The Mechanisms And Biological Significance Of Stomatin Expression Induced By Hypoxia And Glucocorticoid

Alveolar hypoxia occurs in many physiological and pathological conditions. It has a close relationship with pulmonary edema, heart failure and acute lung injury. GC is important stress hormone that not only necessary for the cellular adaptation to hypoxic condition, but it therapeutically used to treat severe pulmonary edema and lung injury in clinical by reducing the leaking of capillaries, accelerating the alveolar space fluid cleaning。Stomatin is an important lipid raft-associated protein, and widely expressed in many cell types, it involved in membrane organization and cholesterol-dependent regulatory processes. But until now only few researches reported its function and regulation. In our previous studies, we found that hypoxia and Dexamethasone (Dex, a synthetic glucorcoticoid) could up-regulates stomatin in lungs of rat and in human lung adenocarcinoma epithelial A549 cells. In this study, we first confirmed that both Dex and hypoxia could induce stomatin expression in vivo and in primary alveolar epithelial cells. On this basis we further focus on the molecular mechanism by which hypoxia and Dex up-regulate transcription of stomatin in A549 cell line (Human lung adenocarcinoma epithelial).We found stomatin mRNA have a long half-life of 32h, both hypoxia and Dex could increase the stability of stomatin mRNA, the half-life of stomatin mRNA was lengthen for 1.22-fold and 1.5-folds separately. Luciferase reporter assays also showed that hypoxia could not induce the promoter activity of stomatin, Dex can induce the promoter activity of stomatin in a time and dose-dependent pattern, this induction mostly happened in early time course and low concentration level, this result indicate Dex also can up-regulate stomatin expression from transcriptional level.To demonstrate which stomatin upstream sequence element was responsive to Dex induction. A 5'deletion series of the stomatin promoter were constructed to localize the potential glucocorticoid responsive region. We found the region -162 to +244 with respect to the transcription start site is essential for the Dex response. This region also contributes to the major basal transcription activity in several kinds of cell lines, so we proposed it is a core promoter of stomatin.We use transcription factor binding sites predict software identified several high-affinity candidate sites in this region, then we constructed the mutational of those potential sites and transfected them into A549 cells separately, cells were exposed to vehicle or 100nM Dex for 24h, We found an imperfect consensus sequence of glucocorticoid response element GRE3, it's mutational construct can abolished the induction effect of Dex, other mutations did not affect the Dex response at all. Those results suggested that the GRE3 sites mediated the promoter response to Dex.Finally we use confocal microscope explored the relationship of stomatin with actin cytoskeleton in A549 cells, we found EGFP-tagged stomatin protein co-localized with membrane-associated actin filaments, inhibiting stomatin expression by RNA interference decreased peripheral actin ring in cells both in normoxia and in hypoxia. These results imply that stomatin may have a role in stabilizing membrane-associated actin, especially under hypoxic condition. Therefore, we hypothesis that the increased expression of stomatin by GC and hypoxia may enhance the function of alveolar epithelial barrier, which is possible one of the mechanisms of cell adaption to hypoxia.