The Role And Mechanism Of PPARγ In Lipopolysaccharide-induced Downregulation Of 11β-HSD2 In Placental Trophoblasts

Lipopolysaccharide（LPS）is the main toxic component of gram-negative bacteria.It is well known that gestational LPS exposure is associated with adverse pregnant outcomes,maternal LPS exposure at late gestational stage caused fetal intrauterine growth restriction（IUGR）.Intrauterine growth restriction（IUGR）refers to the failure of a fetus to reach its genetic growth potential.Clinically,IUGR is defined that the neonate weight is below the 10th percentile or two standard deviations for same baby’s gestational age and gender.IUGR not only is the main cause of high morbidity and mortality of perinatal fetuses,but also increases the risks of chronic diseases during adulthood.At present,the incidence of IUGR is very high in China and all over the world.However,the potential molecular mechanism of LPS-induced IUGR is unclear.Accumulating data demonstrate that over-exposure of the fetus to glucocorticoids not only causes fetal growth restriction but also programs the development of chronic diseases in later life.In many species,including amphibians,reptiles,rodents and birds,corticosterone is a major biologically active glucocorticoid,while in humans,cortisol is the primary biologically active glucocorticoid.GC involves in maintaining normal human pregnancy,and also plays a significant role in development and delivery of fetus.Therefore,it is essential to control glucocorticoids at optimal levels in the fetal circulation to ensure a safe intrauterine development.It is widely accepted that the glucocorticoid inactivating enzyme,11β-hydroxysteroid dehydrogenase 2（11β-HSD2）,acts as the placental glucocorticoid barrier,and it exclusively localized to the syncytiotrophoblast of the chorionic villi.Several studies have indicated that 11β-HSD2 catalyzes active glucocorticoids,such as cortisol,into their inactive products,such as cortisone,thus preventing glucocorticoids from maternal circulation into the fetus.A few studies have found that gestational LPS exposure could inhibit placental 11β-HSD2 expression and activity.11β-HSD2deficiency would destroy placental barrier and induced IUGR.These results indicate maternal gestational LPS exposure causes IUGR through repressing placental 11β-HSD2 and destroys placental glucocorticoid barrier.However,the potential molecular mechanism of LPS-induced reduction of placental 11β-HSD2has not yet been elucidated.1.The effects of LPS exposure on the expression of 11β-HSD2 in mice placentas and human placental trophoblastsICR pregnant mice were divided into five groups:Control group,LPS 2 h group,LPS 6 h group,LPS 12 h group and LPS 24 h group.Pregnant mice received an intraperitoneal（i.p.）injection of LPS（200μg/kg）or normal saline（NS）on gestational day（GD）16.Pregnant mice were anesthetized and placenta tissues were collected at 0 h,2 h,6 h,12 h and 24 h after LPS injection.Placental 11β-HSD2 protein expression and mRNA were detected.Results showed that placental 11β-Hsd2 mRNA was decreased in mice,beginning at 2 h and remained reducing 6 h after LPS injection.The expression of placental 11β-HSD2 protein was accordingly downregulation at 12 h and further reduced at 24 h after LPS injection.Human placental trophoblasts were divided five groups:Control group,LPS 2 h group,LPS 6 h group,LPS 12 h group and LPS 24 h group.Human HTR-8/SVneo cells were incubated with LPS（2μg/mL）or PBS,and collected at 0 h,2 h,6 h,12 h and 24 h after LPS exposure.The levels of 11β-HSD2 protein and mRNA were detected in human placental trophoblasts.Results showed that the level of 11β-HSD2 mRNA in HTR-8/SVneo cells was downregulation,beginning at 2 h and remained decreasing 24 h after LPS exposure.The expression of 11β-HSD2 protein in HTR-8/SVneo cells was downregulation at 12 h and further reduced at 24 h after LPS exposure.2.The effect of PPARγagonist on the expression of 11β-HSD2 in human placental trophoblasts.HTR-8/SVneo cells were divided into three groups:Control group,RSG（10^-77 M）group,RSG（10^-66 M）group.HTR-8/SVneo cells were incubated with different concentrations of RSG for 24 h and harvested.The expressions of PPARγ（Peroxisome proliferator-activated receptor gamma）and 11β-HSD2 were measured.Results showed that pretreatment with RSG significantly elevated the level of nuclear PPARγin HTR-8/SVneo cells in a dose-dependent manner.Accordingly,pretreatment with RSG also markedly upregulated the expression of 11β-HSD2 in HTR-8/SVneo cells in a dose-dependent manner.HTR-8/SVneo cells were incubated with different concentrations of GW9662(0,10^-3,10^-2 M)for 24 h and then treated with RSG(0,10^-6 M)for 24 h.HTR-8/SVneo cells were harvested.The expression of PPARγand 11β-HSD2 were measured.Results showed that GW9662 attenuated RSG-induced elevation of nuclear PPARγin human HTR-8/SVneo cells.Accordingly,GW9662 attenuated RSG-induced upregulation of11β-HSD2 in HTR-8/SVneo cells.3.The effects of LPS on PPARγand its target genes in mice placentas.Pregnant mice received an i.p.injection of LPS（200μg/kg）or NS on GD16.Pregnant mice were anesthetized and placenta tissues were collected at 0 h,2 h,6 h,12h and 24 h after LPS injection.PPARγnuclear translocation and its target genes were analyzed.Results showed that placental PPARγnuclear translocation was reduction,beginning at 12 h and remained decreasing 24 h after LPS injection.The levels of placental Vegf and Cd36 mRNAs,two target genes of PPARγ,were downregulation in mice injected with LPS.The effects of LPS on RSG-induced PPARγnuclear translocation and its target genes in mice were analyzed.All pregnant mice except controls were orally administered with RSG（10 mg/kg）daily from GD13 to GD16.In the LPS and LPS+RSG groups,pregnant mice were i.p.injected with LPS（200μg/kg）on GD16.Placentas were collected at 24 h after LPS injection.Interestingly,a strong PPARγimmunoreactivity was observed in mononuclear sinusoidal trophoblast giant cells of labyrinth zone in mice pretreated with RSG.Accordingly,gestational LPS exposure inhibited RSG-induced nuclear translocation of PPARγin mice placentas.Placental Cd36 mRNA was obviously elevated in mice pretreated with RSG,LPS injection significnaty suppressed RSG-induced elevation of Cd36 mRNA in mice placentas.However,RSG pretreatment had no effect on Vegf mRNA in mice placentas.4.The effect of LPS on nuclear translocation of PPARγin human placental trophoblasts.HTR-8/SVneo cells were incubated with LPS（2μg/mL）and harvested at different time points.PPARγnuclear translocation was measured.The results showed that nuclear translocation of PPARγin LPS-treated HTR-8/SVneo cells,was significantly reduction,beginning at 2 h and further decreased at 24 h after LPS exposure.Immunofluorescent intensity of PPARγwas weakened in the nuclei of LPS-treated HTR-8/SVneo cells.The effect of LPS on RSG-induced PPARγactivation in human placental trophoblasts was then analyzed.HTR-8/SVneo cells were incubated with or without RSG（10^-66 M）for 24 h and cocultured with LPS（2μg/mL）for additional 6 h.PPARγnuclear translocation and immunofluorescent intensity were determined.These results showed that LPS blocked RSG-induced elevation of PPARγnuclear translocation in HTR-8/SVneo cells.5.The effects of LPS on NF-κB signaling in mice placentas and human placental trophoblasts.The effect of gestational LPS exposure on placental NF-κB activation was analyzed.Pregnant mice received an i.p.injection of LPS（200μg/kg）on GD16.The placentas were collected at different time points after LPS injection.NF-κB p65 and p50 were measured.NF-κB p65 nuclear protein in mice placentas was markedly increased at 2 h and remained elevating 6 h after LPS exposure.Moreover,placental NF-κB p50 nuclear protein was increased at 2 h and further elevated 6 h after LPS exposure.The effect of LPS on NF-κB in human placental trophoblasts was then analyzed.HTR-8/SVneo cells were incubated with LPS（2μg/mL）.The cells were harvested at different time points.As expected,NF-κB p65 nuclear protein in HTR-8/SVneo cells was markedly increased at 6 h and remained elevating at 12 h after LPS exposure.Moreover,nuclear NF-κB p50 protein in HTR-8/SVneo cells was elevated at 6 h and further increased at 12 h after LPS exposure.6.The effects of NF-κB p65 inhibition or knockdown on LPS-induced PPARγand 11β-HSD2 downregulation in human placental trophoblasts.To verify the role of NF-κB p65 on LPS-evoked suppression of PPARγ,HTR-8/SVneo cells were treated with PDTC to inhibited NF-κB activation.HTR-8/SVneo cells were incubated with PDTC（100μM）for 1 h before LPS exposure in PDTC and LPS+PDTC group.Then HTR-8/SVneo cells were cocultured with LPS（2μg/mL）in LPS and LPS+PDTC groups.HTR-8/SVneo cells were harvested at 6 h after LPS exposure.As expected,PDTC suppressed LPS-induced elevation of NF-κB p65nuclear protein in HTR-8/SVneo cells.Pretreatment with PDTC also attenuated LPS-induced reduction of PPARγnuclear translocation.To further confirm the role of NF-κB p65 on LPS-induced suppressin of PPARγin HTR-8/SVneo cells,NF-κB p65siRNA was used to repressing NF-κB p65 expression.Cells were transfected with NF-κB p65 siRNA.Twenty-four hours after transfection,HTR-8/SVneo cells were incubated with LPS（2μg/mL）for additional 6 h in the LPS and LPS+p65 siRNA groups.Then the cells were harvested.These results showed that NF-κB p65 siRNA strongly inhibited the expression of NF-κB p65 in HTR-8/SVneo cells.Accordingly,NF-κB p65 knockdown attenuated LPS-induced reduction of PPARγnuclear translocation in HTR-8/SVneo cells.In addition,NF-κB p65 knockdown attenuated LPS-induced downregulation of 11β-HSD2 protein in HTR-8/SVneo cells.7.LPS promotes interaction between NF-κB p65 and PPARγin human placental trophoblasts.The present study further analyzed the interaction between PPARγand NF-κB p65in HTR-8/SVneo cells.HTR-8/SVneo cells were incubated with RSG（10^-66 M）for 24 h and then cocultured with LPS（2μg/mL）for additional 6 h.Cytoplasmic and nuclear proteins were isolated.Cytoplasmic fractions and nuclear fractions were incubated with agarose-conjugated antibody against PPARγ.Then we detected the expression of NF-κB p65 in cytoplasmic and nuclear proteins.The results showed that RSG plus LPS treatment elevated the level of NF-κB p65 protein in cytoplasmic and nuclear immunocomplexes.Immunofluorescence demonstrated that coculture of LPS with RSG promoted co-localization of NF-κB p65 and PPARγin the nuclei of HTR-8/SVneo cells.These results indicated that LPS reinforced that interaction between NF-κB p65 and PPARγin placental trophoblasts in cytoplasm and nucleus.8.Case-control study:The correlation analysis between PPARγ-mediated 11β-HSD2 downregulation and LPS-induced IUGR.Based on established placental specimen bank,the neonate were divided into AGA and SGA groups.SGA was defined that the neonate weight was below the 10th percentile for same baby’s gestational age and gender,AGA weight was higher the standard.We randomly selected 46 pairs of AGA and SGA placentas,and then detected placental 11β-HSD2 expression,the number of NF-κB p65 and PPARγpositive nuclei.These results showed that the expression of placental 11β-HSD2 was significantly down-regulation in SGA subjects compared with AGA subjects.The number of placental PPARγpositive nuclei in SGA subjects was less than in AGA subjects.By contrast,the number of placental NF-κB p65 positive nuclei in SGA subjects was more than in AGA subjects.The associations of neonate weights with placental NF-κB p65,PPARγand11β-HSD2 were analyzed.As expected,there was a positive correlation between neonate weight and placental 11β-HSD2 expression in AGA and SGA subjects.Despite of no significant association between neonate weight and the numbers of placental NF-κB p65 and PPARγpositive nuclei in AGA subjects,neonate weight was positively correlated with the number of placental PPARγpositive nuclei in SGA subjects.By contrast,neonate weight was negatively correlated with the number of placental NF-κB p65 positive nuclei in SGA subjects.The associations among placental NF-κB p65,PPARγand 11β-HSD2 were then analyzed.As expected,there was a positive correlation between PPARγpositive nuclei and 11β-HSD2 expression in placentas of AGA and SGA subjects.Although it wasn’t associated with PPARγand 11β-HSD2 in AGA subjects,placental NF-κB p65 was negatively correlated with placental PPARγnuclei in SGA subjects.In addition,placental NF-κB p65 was negatively correlated with placental 11β-HSD2 expression in SGA subjects.According to the above experimental results,the following conclusions can be drawn:The present study investigated the effects of LPS on the expression of11β-HSD2 in mice placentas and human placental trophoblasts.Our results showed that LPS downregulated 11β-HSD2 via suppressing PPARγnuclear translocation in placental trophoblasts.Then LPS inhibited PPARγtranscriptional activity and downstream 11β-HSD2.Mechanistically,LPS inhibited PPARγthrough promoting the interaction between NF-κB p65 and PPARγin cytoplasm and nucleus of placental trophoblasts.In population-based case-control study,both PPARγand 11β-HSD2 were down-regulation in SGA placentas.These results provide evidence that PPARγ-mediated downregulation of placental 11β-HSD2 may contribute,at least partially,to LPS-induced fetal IUGR.