Genetic and epigenetic regulation of the non-muscle myosin light chain kinase isoform by lung inflammatory factors and mechanical stress

Xiaoguang Sun, Belinda L. Sun, Saad Sammani, Tadeo Bermudez, Steven M. Dudek, Sara M. Camp, Joe G.N. Garcia

Research output: Contribution to journalArticlepeer-review

Abstract

Rationale: The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically 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 (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein-DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2'-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (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, tumor necrosis factor-α (TNF-α), 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 (HIF) expression, 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 (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (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 clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.

Original languageEnglish (US)
Pages (from-to)963-977
Number of pages15
JournalClinical Science
Volume135
Issue number7
DOIs
StatePublished - Apr 2021

ASJC Scopus subject areas

  • Medicine(all)

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