Abstract | RATIONALE: METHODS: Full-length or serially deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein- DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2'-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (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, tumor necrosis factor-α (TNF-α), 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 (HIF) expression, 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 (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (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 clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.
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Authors | Xiaoguang Sun, Belinda L Sun, Saad Sammani, Tadeo Bermudez, Steven M Dudek, Sara M Camp, Joe G N Garcia |
Journal | Clinical science (London, England : 1979)
(Clin Sci (Lond))
Vol. 135
Issue 7
Pg. 963-977
(04 16 2021)
ISSN: 1470-8736 [Electronic] England |
PMID | 33792658
(Publication Type: Journal Article, Research Support, N.I.H., Extramural)
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Copyright | © 2021 The Author(s). |
Chemical References |
- Lipopolysaccharides
- Tumor Necrosis Factor-alpha
- Decitabine
- Myosin-Light-Chain Kinase
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Topics |
- Acute Lung Injury
(chemically induced)
- Animals
- Cells, Cultured
- Decitabine
- Disease Models, Animal
- Endothelial Cells
(metabolism)
- Epigenesis, Genetic
- Humans
- Lipopolysaccharides
(toxicity)
- Lung Injury
(chemically induced)
- Male
- Mice, Inbred C57BL
- Myosin-Light-Chain Kinase
(genetics, metabolism)
- Pneumonia
- Polymorphism, Single Nucleotide
- Respiratory Distress Syndrome
(genetics)
- Stress, Mechanical
- Tumor Necrosis Factor-alpha
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