The nonmuscle (nm) myosin light-chain kinase isoform (MLCK), encoded by the MYLK gene, is a vital participant in regulating vascular barrier responses to mechanical and inflammatory stimuli. We determined that MYLK is alternatively spliced, yielding functionally distinct nmMLCK splice variants including nmMLCK2, a splice variant highly expressed in vascular endothelial cells (EC) and associated with reduced EC barrier integrity. We demonstrated previously that the nmMLCK2 variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y and Y) that influence vascular barrier function during inflammation. In this study, we used minigene constructs and RT-PCR to interrogate biophysical factors (mechanical stress) and genetic variants (MYLK single-nucleotide polymorphisms [SNPs]) that are potentially involved in regulating MYLK alternative splicing and nmMLCK2 generation. Human lung EC exposed to pathologic mechanical stress (18% cyclic stretch) produced increased nmMLCK2 expression relative to levels of nmMLCK1 with alternative splicing significantly influenced by MYLK SNPs rs77323602 and rs147245669. In silico analyses predicted that these variants would alter exon 11 donor and acceptor sites for alternative splicing, computational predictions that were confirmed by minigene studies. The introduction of rs77323602 favored wild-type nmMLCK expression, whereas rs147245669 favored alternative splicing and deletion of exon 11, yielding increased nmMLCK2 expression. Finally, lymphoblastoid cell lines selectively harboring these MYLK SNPs (rs77323602 and rs147245669) directly validated SNP-specific effects on MYLK alternative splicing and nmMLCK2 generation. Together, these studies demonstrate that mechanical stress and MYLK SNPs regulate MYLK alternative splicing and generation of a splice variant, nmMLCK2, that contributes to the severity of inflammatory injury.

Profound lung vascular permeability is a cardinal feature of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI), two syndromes known to centrally involve the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier dysregulation. Two main splice variants, nmMLCK1 and nmMLCK2, are well represented in human lung endothelial cells and encoded by MYLK, and they differ only in the presence of exon 11 in nmMLCK1, which contains critical phosphorylation sites (Y and Y) that influence nmMLCK enzymatic activity, cellular translocation, and localization in response to vascular agonists. We recently demonstrated the functional role of SNPs in altering MYLK splicing, and in the present study we sought to identify the role of splicing factors in the generation of nmMLCK1 and nmMLCK2 spliced variants. Using bioinformatic in silico approaches, we identified a putative binding site for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a recognized splicing factor. We verified hnRNPA1 binding to MYLK by gel shift analyses and that hnRNPA1 gene and protein expression is upregulated in mouse lungs obtained from preclinical models of ARDS and VILI and in human endothelial cells exposed to 18% cyclic stretch, a model that reproduces the excessive mechanical stress observed in VILI. Using an MYLK minigene approach, we established a direct role of hnRNPA1 in MYLK splicing and in the context of 18% cyclic stretch. In summary, these data indicate an important regulatory role for hnRNPA1 in MYLK splicing, and they increase understanding of MYLK splicing in the regulation of lung vascular integrity during acute lung inflammation and excessive mechanical stress, such as that observed in ARDS and VILI.