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 radiationinduced 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.Keywords: bronchoalveolar lavage, nonmuscle myosin light chain kinase, radiation-induced lung injury.Pulm Circ 2016;6(2):234-239. DOI: 10.1086/686491.Radiotherapy, a primary treatment for thoracic malignancies, is often limited by deleterious dose-limiting side effects that manifest themselves as acute or subacute lung injury 6-24 weeks after radiation exposure. 1 Although the exact molecular mechanisms of radiation-induced lung injury (RILI) are controversial and likely multifactorial, a potentially important cellular target in RILI is the lung endothelium, a functionally dynamic tissue that regulates influx of macromolecules and fluid between the vascular compartment and the lung interstitial space. 2 Disruption of endothelial cell (EC) barrier integrity is considered a key feature of pulmonary inflammation in general and RILI in particular, resulting in increased vascular permeability and alveolar flooding. Thus, stabilization or enhanced restoration of lung vascular endothelial integrity is a critical step in preventing or reducing acute or subacute RILI, as we have previously demonstrated. 2We have also reported the role of the nonmuscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory responses, with variants in the MYLK gene contributing to acute lung injury (ALI), ventilation-induced lung injury (VILI) susceptibility, and asthma susceptibility.3,4 Nonmuscle MLCK is a calcium/calmodulin-dependent kinase critically involved in catalyzing the phosphorylation of myosin light chain (MLC), leading to cell contraction and vascular EC barrier disruption, which indicates a critical role for nmMLCK in regulating vascular permeability. 5 We have also reported that genetic variants (single-nucleotide pol...

Myosin Light Chain Kinase: A Potential Target for Treatment of Inflammatory Diseases

“…In inflammatory lung disorders, damage to lung endothelial cell barrier integrity alters vascular permeability, and alveolar flooding often results ( Mao et al, 2015 ). Abnormal expression of MLCK occurs in lung injury, and the MLCK inhibitor ML-7 or deletion of the MLCK gene can attenuate lung injury ( Wang T. et al, 2016 ). MLCK has similar activity in asthmatic and in lung inflammation, and variation of the MYLK gene is strongly associated with acute lung injury and asthma susceptibility ( Wang et al, 2014 , 2015 ; Wang T. et al, 2016 ).…”

Particulate matter disrupts human lung endothelial cell barrier integrity via Rho‐dependent pathways

“…12 Increased lung endothelial cell (EC) monolayer permeability not only exacerbates pulmonary inflammatory responses, but contributes to amplified systemic responses via increased translocation of pollutant particles into the circulation. 12 We have previously demonstrated the critical role of myosin light chain (MLC) phosphorylation in endothelial barrier regulation, and its regulation by key proteins myosin light chain kinase (MLCK) and myosin light chain phosphatase (MYPT), 13,14 in various pulmonary inflammatory diseases. 15,16 MLC phosphorylation, triggered by edemagenic agonists such as thrombin, TNFa, or LPS, facilitates actin stress fiber formation, cell contraction, and creation of paracellular gaps between ECs, thereby increasing lung fluid imbalance and leukocyte infiltration, as well as transport of pollutant particles to the systemic circulation.…”

Genetic knockout of myosin light chain kinase (MLCK210) prevents cerebral microhemorrhages and attenuates neuroinflammation in a mouse model of vascular cognitive impairment and dementia

“…There are multiple MLCK proteins, but the focus of the present study is a 210-kDa MLCK protein encoded by the mylk1 genetic locus, referred to as MLCK210 (for review see Khapchaev and Shirinsky 2016). Prior studies on the role of MLCK210 in tissue barrier dysfunction and the potential of selective inhibitors have largely focused on non-CNS disorders (for recent reviews see Cunningham and Turner 2012; Rigor et al 2013; Khapchaev and Shirinsky 2016; Xiong et al 2017), including acute lung injury models (Wainwright et al 2003; Rossi et al 2007; Mirzapoiazova et al 2011; Usatyuk et al 2012; Fazal et al 2013; Wang et al 2014; Wang et al 2016; Zhou et al 2015), burn injury (Reynoso et al 2007; Guo et al 2012; Zahs et al 2012), acute diarrhea (Clayburgh et al 2005; Clayburgh et al 2006), endotoxic shock (Ralay Ranaivo et al 2007; Gaceb et al 2016), cardiovascular shear stress (Ohlmann et al 2005), atherosclerosis (Sun et al 2011), hypoxia (Arnaud et al 2018), and intestinal injury models (Al-Sadi et al 2012; Gilbert et al 2012; Wu et al 2014; Lorentz et al 2017; Nighot et al 2017; Al-Sadi et al 2019). Additionally, there exists a smaller literature exploring the benefit of inhibition of MLCK in the context of BBB dysfunction.…”