We have demonstrated that the non-muscle isoform of myosin light chain kinase (nmMLCK, 1914 aa, gene code: MYLK) is an essential, multi-functional cytoskeletal effector involved: i) in lung endothelial cell (EC) barrier-disruptive and barrier-restorative processes, ii) in lung inflammatory cell trafficking; and, iii) in lung vascular responses to mechanical stress. Each of these events is critical to the pathobiology of acute respiratory distress syndrome (ARDS) and ventilator?induced lung injury (VILI). We performed MYLK sequencing and identified coding and non-coding MYLK single nucleotide polymorphisms (SNPs) that contribute to ARDS susceptibility and to ARDS outcomes. In addition, many ARDS-associated MYLK SNPs were profoundly over-represented in individuals of African descent (AD), a population at risk for reduced survival in ARDS. As these studies indicate MYLK is a viable ARDS candidate gene, we will address the functionality of 29 methodically- selected MYLK SNPs that are potentially involved in the dual EC barrier-regulatory roles of nmMLCK both in the development of ARDS and VILI as well as in the recovery phase associated with EC barrier restoration. One potential influence of these SNPs is on the regulation of nmMLCK expression. SA #1 will extend our recently published studies to mechanistically characterize the effects of 5' promoter and 3' UTR MYLK SNPs on genetic/epigenetic regulation of nmMLCK expression. Continuing our structure/function interrogation of MYLK, we recently employed RNA sequencing analysis and corroborated our published studies that human lung EC exhibit substantial expression of a unique 1845 aa nmMLCK splice variant, nmMLCK2, generated by a splicing deletion of exon11. Exciting preliminary data demonstrate that lung EC exposure to inflammatory agonists and excessive mechanical stress both selectively increase nmMLCK2 expression with MYLK SNPs dramatically influencing these splicing events. SA #2 will define the impact of MYLK SNPs and splicing factors on nmMLCK mRNA splice variant generation. Effects of coding SNPs on post-translational modifications (PTMs) are yet another potential mechanism to alter barrier-regulatory processes in ARDS patients. Extensively utilizing Cores B & D expertise, SA #3 will evaluate the effects of PTMs (such as phosphorylation of Y464 and Y471 located in the spliced out exon 11) and N-terminal coding SNPs on nmMLCK1 and nmMLCK2 spatially-directed kinase activities, structure/function relationships, and EC barrier responses (peripheral cytoskeletal remodeling, lamellipodia formation, paracellular gap regulation). Finally, with Core C support, SA #4 will validate the in vivo effects of selected nmMLCK coding SNPs and PTMs and define nmMLCK antagonism as a potential novel therapy in preclinical ARDS and VILI models. Thus, via the intense leveraging of highly integrated interactions with each PPG Project and Core, Project #1's system biology approaches will allow us to clarify the contributions of lung EC cytoskeletal variants, PTMs and SNPs to ARDS and VILI pathobiology, enhance therapeutic targeting of lung vascular barrier dysregulation, and increase knowledge of the genetic basis for ARDS health disparities.
Acute Respiratory Distress Syndrome (ARDS) is a devastating consequence of systemic inflammatory conditions (such as sepsis) that afflicts an estimated 200,000 people a year in the US with 75,000 deaths. No specific therapy is available to target the underlying mechanistic causes of this syndrome. This proposal seeks to better understand the role of the MYLK gene, its protein product, nmMLCK, and cytoskeletal contractile mechanisms in regulating the pulmonary vascular leak that occurs in this syndrome in order to develop precise, individualized therapies for the critically ill.
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