pregnancy, Term Labor, and Postpartum

ABSTRACT Pregnancy, spontaneous term labor (TL), and postpartum (PP) involution are associated with changes in the cellular and extracellular matrix composition of the uterus. Both the uterine smooth muscle (myometrium) and the infiltrating peripheral blood leukocytes involved in the activation of labor secrete extracellular matrix-degrading enzymes (matrix metalloproteinases, MMPs) that can modulate cellular behavior and barrier function. MMP expression is induced by mechanical stretch in several tissues. We hypothesized that the expression and activity of myometrial MMPs and their tissue inhibitors (TIMPs) are modulated in preparation for TL and PP involution and are regulated by mechanical stretch of uterine walls imposed by the growing fetus. Myometrial tissues were collected from bilaterally and unilaterally pregnant rats across gestation, TL, and PP. Total RNA and proteins were subjected to real-time PCR and immunoblotting, respectively, and tissue localization and activity was examined by immunohistochemistry and in situ zymography. We found that Mmp7, Mmp11, and Mmp12 mRNA levels were upregulated during TL and PP, while Mmp2, Mmp3, Mmp8, Mmp9, Mmp10, and Mmp13 mRNAs were only upregulated during PP. Timp1–Timp4 were stably expressed throughout gestation with some fluctuations PP. Active MMP2 was induced in the empty uterine horn during gestation and in the gravid PP uterus, suggesting negative regulation by biological mechanical stretch. We conclude that specific subsets of uterine MMPs are differentially regulated in the rat myometrium in preparation for two major events: TL and PP uterine involution.

Progesterone Via its Type-A Receptor Promotes Myometrial Gap Junction Coupling

Effective labour contractions require synchronization of myometrial cells through gap junctions (GJs). Clasically, progesterone (P4) is known to inhibit the expression of connexin-43 (Cx43, major component of GJs) and GJ formation in myometrium. Our current study is based on a striking observation that challenges this dogma. We observed conspicuous differences in the intracellular localization of Cx43 protein in PRA versus PRB expressing myocytes. Thus in P4 stimulated PRA cells Cx43 protein forms GJs, whereas in PRB cells the forward trafficking of Cx43 and GJ formation is inhibited even when Cx43 is overexpressed. We found that P4, via PRA/B, differentially regulates Cx43 translation to generate a Cx43-20u2009K isoform, which facilitates the transport of full length Cx43 to plasma membrane. The P4 mediated regulation of Cx43 trafficking and GJ formation occurs via non-genomic pathway and involves the regulation of mTOR signaling since inhibition of this pathway restored the Cx43 trafficking defect in PRB cells. We propose that PRA is a master regulator of Cx43 expression, GJ formation and myocyte connectivity/synchronization for labour.

Static Mechanical Loading Influences the Expression of Extracellular Matrix and Cell Adhesion Proteins in Vaginal Cells Derived From Premenopausal Women With Severe Pelvic Organ Prolapse

Introduction: Primary human vaginal cells derived from women with severe pelvic organ prolapse (POP-HVCs) demonstrate altered cellular characteristics as compared to cells derived from asymptomatic women (control-HVCs). Using computer-controllable Flexcell stretch unit, we examined whether POP-HVCs react differently to mechanical loading as compared to control-HVCs by the expression of extracellular matrix (ECM) components, cell–ECM adhesion proteins, and ECM degrading and maturating enzymes. Methods: Vaginal tissue biopsies from premenopausal patients with Pelvic Organ Prolapse Quantification System stage ≥3 (n = 8) and asymptomatic controls (n = 7) were collected during vaginal hysterectomy or repair. Human vaginal cells were isolated by enzymatic digestion, seeded on collagen (COLI)-coated plates, and stretched (24 hours, 25% elongation). Total RNA was extracted, and 84 genes were screened using Human ECM and Adhesion Molecules polymerase chain reaction array; selected genes were verified by quantitative reverse transcription-polymerase chain reaction. Stretch-conditioned media (SCM) were collected and analyzed by protein array, immunoblotting, and zymography. Results: In mechanically stretched control-HVCs, transcript levels of integrins (ITGA1, ITGA4, ITGAV, and ITGB1) and matrix metalloproteinases (MMPs) 2, 8, and 13 were downregulated (P < .05); in POP-HVCs, MMP1, MMP3, and MMP10, ADAMTS8 and 13, tissue inhibitor of metalloproteinases (TIMPs) 1 to 3, ITGA2, ITGA4, ITGA6, ITGB1, contactin (CNTN1), catenins (A1 and B1), and laminins (A3 and C1) were significantly upregulated, whereas COLs (1, 4, 5, 6, 11, and 12) and LOXL1 were downregulated. Human vaginal cells massively secrete MMPs and TIMPs proteins; MMP1, MMP8, MMP9 protein expression and MMP2 gelatinase activity were increased, whereas TIMP2 decreased in SCM from POP-HVCs compared to control-HVCs. Conclusions: Primary human vaginal cells derived from women with severe pelvic organ prolapse and control-HVCs react differentially to in vitro mechanical stretch. Risk factors that induce stretch may alter ECM composition and cell–ECM interaction in pelvic floor tissue leading to the abatement of pelvic organ support and subsequent POP development.