Nonmuscle Myosin Light Chain Kinase Research Articles

Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element

The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a β-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.

MicroRNA Regulation of Nonmuscle Myosin Light Chain Kinase Expression in Human Lung Endothelium

Increased lung vascular permeability, the consequence of endothelial cell (EC) barrier dysfunction, is a cardinal feature of inflammatory conditions such as acute lung injury and sepsis and leads to lethal physiological dysfunction characterized by alveolar flooding, hypoxemia, and pulmonary edema. We previously demonstrated that the nonmuscle myosin light chain kinase isoform (nmMLCK) plays a key role in agonist-induced pulmonary EC barrier regulation. The present study evaluated posttranscriptional regulation of MYLK expression, the gene encoding nmMLCK, via 3' untranslated region (UTR) binding by microRNAs (miRNAs) with in silico analysis identifying hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290 as miRNA candidates. We identified increased MYLK gene transcription induced by TNF-α (24 h; 4.7 ± 0.45 fold increase [FI]), LPS (4 h; 2.85 ± 0.15 [FI]), and 18% cyclic stretch (24 h; 4.6 ± 0.24 FI) that was attenuated by transfection of human lung ECs with mimics of hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, or hsa-miR-1290 (20-80% reductions by each miRNA). TNF-α, LPS, and 18% cyclic stretch each increased the activity of a MYLK 3'UTR luciferase reporter (2.5-7.0 FI) with induction reduced by mimics of each miRNA (30-60% reduction). MiRNA inhibitors (antagomirs) for each MYLK miRNA significantly increased 3'UTR luciferase activity (1.2-2.3 FI) and rescued the decreased MLCK-3'UTR reporter activity produced by miRNA mimics (70-110% increases for each miRNA; P < 0.05). These data demonstrate that increased human lung EC expression of MYLK by bioactive agonists (excessive mechanical stress, LPS, TNF-α) is regulated in part by specific miRNAs (hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290), representing a novel therapeutic strategy for reducing inflammatory lung injury.

A potential role for myosin light chain kinase in lung adenocarcinoma tumorigenesis.

e22051 Background: Treatment of non-small cell lung cancer remains a clinical challenge despite the migration from cytotoxic chemotherapies to targeted agents. Myosin light chain kinase (MLCK) is a protein involved in phosphorylation of regulatory chains of myosin, thereby regulating the contraction of myosin II. The 210 KDa non-muscle MLCK isoform (nmMLCK), expressed in endothelial cells, regulates cell shape and motility, and increases EGFR-mediated proliferation of hepatocytes. Furthermore, inhibitors of MLCK such as ML-7 and ML-9 have been shown to decrease invasiveness of pancreatic prostate cancer cell lines. Clinically, levels of MLCK gene (MYLK) mRNA correlate with increased risk of disease recurrence and development of distant metastasis in lung cancer. These data suggest a potential role for MLCK in lung cancer tumorigenesis and metastasis. Methods: H-23 and H-441 human lung adenocarcinoma cell lines were treated for 12hrs with MLCK chemical inhibitor, ML-7 (20uM), and harvested at 24hr, 48hr, and 72hr for MTS Assay. Cell proliferation was assessed with MTS Assay per manufacturer protocol (Promega). H-23 cells were transiently transfected with a FLAG-tagged (WT) nmMLCK overexpression vector using Fugene Transfection Kit per manufacturer protocol (Promega). Results: While no significant difference in proliferation was noted in A-549 cells, both H-23 and H-441 cells showed a decrease in cell proliferation with inhibition of MLCK. H-23 cells also showed an increase in proliferation when transiently transfected with an MLCK-overexpression vector. Conclusions: Inhibition of endogenous nmMLCK decreases H-23 and H-441 proliferation. Conversely, an increase in nmMLCK expression in vitro increases cell proliferation in H23 cells. These results suggest that MLCK/MYLK may participate in regulation of lung adenocarcinoma cell proliferation.

Critical Role of Non-Muscle Myosin Light Chain Kinase in Thrombin-Induced Endothelial Cell Inflammation and Lung PMN Infiltration

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.

Nonmuscle myosin light‐chain kinase mediates microglial migration induced by HIV Tat: Involvement of β1 integrins

One of the hallmark features of HIV-associated neurological disease is increased activation and migration of microglia. HIV transactivator of transcription (Tat) is released from infected cells and has the ability to recruit microglia. The purpose of this study was to investigate molecular mechanisms by which recombinant Tat₁₋₇₂, but not heated-inactive Tat₁₋₇₂,induces migration of rat primary microglia. Using primary microglia in Boyden chambers, we demonstrated the role of nonmuscle myosin light-chain kinase (nmMYLK) in Tat₁₋₇₂ (14.4 nM)-mediated increased microglial migration (up to 171.85%). These findings were validated using microglia isolated from wild-type (WT) or nmMYLK(-/-) mice in Dunn chamber assays. Tat₁₋₇₂-mediated activation of nmMYLK resulted in "inside-out" activation of β1 integrin, followed by "outside-in" activation of c-Src, Pyk2, and Cdc42-GTP (using G-LISA in primary and nmMYLK(-/-) microglia) and, subsequently, actin polymerization (flow cytometry and Western blot assays). In vivo corroboration of these findings demonstrated decreased migration of nmMYLK(-/-) microglia (2 × 10(5) cells transplanted into corpus callosum) compared with WT microglia toward microinjected Tat₁₋₇₂ (2 μg/mouse) in hippocampus. Up-regulation of nmMYLK in microglia was also detected in sections of basal ganglia from humans with HIV-encephalitis compared with uninfected controls. nmMYLK is thus critical for eliciting microglial migration during the innate immune response.

TLR4 activation of TRPC6-dependent calcium signaling mediates endotoxin-induced lung vascular permeability and inflammation

Lung vascular endothelial barrier disruption and the accompanying inflammation are primary pathogenic features of acute lung injury (ALI); however, the basis for the development of both remains unclear. Studies have shown that activation of transient receptor potential canonical (TRPC) channels induces Ca(2+) entry, which is essential for increased endothelial permeability. Here, we addressed the role of Toll-like receptor 4 (TLR4) intersection with TRPC6-dependent Ca(2+) signaling in endothelial cells (ECs) in mediating lung vascular leakage and inflammation. We find that the endotoxin (lipopolysaccharide; LPS) induces Ca(2+) entry in ECs in a TLR4-dependent manner. Moreover, deletion of TRPC6 renders mice resistant to endotoxin-induced barrier dysfunction and inflammation, and protects against sepsis-induced lethality. TRPC6 induces Ca(2+) entry in ECs, which is secondary to the generation of diacylglycerol (DAG) induced by LPS. Ca(2+) entry mediated by TRPC6, in turn, activates the nonmuscle myosin light chain kinase (MYLK), which not only increases lung vascular permeability but also serves as a scaffold to promote the interaction of myeloid differentiation factor 88 and IL-1R-associated kinase 4, which are required for NF-κB activation and lung inflammation. Our findings suggest that TRPC6-dependent Ca(2+) entry into ECs, secondary to TLR4-induced DAG generation, participates in mediating both lung vascular barrier disruption and inflammation induced by endotoxin.

Role of Non-Muscle Myosin Light Chain Kinase in Neutrophil-Mediated Intestinal Barrier Dysfunction During Thermal Injury

Neutrophils and non-muscle myosin light chain kinase (nmMLCK) have been implicated in intestinal microvascular leakage and mucosal hyperpermeability in inflammation and trauma. The aim of this study was to characterize the role of nmMLCK in neutrophil-dependent gut barrier dysfunction following thermal injury, a common form of trauma that typically induces inflammation in multiple organs. Histopathological examination of the small intestine in mice after a full-thickness burn revealed morphological evidence of mucosa inflammation characterized by neutrophil infiltration into the lamina propria, epithelial contraction, and narrow villi with blunt brush borders and loss of goblet cells. Compared with their wild-type counterparts, nmMLCK mice displayed diminished morphological abnormalities. Likewise, intravital microscopic studies showed significant leukocyte adhesion in intestinal microvessels after burn, a response that was blunted in the absence of nmMLCK. Functionally, thermal injury significantly increased the gut lumen-to-blood transport of fluorescein isothiocyanate-dextran (4 kd), and this hyperpermeability was attenuated by either neutrophil depletion or nmMLCK deficiency. Consistent with the in vivo observations, in vitro assays with Caco-2 epithelial cell monolayers revealed a decrease in transcellular electric resistance coupled with myosin light chain phosphorylation, actomyosin ring condensation, and claudin-1 internalization upon stimulation with fMLP (N-formyl-methionyl-leucyl-phenylalanine)-activated neutrophils. Pretreatment of the cells with the MLCK inhibitor ML-7 prevented the tight junction responses. Taken together, the results suggest that nmMLCK plays an important role in neutrophil-dependent intestinal barrier dysfunction during inflammatory injury.

C‐Abl Inhibition Exacerbates Mechanical Ventilation‐Induced Lung Injury In A Murine Model

Endothelial cytoskeleton is a contractile structure responsible for cellular responses to external stimuli and mechanical stress. Nonmuscle myosin light chain kinase (nmMLCK), a critical actin‐binding protein is required for trafficking of inflammatory cellsand alterations in permeability. c‐Abl kinase plays an essential role in barrier regulation via posttranslational modification of nmMLCK. Abl tyrosine kinase phosphorylates nmMLCK and regulates endothelial barrier function. We hypothesized that tyrosine kinase inhibitor imatinib, an anticancer agent, can exacerbate lung injury induced by mechanical ventilation (MV). To explore the role of c‐Abl in lung injury in vivo, mice were tested in our ventilation‐induced acute lung injury (VILI) model after imatinib. Mice were harvested 4h after MV. Imatinib significantly increased bronchoalveolar lavage protein levels (825ug/ml) and total inflammatory cell counts (1.07 × 10□) compared to sham (571ug/ml, 0.75 × 10μ) (p&lt;0.05). These preliminary results indicate that imatinib exacerbates VILI‐injury, likely via c‐Abl blockade. These studies have clinical relevance given the extensive use of imatinib as an anticancer agent and support an important functional role for c‐Abl in lung permeabilityFunded by: NIH grant: P01 HL58064

Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.

An intronic MYLK variant associated with inflammatory lung disease regulates promoter activity of the smooth muscle myosin light chain kinase isoform

Intronic single-nucleotide polymorphisms (SNPs) are commonly associated with complex diseases but exhibit unknown biologic functionality. Myosin light-chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK, plays a key pathophysiological role in complex diseases including acute lung injury (ALI) and asthma. We studied the potential regulatory roles of two intronic MYLK SNPs (rs936170 and rs820336) previously associated with ALI and asthma. Due to their genomic location at the junction encoding the non-muscle and smooth muscle MLCK (smMLCK) isoforms, we first identified the transcription start site (TSS) of the smMLCK isoform, and isolated a 2,954-bp DNA fragment upstream of the smMLCK TSS. Serial 5' deletion of the fragment revealed a proximal promoter region exhibiting strong promoter activity with potential inhibitory elements in the distal region. Site-directed mutageneses and luciferase reporter assays showed no effect of the distal promoter SNP rs936170 on smMLCK promoter activity. In contrast, SNP rs820336, located in an enhancer/repressor region downstream of TSS, was identified to regulate smMLCK promoter activity in an allelic-dependent manner. The A allele interrupted the binding site for Forkhead box protein N1 (FOXN1), a transcription factor governing expression of immune response genes. Silencing of FOXN1 expression (siRNA) reduced FOXN1 interaction with cis-regulatory elements in proximity to rs820336 and significantly decreased smMLCK expression. These functional insights into the involvement of intronic MYLK SNPs further strengthen the concept that MYLK contributes to inflammatory disease susceptibility and represents an attractive molecular target in complex inflammatory disorders.

Nonmuscle Myosin Light-Chain Kinase Deficiency Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice via Reduced Endothelial Barrier Dysfunction and Monocyte Migration

Endothelial dysfunction and monocyte migration are key events in the pathogenesis of atherosclerosis. Nonmuscle myosin light-chain kinase (nmMLCK), the predominant MLCK isoform in endothelial cells, has been shown to contribute to vascular inflammation by altering endothelial barrier function. However, its impact on atherogenesis remains unknown. We investigated the role of nmMLCK in the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE(-/-)) mice fed an atherogenic diet for 12 weeks. Histopathological examination demonstrated that nmMLCK deficiency (apoE(-/-)nmmlck(-/-)) reduced the size of aortic lesions by 53%, lipid contents by 44%, and macrophage deposition by 40%. Western blotting and reverse-transcription polymerase chain reaction revealed the expression of nmMLCK in aortic endothelial cells and peripheral blood monocytes. Measurements of transendothelial electric resistance indicated that nmMLCK deficiency attenuated endothelial barrier dysfunction caused by thrombin, oxidized low-density lipoprotein, and tumor necrosis factor α. In monocytes, nmMLCK deficiency reduced their migration in response to the chemokine monocyte chemoattractant protein-1. Further mechanistic studies showed that nmMLCK acted through both myosin light chain phosphorylation-coupled and -uncoupled pathways; the latter involved Rous sacracoma virus homolog genes-encoded tyrosine kinases (Src) signaling. Moreover, depletion of Src via gene silencing, site-specific mutagenesis, or pharmacological inhibition of Src greatly attenuated nmMLCK-dependent endothelial barrier dysfunction and monocyte migration. Nonmuscle myosin light-chain kinase contributes to atherosclerosis by regulating endothelial barrier function and monocyte migration via mechanisms involving not only kinase-mediated MLC phosphorylation but also Src activation.

A transcribed pseudogene ofMYLKpromotes cell proliferation

Pseudogenes are considered nonfunctional genomic artifacts of catastrophic pathways. Recent evidence, however, indicates novel roles for pseudogenes as regulators of gene expression. We tested the functionality of myosin light chain kinase pseudogene (MYLKP1) in human cells and tissues by RT-PCR, promoter activity, and cell proliferation assays. MYLKP1 is partially duplicated from the original MYLK gene that encodes nonmuscle and smooth muscle myosin light chain kinase (smMLCK) isoforms and regulates cell contractility and cytokinesis. Despite strong homology with the smMLCK promoter (∼ 89.9%), the MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. Moreover, MYLKP1 and smMLCK exhibit negatively correlated transcriptional patterns in normal and cancer cells with MYLKP1 strongly expressed in cancer cells and smMLCK highly expressed in non-neoplastic cells. For instance, expression of smMLCK decreased (19.5 ± 4.7 fold) in colon carcinoma tissues compared to normal colon tissues. Mechanistically, MYLKP1 overexpression inhibits smMLCK expression in cancer cells by decreasing RNA stability, leading to increased cell proliferation. These studies provide strong evidence for the functional involvement of pseudogenes in carcinogenesis and suggest MYLKP1 as a potential novel diagnostic or therapeutic target in human cancers.

Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl-mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.

MLCK210 deficiency ameliorates atherosclerosis in apoE knockout mice

Endothelial barrier dysfunction is a key event in the pathogenesis of atherosclerosis. The objective of this study was to explore the role and mechanisms of MLCK210 (non‐muscle myosin light‐chain kinase) in the development of atherosclerosis in mice with an apoE knockout background. Histopathological examination demonstrated that MLCK210 deficiency reduced atherosclerotic lesion and macrophage deposit. Western blot and RT‐PCR analyses revealed MLCK210 expression in the aortic endothelium and peripheral monocytes. By measuring transendothelial electric resistance, we found that MLCK210 deficiency attenuated aorta endothelial hyperpermeability to pro‐inflammatory stimuli, including thrombin, ox‐LDL and TNF‐alpha. In monocytes, MLCK210 deficiency reduced their transendothelial migration in response to murine MCP‐1. Further mechanistic studies showed that these inflammatory mediators induced Src phosphorylation in endothelial cells and monocytes. The phosphorylation response was attenuated in MLCK210 deficient cells, corresponding to reduced endothelial permeability and monocytes transmigration. Taken together, the data suggest that MLCK210 contributes to the development of atherosclerosis by regulating endothelial barrier function and monocyte migration via a mechanism involving Src signaling. Supported by NIH HL61507, HL84542, HL70752, and HL73324.

Non-muscle myosin light chain kinase isoform is a viable molecular target in acute inflammatory lung injury.

Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non-muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (~50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody-conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6-signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.

Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium

Vascular barrier regulation is intimately linked to alterations in the distribution and configuration of the endothelial cell (EC) cytoskeleton in response to angiogenic and edemagenic agonists. Critical actin cytoskeletal rearrangement includes spatially directed increases in myosin light chain (MLC) phosphorylation, catalyzed by Ca2+/calmodulin-dependent non-muscle myosin light chain kinase variants (nmMLCK1- and -2), as well as association of nmMLCK with the actin-binding protein, cortactin. As these associations have proven difficult to quantify in a spatially specific manner, we now describe the utility of intensity correlation image analysis and the intensity correlation quotient (ICQ) to quantify colocalization in fixed and live cell imaging assays in human pulmonary artery EC. From baseline ICQ values averaging 0.216 reflecting colocalization of cortactin–DsRed with EGFP–nmMLCK fusion proteins in resting EC, thrombin-induced EC contraction significantly reduced cortactin–DsRed–EGFP–nmMLCK colocalization (nmMLCK1: ICQ=0.118; nmMLCK2: ICQ=0.091) whereas the potent EC barrier-protective agonist, sphingosine 1-phosphate (S1P), significantly increased nmMLCK–cortactin colocalization within lamellipodia (nmMLCK1: ICQ=0.275; nmMLCK2: ICQ=0.334). Over-expression of a cortactin–DsRed mutant fusion protein lacking the SH3 domain, known to be essential for cortactin–nmMLCK association, reduced baseline and S1P-mediated live cell colocalization with each nmMLCK variant (nmMLCK1: ICQ=0.160; nmMLCK2: ICQ=0.157). Similarly, expression of a truncated EGFP–nmMLCK2 mutant lacking cortactin- and actin-binding domains, markedly reduced basal localization in lamellipodia and abolished colocalization with cortactin–DsRed in lamellipodia after S1P (ICQ=−0.148). These data provide insights into the molecular basis for vascular barrier-regulatory cytoskeletal responses and support the utility of sophisticated imaging analyses and methodological assessment to quantify the critical nmMLCK and cortactin interaction during vascular barrier regulation.

Abstract 5660: Essential Role of Non-muscle Myosin Light Chain Kinase in Hypoxia-induced Murine Pulmonary Hypertension

Introduction: The pulmonary arterial hypertension (PAH) phenotype shares several pathways involved in cancer pathobiology. We previously demonstrated that the multi-kinase inhibitor, sorafenib, ameliorates rodent PAH (Moreno-Vinasco et al 2008) with genomic signatures which strongly implicated the endothelial cell cytoskeleton in the development of PAH vascular remodeling including non-muscle caldesmon, Rho-kinase and both smooth muscle- and non-muscle myosin light chain kinase (nmMLCK) isoforms. We hypothesized that the acto-myosin machinery present within lung endothelium are critical to the development of PAH in a murine model of hypoxia-induced pulmonary hypertension. Methods: Wild type (WT) and genetically engineered mice with targeted deletion of the nmMLCK allele were exposed to normoxia, or to hypoxia and SU5416, a VEGFR-1,2 inhibitor that induces severe PH (3.5 weeks). WT and nmMLCK KO mice were assessed for hemodynamics and histopathology. Results: Hypoxia-SU5416 WT mice developed pulmonary artery phenotype with significant increases in diphosphorylated MLC levels (2-fold, no change in total MLC), increased mean right ventricular systolic pressures (RVSP) and hypertrophy (RV/LV+S) and increased hematocrit when compared to WT-normoxic controls. However, in response to hypoxia/SU, nmMLCK-KO mice displayed reduced pMLC, significant decreases in right ventricular hypertrophy (27.3% decrease), in RVSP (25% decrease) without changes in hematocrit. Histopathology indicates reduced peri-vascular infiltration and remodeling in the nmMLCK KO mice compared to WT controls. Conclusions: Our results are consistent with the notion that myosin light chains (MLC) are a canonical and downstream mediator of mechanical events underlying the actin-myosin interactions in lung endothelium during PH remodeling and nmMLCK is a potential therapeutic target in PAH-mediated remodeling.

Identification of novelin vitroprotein kinase A phosphorylation sites on recombinant non-muscle myosin light chain kinase: nano-liquid chromatography tandem mass spectrometry methodology

Protein phosphorylation is a key event in endothelial cell signaling and barrier regulation. We previously demonstrated that the non-muscle myosin light chain kinase (nmMLCK) isoform present in endothelium (1914aa) is a multi-functional enzyme which drives the participation of the actin cytoskeleton in vascular barrier regulation, trafficking of inflammatory cells into the lung, and susceptibility to sepsis-induced acute lung injury. The activity of the nmMLCK isoform is differentially regulated by both Ser/Thr and Tyr phosphorylation; however, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA)-mediated nmMLCK phosphorylation exerts paradoxical effects on kinase activity depending on the exact Ser/Thr sites. To address this conundrum, we mapped in vitro phosphorylation sites of nmMLCK catalyzed by the catalytic unit of PKA using data-dependent nano-liquid chromatography tandem mass spectrometry. High mass-accuracy MS and optimized biochemical protocols identified 26 novel nmMLCK1 PKA p...

Nonmuscle myosin light-chain kinase mediates neutrophil transmigration in sepsis-induced lung inflammation by activating β2 integrins

Nonmuscle myosin light-chain kinase (MYLK) mediates increased lung vascular endothelial permeability in lipopolysaccharide-induced lung inflammatory injury, the chief cause of the acute respiratory distress syndrome. In a lung injury model, we demonstrate here that MYLK was also essential for neutrophil transmigration, but that this function was mostly independent of myosin II regulatory light chain, the only known substrate of MYLK. Instead, MYLK in neutrophils was required for the recruitment and activation of the tyrosine kinase Pyk2, which mediated full activation of beta(2) integrins. Our results demonstrate that MYLK-mediated activation of beta(2) integrins through Pyk2 links beta(2) integrin signaling to the actin motile machinery of neutrophils.

Identification of novel in vitro protein kinase A phosphorylation sites on recombinant non-muscle myosin light chain kinase: nano-liquid chromatography tandem mass spectrometry methodology

Identification of novel in vitro protein kinase A phosphorylation sites on recombinant non-muscle myosin light chain kinase: nano-liquid chromatography tandem mass spectrometry methodology