Effects Of PPARγ On Invasion In Human Cytotrophoblast Of Early Pregnancy And Its Mechanism

IntroductionImplantation of the human conceptus involves invasion of the uterine epithelium and the underlying stroma by extraembryonic trophoblastic cells, which undergo a complex process of proliferation,migration, and differentiation. As the fetal placental cells,named trophoblasts,are in direct contact with maternal blood, the human placenta has been classified as hemomonochorial.One particularity of human placentation is therefore the very high degree of trophoblast invasion during the first trimester, unparalleled in other mammals.Indeed, the cytotrophoblasts located at the tip of the villi contacting the uterine wall proliferate to form multilayered columns of cells that rapidly invade the uterus. These cells, named extravillous cytotrophoblasts(EVCT), nvade the deciduas and the upper third of the myometrium.EVCT also invade the uterine arterioles and replace the endothelial lining and most of the musculoelastic tissue of the vessel wall. This arteriole remodeling leads to low resistance vessels that provide an adequate supply of maternal blood to the intervillous space necessary for fetal growth.Defective invasion of the uterine spiral arteries is directly involved in preeclampsia, a major and frequent complication of human pregnancy with serious fetal and maternal consequences. The human trophoblastic invasion, unlike tumor invasion, is precisely regulated. It is temporally restricted to early pregnancy, and it is spatially confined to the endometrium, the first third of the myometrium, and the associated spiral arterioles.Therefore, it offers a unique model of a controlled and oriented cell invasion process, which remains poorly understood.Therefore, the invasion process to proceed smoothly, which is an essensial for development of placenta and maintenance of normal pregnancy. Although molecular mechanism is not clear at present of control invasion of trophoblast, but many study indicate that the invasion process can adjusted fine by trophoblast and the secretion of regional microenvironment, such as hormone,cytokine,growth factor,extracellular matrix glucoprotein and various kinds of transcription factor and so on.Peroxisome proliferators-activated receptors (PPARs) are a family of intracellular ligand-activated nuclear receptors superfamily that controls the expression of a large array of genes in a ligand-dependent manner. To date, three human PPAR isoforms have been identified: PPARα,PPARβand PPARγ. Ligands for PPARγinclude endogenous ligand 15-d-PGJ2, linoleate,fatty acid,oxidized low density lipoprotein compounds et al, as well as its synthetic Ligands, such as the antidiabetic thiazolidinedione drug Troglitazone,Rosiglitazone,non-steroid anti-inflammatory drug as indomethacin and Brufen et al. Investigate indicate that on activation, PPARγheterodimerize with their common nuclear receptor binding partner, the retinoid X receptor(RXR)and activation by their ligands. PPAR/RXR heterodimers bind to a PPAR response element(PPRE) located in the promoter region of target genes and regulate their transcription, and control proliferation,invasion,differentiation and apoptosis.Recent genetic studies performed in mice established that nuclear hormone receptors PPARγare essential for placental development and vasculature. Indeed, PARγis requested not only for trophoblast differentiation,but also trophoblast maturation to establish maternal-fetal transport. These studies suggested that PPARγheterodimers might be essential for implantation and the formation of a functional placenta in mice. Whether PPARγalso play an essential role in the human placental development remains, however, largely unknown. Initial study in the human placenta indicate that there is expression of PPARγin trophoblast,and identified PPARγ expression in JEG-3 that derived from trophoblast of placenta. This provided evidence for deeply reseach effect of PPARγin pregnancy complication as to trophoblast abnormal invasion.The invasion is an initiative biochemistry process, the cell to have invasion,which related with that the cell can secrete the protease and to dissolve extracellular matrix. Trophoblast invasion have not exception.Although its mechanism still to remain not clear, but studies indicate that trophoblast invasion related with the placental development is regulated by MMPs. In the invasion process trophoblast can expression a various kinds of MMPs, but effect of all MMPs on trophoblast invasion is not same essensial. Because MMP-2 and MMP-9 resolve an essential component typeⅣcollagen of basal membrane, it is key enzyme that involved in thophoblast invasion. Many placental hormone and cytokine rerulate thophoblast invasion by control secrete of MMPs.Although some sdudies hint that PPARγligand related with trophoblast invasion, which can represent a promising, novel therapeutic approach for abnormal invasion of trophoblast. But concrete mechanism of action is not clear yet. In a variety of types of cells,there are sdudies indicate that PPARγto produce a marked effect by regulation MMPs. Liu et al research indicate that activation of PPARγby ligands in myeloid leukemia cells K562 and HL-60, and can inhibit K562 and HL-60 cell adhesion to and invasion through the extracellular matrix as well as downregulate MMP-9 and MMP-2 expressions. The data suggest that PPARγligands may serve as potential anti-leukemia reagents. Zafiriou S et al demonstrate that PPARγactivator Piglitazone can significantly improve process of the renal tubule interstitial fibrosis through inhibition the cell growth and down regulation the activation of MMP-9,TIMP-1 and TIMP-2．Chang et al induced the cell differentiation of nonsmall-cell lung cancer and reversed the malignant phenotype of tumor cell with PPARγligands, and showed that inhibit the expression of MMPs and anchoring independent growth of the tumor cell. Therefore,to study and research the relation PPARγwith MMPs, to identify mechanism of action of PPARγin the tissue and cell and will provide valuable scientific research data.ObjectiveThe objectives of our current study were to determine:investigate the relation of PPARγ,MMP-2 and MMP-9 with trophoblast invasion by detection the protein and mRNA location and expression of PPARγ,MMP-2 and MMP-9 in the placenta during week 6 to 8 and 11 to 12 of gestations; The effect of PPARγactivator 15-d-PGJ2 and Troglitazone as well as antagon ligand BADGE on the invasion of cultured human cytotrophoblast cells during the week 6 to 8 and 11 to 12 of gestations; The regulation of PPARγactivator 15-d-PGJ2 and Troglitazone on the expression of MMP-2 and MMP-9 in cultured human cytotrophoblast cells during the week 6 to 8 of gestations,and deeply identify the relationship between PPARγand the cytotrophoblast invasion as well as the mechanism of action,to provide theoretical experiment basement for application of PPARγligand drug to clinic ultimum.MethodsOur research was detected the expression of the protein and mRNA of PPARγ,MMP-2 and MMP-9 in the first trimester pregnancy by immunohistochemistry,Real Time RT-PCR and Western blot technique; Separated cytotrophoblast by cell culture technique and cultured use serum-free medium, observed and detected the expression of PPARγin cultured human cytotrophoblast by immunofluorescence cytochemistry,invadion assay in vitro and electron microscopy technique; Observed and detected the regulation of PPARγligand on MMP-2 and MMP-9 expressed by cultured human cytotrophoblast through immunofluorescence confocal,Real Time RT-PCR and Western blot technique.Rusults1．PPARγprotein is located manly in in the nuclei of cytotrophoblasts from proximal and distal columns of the anchoring villi and villous cytotrophoblast, MMP-2 and MMP-9 is located manly in the intracytoplasm of cytotrophoblast and syncytiocytotrophoblast and the villous stromal core as well. The expression of PPARγwere higher in week 6 to 8 of gestation than in week 11 to 12, and The expression of MMP-2 and MMP-9 more in week 11 to 12 of gestation than in week 6 to 8, there is the negative correlation between PPARγand MMP-2,MMP-9．2．There is the expression of PPARγin surum-free cultured human cytotrophoblast in vitro; Both of PPARγnatural activator 15-d-PGJ2 and synthetical activator Troglitazone at concentrations ranging from 0．1～10μmol/liter inhibit the trophoblast invasion after cultured 48 hours in a concentration-dependent manner, and the effect of 15-d-PGJ2 stronger than Troglitazone. The inhibitory effect of 15-d-PGJ2 (1,10μmol/liter) and Troglitazone (10μmol/liter) on the trophoblast invasion was considered statistically significant. And inhibitory effect of both on cytotrophoblast in gestation week 6 to 8 stroger than in gestation week 11 to 12, and difference between them is significant. PPARγantagonist BADGE at concentrations 0,20,50μmol/liter can promote the trophoblast invasion in a concentration-dependent manner, BADGE (50μmol/liter) increased trophoblast invasion by about 50%, and it must be noted that the antagonist BADGE reversed partly the inhibitory effect of the PPARγagonists. cytotrophoblast invasion was obviously reinforced in combination cultured of BADGE(50μmol/liter) and 15-d-PGJ2 (10μmol/ liter) than in 15-d-PGJ2 (10μmol/liter) cultured alone,and difference is significant. Even if cytotrophoblast invasion was reinforced in combination cultured of BADGE ( 50μmol/liter) and Troglitazone(10μmol/liter) than in Troglitazone (10μmol/liter) cultured alone, but difference was considered not significant.3．Both of PPARγnatural activator 15-d-PGJ2 and synthetical activator Troglitazone at concentrations ranging from 0．1～10μmol/liter inhibit the expression of MMP-2 and MMP-9 mRNA and protein in a concentration-dependent manner after cultured 48 hours. The expression of MMP-2 and MMP-9 mRNA and protein decreased significant, while 15-d-PGJ2 at concentrations 0．1mol/liter,1μmol/liter and 10μmol/liter, Troglitazone at concentrations 1μmol/liter and 10μmol/liter, compared with control difference is clear. The down regulatory effect of 15-d-PGJ2 on the expression of MMP-2 and MMP-9 mRNA and protein obviously stroger than Troglitazone, and was considered statistically significant.Conclusions1．There are the expression PPARγ,MMP-2 and MMP-9 in human trophoblast of early pregnancy, and PPARγexpression decreased gradually while MMP-2 and MMP-9 increased gradually from early stage to late stage of first trimester pregnancy, there is the negative correlation between PPARγand MMP-2,MMP-9．2．Both of PPARγnatural activator 15-d-PGJ2 and synthetical activator Troglitazone inhibit the trophoblast invasion, and the effect of 15d-PGJ2 stronger than Troglitazone. And inhibitory effect of both on cytotrophoblast in gestation week 6 to 8 stroger than in gestation week 11 to 12．PPARγantagonist BADGE can promote the trophoblast invasion, and BADGE can reverse partly the inhibitory effect of the PPARγagonists.3．Both of PPARγnatural activator 15-d-PGJ2 and synthetical activator Troglitazone inhibit the expression of MMP-2 and MMP-9 mRNA and protein. The down regulatory effect of 15-d-PGJ2 on the expression of MMP-2 and MMP-9 mRNA and protein obviously stroger than Troglitazone.4．PPARγplay an important role in the modulation of thophoblast invasion, PPARγligands can inhibit invasion through downregulate MMP-2 and MMP-9 expressions, these provided experiment basement for deeply investigation action of PPARγligands in the mechanism of cytotrophoblast invasion.