GADD45a Promoter Regulation by a Functional Genetic Variant Associated with Acute Lung Injury
GADD45a Promoter Regulation by a Functional Genetic Variant Associated with Acute Lung Injury
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DOI：
10.1371/journal.pone.0100169
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发表时间：
2014-06-18
期刊：
影响因子：
3.7
通讯作者：
Jacobson, Jeffrey R.
中科院分区：
文献类型：
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作者：
Mitra, Sumegha;Wade, Michael S.;Jacobson, Jeffrey R.
理由：生长阻滞DNA损伤诱导α (Growth arrest DNA damage inducible alpha, GADD45a)是一种应激诱导基因，我们已经证明它通过调节机械应激诱导的Akt泛素化和磷酸化参与呼吸机诱导肺损伤（VILI）的病理生理过程。然而，机械应力对GADD45a表达的调控及其与急性肺损伤（ALI）易感性和严重程度的关系尚不清楚。目的：研究GADD45a启动子中的机械应力依赖性调控元件（MSRE），以及启动子多态性在GADD45a表达和ALI易感性中的作用。方法与结果：GADD45a基因敲除和杂合小鼠的初步研究证实了GADD45a基因剂量与VILI严重程度的关系。转染含有全长GADD45a启动子序列（-771至+223）的荧光素酶载体的人肺内皮细胞（EC）在18%的循环拉伸（CS， 4小时）下，与静态对照相比，GADD45a的表达增加了bb40倍，而使用含有GADD45a启动子序列缺失构建的载体鉴定了含有CS依赖性MSRE的特定启动子区域。GADD45a启动子区域（-371至-133）的计算机分析揭示了特异性蛋白1 （SP1）的潜在结合位点，这一发现得到了SP1与GADD45a启动子结合的证实，以及在SP1沉默后cs依赖性GADD45a启动子活性的显著衰减的支持。另外，病例对照关联研究显示GADD45a启动子SNP在-589 （rs581000， G>C）与ALI易感性降低有显著关联。随后，我们发现该SNP的等位基因变异与机械应激EC （18% CS, 4 h）中GADD45a的差异表达以及该位点上干扰素调节因子7 （IRF7）的差异结合位点相关。结论：这些结果有力地支持GADD45a在ALI/VILI中的功能作用，并确定了一个特定的基因变异，赋予ALI风险。
Rationale: Growth arrest DNA damage inducible alpha (GADD45a) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of GADD45a expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown.Objectives: We examined mechanical stress-dependent regulatory elements (MSRE) in the GADD45a promoter and the contribution of promoter polymorphisms in GADD45a expression and ALI susceptibility.Methods and Results: Initial studies in GADD45a knockout and heterozygous mice confirmed the relationship of GADD45a gene dose to VILI severity. Human lung endothelial cells (EC) transfected with a luciferase vector containing the full length GADD45a promoter sequence (-771 to +223) demonstrated a >4 fold increase in GADD45a expression in response to 18% cyclic stretch (CS, 4 h) compared to static controls while specific promoter regions harboring CS-dependent MSRE were identified using vectors containing serial deletion constructs of the GADD45a promoter. In silico analyses of GADD45a promoter region (-371 to -133) revealed a potential binding site for specificity protein 1 (SP1), a finding supported by confirmed SP1 binding with the GADD45a promoter and by the significant attenuation of CS-dependent GADD45a promoter activity in response to SP1 silencing. Separately, case-control association studies revealed a significant association of a GADD45a promoter SNP at -589 (rs581000, G>C) with reduced ALI susceptibility. Subsequently, we found allelic variation of this SNP is associated with both differential GADD45a expression in mechanically stressed EC (18% CS, 4 h) and differential binding site of interferon regulatory factor 7 (IRF7) at this site.Conclusion: These results strongly support a functional role for GADD45a in ALI/VILI and identify a specific gene variant that confers risk for ALI.