scholarly journals Genetic and Epigenetic Regulation of the Non-Muscle Myosin Light Chain Kinase Isoform by Lung Inflammatory Factors and Mechanical Stress

2021 ◽  
Author(s):  
Xiaoguang Sun ◽  
Belinda L Sun ◽  
Saad Sammani ◽  
Tadeo Bermudez ◽  
Steven M. Dudek ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Promoter Activity ◽  
Myosin Light Chain ◽  
Cpg Island ◽  
Cyclic Stretch ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Kinase Gene ◽  
Inflammatory Disorders

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.

ID: 110: GENETIC AND EPIGENETIC REGULATION OF MYOSIN LIGHT CHAIN KINASE BY INFLAMMATORY LUNG DISEASE ASSOCIATED POLYMORPHISMS

2016 ◽  
Vol 64 (4) ◽  
pp. 963.2-964
Author(s):  
X Sun ◽  
R Elangovan ◽  
Y Shimizu ◽  
T Wang ◽  
JG Garcia ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Endothelial Cell ◽  
Epigenetic Regulation ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Promoter Activity ◽  
Myosin Light Chain ◽  
Inflammatory Diseases ◽  
Inflammatory Factors ◽  
Luciferase Reporter

RationaleMyosin light chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK gene, regulates muscle contraction, cell migration, endothelial cell–cell adhesion, and barrier function, thereby playing key pathophysiological roles in lung inflammatory diseases. We previously identified that MYLK single nucleotide polymorphisms (SNPs) as well as haplotypes are significantly associated with severe sepsis, acute lung injury and asthma in African Americans (AA) and European Americans (EA). Here we examined genetic and epigenetic regulation of the MYLK promoter as well as the effects of SNPs on MLCK expression and activity, thereby influencing cytoskeletal balance and cell integrity.MethodsA series of nested deletions from the ∼2.5 kb putative promoter fragment were fused to luciferase reporter vectors, and transfected into human lung endothelium. We next evaluated the influence of ARDS and asthma associated SNPs on transcription factor (TF) binding and promoter activity. Exon SNP rs2700408 and intronic SNP rs11714297 were associated with ARDS in AA (GWAS) and EA (Gao et al, 2006), respectively. Rs57186134 has high LD with rs936170, one asthma associated SNP in AA (Gao et al, 2007). The DNA fragments containing SNPs were generated by site-directed mutagenesis. Transcription factor binding to the MYLK promoter was detected by protein-DNA electrophoretic mobility shift assay. Genetic regulation of MYLK and influences of disease-associated SNPs from previous studies were measured by luciferase promoter activity assays following challenge with inflammatory factors and mechanical stretch.ResultsDeletion construct luciferase reporter analysis revealed that the MYLK promoter for nmMLCK contains distal inhibitory and proximal enhancing regulatory regions. Human endothelial cell challenge with either 18% cyclic stretch, demethylation agents (5-Aza), or inflammatory factor TNFα and IL-4 significantly up-regulate MYLK promoter activities (p<0.05). rs2700408 and rs11714297 altered MYLK binding to TFs GCM and ISX, respectively, and two SNPs significantly increased MYLK promoter reporter activities by 1.8- and 1.3-folds, respectively (p<0.05). Rs57186134 interrupted MYLK binding to transcription repressor GFI1, and significantly increased MYLK promoter activity in endothelial cells (p<0.05). Finally, we evaluated MYLK SNP rs78755744 (-261G/A) that resides directly within a CpG island within the MYLK promoter, significantly interrupted demethylating agent 5-aza- induced up-regulation of MYLKpromoter activity (p<0.05).ConclusionThese findings suggest that the MYLK gene is transcriptionally regulated by mechanical stress and inflammatory factors, and modulated by SNPs associated with lung inflammatory diseases. These functional insights further strengthen the concept that MYLKcontributes to inflammatory disease susceptibility and represents a molecular target in complex lung disorders.

The Myosin Light Chain Kinase Gene Is Not Duplicated in Mouse: Partial Structure and Chromosomal Localization of Mylk

Genomics ◽  
2001 ◽  
Vol 75 (1-3) ◽  
pp. 49-56 ◽  
Author(s):  
Dominique Giorgi ◽  
Concepcion Ferraz ◽  
Marie-Geneviève Mattéi ◽  
Jacques Demaille ◽  
Sylvie Rouquier
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Chromosomal Localization ◽  
Partial Structure ◽  
Kinase Gene

scholarly journals Naturally Extended CT · AG Repeats Increase H-DNA Structures and Promoter Activity in the Smooth Muscle Myosin Light Chain Kinase Gene

2007 ◽  
Vol 28 (2) ◽  
pp. 863-872 ◽  
Author(s):  
Yoo-Jeong Han ◽  
Primal de Lanerolle
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Promoter Activity ◽  
Myosin Light Chain ◽  
Smooth Muscle Myosin ◽  
Insertion Mutation ◽  
Hypertensive Rats ◽  
Dna Structures ◽  
Muscle Myosin

ABSTRACT Naturally occurring repeat sequences capable of adopting H-DNA structures are abundant in promoters of disease-related genes. In support of this, we found (CT)22 · (AG)22 repeats in the promoter of smooth muscle myosin light chain kinase (smMLCK), a key regulator of vascular smooth muscle function. We also found an insertion mutation that adds another six pairs of CT · AG repeats and increases smMLCK promoter activity in spontaneously hypertensive rats (SHR). Therefore, we used the smMLCK promoters from normotensive and hypertensive rats as a model system to determine how CT · AG repeats form H-DNA, an intramolecular triplex, and regulate promoter activity. High-resolution mapping with a chemical probe selective for H-DNA showed that the CT · AG repeats adopt H-DNA structures at a neutral pH. Importantly, the SHR promoter forms longer H-DNA structures than the promoter from normotensive rats. Reconstituting nucleosomes on the promoters, in vitro, showed no difference in nucleosome positioning between the two promoters. However, chromatin immunoprecipitation analyses revealed that histone acetylations are greater in the hypertensive promoter. Thus, our findings suggest that the extended CT · AG repeats in the SHR promoter increase H-DNA structures, histone modifications, and promoter activity of the smMLCK, perhaps contributing to vascular disorders in hypertension.

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