The Study Of Steroid Regulations Of Placental CRH Gene Transcription And Their Mechanisms

Corticotropin-releasing hormone (CRH), first indentificd in the hypothalamus, is also produced by the human placenta. During human pregnancy, placenta! syncytiotrophoblasts secrete large amounts of CRH into the maternal and fetal circulations. Several studies strongly suggest that placenta! CRH production is linked to the length of human pregnancy and abnormal elevated CRH production may lead to preterm delivery. Thus the mechanisms govering CRH synthesis have become of considerable interest.A variety of endogenous factors are known to regulate placental CRH production. The steroids which play pivotal roles in the pregnancy could also influence placental CRH gene expression. The mechanism by which the estrogen and progesterone regulating CRH gene transcription is unclear. In this study we firstly observed the effect of estrogen on the CRH gene transcription and determined which estrogen receptor mediating estrogen regulation of CRH gene expression and the sequences in CRH promoter responsible for estrogen response. Secondly, we examined the expression of progesterone receptor (PR) and glococorticoid receptor (GR) in the trophoblast and observed the effect of progesterone on the CRH gene transcription, and the receptor and regulatory elements responsible for progesterone response. Finally we studied the regulation of CRH gene promoter activity by cAMP and PMA and the effects of estrogen and porgesterone on cAMP- or PMA-induced CRH promoter activity.Main results and conclusions are as followings:1. E2 represssed CRH gene promoter activity in a time and dose-dependent manner. ICI, a pure estrogen receptor antagonist, blocked E2 effects. Morever, ICI itself stimulated CRH gene transcription, suggesting that ER mediate E2 action andendogenous estrogen has a tonic inhibitory effect on CRH gene transcription. The human ERa and ERp expression constructs were cotransfected with CRH reporter genes into placental cells. Significant changes in promoter activity were achieved upon E2 or ICI treatmen after cotransfection with ERa. Overexpression of human ERp in the cells, no significant changes were found by the exposure to E, or ICI. These suggest that ERa mediate the E2 and ICI transcriptional regulation. The analysis of 5'-deletion of CRH promoter sequences indicates that the region between -248 and -213 bp be essential for estrogen responsiveness. Further studies had identified the consensus cAMP regulatory element (CRE), located in this region, was required for estrogen actions since the mutation of these motifs abolished the estrogen effects. It was also shown that the CRE sequence could confer estrogen inhibition upon a heterologous promoter (rabbit (3-globin).2.Western blots results showed that placental trophoblasts expressed GR, PRA and PRB, but GR predominant and PRB is more than PRA. RU38486(106M), a progesterone receptor antagonist, and trilostane(10-6M) , a 3-PHSD inhibitor, could stimulate placental CRH gene promoter activity. P4(10-7M) reversed RU or trilostane effects. The GR, PRA and PRB expression vectors were cotransfected with CRH reporter genes into the placental cells respectively. When GR or PRA is overexpressed in the cells, RU(106M) or trilostane (10-6M) upregulated the CRH promoter activity and P4(10-7M) blocked the upregulation by RU and trilostane. When the PRB overexpressed, RU(10"6M) or trilostane (10"6M) inhibited the CRH promoter activity and P4(10-7M) blocked the inhibition. These results suggest that the regulation of CRH gene transcriptional activity by P4 is the net result of P4 interacting with PRA, PRB and GR. Further studies showed that CRE is also required for progesterone actions.3. 8-Br-cAMP, a PKA activator, and PMA, a PKC activator, could stimulate CRH gene promoter activity through CRE. E, decreased cAMP- or PMA-stimulated promoter activity, whereas ICI exhibited an enhancing effect on cAMP- or PMA-stimulated promoter activity. When the cells overexpressed GR or PRA, progesteronetreatment decreased cAMP- or PMA-stimulated promoter activity, whereas progesteron...