Abstract
RationaleGrowth 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.ObjectivesWe 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 ResultsInitial 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.ConclusionThese results strongly support a functional role for GADD45a in ALI/VILI and identify a specific gene variant that confers risk for ALI.
Highlights
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are complex disorders that are precipitated by the interplay of both environmental factors and genetic factors
We subsequently reported that mice lacking GADD45a gene (GADD45a2/2) demonstrated significantly increased ventilator-induced lung injury (VILI) susceptibility [10] and linked this observation to effects of GADD45a depletion on the differential ubiquitination of Akt resulting in both increased proteasomal degradation of Akt and decreased Akt phosphorylation in response to mechanical stress [11]
Our results provide evidence for ALI/VILI susceptibility conferred by specific GADD45a genetic variants that further supports an important role for GADD45a in susceptibility to inflammatory lung injury
Summary
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are complex disorders that are precipitated by the interplay of both environmental factors (such as mechanical ventilation) and genetic factors. Several case-control association studies have identified specific single nucleotide polymorphisms (SNPs) that contribute to ALI susceptibility and survival [1,2,3] In this regard, we have previously employed preclinical models of ALI and global gene expression profiling to identify several ALI candidate genes, including GADD45a, and ALI-associated SNPs [4,5,6,7,8]. We have previously employed preclinical models of ALI and global gene expression profiling to identify several ALI candidate genes, including GADD45a, and ALI-associated SNPs [4,5,6,7,8] As these studies have yielded important insights into ALI pathobiology and implicated specific genetic variants associated with ALI risk and severity, further research may lead to novel therapeutic targets that bring personalized medicine to the fore in strategies aimed at treating or preventing ALI. The regulation of GADD45a expression in response to mechanical stress and the association of GADD45a genetic variants with ALI/VILI susceptibility are largely unknown
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