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Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

ABSTRACT: Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK(-/-) mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK.

SUBMITTER: Wang T

PROVIDER: S-EPMC4869928 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

Wang Ting T Mathew Biji B Wu Xiaomin X Shimizu Yuka Y Rizzo Alicia N AN Dudek Steven M SM Weichselbaum Ralph R RR Jacobson Jeffrey R JR Hecker Louise L Garcia Joe G N JG

Pulmonary circulation 20160601 2

Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regul ...[more]

PMID: 27252850

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Project description: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 ventilator-induced 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 nmMLCK may represent an attractive therapeutic strategy to reduce lung inflammation and both lung and systemic vascular permeability.

Project description:Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.Significance of the studyPhosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Project description:Acute lung injury (ALI), a major cause of acute respiratory failure with high morbidity and mortality, isare characterized by significant pulmonary inflammation and both alveolar and vascular barriers dysfunction. In Pprior studies have highlighted the role of nonmuscle myosin light chain kinase (nmMLCK) as an essential element of inflammatory response with MYLK polymorphisms associated withwhich alters ALI susceptibility. In the present study we sought to further define nmMLCK in acute inflammatory syndromes and examined We examined nmMLCK as a molecular target involved in increase of lung epithelial and endothelial barrier permeability. We utilized in two muirine models of inflammatory lung injury: intratracheal administration of endotoxin/lipopolysaccharide (LPS, 2.5 mg/kg) and VILI (ventilator-induced lung injury, tidal volume 40ml/kg). Two complementary strategies were used to reduce nmMLCK activity or expression. We found that membrane permeant oligopeptide, PIK, inhibited MLC kinase activity in vitro in aand displayed dose-dependent mannerinhibition of MLC kinase activity.. Intravenous delivery of PIK significantly attenuated LPS-induced lung inflammation reflected by decreasing accumulation of bronchoalveolar lavage (BAL) albumin (~ 50% reduction) as well as reduction in BAL cells, tissue MPO activity and tissue albumin in lung homogenates. A second regulatory approach explored targeting murine nmMLCK by administration of siRNA (5mg/kg) 3 days prior to LPS challenge. siRNA decreased of nmMLCK expression in lungs (~ 70% reduction) and resulted in significant attenuation LPS-induced lung inflammation (~ 40% reduction) as reflected by decreased BAL protein level and BAL cells. For targeting pulmonary vessels nmMLCK we used ACE antibody-conjugated liposomes with nmMLCK siRNA in murine ventilator-induced lung injury (VILI) model. Protein silencing of nmMLCK was evident by immunohistochemical analysis with a decrease in relative intensity of fluorescence in lung vessels compared with control animals. Furthermore, the inhibition of nmMLCK expression by siRNA in vessels significantly attenuated VILI lung injury as reflected by decreased BAL protein level (40% reduction). Finally, MLCK knockout mice were significantly protected (reduced BAL protein and albumin) when exposed to a model of severe VILI (4h, 40ml/kg tidal volume). Conclusion: the MLCK gene KO and chemical biology results indicate that the targeting of nmMLCK in vivo attenuate the severity of LPS-induced or VILI acute lung injury. We used microarrays to detail the global programme of gene expression induced by VILI in Wild type and nmMLCK-/- mouse.

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Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

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Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

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