Altered expression of histone deacetylases, inflammatory cytokines and contractile-associated factors in uterine myometrium of Long Evans rats gestationally exposed to benzo[a]pyrene.

Etiology of preterm birth (PTB) is multifactorial; therefore, decreasing the incidence of PTB is a major challenge in the field of obstetrics. Epidemiological studies have reported an association between toxicants and PTB. However, there are no studies on the role of benzo[a]pyrene (BaP), an environmental toxicant, in the incidence of PTB. We first assessed the effects of BaP (150 and 300 µg kg(-1) body weight) dosed via gavage from day 14 to 17 of pregnancy on gestation length in Long Evans rats. We further assessed the histopathology of the uterus, expression of inflammatory cytokines, contractile-associated factors, histone deacetylases (HDACs) and NFқB-p65 in myometrium collected on day 22 postpartum versus vehicle-treated controls. In our study, rats exposed to BaP delivered prematurely (P < 0.05) compared to control. Hematoxylin and eosin staining of uterus showed squamous metaplasia, glandular and stromal hyperplasia in BaP-exposed rats versus control. The concentrations of BaP metabolites measured by high-pressure liquid chromatography were higher in uterine myometrium of BaP-exposed rats while they were undetectable in controls. Quantitative real-time polymerase chain reaction showed significant increases in mRNA expression of interleukin-1ß and -8, tumor necrosis factor-α, connexin 43, cyclo-oxygenase-2 and prostaglandin F2α receptor as compared to controls (P < 0.05). Western blot analysis revealed that BaP exposure caused decreases in class I HDACs 1 and 3 and increases in class II HDAC 5, cyclo-oxygenase-2 and nuclear translocation of NFκB-p65 relative to controls. Our results suggest that gestational exposure to BaP increases incidence of PTB through epigenetic changes that causes increases in the expression of contractile-associated factors through the NFκB pathway. Copyright © 2015 John Wiley & Sons, Ltd.

Vitamin D regulates contractile profile in human uterine myometrial cells via NF-κB pathway.

OBJECTIVE: Infection triggers inflammation that, in turn, enhances the expression of contractile-associated factors in myometrium and increases the risk of preterm delivery. In this study, we assessed vitamin D regulation of inflammatory markers, contractile-associated factors, steroid hormone receptors, and NFκB pathway proteins in human uterine myometrial smooth muscle (UtSM) cells that were cultured in an inflammatory environment. STUDY DESIGN: Inflammatory environment was simulated for UtSM cells by coculturing them with monocyte lineage (THP1) cells. We measured the expression of inflammatory markers, contractile-associated factors, steroid hormone receptors, and NFκB pathway proteins in UtSM cells that were cultured with THP1 cells in the presence and absence of vitamin D by real time polymerase chain reaction and Western blot analysis. RESULTS: Monocytes secreted monocyte inflammatory protein-1α and -1ß, interleukin (IL)-1ß and 6, and tumor necrosis factor-α into the conditioned medium. In the UtSM cells that had been cocultured with THP1 cells, there was a significant (P < .05) increase in the expression of inflammatory markers IL-1ß, -6, and -13 and tumor necrosis factor-α; the contractile-associated factors connexin-43, Cox-2, and prostaglandin F2α receptor; the estrogen receptor α, and progesterone receptors A and B. Vitamin D treatment of cocultures decreased (P < .05) the expression of inflammatory markers and contractile-associated factors in UtSM cells. Similarly, vitamin D decreased estrogen receptor α and progesterone receptors A-to-B ratio in UtSM cells that were cocultured with THP1 cells. In addition, vitamin D treatment significantly (P < .05) decreased monocyte-induced p-IκBα in cytosol and NFκB-p65 in the nucleus and increased IκBα in cytosol in UtSM cells. CONCLUSION: Our results suggest that vitamin D treatment decreases inflammation-induced cytokines and contractile-associated factors in the uterine myometrial smooth muscle cells through the NFκB pathway.

1,25-Dihydroxyvitamin D deficiency is associated with preterm birth in African American and Caucasian women.

Vitamin (vit) D deficiency and preterm birth (PTB) are more prevalent among African American (AA) women compared to caucasian (Cau) women. Because vit D is important in regulating cell-mediated immune responses, vit D insufficiency or deficiency during pregnancy may enhance inflammation in pregnant women and increase the risk of PTB. In this study, circulatory levels of 25-hydroxy (OH) and 1,25-dihydroxy (OH)2 vit D were measured using chemiluminescence and radioimmunoassay techniques, respectively, in AA (n = 108) and Cau (n = 84) women who delivered at term and preterm. The results from this study suggest that the serum levels of the 25-(OH) vit D concentrations tend to decrease (P = .06) in the Cau women who delivered at preterm compared to those delivering at term. However, the 25-(OH) vit D levels in Cau and AA between term and preterm deliveries were not significantly different. The serum levels of 1,25-(OH)2 vit D were found to be significantly lower in AA women compared to Cau women (P < .02) at term, and in the Cau (P < .01) and AA (P < .04) women delivering at preterm compared to those delivering at term. One-way analysis of variance demonstrated that 1,25-(OH)2 vit D levels were significantly lower in participants delivering at preterm (<34 weeks and between 34 and 37 weeks) compared to those delivering at term (>37 weeks).These results suggest that low levels of serum 1,25-(OH)2 vit D are associated with PTB, and vit D can potentially be used as a novel diagnostic marker in the detection of PTB.

Vitamin D elicits anti-inflammatory response, inhibits contractile-associated proteins, and modulates Toll-like receptors in human myometrial cells.

Infection during pregnancy triggers inflammation, which can increase myometrial contractions and the risk of premature labor and delivery. In this study, we assessed the effects of vitamin D, an anti-inflammatory ligand on cytokines, chemokines, toll-like receptors, and contractile-associated proteins on immortalized human myometrial smooth muscle (UtSM) cells stimulated with lipopolysaccharide (LPS), a bacterial endotoxin, or interleukin (IL)-1ß and measured Toll-like receptor (TLR)-10 expression in pregnant myometrial tissues. A superarray analysis revealed downregulation of the chemokines monocyte chemoattractant protein (MCP)-1, Chemokine (C-X-C motif) ligand (CXCL)-10, CXCL-11, and chemokine (C-X3-C motif) ligand (CX3CL)-1; the proinflammatory cytokines IL-13 and tumor necrosis factor (TNF)-α; the TLR-4 and -5 and triggering receptor expressed on myeloid cells (TREM)-2 and upregulation of the anti-inflammatory cytokine IL-10, as well as Toll interacting protein (TOLLIP) and TREM-1 in vitamin D-treated UtSM cells. In the presence of LPS, vitamin D caused dose-dependent decreases in the messenger RNA expression of MCP-1, IL-1ß, IL-13, TNF-α, TLR-4, and TLR-5, the contractile-associated proteins connexin 43, the oxytocin receptor, and the prostaglandin receptor but caused increases in IL-10 and TLR-10 in UtSM cells. The TLR-10 expression was higher in human myometrial tissue obtained from women at term not in labor compared to labor. Vitamin D also attenuated IL-1ß-induced MCP-1, IL-6, connexin 43, cyclooxygenase (COX)-2, and prostaglandin receptor expression. Western analysis showed that vitamin D decreased MCP-1, TLR-4, and connexin 43 in the presence of LPS and decreased connexin 43 in the presence of IL-1ß. Our results suggest that vitamin D can potentially decrease infection-induced increases in cytokines and contractile-associated proteins in the myometrium.

A single-nucleotide polymorphism in the fetal catechol-O-methyltransferase gene is associated with spontaneous preterm birth in African Americans.

Catechol-O-methyltransferase (COMT) activity has been reported to be higher in African Americans (AA) than Caucasians (Cau). COMT converts 2- and 4-hydroxy (OH) estrogens to 2- and 4-methoxyestrogens, respectively, and can increase estrogenic milieu locally in tissues. To assess whether the increased incidence of preterm birth (PTB) among AA women is associated with single-nucleotide polymorphism (SNP) in the COMT gene, we examined variations in maternal and fetal COMT genes and their association with pregnancy outcomes (term vs preterm pregnancies) using 4 functional SNPs: rs4633, rs4680, rs4818, and rs6269 in both AA and Cau. We analyzed samples from 267 AA women (191 term and 76 preterm pregnancies) and 339 Cau (194 term and 145 preterm pregnancies) in this study. The results showed a significant difference (P < .05) in allele and genotype frequencies between term and preterm AA and Cau women in 3 SNPs in both maternal and fetal DNA. The analysis revealed that in AA fetal COMT genes, SNP rs4818 is associated with PTB at the allele (C; P < .001), genotype (C/C; P < .01), and 2- (P < .03) and 3 (P < .04)-window haplotype levels. Multidimensionality reduction analysis also showed a significant (P < .01) association between rs4818 and PTB. In conclusion, our study demonstrated that a synonymous polymorphism, rs4818 in the fetal COMT gene, is associated with PTB in AA.

Vitamin D inhibits proliferation of human uterine leiomyoma cells via catechol-O-methyltransferase.

OBJECTIVE: To evaluate the effects and mechanisms of action of vitamin D on human uterine leiomyoma (HuLM) cell proliferation in vitro. DESIGN: Laboratory study. SETTING: University hospitals. PATIENTS(S): Not applicable. INTERVENTIONS(S): Not applicable. MAIN OUTCOME MEASURE(S): HuLM cells were treated with 1,25-dihydroxyvitamin D3 (vitamin D), and cell proliferation was assayed by the methylthiazolyl tetrazolium technique. proliferating cell nuclear antigen (PCNA), BCL-2, BCL-w, cyclin-dependent kinase (CDK) 1, and catechol-O-methyltransferase (COMT) protein levels were analyzed by Western blotting. COMT mRNA and enzyme activity were assayed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and high-performance liquid chromatography analysis, respectively. The role of COMT was evaluated in stable HuLM cells by silencing COMT expression. RESULT(S): Vitamin D inhibited the growth of HuLM cells by 47±0.03% at 1 µM and by 38±0.02% at 0.1 µM compared with control cells at 120 hours of treatment. Vitamin D inhibited extracellular signal-regulated kinase activation and down-regulated the expression of BCL-2, BCL-w, CDK1, and PCNA. Western blot, RT-PCR, and enzyme assay of COMT demonstrated inhibitory effects of vitamin D on COMT expression and enzyme activity. Silencing endogenous COMT expression abolished vitamin D-mediated inhibition of HuLM cell proliferation. CONCLUSION(S): Vitamin D inhibits growth of HuLM cells through the down-regulation of PCNA, CDK1, and BCL-2 and suppresses COMT expression and activity in HuLM cells. Thus, hypovitaminosis D appears to be a risk factor for uterine fibroids.

Elevated expression of catechol-O-methyltransferase is associated with labor and increased prostaglandin E(2) production by human fetal membranes.

OBJECTIVE: The purpose of this study was to evaluate the expression and function of catechol-O-methyltransferase in human fetal membranes at term. STUDY DESIGN: Fetal membranes obtained from women between 38-42 weeks of gestation, after (1) vaginal delivery with spontaneous labor and (2) prelabor elective cesarean section (no labor), were assayed for catechol-O-methyltransferase expression using quantitative real-time polymerase chain reaction analysis, immunohistochemistry, and Western blot analysis. Prostaglandin E(2) secretion from amnion and choriodecidua explants treated with or without catechol-O-methyltransferase inhibitor was assayed by enzyme-linked immunosorbent analysis. RESULTS: Amnion layer of fetal membranes from laboring women expressed significantly higher levels of catechol-O-methyltransferase, compared with those from women with no labor. Catechol-O-methyltransferase was higher in the amnion layer than in choriodecidua. Selective catechol-O-methyltransferase inhibition significantly decreased prostaglandin E(2) production from fetal membranes. CONCLUSION: Labor increases catechol-O-methyltransferase expression in the amnion of human fetal membranes. Selective catechol-O-methyltransferase inhibition decreased prostaglandin E(2) secretion in fetal explant cultures, suggesting a role for catechol-O-methyltransferase in human labor and delivery.

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.

Evidence for decreased calcitonin gene-related peptide (CGRP) receptors and compromised responsiveness to CGRP of fetoplacental vessels in preeclamptic pregnancies.

Calcitonin gene-related peptide (CGRP) is a potent vasodilatory peptide, and its concentration is increased in both maternal and fetal circulation during late pregnancy. The present study was designed to investigate the expression of CGRP receptor components, calcitonin receptor-like receptor (CRLR), and receptor activity modifying protein 1 (RAMP1), and the relaxation response to CGRP in fetoplacental vessels from normotensive pregnant women and women with preeclampsia. Results showed that: 1) mRNA for both CRLR and RAMP1 was expressed in fetoplacental vessels from normal pregnancies; however, these mRNA expressions were substantially reduced in the vessels from preeclamptic women; 2) CRLR and RAMP1 proteins were abundantly expressed in the endothelium and smooth muscle layer of the fetoplacental vessels, as well as the trophoblast cells in normal placentas. In contrast, both vascular tissues and trophoblasts showed decreased expressions for CRLR and RAMP1 proteins and declined CGRP binding sites in preeclamptic placentas; and 3) CGRP produced a dose-dependent relaxation of serotonin-induced contraction of umbilical and chorionic arteries from normal pregnancies, but the response to CGRP was significantly attenuated in the vessels from preeclampsia. We concluded that CGRP may contribute to the low fetoplacental vascular resistance in normal pregnancies; however, CGRP-dependent vascular relaxation appears to be compromised in preeclamptic pregnancies.