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The Mechanisms and Biological Significance of Stomatin Expression Induced by Hypoxia and Glucocorticoid

Author: ChenJiCheng
Tutor: LuJian
School: Second Military Medical University
Course: Pathology and Pathophysiology
Keywords: Glucocorticoid Glucocorticoid receptor Hypoxia stomatin actin cytoskeleton
CLC: R363
Type: Master's thesis
Year: 2011
Downloads: 24
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Abstract

Hypoxic stress response is the most important one, is also the most common clinical pathophysiological process, closely associated with the incidence of cardiovascular disease and high altitude pulmonary edema. As the body's most important glucocorticoid stress hormones play an important role in the body to adapt to hypoxia. Synthetic glucocorticoid - dexamethasone (Dex) in clinical been widely used for the treatment of pulmonary edema, and lung injury, and it is this effect is to reduce the permeability of vascular endothelial cells, accelerating the removal of the liquid in the alveolar space. and enhancement of lung epithelial barrier function to achieve. Stomatin lipid rafts is an important marker protein, it is widely expressed in each of the organizations of the human and mouse cells, and to participate in the structure of the assembly of the lipid rafts, vesicle transport, the transmembrane transport of substances, as well as regulation of ion channels and cytoskeleton and other physiological processes. But so far on the function of stomatin to know less of its regulation under physiological and pathological conditions. Our previous studies of hypoxia and dexamethasone (Dex) separate and combined effects of rat lung and human lung adenocarcinoma A549 cells stomatin expression. Found that hypoxia and Dex stomatin can upregulate the expression both in vivo and in vitro. This topic further determine from the cells and the overall level of the glucocorticoids and hypoxia alone or combined action of stomatin gene expression, on this basis, focusing on hypoxia and dexamethasone raised stomatin expression mechanism, and to explore the stomatin upregulation of biological significance. Real time-PCR and Western Blot method, we first confirmed in rat lung and alveolar epithelial cells in primary culture level of dexamethasone and hypoxia not only alone can induce the expression of stomatin both can jointly raised stomatin expressed. Next, we studied hypoxia and dexamethasone raised the stomatin the mechanism. The study found that stomatin mRNA in a fairly long half-life of about 32 hours, hypoxia and dexamethasone treatment can enhance stomatin mRNA stability, its half-life extended to 1.22-fold and 1.5-fold respectively. Luciferase reporter gene experiments found that not hypoxia induced the stomatin promoter transcriptional activity, and hypoxia the dexamethasone to time-and concentration-dependent manner induced the stomatin promoter activity, this result show that Dexamethasone can also be directly stomatin mRNA expression induced at the transcriptional level. To further positioning of glucocorticoid-induced reaction stomatin promoter sites, we constructed a series of 5 'end truncated form containing stomatin promoter reporter vector, luciferase activity tests showed promoter -162 to 244 regions of glucocorticoid-induced reactions are indispensable in a variety of cell types, and this region contributed the vast majority of the stomatin gene basal transcription activity, so we speculated that this region is its core promoter zone. Subsequently, we used the software prediction of transcription factor binding sites contained in this region may be mediated by glucocorticoid-induced reaction, and constructed these loci corresponding mutant reporter gene vector. Results a site GRE3, mutations enables stomatin promoter loses dexamethasone-induced reaction, this description GRE3 mediated dexamethasone induction of stomatin reaction. We therefore believe that the the the dexamethasone induction at the transcriptional level stomatin is achieved through activation of the glucocorticoid receptor binding GRE3 locus. Finally, we used laser scanning confocal microscope observed in A549 cells stomatin expression of actin cytoskeleton relationship. The results found that, stomatin colocalization exist in the film week with the actin cytoskeleton. Hypoxic exposure or Dex processing make the film weeks actin, interfere with endogenous stomatin expression after hypoxia exposure or Dex treatment of A549 cells can make the actin cytoskeleton distribution in the membrane weeks significantly reduced, indicating that hypoxia and Dex through raised stomatin expression, increase the connection cytoskeleton and cell membrane, thereby enhancing the stability of the alveolar epithelial membrane. This may enhance the barrier function of alveolar epithelial cells, hypoxia and GC allowing the body to produce one of the mechanisms of the adaptive response to hypoxia.

The Mechanisms and Biological Significance of Stomatin Expression Induced by Hypoxia and Glucocorticoid

Abstract

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