Genetic and Epigenetic Regulation of the Non-Muscle Myosin Light Chain Kinase Isoform by Lung Inflammatory Factors and Mechanical Stress.

RATIONALE The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle MLCK isoform (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, ARDS), we explored clinical-relevant inflammatory stimuli and promoter SNPs in nmMLCK promoter regulation. METHODS Full-length or serially--deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (EC). SNP-containing nmMYLK DNA fragments were generated and nmMYLK promoter binding by transcription factors detected by protein-DNA electrophoretic mobility shift assay. Promoter demethylation was evaluated by 5-Aza. A preclinical mouse model of LPS-induced acute lung injury (ALI) was utilized for nmMLCK validation. RESULTS Lung EC levels of nmMLCK were significantly increased in LPS-challenged mice and  LPS, TNFα,  18% cyclic stretch (CS) and 5-Aza each significantly up-regulated EC nmMYLK promoter activities. EC exposure to FG-4592, a prolyl hydroxylase inhibitor that increases hypoxia-inducible factor expression (HIFs), increased nmMYLK promoter activity, confirmed by HIF1α/HIF2α silencing. nmMYLK promoter deletion studies identified distal inhibitory and proximal enhancing promoter regions as well as mechanical stretch-, LPS-, and TNFα-inducible regions. Insertion of ARDS-associated SNPs (rs2700408, rs11714297) significantly increased nmMYLK promoter activity via increased transcription binding (GCM1 and ISX, respectively). Finally, the MYLK rs78755744 SNP (-261G/A), residing within a nmMYLK CpG island, significantly attenuated 5-Aza -induced promoter activity. CONCLUSION These findings indicate nmMYLK transcriptional regulation by clinical-relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.