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

Abstract

Rationale: The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle MLCK isoform (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, ARDS), we explored clinical-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 (EC). SNP-containing nmMYLK DNA fragments were generated and nmMYLK promoter binding by transcription factors detected by protein-DNA electrophoretic mobility shift assay. Promoter demethylation was evaluated by 5-Aza. A preclinical mouse model of 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, TNFα, 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 expression (HIFs), 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 (GCM1 and 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 clinical-relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.

Endothelial upregulation of mechanosensitive channel Piezo1 in pulmonary hypertension

“…Mannitol (100 mM) was added to maintain the osmolality constant at 310 mOsm/kg, whereas the hypo-osmotic solution (200 mOsm/ kg) and hyperosmotic solution (400 mOsm/kg) were prepared by omitting or adding mannitol in the solutions, respectively. For cyclic stretch (CS) experiments, PAECs were plated onto Bioflex collagen I type cell culture plates (FlexCell International, Hillsborough, NC) and stimulated for 15 min at 18% CS on the FlexCell FX-5000 System (FlexCell International), mimicking high-tidal volume ventilation (40,41).…”

“…Decreasing extracellular osmolality is used to result in swelling-mediated membrane stretch that activates mechanosensitive channels (51)(52)(53). Membrane stretch using a cyclic stretch (CS 18%) device has been used to acutely activate mechanosensitive events in lung EC (40,41). In human PAEC, membrane stretch by decreasing extracellular osmolality from 310 mOsm/kg using the iso-osmotic solution (Iso-Osm) to 200 mOsm/kg using the hypo-osmotic solution (Hypo-Osm, 15 min) (Fig.…”

Targeting SELPLG/P‐selectin glycoprotein ligand 1 in preclinical ARDS: Genetic and epigenetic regulation of the SELPLG promoter

“…HIF-1α drives LPSinduced IL-1β, 40 tumor necrosis factor, IL-12, and VEGF expression 41 and nmMLCK expression. 39 We have shown HIF-2α, and to a lesser extent HIF-1α, are involved in transcriptional regulation of the ARDS/VILI DAMP, eNAMPT 42 and cytoskeletal element gene, MYLK. 39 Our current studies demonstrate that HIF-1α and HIF-2α accumulation (by PHD2 inhibition) also increase SELPLG promoter activity.…”

Frail Silk: Is the Hughes-Stovin Syndrome a Behçet Syndrome Subtype with Aneurysm-Involved Gene Variants?

“…The MYLK gene (OMIM 600922), located on 3q21, encodes at least 3 proteins ( Figure 2 ) via different unique promoters: non-muscle MLCK 210 (nmMLCK), smooth muscle MLCK 108, and telokin/KRP [ 28 , 40 , 41 ]. MLCK plays an important role in maintaining SMC contractility and cell survival, but also in cell division, cell migration, and cell–matrix adhesion [ 42 , 43 , 44 ].…”