Urocortins (UCNs), belonging to corticotropin-releasing hormone (CRH) family, exert their function via CRH receptor type 1(CRHR1) and 2 (CRHR2). Our previous studies have demonstrated that CRH acts on CRHR1 to potentiate prostaglandin (PG) output induced by inflammatory stimuli in myometrial cells. In the present study, we sought to investigate the effects of UCNs on prostaglandin (PG) output via CRHR2 in cultured human uterine smooth muscle cells (HUSMCs) from human term myometrium. We found that UCN and UCN3 treatment promoted PGE2 and PGF2α secretion in a dose-dependent manner. In contrast, UCN2 dose-dependently inhibited PGE2 and PGF2α secretion. Their effects could be reversed by CRHR2 antagonist and CRHR2 siRNA. Mechanically, we showed that UCN and UCN3 suppressed cAMP production and led to Gi activation, while UCN2 promoted cAMP production and activated Gs signaling. Further, UCN and UCN3 could activate NF-κB and MAPK signaling pathways. These effects were dependent on Gi signaling. In contrast, UCN did not activate MAPK and NF-κB signaling. UCN and UCN3 stimulation of PG secretion was dependent on Gi/adenylyl cyclase (AC)/cAMP, NF-κB and MAPK signaling pathways, while UCN2 suppression of PG output was through Gs/AC/cAMP signaling pathways. Our data suggest that UCN, UCN2 and UCN3 can finely regulate the secretion of PGs via CRHR2, which facilities the functional status of uterus during pregnancy.
Xingji You, Zixi Chen, Qianqian Sun, Ruojing Yao, Hang Gu, and Xin Ni
Chen Xu, Xingji You, Weina Liu, Qianqian Sun, Xiaoying Ding, Ying Huang, and Xin Ni
Prostaglandin F2α (PGF2A) has multiple roles in the birth process in addition to its vital contractile role. Our previous study has demonstrated that PGF2A can modulate uterine activation proteins (UAPs) in cultured pregnant human myometrial smooth muscle cells (HMSMCs). The objective of this study was to define the signalling pathways responsible for PGF2A modulation of UAPs in myometrium. It was found that PGF2A stimulated the expression of (GJA1) connexin 43 (CX43), prostaglandin endoperoxide synthase 2 (PTGS2) and oxytocin receptor (OTR) in cultured HMSMCs. The inhibitors of phospholipase C (PLC) and protein kinase C (PKC) blocked PGF2A-stimulated expression of CX43. The inhibitors of ERK, P38 and NFκB also blocked the effect of PGF2A on CX43 expression, whereas PI3K and calcineurin/nuclear factor of activated T-cells (NFAT) pathway inhibitors did not reverse the effect of PGF2A on CX43. For PTGS2 and OTR, PLC, PI3K, P38 and calcineurin/NFAT signalling pathways were involved in PGF2A action, whereas PKC and NFκB signalling were not involved. In addition, PGF2A activated NFAT, PI3K, NFκB, ERK and P38 signalling pathways. Our data suggest that PGF2A stimulates CX43, PTGS2 and OTR through divergent signalling pathways.
Xingji You, Zixi Chen, Huina Zhao, Chen Xu, Weina Liu, Qianqian Sun, Ping He, Hang Gu, and Xin Ni
Recent evidence suggests that uterine activation for labor is associated with inflammation within uterine tissues. Hydrogen sulfide (H2S) plays a critical role in inflammatory responses in various tissues. Our previous study has shown that human myometrium produces H2S via its generating enzymes cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) during pregnancy. We therefore explored whether H2S plays a role in the maintenance of uterine quiescence during pregnancy. Human myometrial biopsies were obtained from pregnant women at term. Uterine smooth muscle cells (UMSCs) isolated from myometrial tissues were treated with various reagents including H2S. The protein expression of CSE, CBS and contraction-associated proteins (CAPs) including connexin 43, oxytocin receptor and prostaglandin F2α receptor determined by Western blot. The levels of cytokines were measured by ELISA. The results showed that CSE and CBS expression inversely correlated to the levels of CAPs and activated NF-κB in pregnant myometrial tissues. H2S inhibited the expression of CAPs, NF-κB activation and the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα) in cultured USMCs. IL-1β treatment reversed H2S inhibition of CAPs. Knockdown of CSE and CBS prevented H2S suppression of inflammation. H2S modulation of inflammation is through KATP channels and phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways. H2S activation of PI3K and ERK signaling is dependent on KATP channels. Our data suggest that H2S suppresses the expression of CAPs via inhibition of inflammation in myometrium. Endogenous H2S is one of the key factors in maintenance of uterine quiescence during pregnancy.