Rationale Myosin light chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK gene, regulates muscle contraction, cell migration, endothelial cell–cell adhesion, and barrier function, thereby playing key pathophysiological roles in lung inflammatory diseases. We previously identified that MYLK single nucleotide polymorphisms (SNPs) as well as haplotypes are significantly associated with severe sepsis, acute lung injury and asthma in African Americans (AA) and European Americans (EA). Here we examined genetic and epigenetic regulation of the MYLK promoter as well as the effects of SNPs on MLCK expression and activity, thereby influencing cytoskeletal balance and cell integrity.
Methods A series of nested deletions from the ∼2.5 kb putative promoter fragment were fused to luciferase reporter vectors, and transfected into human lung endothelium. We next evaluated the influence of ARDS and asthma associated SNPs on transcription factor (TF) binding and promoter activity. Exon SNP rs2700408 and intronic SNP rs11714297 were associated with ARDS in AA (GWAS) and EA (Gao et al, 2006), respectively. Rs57186134 has high LD with rs936170, one asthma associated SNP in AA (Gao et al, 2007). The DNA fragments containing SNPs were generated by site-directed mutagenesis. Transcription factor binding to the MYLK promoter was detected by protein-DNA electrophoretic mobility shift assay. Genetic regulation of MYLK and influences of disease-associated SNPs from previous studies were measured by luciferase promoter activity assays following challenge with inflammatory factors and mechanical stretch.
Results Deletion construct luciferase reporter analysis revealed that the MYLK promoter for nmMLCK contains distal inhibitory and proximal enhancing regulatory regions. Human endothelial cell challenge with either 18% cyclic stretch, demethylation agents (5-Aza), or inflammatory factor TNFα and IL-4 significantly up-regulate MYLK promoter activities (p<0.05). rs2700408 and rs11714297 altered MYLK binding to TFs GCM and ISX, respectively, and two SNPs significantly increased MYLK promoter reporter activities by 1.8- and 1.3-folds, respectively (p<0.05). Rs57186134 interrupted MYLK binding to transcription repressor GFI1, and significantly increased MYLK promoter activity in endothelial cells (p<0.05). Finally, we evaluated MYLK SNP rs78755744 (-261G/A) that resides directly within a CpG island within the MYLK promoter, significantly interrupted demethylating agent 5-aza- induced up-regulation of MYLKpromoter activity (p<0.05).
Conclusion These findings suggest that the MYLK gene is transcriptionally regulated by mechanical stress and inflammatory factors, and modulated by SNPs associated with lung inflammatory diseases. These functional insights further strengthen the concept that MYLKcontributes to inflammatory disease susceptibility and represents a molecular target in complex lung disorders.
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