Vitamin D stimulates multiple microRNAs to inhibit CRH and other pro-labor genes in human placenta

Maternal vitamin D deficiency is linked to adverse pregnancy outcomes including spontaneous preterm birth (SPB). Placental corticotropin-releasing hormone (CRH) has been proposed to be part of a clock that governs the length of gestation in humans, with elevated maternal serum levels predicting early delivery. In this study, we test the hypothesis that vitamin D could contribute to the prevention of preterm labor by inhibiting CRH and other pro-labor mediators. The biological activity of vitamin D occurs via two pathways: non-genomic and genomic responses, both of which involve binding of 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D binding to the vitamin D receptor (VDR). By using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we found that 1,25(OH)2D stimulates association of VDR with a number of miRNA genes including MIR181B2 and MIR26B, and their mature products miR-181b-5p and miR-26b-5p are predicted to target CRH and cyclooxygenase-2 (COX-2) mRNA at 3′-untranslated region (UTR), respectively. We performed RT-qPCR analysis to validate that expression of mature miR-181b-5p and miR-26b-5p in term human syncytiotrophoblast increased in response to treatment with 1,25(OH)2D. miR-181b-5p- or miR-26b-5p-mediated inhibition of CRH or COX-2 was further assessed by the use of miRNA mimics/inhibitors and a luciferase reporter assay. Taken together, this study has identified novel mechanisms by which vitamin D downregulates pro-labor genes and could lower the risk of preterm delivery

Fetal lung C4BPA induces p100 processing in human placenta

Abstract The non-canonical NF-κB signaling may be a central integrator of a placental clock that governs the length of human pregnancy. We sought to identify fetal signals that could activate this NF-κB pathway in the placenta, and in turn, contribute to the onset of labor. Proteomics analysis of exosomes purified from fetal cord arterial blood revealed a total of 328 proteins, among which 48 were more significantly abundant (p < 0.01) in samples from women who delivered following elective Cesarean-section at term (39 to 40 weeks of estimated gestational age, EGA) compared to those who had elective Cesarean deliveries near term (35 to 36 weeks of EGA). Computational, crystal structural, and gene functional analyses showed that one of these 48 proteins, C4BPA, binds to CD40 of placental villous trophoblast to activate p100 processing to p52, and in turn, pro-labor genes. These results suggest that fetal C4BPA-induced activation of non-canonical NF-κB in human placenta may play a critical role in processes of term or preterm labor

Mono-(2-Ethylhexyl) Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta.

Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2) are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52) signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl)-phthalate (MEHP), increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth

MEHP upregulates expression of CRH and COX-2 in primary cultures of human cytotrophoblast.

<p>Primary cultures of term cytotrophoblast were exposed to different concentrations of MEHP for 24 hours with DMSO as the vehicle control. (<b>A</b>) Trypan blue exclusion test of cell viability for primary cytototrophoblast exposed to MEHP at concentrations as indicated (N = 3 independent experiments). (<b>B</b>) Representative gel patterns and analysis in whole cell lysates from three independent term placentas by Western blot assay with use of antibody as indicated. (<b>C</b>) Total RNAs were extracted and RT-qPCR was performed for assessment of CRH and COX-2 mRNA levels with normalization to GAPDH mRNA level. Bars represent the average of relative mRNA levels and error bars represent standard deviation from experiments performed in three independent term placentas. * p < 0.05; ** p < 0.01.</p

The effects of MEHP exposure on interaction of RelB/p52 with the <i>CRH/COX-2</i> gene promoters.

<p>Term cytotrophoblast were exposed to MEHP for 24 hrs at concentrations as indicated. ChIP assays were performed to determine occupancy of RelB/p52 at <i>CRH</i> (<b>A</b>) and <i>COX-2</i> (<b>B</b>), and also occupancy of RelA at the <i>CRH</i> (<b>C</b>) and <i>COX-2</i> (<b>D</b>) gene promoter. Fold enrichment was derived with normalization to a human DNA a satellite, a non-coding DNA sequence to which no transcriptional factors should bind. Rabbit IgG was used as a non-specific control. The bars indicate the average of fold enrichment with error bars representing the standard deviation from three independent experiments. * p < 0.01.</p

NIK knockdown attenuates MEHP-induced upregulation of <i>CRH</i> and <i>COX-2</i> in the human placenta.

<p>Primary term cytotrophoblast were incubated with siRNAs targeting NIK (siNIK) for 24 hrs, and then treated with MEHP for an additional 24 hrs. Scramble siRNA (Scr-siRNA) was used as the non-targeting control. Total RNAs were extracted and RT-qPCR was performed to determine mRNA levels of CRH (<b>A</b>), COX-2 (<b>B</b>), or NIK (<b>C</b>) (N = 3 independent experiments). * p < 0.05 (compared to DMSO). ** p< 0.01 (compared to DMSO). ## p < 0.01 (compared to Scr-siRNA). (<b>D</b>) Representative gel pattern of Western blot analysis on whole cell lysates from three independent experiments. (<b>E</b>) IF staining was performed to directly assess intracellular distribution of RelB. These images represent data obtained from three independent placentas. Original magnification, 200Χ.</p