Myosin Light Chain Kinase Isoform Research Articles

Characterisation of the myosin light chain kinase (MLCK) gene of Locusta migratoria and the encoded MLCK.

Myosin light chain kinase (MLCK) is a dedicated kinase of myosin regulatory light chain (RLC), playing an essential role in the regulation of muscle contraction and cell motility. Much of the knowledge about MLCK comes from the study of vertebrate MLCK, and little is known about insect MLCK. Here, we identified the single MLCK gene in the locust Locusta migratoria, which spans over 1400 kb, includes 62 exons and accounts for at least five transcripts. We found that the five distinct transcripts of the locust MLCK gene are expressed in a tissue-specific manner, including three muscle-specific isoforms and two generic isoforms. To characterise the kinase activity of locust MLCK, we recombinantly expressed LmMLCK-G, the smallest locust MLCK isoform, in insect Sf9 cells. We demonstrated that LmMLCK-G is a Ca2+ /calmodulin-dependent kinase that specifically phosphorylates serine 50 of locust muscle myosin RLC (LmRLC). Additionally, we found that almost all LmRLC molecules in the flight muscle and the hindleg muscles of adult locusts are phosphorylated.

Molecular Insights into the MLCK Activation by CaM.

Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca2+) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally, we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a de novo molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.

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

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.

Phosphorylation of MYL12 by Myosin Light Chain Kinase Regulates Cellular Shape Changes in Cochlear Hair Cells.

The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape changes of HCs are important for the development of auditory epithelia and hearing function. It was previously observed that HCs and inner sulcus cells (ISCs) demonstrate cellular shape changes similar to the apical constriction of the neural epithelia. Apical constriction is induced via actomyosin cable contraction in the apical junctional complex and necessary for the physiological function of the epithelium. Actomyosin cable contraction is mainly regulated by myosin regulatory light chain (MRLC) phosphorylation by myosin light chain kinase (MLCK). However, MRLC and MLCK isoforms expressed in HCs and ISCs are unknown. Hence, we investigated the expression patterns and roles of MRLCs and MLCKs in HCs. Droplet digital PCR revealed that HCs expressed MYL12A/B and MYL9, which are non-muscle MRLC and smooth muscle MLCK (smMLCK), respectively. Immunofluorescence staining throughout the organ of Corti demonstrated that only MYL12 was expressed in the apical portion of HCs, whereas MYL12 and MYL9 were expressed on ISCs. In addition, purified MYL12B was phosphorylated by smMLCK in vitro, and the harvested HCs contained phosphorylated MYL12. Furthermore, accompanied by the expansion of the cell area of outer HCs, MYL12 phosphorylation was reduced by ML-7, which is an inhibitor of smMLCK. In conclusion, MYL12 phosphorylation by smMLCK contributed to the apical constriction-like cellular shape change of HCs possibly relating to the development of auditory epithelia and hearing function.

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

Rationale: The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically 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 (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein–DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2′-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (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, tumor necrosis factor-α (TNF-α), 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 (HIF) expression, 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 (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (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 clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.

Gut microbial transcytosis induced by tumor necrosis factor-like 1A-dependent activation of a myosin light chain kinase splice variant contributes to IBD.

Inflammatory bowel disease (IBD) is characterized by abnormal host-microbe interactions. Proinflammatory cytokine IFNγ and a novel TNF superfamily member, TL1A, have been implicated in epithelial barrier dysfunction. The divergent regulatory mechanisms of transcellular versus paracellular hyperpermeability remain poorly understood. Intestinal epithelia express two splice variants of long myosin light chain kinase (MLCK), of which the full-length MLCK1 differ from the shorter isoform MLCK2 by a Src kinase phosphorylation site. The aim was to investigate the roles of MLCK splice variants in gut barrier defects under proinflammatory stress. Upregulated expression of TL1A, IFNγ, and two MLCK variants was observed in human IBD biopsy specimens. The presence of intraepithelial bacteria preceded tight junction (TJ) damage in dextran sodium sulfate-treated and TL1A-transgenic mouse models. Lack of barrier defects was observed in long MLCK(-/-) mice. TL1A induced MLCK-dependent terminal web (TW) contraction, brush border fanning, and transepithelial bacterial internalization. The bacterial taxa identified in the inflamed colonocytes included Escherichia, Enterococcus, Staphylococcus,and Lactobacillus. Recombinant TL1A and IFNγ at low doses induced PI3K/Akt/MLCK2-dependent bacterial endocytosis, whereas high-dose IFNγ caused TJ opening via the iNOS/Src/MLCK1 axis. Bacterial internalization was recapitulated in MLCK-knockout cells individually expressing MLCK2 but not MLCK1. Immunostaining showed different subcellular sites of phosphorylated MLC localized to the TJ and TW in the MLCK1- and MLCK2-expressing cells, respectively. In conclusion, proinflammatory cytokines induced bacterial influx through transcellular and paracellular routes via divergent pathways orchestrated by distinct MLCK isoforms. Bacterial transcytosis induced by TL1A may be an alternative route causing symptom flares in IBD.

Tu1751 - Distinct Roles of Myosin Light Chain Kinase Isoforms in Bacterial Endocytosis and Tight Junctional Defects in Mouse Models of Inflammatory Bowel Disease

Tu1751 - Distinct Roles of Myosin Light Chain Kinase Isoforms in Bacterial Endocytosis and Tight Junctional Defects in Mouse Models of Inflammatory Bowel Disease

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

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.

Nonmuscle Myosin Light Chain Kinase: A Key Player in Intermittent Hypoxia-Induced Vascular Alterations.

BackgroundObstructive sleep apnea is characterized by repetitive pharyngeal collapses during sleep, leading to intermittent hypoxia (IH), the main contributor of obstructive sleep apnea–related cardiovascular morbidity. In patients and rodents with obstructive sleep apnea exposed to IH, vascular inflammation and remodeling, endothelial dysfunction, and circulating inflammatory markers are linked with IH severity. The nonmuscle myosin light chain kinase (nmMLCK) isoform contributes to vascular inflammation and oxidative stress in different cardiovascular and inflammatory diseases. Thus, in the present study, we hypothesized that nmMLCK plays a key role in the IH‐induced vascular dysfunctions and inflammatory remodeling.Methods and ResultsTwelve‐week‐old nmMLCK +/+ or nmMLCK −/− mice were exposed to 14‐day IH or normoxia. IH was associated with functional alterations characterized by an elevation of arterial blood pressure and stiffness and perturbations of NO signaling. IH caused endothelial barrier dysfunction (ie, reduced transendothelial resistance in vitro) and induced vascular oxidative stress associated with an inflammatory remodeling, characterized by an increased intima‐media thickness and an increased expression and activity of inflammatory markers, such as interferon‐γ and nuclear factor‐κB, in the vascular wall. Interestingly, nmMLCK deletion prevented all IH‐induced functional and structural alterations, including the restoration of NO signaling, correction of endothelial barrier integrity, and reduction of both oxidative stress and associated inflammatory response.ConclusionsnmMLCK is a key mechanism in IH‐induced vascular oxidative stress and inflammation and both functional and structural remodeling.

Non-muscular myosin light chain kinase triggers intermittent hypoxia-induced interleukin-6 release, endothelial dysfunction and permeability

Obstructive sleep apnea is characterized by intermittent hypoxia (IH) which alters endothelial function, induces inflammation and accelerates atherosclerosis-induced cardiovascular diseases. The non-muscular myosin light chain kinase (nmMLCK) isoform contributes to endothelial cell-cell junction opening. Deletion of nmMLCK protects mice from death in septic shock models and prevents atherosclerosis in high-fat diet-fed mice. The aim of the study was to analyze the implication of nmMLCK in IH-induced vascular inflammation. Human aortic endothelial cells were exposed to 6 hours of IH in absence or presence of nmMLCK inhibitors, ML-7 (5 µM) or PIK (150 µM). IH increased reactive oxygen species (ROS) and nitric oxide (NO) production, p65-NFκB activation and IL-6 secretion. While nmMLCK inhibition did not prevent IH-induced ROS production and p65-NFκB activation, it decreased NO production and partially prevented IL-6 secretion. IH-induced IL-6 secretion and vesicle-associated membrane protein-associated vesicles re-organization were inhibited in presence of the inhibitor of protein secretion, brefeldin A, or ML-7. IH increased monocytes transendothelial migration that was partially prevented by ML-7. Finally, IH reduced endothelium-dependent relaxation to acetylcholine of aortas from wild-type but not those taken from nmMLCK-deficient mice. These results suggest that nmMLCK participates to IH-induced endothelial dysfunction resulting from cytokines secretion and endothelial permeability.

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

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 (Y464 and Y471) 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.

0141 : NmMLCK a key player in intermittent hypoxia-induced inflammatory vascular remodeling?

Obstructive sleep apnea (OSA) is characterized by repetitive pharyngeal collapses during sleep leading to intermittent hypoxia (IH), the main contributor of OSA-related cardiovascular morbidity. OSA patients exhibit increased intima-media thickness (IMT) that correlates with circulating inflammatory markers and severity of nocturnal oxygen desaturation. In mice models, IH also induces hemodynamic alterations and cardiovascular inflammatory remodeling. The non-muscle myosin light chain kinase (nmMLCK) isoform contributes to endothelial cell-cell junction opening, monocyte migration and thus participates to inflammation. nmMLCK deficiency or inhibition has been reported to attenuate systemic, lung and atherosclerotic inflammation. The aim of the present study was to investigate the role of nmMLCK in the IH-induced inflammatory vascular remodeling. We assess vascular remodeling/inflammation in different vascular beds (aorta, carotid and mesenteric arteries). 12 week-old C57bl6 or nmMLCK knockout mice were exposed to 14-days IH or normoxia. IH was associated with an elevation of mean arterial pressure, which was prevented by the nmMLCK deletion. We demonstrated that IH induced an inflammatory response in the three vascular beds, characterized by an increased mRNA expression of CD45 and IFNγ, an increased expression of the macrophage marker F4/80 and an increased expression and activity of NFkB. Interestingly nmMLCK deletion prevented the IH-induced vascular inflammatory responses. On-going experiments are characterizing the effect of nmMLCK deletion on IH-induced structural vascular remodeling. These results strongly suggest that nmMLCK participates to IH-induced vascular inflammatory remodeling and might represent an attractive target to fight against the occurrence of inflammation and the vascular outcomes of OSA. The author hereby declares no conflict of interest

Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

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 effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

A MYLK variant regulates asthmatic inflammation via alterations in mRNA secondary structure.

Myosin light-chain kinase (MYLK) is a gene known to be significantly associated with severe asthma in African Americans. Here we further examine the molecular function of a single-nucleotide polymorphism (SNP), located in the non-muscle myosin light-chain kinase isoform (nmMLCK), in asthma susceptibility and pathobiology. We identified nmMLCK variant (reference SNP: rs9840993, NM_053025: 721C>T, c.439C>T) with a distinct mRNA secondary structure from the other variants. The nmMLCK variant (721C) secondary structure exhibits increased stability with an elongated half-life in the human endothelial cell, and greater efficiency in protein translation initiation owing to an increased accessibility to translation start site. Finally, nmMLCK expression of 721C- and 721T-containing MYLK transgenes were compared in nmMLCK−/− mice and confirmed deleterious effects of nmMLCK expression on asthmatic indices and implicated the augmented influence of MYLK 721C>T (c.439C>T) SNP on asthma severity. The confirmation of the novel mechanism of the regulation of asthmatic inflammation by a MYLK advances knowledge of the genetic basis for asthma disparities, and further suggests the potential of nmMLCK as a therapeutic target. Our study suggests that in addition to altering protein structure and function, non-synonymous SNPs may also lead to phenotypic disparity by altering protein expression.

Role of c-Met/Phosphatidylinositol 3-Kinase (PI3k)/Akt Signaling in Hepatocyte Growth Factor (HGF)-mediated Lamellipodia Formation, Reactive Oxygen Species (ROS) Generation, and Motility of Lung Endothelial Cells

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

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.

TNFR2 Activates MLCK-Dependent Tight Junction Dysregulation to Cause Apoptosis-Mediated Barrier Loss and Experimental Colitis

Tight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease. However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are not completely understood. We investigated these processes using colitis models in mice. We induced colitis by adoptive transfer of CD4(+)CD45RB(hi) cells or administration of dextran sulfate sodium to mice, including those deficient in tumor necrosis factor receptor (TNFR) 1, TNFR2, or the long isoform of myosin light chain kinase (MLCK). Intestinal tissues and isolated epithelial cells were analyzed by immunoblot, immunofluorescence, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction assays. Induction of immune-mediated colitis by CD4(+)CD45RB(hi) adoptive transfer increased intestinal permeability, epithelial expression of claudin-2, the long isoform of MLCK, and TNFR2 (but not TNFR1) and phosphorylation of the myosin II light chain. Long MLCK upregulation, myosin II light chain phosphorylation, barrier loss, and weight loss were attenuated in TNFR2(-/-) , but not TNFR1(-/-) , recipients of wild-type CD4(+)CD45RB(hi) cells. Similarly, long MLCK(-/-) mice had limited increases in myosin II light chain phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of adoptive transfer-induced colitis. However, coincident with onset of epithelial apoptosis, long MLCK(-/-) mice ultimately developed colitis. This indicates that disease progresses via apoptosis in the absence of MLCK-dependent tight junction regulation. In support of this conclusion, long MLCK(-/-) mice were not protected from epithelial apoptosis-mediated, damage-dependent dextran sulfate sodium colitis. In immune-mediated inflammatory bowel disease models, TNFR2 signaling increases long MLCK expression, resulting in tight junction dysregulation, barrier loss, and induction of colitis. At advanced stages, colitis progresses by apoptosis and mucosal damage that result in tight junction- and MLCK-independent barrier loss. Therefore, barrier loss in immune-mediated colitis occurs via two temporally and morphologically distinct mechanisms. Differential targeting of these mechanisms can lead to improved inflammatory bowel disease therapies.

Long Isoform of Myosin Light Chain Kinase Interacts with Calcium Release‐Activated Calcium Channel Constituents to Induce an Amplified and Protracted Increase in Intracellular Calcium

Stromal interaction molecule 1 (STIM1) induces calcium (Ca2+) entry by activating transient receptor potential canonical (TRPC) channel‐mediated store‐operated calcium (SOC) entry or ORAI1 mediated Ca2+ release‐activated Ca2+(CRAC) entry. However, mechanisms regulating STIM1 interaction with ORAI1/TRPC1 remains unclear. Myosin light chain kinase (MLCK)‐210, a Ca2+‐calmodulin dependent long isoform of MLCK, contains domains at N‐terminus which mediates protein‐protein interaction. We tested the hypothesis that MLCK through its N‐terminus interacts with STIM1, ORAI1 and TRPC1 leading to amplified and protracted increase in Ca2+ entry. We show that thapsigargin, which activates SOC as well as CRAC channels, induce markedly attenuated increase in Ca2+ entry in MLCK‐210−/− endothelial cells (ECs). Restoring N‐terminus but not C‐terminus of MLCK‐210 rescued Ca2+ entry in MLCK‐210−/− ECs. Over expression of N‐MLCK‐210 mutant in wild type ECs rapidly enhanced Ca2+ entry which had a steep slope quite distinct than vector expressing ECs. Interestingly, MLCK‐210 interacted with ORAI1, STIM1 and TRPC1 under basal condition and the interaction further increased following thapsigargin stimulation of ECs. These observations suggest that MLCK‐210 may facilitate STIM1 interaction with ORAI1 and TRPC1 to amplify and protract Ca2+entry in ECs

Myosin Light Chain Kinase Signaling in Endothelial Barrier Dysfunction

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia-reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell-cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca(++) , protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability.