1.Studies On Synergistic Effect And Mechanism Of Up-regulation Of Plasminogen Activator Inhibitor-1 By Glucocorticoid And Transforming Growth Factor-β1 In Ovarian Cancer HO-8910 Cells 2.Hypoxia Up-regulates The Expression Of Stomation In Cerebral Cortex

PartⅠStudies on Synergistic Effect and Mechanism of Up-regulation of Plasminogen Activator Inhibitor-1 by Glucocorticoid and Transforming Growth Factor-β1 in Ovarian Cancer HO-8910 CellsBoth of Glucocorticoid (GC) and Transforming growth factorβ1 (TGFβ1) have multiply biology effects. According to the recent studies, there are interaction between these two pathways and GC enhances TGFβ1 signaling pathway and its effect in several cell lines. Our previous studies demonstrated that the co-treatment of HO-8910 cells with dexamethasone (Dex) and TGFβ1 significantly enhanced the adhesion of HO-8910 cells to ECM and increased the synthesis of extracellular matrix (ECM). Plasminogen activator inhibitor-1 (PAI-1) plays a key role in the regulation of fibrinolysis and cellular adhesion by suppressing plasminogen activator action. It has been reported that PAI-1 is a endogenous target gene of TGFβ1, and Dex and TGFβ1 have a synergistic effect on inducing PAI-1 expression. The purpose of this study was to elucidate the mechanism of synergistic up-regulation of the expression of PAI-1 by GC and TGFβ1 in human ovarian cancer HO-8910 cells.We at first demonstrated that both the 10ng/ml TGFβ1 and 100 nM Dex alone could up-regulate the expression of PAI-1, however the inducing effect of TGFβ1 was stronger than that of Dex. Combined treatment of cells with Dex and TGFβ1 for 2 to 4 hours showed obvious synergistic effects on up-regulation of the expression of PAI-1 in HO-8910 cells. Therefore, we mainly focus our study on the mechanism of the synergistic effects of Dex and TGFβ1 at 4 hours.Using Act D, which can inhibit transcription, we tested the PAI-1 mRNA stabilize change after the treatment with Dex, TGFβ1 or Dex plus TGFβ1 for 4 hours. The results demonstrated that Dex and TGFβ1 alone or both increased PAI-1 mRNA level by transcriptional mechanism, but not by stabilizing PAI-1 mRNA. RU486, a specific inhibitor of GR, could not only block the up-regulate effect of Dex alone , but also obviously block the synergistic effect of up-regulating the expression of PAI-1 by GC and TGFβ1, indicating that the synergistic effect was mediated by glucocorticoid receptor(GR). We then examined the effect of TGFβ1 on the expression of GR by real time-RT-PCR and GR transcriptional activity determined by GR responsible reporter plasmid( MMTV-Luc). We found that TGFβ1 could significantly enhance GR transcriptional activity although it had no effect on the expression of GR. We further observed the effects of 100μg/ml TGFβ1 antibody on the up-regulation of PAI-1 by TGFβ1 alone or combined with Dex, and found that TGFβ1 antibody not only blocked the effect of TGFβ1, but also obviously blocked the synergistic effects of GC and TGFβ1 on up-regulation of the expression of PAI-1, indicating that Dex could also enhance TGFβ1 signal pathway in HO-8910 cells. Finally, we demonstrated that SB203580, a inhibitor of p38MAPK pathway not only inhibit the effect of TGFβ1, but also significantly inhibit the synergistic effects of GC and TGFβ1 on up-regulation of the expression of PAI-1.In summary, in this study we demonstrated that combined treatment of HO-8910 cells with Dex and TGFβ1 resulted in an obvious synergistic up-regulation of PAI-1 expression. The synergistic effect occurs at transcription level mediated by GR. We further found that TGFβ1 increased GR transcription activity and Dex enhanced the effect of TGFβ1 on the expression of PAI-1. Furthermore, p38MAPK pathway also involved in the effect of TGFβ1 alone or synergistic effect of TGFβ1 and Dex on up-regulation of PAI-1 expression. PartⅡHypoxia Up-regulates the Expression of Stomatin in Cerebral Cortex of RatsStomatin, a kind of lipid raft proteins, can combine with various membrane proteins and impact their functions. In this study, we investigated the effects of hypoxia exposure alone and combination with glucocorticoid on the expression of stomatin in cerebral cortex of rat in order to know if it plays role in hypoxia adaptation.Adult male Sprague-Dawley rats were put into hypoxia chamber (filled with 8% O2 and 92% N2 ) for different time (8h,12h,16h). Rats at normal oxygen was used as control. Stomatin mRNA and protein levels were determined by RT-real-time PCR and Western blot. We found that hypoxia up-regulated stomatin expression. After hypoxia treatment for 12h, stomatin mRNA and protein expression in rat cerebral cortex significantly increased (2.2 fold and 1.5 fold pared with control respectively , p<0.05). Further experiment showed that hypoxia could up-regulate stomatin expression of rats underwent adrenalectomy, indicating that hypoxia could up-regulate stomatin expression independent of adrenocortical hormone. Our results also showed that stomatin expression level of rats underwent adrenalectomy decreased obviously compared with sham-operated rats, supplement with dexamethasone(Dex) alone could not increase stomatin expression of rats underwent adrenalectomy. However Dex could enhance hypoxia-induced stomatin expression.Conclusions: this study found that hypoxia could increase stomatin expression in rat cerebral cortex independent of adrenocortical hormone. Glucocorticoid could not influence stomatin expression alone but it could increase hypoxia induced stomatin expression.