Regulation Of 11β-HSD2 And Responsible For H₂S Production Expression In Human Placenta By Peroxisome Proliferation-activated Receptors

Glococorticoids are essential for normal fetal organ growth and maturation. However, excessive glucocorticoid exposure in uterus leads to intrauterine growth restriction (IUGR) and pregnancy-induced hypertension (PIH). The placental enzyme 11β- hydroxysteroid dehydrogenase type 2 (11β-HSD2) is believed to play a key role in protecting the fetus from exposure to high levels of maternal glucocorticoid by converting maternal cortisol to its inactive metabolite, cortisone. Indeed, placental 11β-HSD2 activity is correlated with birth weight and is attenuated in pregnancies complicated with IUGR. Thus, the precise control of placental 11β-HSD2 expression and activity appears to be critical to normal fetal development. However, the understanding of placental 11β-HSD2 regulation is incomplete.Peroxisome proliferatoractivated receptors (PPARs) are members of the nuclear receptor superfamily. Three distinct PPAR subtypes have been identified, namely, PPAR?, PPAR??(also known as PPAR?) and PPAR?, with unique tissue distributions and physiological functions. Three PPAR subtypes have been found to express in the human placenta, which function in this organ is unclear. Recently PPARs-null mice exhibit placental defects and reduced birth weight, indicating a pivotal role for this nuclear receptor in placental function and fetal development. We hypothesized that this nuclear receptor may regulate human placental function in part by targeting 11β-HSD2. In the first part of present study, we conducted experiments to determine the modulation and mechanism of PPARs on 11β-HSD2 using cultured human placental trophoblast cells as a model system.The pharmacological, physiological and pathological roles of gasotransmitters nitric oxide (NO) and carbon monoxide (CO) have been extensively researched in the reproductive system. Hydrogen sulphide (H₂S) is another gasotransmitter that has many parallels with NO and CO. However there are few reports to date on the production and function of H₂S in reproductive tissues. H₂S is endogenously produced from L-cysteine by two pyridoxal 5' phosphate-dependent enzymes cystathionineβ-synthase (CBS) and cystathionineγ-lyase (CSE). Recently the presence of CBS and CSE enzymes was demonstrated in human intrauterine tissues including placenta. However the role and regulation of H₂S in the placenta are unclear. In the second part, we conducted experiments to observe expression level of H₂S synthetases in normal placenta and PIH placenta; to determine if PPARs modulate the expression of CBS and CSE in human placental trophoblast. As we known, Corticotropin-Releasing Hormone (CRH) takes a vital part in the progression of pregnancy and initiation of labor in human. However, the mechanism of CRH regulation is fully uncovered. So further experiment was conducted to observe whether H₂S and PPARs influence the synthesis and release of CRH in human placenta.Main results and conclusions are as follows:1 . Regulation and Mechanism of 11β-HSD2 Expression in Human Placenta Syncytiotrophoblasts by PPARs1) Treatment of placental cells with PPARγagonist GW7647 caused a decrease in the mRNA and protein expression as well as the activity of 11β-HSD2. These effects could be blocked by PPARγantagonist GW6471. PPARγagonist rosiglitazone dose-dependently stimulated the expression and activity of 11β-HSD2. PPARγantagonist GW9662 reversed the effects of rosiglitazone.2) Placental Cells were treated with PPARs agonists in the absence and presence of CHX, a protein synthesis inhibitor. It was found that GW7647 and rosiglitazone were equally effective in modulating 11β-HSD2 mRNA in the absence and presence of CHX, indicating that de novo protein synthesis was not required.3) RXR ligand 9-cis-RA alone could increase the expression of 11β-HSD2 in the placental cells. And 9-cis-RA potentiated the effect induced by PPAR? agonist. Transfected the placental cells with RXRαsiRNA abolished the effect by rosiglitazone, whereas the effect of GW7647 was not influenced. The results suggested that the modulation of PPARγnot PPAR? depended on RXRα.4) To elucidate the molecular mechanisms by which PPARs regulated 11β-HSD2 mRNA, cells were treated with DRB, an inhibitor of mRNA synthesis. The results showed that GW7647 and rosiglitazone did not alter the half-life of 11β-HSD2 mRNA. 5) GW7647 repressed whereas rosiglitazone stimulated the promoter activity of 11β-HSD2 gene. GW6471 and GW9662 could reverse the effects of GW7647 and rosiglitazone respectively. The results suggested that GW7647 and rosiglitazone could regulate genetic transcription of 11β-HSD2.6) Western blot hybridization and real-time PCR showed that the expression of 11β-HSD2, ?and PPARγwere significantly lower in placentas from women with PIH compared with those with normal pregnant, while the expression of PPARγand PPARγwas opposite. Moreover, Statistical analyses showed that there is a negative correlation between the expression of 11β-HSD2 and PPARγ, whereas a positive correlation existed between the expression of 11β-HSD2 and PPARγin human placenta. Results suggested that PPARs involve in maintain of 11β-HSD2 expression in placenta.2.Regulation of Synthetases Responsible for H₂S Production in Human Placenta Syncytiotrophoblasts by PPARs.1) Both H₂S synthetases, CBS and CSE, express in human placenta showed by immunochemistry.2) Treatment the placental cells with L-cysteine or sodium hydrosulfide, CRH mRNA expression and CRH IR concentration in media and cell lysis of cells decreased. The effect of L-cycsteine was blocked in the presence of CSE antagonist PAG or CBS antagonist AOAA. Meanwhile D-cysteine had no effect on CRH mRNA and IR concentration. The results suggested that H₂S regulate CRH synthesis and secretion in placenta.3) PPARγagonist GW7647 and PPARβagonist GW0742 significantly down-regulated the mRNA and protein expression of CBS and CSE in placental trophoblast cells, while PPARγagonist rosiglitazone stimulated the expression of CBS and CSE. The above effects of PPARs were reversed by PPARs subtypes antagonists repectively.4) Treatment the placental cells with PPARαand PPARβagonists, CRH mRNA expression and IR concentration in media and cell lysis of cells increased. The effects of PPARγand PPARγwere reversed by PPARγand PPARγantagonist respectively. Meanwhile PPARγhad down-regulation on CRH mRNA expression and IR concentration.5) The mRNA and protein expression of CBS and CSE were significantly decreased in PIH placental biopsies compared to those of normal samples. Statistical analyses showed that there is a negative correlation between PPARγand CBS, PPARγand CSE, while a positive correlation existed between CSE and PPARγin human placenta. To define the real-time kinetics of H₂S production by placenta, we used miniaturized H₂S micro-respiration sensor to measure its production. Results showed that the level of H₂S production in PIH placenta significantly decreased compared to normal placenta. It suggested that H₂S maybe involve in the pathological progress of PIH.In summary, the above results demonstrated:These data suggested that PPARγand PPARγdifferentially modulate the expression and activity of 11β-HSD2 in human trophoblast cells, these effects are mediated primarily at transcriptional level. Moreover, the results suggested that PPARs regulate the expression of CBS and CSE in human placenta, which effects were different. Furthermore, the above effects could modulate the production of H₂S in placenta, which further effect the synthesis and secrete of CRH in placenta. In fact the results showed that both H2S and PPARs subtypes coulud modulate the synthsis and secretion of CRH in placnta.Also the results showed that there is a correlation between the expression of 11β-HSD2 and PPARs, H2S Synthetases and PPARs in human placenta. Thus, the present study indicates that PPARs may modulate the human placental function, and, consequently, fetal development.