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 MTT technique. PCNA, BCL-2, BCL-w, CDK-1 and COMT protein levels were analyzed by Western blotting. COMT mRNA and enzyme activity were assayed by quantitative RT-PCR and HPLC 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 to control cells at 120 hours of treatment (P < 0.05). Vitamin D inhibited ERK activation and downregulated 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.

OBJECTIVE Infection triggers inflammation which 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 cultured in an inflammatory environment. STUDY DESIGN Inflammatory environment was simulated for UtSM cells by co-culturing 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 cultured with THP1 cells in the presence and absence of vitamin D by RT-PCR and western analysis. RESULTS Monocytes secreted MIP-1α, MIP-1 β, IL-1β, IL6, and TNFα into the conditioned medium. In UtSM cells co-cultured with THP1 cells there was a significant (p<0.05) increase in the expression of inflammatory markers IL-1β, IL6, IL13 and TNFα; 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 co-cultures decreased (p<0.05) 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 co-cultured with THP1 cells. In addition, vitamin D treatment significantly (p<0.05) decreased monocyte induced p-IκBα in cytosol, 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 via NFκB pathway.

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)-1β 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-1β, 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-1β-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-1β. Our results suggest that vitamin D can potentially decrease infection-induced increases in cytokines and contractile-associated proteins in the myometrium.

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.

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 ¼ 108) and Cau (n ¼ 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 ¼ .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.