Myosin Light Chain Kinase Protein Research Articles

Effects of berberine on rat jejunal motility

The aim of the study was to evaluate berberine-induced bidirectional regulation on the contractility of jejunum. Different low and high contractile states of isolated jejunal segment from rat were established to investigate the effects of berberine. Stimulatory effects on jejunal segment were exerted by berberine in six low contractile states and inhibitory effects were produced on jejunal segment in six high contractile states. The effects of berberine on myosin light chain kinase (MLCK) mRNA expression, MLCK protein content, and myosin phosphorylation in jejunum were also bidirectional. Bidirectional regulation was not observed in the presence of tetrodotoxin. No regulatory effects of berberine on jejunal contractility were observed in the presence of verapamil. The stimulatory effects of berberine on jejunal contractility were blocked by atropine. The inhibitory effects of berberine on jejunal contractility were abolished by phentolamine, propranolol and L-NG-nitro-arginine, respectively. Berberine-induced bidirectional regulation needed the presence of the enteric nervous system, and depended on the influx of extracellular Ca(2+) , related to the cholinergic system while jejunum was in low contractile states, and related to the adrenergic system and nitric oxide relaxing mechanism while jejunum was in high contractile states. The results suggested the potential clinical implication of berberine for alternating-type irritable bowel syndrome.

Myosin Light Chain Kinase Inhibitor Inhibits Dextran Sulfate Sodium-Induced Colitis in Mice

Myosin light chain kinase (MLCK) plays a central role in the mechanisms of barrier dysfunction, and intestinal epithelial MLCK protein expression is upregulated in active ulcerative colitis (UC). ML-7, a MLCK inhibitor, has been used in many MLCK studies. However, the effect of ML-7 has never been estimated in colitis models. The aim of this study was to determine whether ML-7 can treat UC. Experimental colitis was induced and ML-7 was administered by intraperitoneal injection. The disease activity index (DAI) scores were evaluated and colon tissue was collected for the assessment of histological changes, myeloperoxidase (MPO) activity, and tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-13 and interleukin (IL)-17 levels. The small intestinal mucosa was ultrastructurally examined, epithelial MLCK protein expression and enzymatic activity were determined, and intestinal permeability was assayed using FITC-dextran 4000 (FD-4) and Evans blue (EB). ML-7 was found to be significantly effective in reducing the DAI scores and histological index scores, and decreasing MPO activity and TNF-α, IFN-γ, IL-13 and IL-17 levels. The small intestinal epithelial MLCK protein expression and enzymatic activity were downregulated by ML-7. The epithelial cells and intercellular tight junctions were ameliorated, and the amount of FD-4 in blood and EB permeating into the intestine were decreased by ML-7 in colitis mice. ML-7 has a significant anti-colitis effect in colitis mice. It is mainly associated with the inhibition of the epithelial MLCK protein expression, resulting in ameliorated intestinal mucosal permeability.

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.

Cellular and molecular mechanism of interleukin-1β modulation of Caco-2 intestinal epithelial tight junction barrier

Interleukin-1β (IL-1β) is a prototypical multifunctional cytokine that plays an important role in intestinal inflammation of Crohn’s disease and other inflammatory conditions of the gut. Previous studies have shown that IL-1β causes an increase in intestinal epithelial tight junction (TJ) permeability both in in vivo animal and in vitro cell culture model systems. The IL-1β-induced increase in intestinal epithelial TJ permeability has been postulated to be an important pathogenic mechanism contributing to intestinal inflammation. However, the signalling pathways and the molecular processes that mediate the IL-1β modulation of intestinal epithelial TJ barrier remain unclear. Here, we show that the IL-1β-induced increase in Caco-2 monolayer TJ permeability was mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2) signalling pathway and that inhibition of ERK1/2 activity inhibits the IL-1β-induced increase in Caco-2 TJ permeability. The activation of ERK1/2 pathway caused a downstream activation of nuclear transcription factor Elk-1. The activated Elk-1 translocated to the nucleus and binds to the cis-binding motif on myosin light chain kinase (MLCK) promoter region, triggering MLCK gene activation, MLCK mRNA transcription and MLCK protein synthesis and MLCK catalysed opening of the intestinal epithelial TJ barrier. These studies provide novel insight into the cellular and molecular processes that mediate the IL-1β-induced increase in intestinal epithelial TJ permeability.

Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light‐chain kinase (MLCK) and MLC phosphatase (MLCP)

To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of β-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to β-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone.

Role of p38 MAPK in Burn-Induced Intestinal Barrier Breakdown

Severe burn results in intestinal barrier breakdown, which may lead to the generation of a systemic inflammatory response and distant organ injury. Intestinal barrier integrity is regulated, in part, by the tight junction protein myosin light chain kinase (MLCK). Previous studies in cell culture have shown that activation of p38 MAPK plays an important role in modulating intestinal barrier function. We hypothesized that (1) severe burn up-regulates p38 MAPK activation and results in increased intestinal permeability via augmented expression of MLCK, and (2) inhibition of p38 MAPK will prevent the burn-induced increase in MLCK expression, resulting in improved intestinal barrier integrity. Male Balb/c mice were subjected to a 30% total body surface area (TBSA) full thickness steam burn, then randomized to receive an intraperitoneal injection of a p38 MAPK inhibitor (SB203580, 25 mg/kg) or vehicle. In vivo intestinal permeability to 4kDa FITC-Dextran was measured. Expression of phosphorylated p38 MAPK, total p38 MAPK, MLCK, and phosphorylated MLC from intestinal extracts was assessed by immunoblotting. Severe burn increased intestinal permeability, which was associated with activation of p38 MAPK, and increased expression of MLCK. Treatment with SB203580 significantly attenuated burn-induced intestinal permeability (212 microg/mL versus 81 microg/mL, P<0.05), and decreased expression of intestinal MLCK resulting in decreased phosphorylation of MLC. p38 MAPK plays an important role in regulating burn-induced intestinal permeability through activation of MLCK. Inhibition of p38 MAPK may be an important therapeutic target aimed at attenuating intestinal barrier breakdown by preventing the burn-induced alterations in tight junction proteins.

Protective effect of myosin light-chain kinase inhibitor on acute lung injury

To investigate the influence of inhibitor of myosin light-chain kinase (MLCK) on the human pulmonary arterial endothelial cell (HPAEC) challenged with lipopolysaccharide (LPS) and LPS induced of acute lung injury (ALI) in mice. HPAECs were cultured in ECM medium and its passages 4-6 were used. After treatment with inhibitor of MLCK (ML-7) for 60 minutes, the HPAECs were incubated in LPS for another 60 minutes, and then cell viability was measured by the methyl thiazolyl tetrazolium (MTT) assay. Immunofluorescence microscope was used to detect phosphorylated-MLCK (p-MLCK) immunoreactive cells. Twenty female BALB/c mice were randomly divided into two groups. The mice of LPS group were exposed to LPS (1 microg/g) through nasal instillation, and the mice of ML-7 group were pretreated with ML-7 before intranasal instillation of LPS. Wet/dry weight (W/D) ratio of lung, bronchoalveolar lavage fluid (BALF) protein content, myeloperoxidase (MPO) activity and histopathological changes of lung tissue were observed. Immunohistochemistry assays were used to determine the status of MLCK and CD11b immunoreactive cells in lung tissue, and expression of MLCK mRNA in lung tissue was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Expression of MLCK protein in lungs was assayed by Western blotting. Compared with LPS group, increased absorbance (A) value of HPAEC was found in ML-7 group (P<0.01). Immunoreactive cells of p-MLCK were more reduced in the ML-7 group (P<0.05), and W/D ratio of lung, MPO activity and BALF protein content of lung tissue were decreased in ML-7 group (P<0.05 or P<0.01). Histological examination showed that an extensive lung inflammation was seen in mice of LPS group, with an accumulation of a large number of neutrophils, marked pulmonary edema and hemorrhage, but the inflammation and parenchymal hemorrhage was significantly alleviated in ML-7 group. Both MLCK immunoreactive cells located in endothelium and CD11b in infiltrated inflammatory cells were decreased in ML-7 group compared with those in LPS group. Compared with LPS group, MLCK mRNA and protein expressions (A) in ML-7 group were significantly decreased (both P<0.05). ML-7, an MLCK inhibitor, enhances activity of HPAEC induced by LPS and reduces expression of p-MLCK. It also reduces the LPS-induced infiltration of neutrophils in lung tissues, pulmonary edema and expression of MLCK and CD11b protein and MLCK mRNA in lung tissues, demonstrating that inhibition of activation of MLCK, leading to an abatement of phosphorylation of myosin light chain or MLCK, resulting in stabilization of vascular barrier function. The results suggest that MLCK has a crucial role in the pathogenesis of ALI.

90. Role of P38 MAPK Signaling in Burn-Induced Intestinal Barrier Breakdown

Severe burn injury results in intestinal barrier breakdown, which may lead to the generation of a systemic inflammatory response and distant organ injury. Intestinal barrier integrity is regulated, in part, by the tight junction protein myosin light chain kinase (MLCK). Activation of MLCK is known to increase tight junction breakdown through phosphorylation of myosin light chain (MLC). Previous studies in cell culture have shown that activation of p38 MAPK plays an important role in modulating intestinal barrier function.

Pentoxifylline Modulates Intestinal Tight Junction Signaling After Burn Injury: Effects on Myosin Light Chain Kinase

Burn injury can result in loss of intestinal barrier function, leading to systemic inflammatory response syndrome and multiorgan failure. Myosin light chain kinase (MLCK), a tight junction protein involved in the regulation of barrier function, increases intestinal epithelial permeability when activated. Prior studies have shown that tumor necrosis factor (TNF)-alpha activates MLCK, in part through a nuclear factor (NF)-kappa B-dependent pathway. We have previously shown that pentoxifylline (PTX) decreases both TNF-alpha synthesis and NF-kappaB activation in models of shock. Therefore, we postulate that PTX will attenuate activation of the tight junction protein MLCK, which may decrease intestinal tight junction permeability after severe burn. Male balb/c mice undergoing a severe burn were randomized to resuscitation with normal saline (NS) or NS + PTX (12.5 mg/kg). Intestinal TNF-alpha levels were evaluated using enzyme linked immunosorbent assay. Gut extracts were obtained to assess MLCK, phosphorylated IKK, IkappaB-alpha, and NF-kappaB p65 levels by immunoblotting. Burn injury increased intestinal MLCK protein levels threefold in animals resuscitated with NS, whereas those receiving PTX had MLCK levels similar to control (p < 0.01). Treatment with PTX attenuated burn-induced intestinal permeability. PTX decreased cytoplasmic IKK, IkappaB-alpha phosphorylation, and nuclear NF-kappaB p65 translocation to sham levels (p < 0.05 vs. NS). Treatment with PTX attenuates activation of the tight junction protein MLCK, likely through its ability to decrease local TNF-alpha synthesis and NF-kappaB activation after burn. PTX may have therapeutic utility by decreasing intestinal barrier breakdown after burn.

Identification of Cardiac-Specific Myosin Light Chain Kinase

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca(2+)/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.

Abstract 1102: Cardiac-Specific Myosin Light Chain Kinase (MLCK), a Third Member of MLCK Family, Potentiates Sarcomere Formation and Cardiac Contraction

Background: Two myosin light chain kinase (MLCK) proteins, skeletal (encoded by mylk2 gene) and smooth muscle MLCK (encoded by mylk1 gene) have been shown to be expressed in mammals. Human mylk2 has been mapped as a disease locus for familial cardiac hypertrophy (OMIM 606566 ), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. While phosphorylation of the putative substrate of skeletal MLCK, myosin light chain 2 (MLC2), is recognized as a key regulator of cardiac contraction, the abundance of skeletal MLCK in the heart is controversial, suggesting the existence of an additional MLCK that is preferentially expressed in cardiac muscle. Methods and Results: We characterized a new kinase named cardiac MLCK that is encoded by a gene homologous to mylk1 and 2 and is specifically expressed in the heart in both atrium and ventricle. Expression of cardiac MLCK was highly regulated by the cardiac homeobox transcription factor, Nkx2.5, in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK including putative catalytic and adjacent Ca2+/calmodulin binding domains at the carboxyl-terminus. The amino-terminus is unique without significant homology to other known proteins. Cardiac MLCK phosphorylated MLC2v with a catalytic value of Km=4.3 micro M (Lineweaver-Burk analysis) indicating high affinity of cardiac MLCK to MLC2v, similar to the affinity of skeletal muscle MLCK to skeletal muscle MLC2 and smooth muscle MLCK to smooth muscle MLC2. Adenoviral-mediated overexpression of cardiac MLCK and knockdown of cardiac MLCK using RNAi in cultured cardiomyocytes revealed that cardiac MLCK regulates MLC2v phosphorylation, sarcomere organization and cardiac myocyte contraction. Expression of cardiac MLCK protein was significantly decreased in severe heart failure in vivo (post-myocardial infarction heart failure mouse model). Conclusion: Cardiac MLCK is a new key regulator of cardiac contraction and sarcomere organization. Reduction of cardiac MLCK function leading to decreased phosphorylation of MLC2v may contribute to compromised contractile function in the failing heart.

Comparison of gel contraction mediated by airway smooth muscle cells from patients with and without asthma

Exaggerated bronchial constriction is the most significant and life threatening response of patients with asthma to inhaled stimuli. However, few studies have investigated the contractility of airway smooth muscle (ASM) from these patients. The purpose of this study was to establish a method to measure contraction of ASM cells by embedding them into a collagen gel, and to compare the contraction between subjects with and without asthma. Gel contraction to histamine was examined in floating gels containing cultured ASM cells from subjects with and without asthma following overnight incubation while unattached (method 1) or attached (method 2) to casting plates. Smooth muscle myosin light chain kinase protein levels were also examined. Collagen gels containing ASM cells reduced in size when stimulated with histamine in a concentration-dependent manner and reached a maximum at a mean (SE) of 15.7 (1.2) min. This gel contraction was decreased by inhibitors for phospholipase C (U73122), myosin light chain kinase (ML-7) and Rho kinase (Y27632). When comparing the two patient groups, the maximal decreased area of gels containing ASM cells from patients with asthma was 19 (2)% (n = 8) using method 1 and 22 (3)% (n = 6) using method 2, both of which were greater than that of cells from patients without asthma: 13 (2)% (n = 9, p = 0.05) and 10 (4)% (n = 5, p = 0.024), respectively. Smooth muscle myosin light chain kinase levels were not different between the two groups. The increased contraction of asthmatic ASM cells may be responsible for exaggerated bronchial constriction in asthma.

Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-alpha-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-alpha-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-alpha-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-alpha-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-alpha increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-alpha-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-alpha-induced activation. Glucocorticoids inhibit the TNF-alpha-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-alpha-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier.

Molecular mechanism of tumor necrosis factor-α modulation of intestinal epithelial tight junction barrier

A TNF-alpha-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions. Previous studies from our laboratory suggested that the TNF-alpha-induced increase in intestinal TJ permeability was mediated by an increase in myosin light chain kinase (MLCK) protein expression. However, the molecular mechanisms that mediate the TNF-alpha increase in intestinal TJ permeability and MLCK protein expression remain unknown. The purpose of this study was to delineate the intracellular and molecular mechanisms that mediate the TNF-alpha-induced increase in intestinal TJ permeability; using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. To examine the molecular mechanisms involved in the TNF-alpha regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-alpha treatment of filter-grown Caco-2 monolayers transfected with plasmid vector containing the MLCK promoter region produced an increase in MLCK promoter activity and MLCK transcription. The TNF-alpha-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation, and the inhibition of NF-kappaB activation prevented the TNF-alpha-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-alpha-induced activation of MLCK promoter was mediated by binding of the activated NF-kappaB p50/p65 dimer to the downstream kappaB binding site (-84 to -75) on the MLCK promoter region; deletion of the kappaB binding site prevented the TNF-alpha increase in promoter activity. Additionally, siRNA silencing of NF-kappaB p65 also prevented the TNF-alpha increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was mediated by NF-kappaB p50/p65 binding and activation of the MLCK promoter. NF-kappaB p50/p65 activation of the MLCK promoter then leads to a stepwise increase in MLCK transcription, expression and activity, and MLCK-mediated opening of the intestinal TJ barrier.

Myosin light chain kinase colocalizes with nonmuscle myosin IIB in myofibril precursors and sarcomeric Z-lines of cardiomyocytes

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the above mentioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB.

Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression

TNF-alpha plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-alpha-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-alpha-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-alpha (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-alpha increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-alpha-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-alpha increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-alpha increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-alpha increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-alpha increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg(2+)-myosin ATPase, and metabolic energy prevented the TNF-alpha increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-alpha-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-alpha increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

Ontogenesis of myosin light chain kinase mRNA and protein content in guinea pig tracheal smooth muscle.

We previously reported in guinea pig tracheal smooth muscle that maximal shortening velocity decreases from 3 weeks of age to adulthood. It is not known whether myosin light chain kinase (MLCK), a key enzyme determining the velocity of smooth muscle contraction, undergoes maturational changes. In the present work, we investigated MLCK protein content and mRNA expression in 1-week-old, 3-week-old, and adult guinea pigs. We extracted either proteins or RNA from isolated tracheal smooth muscle. The content of MLCK was assessed by Western immunoblots. MLCK mRNA was evaluated by Northern analysis and by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). The content of MLCK increased 3-fold at 3 weeks of age and then decreased in adults, being 0.116 +/- 0.042, 0.330 +/- 0.125 (P < 0.05), and 0.153 +/- 0.054 microg/mg of total protein, respectively, in 1-week, 3-week, and adult animals. Quantitative RT-PCR revealed that MLCK mRNA increased with age to 135 +/- 35% and 177 +/- 23% (P < 0.01) in 3-week and adult animals, respectively, compared to 1-week animals. The transient increase of MLCK content in juvenile guinea pig tracheal smooth muscle may contribute to the increased shortening velocity at this age. We suggest that this increased content of MLCK is one of the mechanisms leading to maturation of airway smooth muscle contractility, which in turn contributes to the airway hyperresponsiveness reported in children and young animals.

Gut edema alters expression of tight junction proteins

Abstract Introduction: We have previously shown that gut edema without ischemia/reperfusion injury increases intestinal permeability in the ileum. Intestinal permeability is regulated by tight junction proteins which control the movement of macromolecules between epithelial cells. The proteins occludin and claudin-1 are the structural components and myosin light chain kinase is the regulatory component of the tight junction which is reinforced by the actin cytoskeleton. We hypothesized that gut edema increases permeability by decreasing expression of tight junction proteins. Methods: At laparotomy, rats were given 80cc/kg of 0.9% saline + superior mesenteric vein partial occlusion (Edema) or sham surgery. At 12 hrs, ileum was harvested for evaluation of occludin, claudin-1, myosin light chain kinase (MLCK) and mature, filamentous actin (F-actin) by deconvolution microscopy. Tissue water was determined using wet to dry weight ratio. Data are expressed as mean +/− SEM, t-test, 4–5/group. Results: Gut edema significantly increased the wet to dry ratio compared to sham and was associated with a significant increase in occludin and claudin-1 protein expression. MLCK protein expression and F-actin were significantly decreased. ∗Wet to dry ratioOccludin (pixel units)Claudin-1 (pixel units)F-actin (pixel units)MLCK (pixel units)Sham3.22 ± 0.092251 ± 291906 ± 211973 ± 1151962 ± 307Edema3.69 ± 0.07∗3786 ± 330∗3356 ± 777∗602 ± 83∗666 ± 113∗∗Denotes a P Conclusions: Gut edema disrupts the cytoskeleton ring by decreasing the presence of mature, filamentous actin. This disruption does not rely on increased expression of MLCK, but may contribute to the increased expression of structural proteins claudin and occludin seen in gut edema.

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues.

The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca(2+)-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM-MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM-CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM.

Ethanol modulation of intestinal epithelial tight junction barrier.

Previous studies have shown that high concentrations of ethanol (>/=40%) cause functional damage of the gastrointestinal epithelial barrier by direct cytotoxic effect on the epithelial cells. The effects of lower noncytotoxic doses of ethanol on epithelial barrier function are unknown. A major function of gastrointestinal epithelial cells is to provide a barrier against the hostile substances in the gastrointestinal lumen. The apicolaterally located tight junctions (TJs) form a paracellular seal between the lateral membranes of adjacent cells and act as a paracellular barrier. In this study, we investigated the effects of lower doses of ethanol on intestinal epithelial TJ barrier function using filter-grown Caco-2 intestinal epithelial monolayers. The Caco-2 TJ barrier function was assessed by measuring epithelial resistance or paracellular permeability of the filter-grown monolayers. Ethanol (0, 1, 2.5, 5, 7.5, and 10%) produced a dose-related drop in Caco-2 epithelial resistance and increase in paracellular permeability. Ethanol also produced a progressive disruption of TJ protein (ZO-1) with separation of ZO-1 proteins from the cellular junctions and formation of large gaps between the adjacent cells. Ethanol, at the doses used (</=10%), did not cause cytotoxicity (lactate dehydrogenase release) to the Caco-2 cells. Ethanol produced a disassembly and displacement of perijunctional actin and myosin filaments from the perijunctional areas. On ethanol removal, actin and myosin filaments rapidly reassembled at the cellular borders. Ethanol stimulated the Caco-2 myosin light chain kinase (MLCK) activity but did not affect the MLCK protein levels. Specific MLCK inhibitor ML-7 inhibited both ethanol increases in MLCK activity and TJ permeability without affecting the MLCK protein levels. Consistent with these findings, metabolic inhibitors sodium azide and 2,4-dinitrophenol significantly prevented ethanol-induced increase in Caco-2 TJ permeability, whereas cycloheximide or actinomycin D had no effect. The results of this study indicate that ethanol at low noncytotoxic doses causes a functional and structural opening of the Caco-2 intestinal epithelial TJ barrier by activating MLCK.