No abstract

Differentiation of human endometrial stromal cells into specialized decidual cells is critical for embryo implantation and survival of the conceptus. Initiation of this differentiation process is strictly dependent on elevated cAMP levels, but the signal intermediates that control the expression of decidual marker genes, such as prolactin (PRL) and IGFBP1, remain poorly characterized. Here we show that cAMP-dependent decidualization can be attenuated or enhanced upon treatment of primary cultures with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenylen iodonium) or activator (apocynin), respectively. Time-course analysis demonstrated that cAMP enhances endogenous reactive oxygen species production, apparent after 12 h of stimulation, which coincides with a dramatic increase in decidual PRL and IGFBP1 expression. Knockdown of the Rho GTPase RAC1, which disables activation of the NADPH oxidase homologs NADPH oxidase (NOX)-1, NOX-2, and NOX-3, had no effect on PRL or IGFBP1 expression. In contrast, silencing of NOX-4, or its cofactor p22(PHOX), inhibited the expression of both decidual markers. Finally, we show that the NOX-4/p22(PHOX) complex regulates the DNA-binding activity of CCAAT/enhancer binding protein-β, a key regulator of human endometrial stromal cell differentiation. Thus, NOX-4 activation and reactive oxygen species signaling play an integral role in initiating the endometrial decidual response in preparation of pregnancy.

Progesterone is widely used to prolong gestation in women at risk of preterm labour (PTL), and acts at least in part via the inhibition of inflammatory cytokine-induced prostaglandin synthesis. This study investigates the mechanisms responsible for this inhibition in human myometrial cells. We used reporter constructs to demonstrate that interleukin 1beta (IL-1β) inhibits progesterone driven PRE activation via p65 activation and that IL-1β reduced progesterone driven gene expression (FKBP5). Conversely, we found that the activity of a p65-driven NFκB reporter construct was reduced by overexpression of progesterone receptor B (PRB) alone and that this was enhanced by the addition of MPA and that both MPA and progesterone suppressed IL-1β-driven cyclo-oxygenase-2 (COX-2) expression. We found that over-expressed Halo-tagged PRB, but not PRA, bound to p65 and that in IL-1β-treated cells, with no overexpression of either PR or p65, activated p65 bound to PR. However, we found that the ability of MPA to repress IL-1β-driven COX-2 expression was not enhanced by overexpression of either PRB or PRA and that although the combined PR and GR antagonist Ru486 blocked the effects of progesterone and MPA, the specific PR antagonist, Org31710, did not, suggesting that progesterone and MPA act via GR and not PR. Knockdown using siRNA confirmed that both MPA and progesterone acted via GR and not PR or AR to repress IL-1β-driven COX-2 expression. We conclude that progesterone acts via GR to repress IL-1β-driven COX-2 activation and that although the interaction between p65 and PRB may be involved in the repression of progesterone driven gene expression it does not seem to be responsible for progesterone repression of IL-1β-induced COX-2 expression.

The process of parturition involves the transformation of the quiescent myometrium (uterine smooth muscle) to the highly contractile laboring state. This is thought to be driven by changes in gene expression in myometrial cells. Despite the existence of multiple myometrial gene expression studies, the transcriptional programs that initiate labor are not known. Here, we integrated three transcriptome datasets, one novel (NCBI Gene Expression Ominibus: GSE80172) and two existing, to characterize the gene expression changes in myometrium associated with the onset of labor at term. Computational analyses including classification, singular value decomposition, pathway enrichment, and network inference were applied to individual and combined datasets. Outcomes across studies were integrated with multiple protein and pathway databases to build a myometrial parturition signaling network. A high-confidence (significant across all studies) set of 126 labor genes were identified and machine learning models exhibited high reproducibility between studies. Labor signatures included both known (interleukins, cytokines) and unknown (apoptosis, MYC , cell proliferation/differentiation) pathways while cyclic AMP signaling and muscle relaxation were associated with non-labor. These signatures accurately classified and characterized the stages of labor. The data-derived parturition signaling networks provide new genes/signaling interactions to understand phenotype-specific processes and aid in future studies of parturition.