Fine mapping of the myosin light chain kinase (MYLK) gene replicates the association with asthma in populations of Spanish descent

Acosta‐Herrera, Marialbert; Pino-Yanes, María; Fan, Shwu; Barreto-Luis, Amalia; Corrales, Almudena; Cumplido, José; Perez-Rodriguez, Eva; Campo, Paloma; Eng, Celeste; García-Robaina, José Carlos; Quintela, Inés; Villar, Jesús; Blanca, Miguel; Carracedo, Ángel; Carrillo, Teresa; Garcia, Joe G.N.; Torgerson, Dara G.; Burchard, Esteban G.; Flores, Carlos

doi:10.1016/j.jaci.2015.04.025

Cited by 8 publications

(17 citation statements)

References 34 publications

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“…nmMLCK2, has been characterized as a candidate gene whose SNPs influence the susceptibility to and the severity of inflammatory lung disorders such as sepsis-and trauma-induced ARDS and asthma (13,(16)(17)(18)(19)(20)(21). We confirmed the role of MYLK SNPs in alternative splicing and generation of mMLCK2 by designing a minigene comprising exons 10-12 of the MYLK gene.…”

Section: Genotype At Rs147245669mentioning

confidence: 54%

“…EC activation in response to S1P and hepatocyte growth factor drives nmMLCK translocation to the cell periphery and into lamellipodia to accelerate paracellular gap closure during the recovery phase of acute lung injury (14,15). Consistent with MYLK as a candidate gene in inflammatory lung processes, we have identified coding and promoter MYLK polymorphisms that confer increased risk of sepsis, ARDS (16)(17)(18)(19), and asthma (19)(20)(21) and that influence the severity of disease, particularly in patients of African descent (16)(17)(18)(19)(20)(21).We have determined that these single nucleotide polymorphisms (SNPs) functionally alter MYLK promoter activity, alter secondary messenger RNA (mRNA) structure, and influence nmMLCK translocation to lamellipodia to restore vascular barrier integrity (21)(22)(23).…”

Section: Clinical Relevancementioning

confidence: 62%

“…In severe acute lung injury such as ARDS, increased vascular hyperpermeability produces persistent lifethreatening alveolar flooding contributing to the observed mortality rates of 30-40% (1,35,36). This lung endothelial hyperpermeability reflects loss of EC barrier integrity caused by actin cytoskeletal remodeling and an imbalance between barrier-disruptive stress fibers and protective peripheral cortical actin (6), processes tightly regulated by nmMLCK activity and spatial-specific myosin lightchain phosphorylation (Ser 18 and Thr 19 ). We have shown that mice deficient in nmMLCK, the primary isoform present in human endothelium (27), are protected against the injurious effects of excessive mechanical stress produced in vitro or by mechanical ventilation in preclinical models of ventilator-induced lung injury (8) and have provided substantial insights into the mechanisms underlying nmMLCK-mediated barrier regulation (7,14,25,26,(29)(30)(31)(37)(38)(39)(40)(41)(42)(43)(44).…”

Section: Discussionmentioning

confidence: 99%

“…The lung endothelial cell (EC) cytoskeleton, a dynamic network of proteins involved in cell shape maintenance and cell-cell interactions, is highly responsive to changes in mechanical stress and is centrally involved in lung vascular barrier regulation and maintenance of vascular integrity (2,(5)(6)(7)(8). The balance between actomyosin cytoskeleton contractile forces and cell-cell and cell-matrix interactions is dependent on the activities of a central bioregulatory cytoskeletal effector protein, myosin light-chain kinase (MLCK, gene code MYLK) (6) that phosphorylates myosin light chains on Ser 18 and Thr 19 to produce localized contraction. We have shown previously that lung vascular endothelium express the nonmuscle (nm) isoform of MLCK (nmMLCK) and that nmMLCK enzymatic activity is increased by permeability-enhancing agents such as thrombin (7) and TNF-a (9), resulting in EC stress fiber formation, cellular contraction, paracellular gap formation, and loss of vascular integrity (10).…”

Section: Clinical Relevancementioning

confidence: 99%

“…As noted above, we have previously identified MYLK SNPs that increase the risk and severity of inflammatory lung diseases such as asthma, sepsis, and ARDS (13,(16)(17)(18)(19)(20)(21). We next used in silico bioinformatic approaches to identify 19 SNPs within exon 11 and 10 SNPs within exonflanking nucleotide sequences that potentially regulate MYLK alternative splicing.…”

Section: In Silico Analysis To Identify Mylk Snps That Influence Altementioning

confidence: 99%

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Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene

Mascarenhas¹,

Tchourbanov

Fan

et al. 2017

Am J Respir Cell Mol Biol

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The nonmuscle (nm) myosin light-chain kinase isoform (MLCK), encoded by the MYLK gene, is a vital participant in regulating vascular barrier responses to mechanical and inflammatory stimuli. We determined that MYLK is alternatively spliced, yielding functionally distinct nmMLCK splice variants including nmMLCK2, a splice variant highly expressed in vascular endothelial cells (EC) and associated with reduced EC barrier integrity. We demonstrated previously that the nmMLCK2 variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y and Y) that influence vascular barrier function during inflammation. In this study, we used minigene constructs and RT-PCR to interrogate biophysical factors (mechanical stress) and genetic variants (MYLK single-nucleotide polymorphisms [SNPs]) that are potentially involved in regulating MYLK alternative splicing and nmMLCK2 generation. Human lung EC exposed to pathologic mechanical stress (18% cyclic stretch) produced increased nmMLCK2 expression relative to levels of nmMLCK1 with alternative splicing significantly influenced by MYLK SNPs rs77323602 and rs147245669. In silico analyses predicted that these variants would alter exon 11 donor and acceptor sites for alternative splicing, computational predictions that were confirmed by minigene studies. The introduction of rs77323602 favored wild-type nmMLCK expression, whereas rs147245669 favored alternative splicing and deletion of exon 11, yielding increased nmMLCK2 expression. Finally, lymphoblastoid cell lines selectively harboring these MYLK SNPs (rs77323602 and rs147245669) directly validated SNP-specific effects on MYLK alternative splicing and nmMLCK2 generation. Together, these studies demonstrate that mechanical stress and MYLK SNPs regulate MYLK alternative splicing and generation of a splice variant, nmMLCK2, that contributes to the severity of inflammatory injury.

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Section: Genotype At Rs147245669mentioning

confidence: 54%

Section: Clinical Relevancementioning

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Clinical Relevancementioning

confidence: 99%

Section: In Silico Analysis To Identify Mylk Snps That Influence Altementioning

confidence: 99%

See 3 more Smart Citations

Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene

Mascarenhas¹,

Tchourbanov

Fan

et al. 2017

Am J Respir Cell Mol Biol

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Critical role for the lung endothelial nonmuscle myosin light‐chain kinase isoform in the severity of inflammatory murine lung injury

et al. 2022

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Global knockout of the nonmuscle isoform of myosin light-chain kinase (nmMLCK), a primary cellular regulator of cytoskeletal machinery, is strongly protective in preclinical murine models of inflammatory lung injury. The current study was designed to assess the specific contribution of endothelial cell (EC) nmMLCK to the severity of murine inflammatory lung injury produced by lipopolysaccharide (LPS) and mechanical ventilation ventilatorinduced lung injury or ventilation (VILI). Responses to combined LPS/VILI exposure were assessed in: (i) wild-type (WT) C57BL/6J mice; (ii) transgenic mice with global deletion of nmMLCK (nmMylk −/− ); (iii) transgenic nmMylk −/− mice with overexpression of nmMLCK restricted to the endothelium (nmMylk −/−/ec-tg+ ). Lung inflammation indices included lung histology, bronchoalveolar lavage (BAL) polymorphonuclear leukocytes (PMNs), lung protein biochemistry, tissue albumin levels, Evans blue dye (EBD) lung extravasation, and plasma cytokines (interleukin-6 [IL-6],keratinocyte chemoattractant [KC]/IL-8, IL-1bβ, extracellular nicotinamide phosphoribosyltransferase, tumor necrosis factor-α). Compared to WT C57BL/ 6J mice, the severity of LPS/VILI-induced lung injury was markedly reduced in mice with global nmMLCK deletion reflected by reductions in histologic inflammatory lung injury, BAL PMN counts, mitogen-activated protein kinase, and NF-kB pathway activation in lung homogenates, plasma cytokine levels, and parameters of lung permeability (increased BAL protein, tissue albumin levels, EBD lung extravasation). In contrast, mice with restricted overexpression of nmMLCK in EC (nmMylk −/−/ec-tg+ ) showed significant persistence of LPS/VILI-induced lung injury severity compared to WT mice. In conclusion, these studies strongly endorse the role of EC nmMLCK in driving the severity of preclinical inflammatory lung injury. Precise targeting of EC

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

Sun

Sammani

et al. 2021

Clinical Science

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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.

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Fine mapping of the myosin light chain kinase (MYLK) gene replicates the association with asthma in populations of Spanish descent

Cited by 8 publications

References 34 publications

Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene

Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene

Critical role for the lung endothelial nonmuscle myosin light‐chain kinase isoform in the severity of inflammatory murine lung injury

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

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