Abstract 20441: Mechanism of Mechanical Stress-Regulated Non-Muscle Myosin Light Chain Kinase Alternative Splicing, Implication for Acute Lung Inflammation
MYLK, the gene encoding the critical cytoskeletal effector, myosin light chain kinase (MLCK), is a compelling candidate gene in the inflammatory lung injuries observed in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We demonstrated that nmMLCK exists as two highly expressed proteins in lung endothelium: wild type nmMLCK1 (1914 aa) and the spliced variant, nmMLCK2 (1845 aa) that shares a primary structure that is identical to nmMLCK1 except for the absence of 69 amino acids encoded by the spliced out exon 11. Both edemagenic and barrier-enhancing agonists stimulate nmMLCK1 phosphorylation at Y464 & Y471, to promote nmMLCK1 enzymatic activity and cellular localization. The regulatory mechanisms governing MYLK expression and nmMLCK1 and nmMLCK2 EC barrier response activities are poorly understood. We studied nmMLCK1/nmMLCK2 mRNA ratios from human lung EC exposed to pathological 18% cyclic stretch (18% CS, 4 hr) by RNA sequencing (RNA Seq, BGI America) and identified increased nmMLCK transcript levels elicited by 18% CS. Specifically, nmMLCK2, but not nmMLCK1 was significantly elevated by 18% CS, results confirmed by qPCR. In silico evaluation identified an intronic splicing inhibitory element (TAGGGAG) in the boundary region in intron 10 as a potential binding site for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a nuclear protein known to regulate alternate splicing (Splice Scan II analysis). Consistent with hnRNPA1 modulation of splicing, we found hnRNPA1 upregulation in EC exposed to 18% CS, and in lung tissues from VILI- or LPS-challenged mice, highly suggesting a regulatory role of hnRNPA1 in nmMLCK splicing during inflammatory lung injury. Our study further confirmed a dysregulated alternative splicing pattern of non-muscle variant of MYLK by splicing factor hnRNPA1 upon inflammation stimuli. We believe these studies contribute to the understanding of the complex and functional role of nmMLCK in endothelial barrier regulation during ARDS or VILI.
Author Disclosures: J.B. Mascarenhas: None. S.S. Lau: None. S.M. Danilov: None. A.Y. Tchourbanov: None. H.L. Granzier: None. V. Ramamoorthi Elangovan: None. T. Wang: None. J.G. Garcia: None.
- © 2014 by American Heart Association, Inc.