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Myosin light chain kinase ( MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

Published on Feb 1, 2018in Pulmonary circulation2.075
· DOI :10.1177/2045894018764171
Ting Wang20
Estimated H-index: 20
(UA: University of Arizona),
Mary E. Brown18
Estimated H-index: 18
(UIC: University of Illinois at Chicago)
+ 5 AuthorsJoe G.N. Garcia89
Estimated H-index: 89
(UA: University of Arizona)
Abstract
Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y464 and Y471 phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y464 phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y464 phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.
  • References (31)
  • Citations (4)
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References31
Newest
#2Alex Y. Tchourbanov (UA: University of Arizona)H-Index: 2
Last. Joe G.N. GarciaH-Index: 89
view all 6 authors...
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, ...
6 CitationsSource
#1P. Viswanathan (UIC: University of Illinois at Chicago)H-Index: 1
#2Y. Ephstein (UIC: University of Illinois at Chicago)H-Index: 1
Last. Steven M. Dudek (UIC: University of Illinois at Chicago)H-Index: 35
view all 5 authors...
Abstract Vascular integrity is primarily determined by endothelial cell (EC) cytoskeletal structure that is differentially regulated by various stimuli. In this study, atomic force microscopy (AFM) was used to characterize structural and mechanical properties in the cytoskeleton of cultured human pulmonary artery EC (HPAEC) and human lung microvascular EC (HLMVEC) by determining elastic properties (Young's modulus) in response to endogenous barrier protective agents sphingosine 1-phosphate (S1P)...
2 CitationsSource
#1Marialbert Acosta-Herrera (ISCIII: Carlos III Health Institute)H-Index: 6
#2Maria Pino-Yanes (UCSF: University of California, San Francisco)H-Index: 20
Last. Carlos Flores (ULL: University of La Laguna)H-Index: 36
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4 CitationsSource
#1Kui Shen (UIC: University of Illinois at Chicago)H-Index: 1
#2Benjamin E. Ramirez (UIC: University of Illinois at Chicago)H-Index: 7
Last. Joe G.N. Garcia (UA: University of Arizona)H-Index: 89
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The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effe...
5 CitationsSource
#1Xin Wang (NU: Northwestern University)H-Index: 12
#2Reiner Bleher (NU: Northwestern University)H-Index: 16
Last. Vinayak P. Dravid (NU: Northwestern University)H-Index: 78
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The endothelial cell (EC) lining of the pulmonary vascular system forms a semipermeable barrier between blood and the interstitium and regulates various critical biochemical functions. Collectively, it represents a prototypical biomechanical system, where the complex hierarchical architecture, from the molecular scale to the cellular and tissue level, has an intimate and intricate relationship with its biological functions. We investigated the mechanical properties of human pulmonary artery endo...
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#1Ting WangH-Index: 20
#2Tong ZhouH-Index: 27
Last. Joe G.N. GarciaH-Index: 89
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A MYLK variant regulates asthmatic inflammation via alterations in mRNA secondary structure
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#1Patrick Belvitch (UIUC: University of Illinois at Urbana–Champaign)H-Index: 5
#2Djanybek Adyshev (UIUC: University of Illinois at Urbana–Champaign)H-Index: 16
Last. Steven M. Dudek (UIUC: University of Illinois at Urbana–Champaign)H-Index: 35
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Abstract Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characteri...
7 CitationsSource
#1Ting Wang (UA: University of Arizona)H-Index: 20
#2Liliana Moreno-Vinasco (UIC: University of Illinois at Chicago)H-Index: 26
Last. Joe G.N. Garcia (UA: University of Arizona)H-Index: 89
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Myosin light chain kinase (MLCK; gene code, MYLK) is a multifunctional enzyme involved in isoform-specific nonmuscle (nm) and smooth muscle contraction, inflammation, and vascular permeability, processes directly relevant to asthma pathobiology. In this report, we highlight the contribution of the nm isoform (nmMLCK) to asthma susceptibility and severity, supported by studies in two lines of transgenic mice with knocking out nmMLCK or selectively overexpressing nmMLCK in endothelium. These mice ...
11 CitationsSource
This review provides an update on asthma in Hispanics, a diverse group tracing their ancestry to countries previously under Spanish rule. A marked variability in the prevalence and morbidity from asthma remains among Hispanic subgroups in the United States and Hispanic America. In the United States, Puerto Ricans and Mexican Americans have high and low burdens of asthma, respectively (the “Hispanic Paradox”). This wide divergence in asthma morbidity among Hispanic subgroups is multifactorial, li...
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#1Fernando Teran Arce (UCSD: University of California, San Diego)H-Index: 26
#2Brian Meckes (UCSD: University of California, San Diego)H-Index: 8
Last. Ratnesh Lal (UCSD: University of California, San Diego)H-Index: 37
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Abstract In this study we employ atomic force microscopy, supported by finite element analysis and fluorescence microscopy, to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli. Statistical analysis of elasticity data obtained from multiple cells demonstrates a heterogeneous cellular elastic response to barrier-enhancing and barrier-disrupting agents; sphingosine 1-phosphate (S1P...
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#1Stephanie E. Wallace (University of Texas Health Science Center at Houston)H-Index: 3
#2Ellen S. Regalado (University of Texas Health Science Center at Houston)H-Index: 25
Last. Dianna M. Milewicz (University of Texas Health Science Center at Houston)H-Index: 58
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Heritable thoracic aortic disease can result from null variants in MYLK, which encodes myosin light-chain kinase (MLCK). Data on which MYLK missense variants are pathogenic and information to guide aortic disease management are limited. Clinical data from 60 cases with MYLK pathogenic variants were analyzed (five null and two missense variants), and the effect of missense variants on kinase activity was assessed. Twenty-three individuals (39%) experienced an aortic event (defined as aneurysm rep...
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#1Patrick Belvitch (UIC: University of Illinois at Chicago)H-Index: 5
#2Yu Maw Htwe (UIC: University of Illinois at Chicago)H-Index: 1
Last. Steven M. Dudek (UIC: University of Illinois at Chicago)H-Index: 35
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Abstract The pulmonary endothelial cell forms a critical semi-permeable barrier between the vascular and interstitial space. As part of the blood-gas barrier in the lung, the endothelium plays a key role in normal physiologic function and pathologic disease. Changes in endothelial cell shape, defined by its plasma membrane, determine barrier integrity. A number of key cytoskeletal regulatory and effector proteins including non-muscle myosin light chain kinase, cortactin, and Arp 2/3 mediate acti...
Source
#2Alex Y. Tchourbanov (UA: University of Arizona)H-Index: 2
Last. Joe G.N. GarciaH-Index: 89
view all 6 authors...
Profound lung vascular permeability is a cardinal feature of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI), two syndromes known to centrally involve the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier dysregulation. Two main splice variants, nmMLCK1 and nmMLCK2, are well represented in human lung endothelial cells and encoded by MYLK, and they differ only in the presence of exon 11 in nmMLCK1, which contains critical phosphorylat...
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