Mechanisms Involved In The Regulation Of Human Chorion PGDH By Steroid Hormone And PPARs

Prostaglandin(PG) produced within the intrauterine tissues play a central role in theinitiation and progression of labor in human. It has been shown that PG maintain uterineand placental blood flow, regulate the cervical ripening, membrane rupture and induceuterine contractility. PG is synthetized by Prostaglandin-H-synthase (PGHS), whilemetabolised by15-hydroxyprostaglandin dehydrogenase (PGDH). Not only PGHS but alsoPGDH is expressed in chorion during pregnancy. However a very high concentration ofPGDH has been localized in chorion.So high level PG is synthetized in amnion,metabolised in chorion,and after acrossing full thickness membranes becomes lower inplacenta and myometrium. Several reports have indicated that chorion has been describedas a protective barrier to prevent the passage of primary PG synthesized within the amnionor chorion from reaching the myometrium and stimulating the onset of preterm or termdelivery. But the regulation of chorion PGDH has not yet been elucidated.Although glucocorticoids and progesterone were demonstrated that could regulate theexpression and activity of PGDH in chorion via bingding glucocorticoid receptor(GR) orprogesterone receptor(PR),the mechanism of their effect is unclear. Chorion expresses bothGR, PRA and PRB—two types of PR.We conducted expreriments to determine thereceptors mediating the effect of GC and P4on PGDH in chorion.We treated choriontrophoblast cells with dexamethasone, progesterone and trilostane (an inhibitor of3β-HSD), to determine PGDH activity and expression. To examine the role of GR and PR onPGDH, we constructed GR siRNA, PR siRNA and their overexpression plasmid.Peroxisome proliferator-activated receptors (PPARs) are transcription factorsbelonging to the ligand-activated nuclear hormone receptor superfamily,and present threeisotypes:α,β andγ. High expression of PPARs has been localized in human intrauterinetissues,such as amnion,chorion,placenta.PPARs are known not only to be involved in thephysiological and pathological events occurring during the placentation, but also contributeto the control of parturition in humans. term human labour is associated with changes inexpression and activity of PPAR isoforms. The production of prostaglandins by the fetalmembranes induces the contraction of the myometrium during labor. This generation ofuterotonic prostaglandins correlates with the increased COX-2activity and the increasedsecretory sPLA2mRNA, proteins and activities. By inhibiting the production of theCOX-2and sPLA2in fetal membranes, PPARs promotes the quiescence of the uterusduring gestation.In the second part of this subject, We culture chorion trophoblast cells to examine the regulation and its mechanism of PPARs on PGDH.The gas hydrogen sulfide (H2S) is emerging as a novel regulator of importantphysiologic functions such as arterial diameter, blood flow and leukocyte adhesion. Inaddition, it may have anti-inflammatory and anti-apoptotic effects. H2S has recentlyattracted much interest as a new gaseous signal molecule.However there is few reportsabout physiological and pathological events caused by H2S in the reproductive system.In2009,Patel et al published the first study to report the detection of both CBS and CSE inhuman intrauterine tissues and the production of H2S by these tissues.They considered thatendogenously produced H2S could possibly have a role in the pathology of pre-eclampsia.However further investigation of the role of H2S in the reproductive system is required.Inthis part,we examined the expression of H2S synthesis enzyme CSE,CBS in fetalmembranes (chorion and amnion).The main results:1.Regulation of PGDH by Glucocorticoid and Progesterone in human Chorion1). In nonlabor chorion tissue, PGDH/PRB mRNA and protein expression issignificantly higher than those in labor tissue,however PRA/GR mRNA and proteinexpression in nonlabor chorion tissue is much lower than in labor. Moreover, Statisticalanalyses showed that there is a negative correlation between the expression of PGDH andGR.2). PGDH protein and mRNA expression levels were detected by Western blot andreal-time quantitative PCR assay respective. DEX and Trilostane dose dependentlydown-regulated the mRNA and protein expression of PGDH. Treatment of chorion cellswith P4caused a dose-dependently increase in the mRNA and protein expression ofPGDH.3) Using RNA interference and over-expression technology to prevent and promoteGR and PR expression respectively,then treated cells with Dex,Tril and P4, PGDH proteinlevels were detected by Western blot.PGDH activity and protein expression is decreased byPR interference and GR/PRA over-expression,while is increased by GR interference andPRB over-expression.Dex decrease effect is also exist moderately with GR interferenceand is doubled with GR over-expression.PR interference and PRa/PRb over-expressioncannot change the effect strength of Dex on PGDH. P4promoted the PGDH activity andprotein expression further with GR interference and PRB over-expression,while inhibitedwith PR interference and GR/PRA over-expression.Trilostane down-regulated the protein level and activity of PGDH.4) With the regulation of steroids on chorion PGDH gene transcription activity ofrelated D NA sequences identified by5’ terminal deletion methods obtained with differentlength of PGDH gene luciferase reporter gene expression vector. Dex, P4and Tril activityhas obvious inhibition or activation effect to PGDH promoter transcription of a reportergene containing-2368bp and-2038bp, and has no moderating effect activity to the onecontaining-1024bp,-388bp and-233bp.2.Regulation and mechanism of PGDH by Peroxisome proliferator-activatedreceptors (PPARs) in human Chorion1) A increase in the mRNA and protein expression of PGDH is caused by thetreatment of chorion cells with PPARα、PPARβ、PPARγ agonist GW7467、GW0742、Rosiglitazone（10-9～10-6M）and a decrease is caused by the antagonist GW6471、GSK0661、GW9662（10-9～10-6M）.These increase effects could be blocked by theantagonists.2) Using RNA interference technology to prevent PPARα、PPARβ、PPARγ expressionrespectively,then treated cells with GW7467、GW0742、Rosiglitazone, PGDH proteinlevels were detected by Western blot. PGDH protein expression is decreased by PPARsinterference.However GW7467, GW0742and Rosiglitazon can not continue to increasethe PGDH protein expression.The results showed that the upregulation of PPARs onPGDH expression is not an drug effects of agonists,but is the effects of PPARs own.3)RXR ligand9-cis-RA alone could increase the expression of PGDH in the chorioncells. And9-cis-RA potentiated the effect induced by PPARs agonists.4) Cells were treated with PPARs agonists in the absence and presence of CHX, aprotein synthesis inhibitor. It was found that GW7467,GW0742and Rosiglitazone wereequally effective in modulating PGDH mRNA in the absence and presence of CHX,indicating that denovo protein synthesis was not required.4) To elucidate the molecular mechanisms by which PPARs regulated PGDH mRNA,cells were treated with DRB, an inhibitor of mRNA synthesis. The results showed thatGW7467,GW0742and Rosiglitazone did not alter the half-life of PGDH mRNA.6) Western blot hybridization and real-time PCR showed that the expression ofPPARγ were significantly lower in labor chorion tissue compared with those in nonlabor,while the expression of PPARα and PPARβ had no change. Results suggested that PPARγinvolve in maintain of PGDH expression in chorion. 3. Expression of synthetases responsible for H2S production in huamanlabor,nonlabor and preeclampsia fetal membranes1). Both H2S synthetases,CBS and CSE，express in human amnion epithelial cells andchorion trophoblast cells showed by immunochemistry．2). RT-PCR and Western-blot were used to define the mRNA and protein expressionof CSE and CBS.To define the real-time kinetics of H2S production by fetal membranes，we used miniaturized H2S micro-respiration sensor to measure its production.Resultsshowed that lower level of H2S production,mRNA and protein expression of CSE and CBSin both labor chorion and amnion compared with nonlabor tissue,and higher level innormal chorion and amnion compared with preeclampsia tissue. It suggests that H2S mayinvolves in pregnancy progress and pregnant blood pressure control.Conclusion:1.In vitro human chorion trophoblast cells, the endogenic Progesterone maintain theexpression of PGDH. Howeve the regulation of exogenetic Progesterone on PGDH isdifferent via PR or GR. Exogenetic Progesterone promotes PGDH via PRb mainly, whileinhibit PGDH via GR and PRa. Glucocorticoid inhibit PGDH via GR. They worktogether to contribute to the normal concentration of PGDH in chorion.And there is areporter gene tips-2038to-1024between the base sequence containing the key sequenceof Dex, P4and Tril regulation of PGDH gene expression.2. PPARs modulate the PGDH expression in human chorion trophoblast cells, theseeffects are mediated primarily at transcriptional level. PPARγ involve in maintain ofPGDH expression in chorion tissue.3. H2S and its synthetic enzymes CBS, CBS expressed significantly different in humanfetal membranes of different pregnancy status.H2S may be involved in the parturition andthe of pathological process of preeclampsia.