MicroRNA Regulation of Nonmuscle Myosin Light Chain Kinase Expression in Human Lung Endothelium

Abstract

Increased lung vascular permeability, the consequence of endothelial cell (EC) barrier dysfunction, is a cardinal feature of inflammatory conditions such as acute lung injury and sepsis and leads to lethal physiological dysfunction characterized by alveolar flooding, hypoxemia, and pulmonary edema. We previously demonstrated that the nonmuscle myosin light chain kinase isoform (nmMLCK) plays a key role in agonist-induced pulmonary EC barrier regulation. The present study evaluated posttranscriptional regulation of MYLK expression, the gene encoding nmMLCK, via 39 untranslated region (UTR) binding by microRNAs (miRNAs) with in silico analysis identifying hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290 as miRNA candidates. We identified increased MYLK gene transcription induced by TNF-a (24 h; 4.7 6 0.45 fold increase [FI]), LPS (4 h; 2.85 6 0.15 [FI]), and 18% cyclic stretch (24 h; 4.6 6 0.24 FI) that was attenuated by transfection of human lung ECs with mimics of hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, or hsa-miR-1290 (20-80% reductions by each miRNA). TNF-a, LPS, and 18% cyclic stretch each increased the activity of a MYLK 39UTR luciferase reporter (2.5-7.0 FI) with induction reduced by mimics of each miRNA (30-60% reduction). MiRNA inhibitors (antagomirs) for each MYLK miRNA significantly increased 39UTR luciferase activity (1.2-2.3 FI) and rescued the decreased MLCK-39UTR reporter activity produced by miRNA mimics (70-110% increases for each miRNA; P , 0.05). These data demonstrate that increased human lung EC expression of MYLK by bioactive agonists (excessive mechanical stress, LPS, TNF-a) is regulated in part by specific miRNAs (hsa-miR-374a, hsamiR-374b, hsa-miR-520c-3p, and hsa-miR-1290), representing a novel therapeutic strategy for reducing inflammatory lung injury.Keywords: miRNA; MLCK; acute lung injury; ventilator-induced lung injury; endothelial cellsThe pulmonary vascular endothelium serves as a semiselective barrier between circulating blood and surrounding tissues, with endothelial cell (EC) integrity being critical to tissue and organ function. Disruption of this vascular barrier induced by inflammatory agonists, such as IL-1b, TNF-a, and LPS, and by excessive mechanical stress, such as produced by shear stress or mechanical ventilation, leads to potentially lethal physiological dysfunction, such as hypoxemia and severe lung edema, which are hallmarks of acute inflammatory lung injury (ALI) (1-3). We previously demonstrated that MYLK, the gene encoding the critical cytoskeletal effector myosin light chain kinase (MLCK), is a compelling candidate gene in inflammatory lung injuries with critical modulation of vascular EC barrier integrity such as ALI and asthma (4-6). We cloned the full-length human MYLK gene encoding a novel nonmuscle MLCK isoform (nmMLCK) (210 kD) (7) and several nmMLCK splice variants that are also highly expressed in endothelium (8) and convincingly demonstrated nmMLCK as a multifunctional enzyme driving cytoskeletal participation in vascular barrier disrup...

Mechanical Stress Induces Pre–B-cell Colony-Enhancing Factor/NAMPT Expression via Epigenetic Regulation by miR-374a and miR-568 in Human Lung Endothelium

“…Previously, we indicated that expression of the broadly conserved mature miR-374a (50) is significantly decreased by 18% CS, TNF-a, or LPS (37). We now demonstrate that miR-568 is expressed in ECs with significant down-regulation by 18% CS and LPS (Figure 2).…”

“…In the present study, we found that PBEF/NAMPT expression in ECs is markedly up-regulated at mRNA and protein levels in response to 18% CS and LPS (Figure 1). Our combined data (37,39,51) demonstrate the negative correlation between expression levels of putative miRNA and mRNA/protein and suggest that down-regulation of these miRNAs may contribute to augmented PBEF/NAMPT gene expression in ECs treated with these proinflammatory agonists. To further evaluate whether miR-374a and miR-568 specifically regulate PBEF/NAMPT expression in ECs, transfection with synthetic miRNA mimics and antagomirs was used.…”

“…As predicted, the potential binding site on PBEF/NAMPT 39-UTR for miR-374a is broadly conserved among mammals. Moreover, we noted that MYLK, an ARDS candidate gene (36) encoding myosin light chain kinase, is a potential target of miR-374a (37). In recent studies, we noted that expression of the mature miR-374a is significantly decreased by Figure 2.…”

Effect of Inulin on Proteome Changes Induced by Pathogenic Lipopolysaccharide in Human Colon

“…Even if these data are referred to the mucosal level of the enzymes, we cannot exclude that a similar mechanism could be present in the muscle layer (which include several cell types) and account, together with protection of muscle cells from oxidative stress, for the prevention of contractility impairment of the entire muscle strips. The rapid increase in MLCK transcript is in agreement with previous reports, showing that LPS treatment leads to a quick induction of MLCK mRNA in lung endothelial cells [49]. …”

MicroRNA‐92a promotes vascular smooth muscle cell proliferation and migration through the ROCK/MLCK signalling pathway

“…33 Loyer et al clearly demonstrated that in vivo inhibition of miR-92a in ldlr −/− mice restricted the development of AS. 33 Loyer et al clearly demonstrated that in vivo inhibition of miR-92a in ldlr −/− mice restricted the development of AS.…”