Myosin Light Chain Kinase Levels Research Articles

Active components of Descurainia sophia improve lung permeability in rats with allergic asthma by regulating airway inflammation and epithelial damage

The present study investigated the effect of active components of Descurainia sophia on allergic asthma and explored the underlying mechanism. SD male rats were randomly divided into a normal group(NC), a model group(M), a D. sophia decoction group(DS), a D. sophia fatty oil group(FO), a D. sophia flavonoid glycoside group(FG), a D. sophia oligosaccharide group(Oli), and a positive drug dexamethasone group(Y). The allergic asthma model was induced in rats by intraperitoneal injection of ovalbumin(OVA) and aluminum hydroxide gel adjuvant(sensitization) and atomization of OVA solution(excitation). After modeling, asthma-related indicators, tracheal phenol red excretion, inflammatory cell levels in the peripheral blood, lung permeability index(LPI), and oxygenation index(OI) of rats were detected. The pathological changes of lung tissues were observed by HE staining. Enzyme-linked immunosorbent assay(ELISA) was used to detect the content of inflammatory factors immunoglobulin E(IgE), interleukin-4(IL-4), and interferon-γ(IFN-γ) in the bronchoalveolar lavage fluid(BALF) and the content of endothelin-1(ET-1) and angiotensin-converting enzyme(ACE) in lung tissue homogenate. The serum content of nitric oxide(NO) was detected by colorimetry. Western blot was employed to determine the protein expression of Toll-like receptor 4(TLR4), nuclear factor κB-p65(NF-κB-p65), phosphorylated NF-κB-p65(p-NF-κB-p65), myosin light chain kinase(MLCK), vascular endothelial cadherin(VE cadherin), connexin 43, and claudin 5, and the mechanism of active components of D. sophia on allergic asthma was explored. As revealed by the results, the M group showed extensive infiltration of inflammatory cells around the bronchus of the lung tissues of the allergic asthma rats, thickened bronchial wall, severely deformed alveolar structure, increased number of wheezes, the content of IgE, IL-4, ET-1, and ACE, inflammatory cells, and LPI, and reduced latency of asthma, tracheal phenol red excretion, IFN-γ, NO content, and OI. After the intervention of the active components of D. sophia, the DS, FO, FG, Oli, and Y groups showed improved asthma-related indicators, tracheal phenol red excretion, and lung tissue lesions in allergic asthma rats, and the effects in the FO and Oli groups were superior. The content of inflammatory factors in BALF was recovered in the DS, FO, and Y groups and the FG and Oli groups. The number of inflammatory cells in rats was reduced in the DS and FO groups, and the FG, Oli, and Y groups to varying degrees, and the effect in the FO group was superior. DS, FO, Oli, and Y reduced ET-1, ACE, and LPI and increased NO and OI. FG recovered NO, ET-1, ACE, LPI, and OI to improve lung epithelial damage and permeability. Further investigation of inflammation-related TLR4/NF-κB pathways, MLCK, and related skeleton protein levels showed that TLR4, NF-κB-p65, p-NF-κB-p65, and MLCK levels were increased, and VE cadherin, connexin 43, and claudin 5 were reduced in the M group. DS, FO, FG, Oli, and Y could reduce the protein expression related to the TLR4 pathway to varying degrees, and regulate the protein expression of MLCK, VE cadherin, connexin 43, and claudin 5. It is inferred that the active components of D. sophia improve lung permeability in rats with allergic asthma presumedly by regulating the TLR4/NF-κB signaling pathway to improve airway inflammation, mediating MLCK and connexin, and regulating epithelial damage.

The Effect of Sagunja Decoction on Functional Dyspepsia - A Systematic Review and Meta-Analysis

Objectives: This study examines the effect of Sagunja-tang on functional dyspepsia (FD) through a systematic review and meta-analysis of a randomized controlled trial (RCT). Methods: A search for RCTs that tested the effect of Sagunja-tang on functional dyspepsia was conducted in Medline, Embase, PubMed, CENTRAL, CiNii, CNKI, NDSL, RISS, OASIS, and KISS databases on November 8, 2020, with no limit on the year of publication. A meta-analysis was performed by synthesizing the findings, including total efficiency, clinical symptom score, myosin light-chain kinase (MLCK) level (pg/mL, and gastric half-emptying time (min). RevMan 5.4.1 software was used for data analysis. The quality of the literature was evaluated using Cochrane’s risk of bias (RoB) tool. Results: A total of 14 RCTs met the selection criteria. As a result of the meta-analysis, the treatment group had higher total efficacy and MLCK levels (gastric antrum, jejunum) than the control group, and the clinical symptom score and gastric half-emptying time were lower. However, due to the low quality of the included RCT and the small sample size, the results may be slightly biased.

Association of actin-myosin interaction biomarkers and fibrosis with myocardial inflammatory changes in patients with idiopathic atrial fibrillation

Abstract Introduction Myocarditis and inflammatory changes in the myocardium are causes of the development and progression of atrial fibrillation (AF). Inflammatory processes may also lead to the development of fibrosis, electrical and structural myocardial remodeling, adversely affecting the effectiveness of interventional treatment. Purpose To assess the dynamics and to identify relationships between serum levels of tropomyosin I, myosin light chain kinase (MLCK), myosin heavy (MYH7) and light chains (CMLC), transforming growth factor (TGF-β1) and myocardial inflammatory changes in patients with idiopathic AF. Methods Subjects were 40 patients (41.0±9.2 y.o.) with idiopathic AF (arrhythmic history constituted 4.9±3.9 years). Paroxysmal AF (ParAF) was detected in 37% (n=15), persistent AF (PerAF)–30% (n=12), and long-standing persistent (LPerAF)–33% (n=13). All patients underwent radiofrequency ablation (RFA) of the pulmonary veins and endomyocardial byopsy with histologic and immunohistochemical studies. Depending on the results of histological study, the patients were divided into 2 groups: 1–with active lymphocytic myocarditis (ALM) (n=22); 2–with lymphocytic infiltration (IL) (n=18). Blood for biomarkers evaluation was collected prior to RFA and in 6 months after RFA. Results In patients with ALM levels of biomarkers significant differed depending on the form of AF. In patients with PerAF, the levels of MYH7, MLCK and tropomyosin I were significantly lower than in patients with ParAF (p<0.005). In 6 months the levels of MLCK, tropomyosin I, and CMLC significantly differed in patients with ALM depending on the form of AF (p<0.05). In patients with PerAF in the group 1, the concentrations of MYH7, CMLC, and tropomyosin I were lower, than the group 2 (p<0.05). In 6 months in patients with PerAF, the TGF-β1 levels also differed between groups 1 and 2 (Me: 24.09 and 40.34 ng/ml, p<0.05). The content of TGF-β1 before the RFA and in 6 months in patients with PerAF was associated with the development of infiltration in 6 months after RFA (R=0.696, p=0.025). In LPerAF patients we showed correlation between the content of TGF-β1 and the severity of fibrosis in 6 months after RFA (R=0.607, p=0.047). At LPerAF TGF-β1 content correlated with degree of fibrosis in 6 months after RFA (R=0.607, p=0.047). The relationship between the form of AF and the inflammatory changes were identified (r=0.32, p=0.049). Thus, in patients with LPerAF and PerAF, ALM was diagnosed in 69% and 50% of cases, while in patients with ParAF the frequency of ALM detection constituted 33%. Conclusion Our study showed that ALM was detected in 55% of patients, and IL–in 45% of cases. Significant differences in the levels of MYH7, MLCK, CLCK, tropomyosin I in patients with PerAF and various forms of inflammatory myocardial changes were revealed. The content of the TGF-β1 is reduced in ALM in patients with PerAFP. The incidence of ALM was higher in patients with long-term persistent AF. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Cardiology Research Institute, Tomsk NRMC

β-adrenergic activation may promote myosin light chain kinase degradation through calpain in pressure overload-induced cardiac hypertrophy: β-adrenergic activation results in MLCK degradation

Backgroundβ-adrenergic activation is able to exacerbate cardiac hypertrophy. Myosin light chain kinase (MLCK) and its phosphorylated substrate, phospho-myosin light chain 2 (p-MLC2), play vital roles in regulating cardiac hypertrophy. However, it is not yet clear whether there is a relationship between β-adrenergic activation and MLCK in the progression of cardiac hypertrophy. Therefore, we explored this relationship and the underlying mechanisms in this work. MethodsCardiac hypertrophy and cardiomyocyte hypertrophy were induced by pressure overload and isoproterenol (ISO) stimulation, respectively. Echocardiography, histological analysis, immunofluorescence and qRT-PCR were used to confirm the successful establishment of the models. A β-blocker (metoprolol) and a calpain inhibitor (calpeptin) were administered to inhibit β-adrenergic activity in rats and calpain in cardiomyocytes, respectively. The protein expression levels of MLCK, myosin light chain 2 (MLC2), p-MLC2, myosin phosphatase 2 (MYPT2), calmodulin (CaM) and calpain were measured using western blotting. A cleavage assay was performed to assess the degradation of recombinant human MLCK by recombinant human calpain. ResultsThe β-blocker alleviated cardiac hypertrophy and dysfunction, increased MLCK and MLC2 phosphorylation and decreased calpain expression in pressure overload-induced cardiac hypertrophy. Additionally, the calpain inhibitor calpeptin attenuated cardiomyocyte hypertrophy, upregulated MLCK and p-MLC2 and reduced MLCK degradation in ISO-induced cardiomyocyte hypertrophy. Recombinant human calpain degraded recombinant human MLCK in vitro in concentration- and time-dependent manners, and this degradation was inhibited by the calpain inhibitor calpeptin. ConclusionOur study suggested that β-adrenergic activation may promote the degradation of MLCK through calpain in pressure overload-induced cardiac hypertrophy.

Chang'an II Decoction ( II )-Containing Serum Ameliorates Tumor Necrosis Factor-α-Induced Intestinal Epithelial Barrier Dysfunction via MLCK-MLC Signaling Pathway in Rats.

To investigate the effect of Chang'an II Decoction ( II ))-containing serum on intestinal epithelial barrier dysfunction in rats. Tumor necrosis factor (TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium. Caco-2 monolayers were treated with blank serum and Chang'an II Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang'an II Decoction intragastrically at doses of 0.49, 0.98, 1.96 g/(kg·d) for 1 week, respectively. After preparation of containing serum, cells were divided into the normal group, the model group, the Chang'an II-H, M, and L groups (treated with 30 ng/mL TNF-α and medium plus 10% high, middle-, and low-doses Chang'an II serum, respectively). Epithelial barrier function was assessed by transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate (FITC)-labeled dextran. Transmission electron microscopy was used to observe the ultrastructure of tight junctions (TJs). Immunofluorescence of zonula occludens-1 (ZO-1), claudin-1 and nuclear transcription factor-kappa p65 (NF-κ Bp65) were measured to determine the protein distribution. The mRNA expression of myosin light chain kinase (MLCK) was measured by real-time polymerase chain reaction. The expression levels of MLCK, myosin light chain (MLC) and p-MLC were determined by Western blot. Chang'an II Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α. It alleviated TNF-α-induced morphological alterations in TJ proteins. The increases in MLCK mRNA and MLCK, MLC and p-MLC protein expressions induced by TNF-α were significantly inhibited in the Chang'an II-H group. Additionally, Chang'an II Decoction significantly attenuated translocation of NF-κ Bp65 into the nucleus. High-dose Chang'an II-containing serum attenuates TNF-α-induced intestinal barrier dysfunction. The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κ Bp65.

Contractile Properties of Intrapulmonary Airway Smooth Muscle in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal-recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Many patients with CF have asthma-like symptoms and airway hyperresponsiveness, which are potentially associated with altered airway smooth muscle (ASM) contractility. Our goal in this study was to assess the contractility of the CF intrapulmonary ASM. ASM strips were dissected from human control and CF intrapulmonary airways, and assessed for methacholine-induced shortening velocity, maximal force, and stress. We also assessed isoproterenol responses in maximally methacholine-contracted ASM. ASM strips were then incubated for 16 hours with IL-13 and measurements were repeated. Myosin light chain kinase (MLCK) expression was assessed by Western blotting. Airways were immunostained for morphometry. ASM mass was increased in CF airways, which likely contributes to airway hyperresponsiveness. Although ASM contractile properties were not intrinsically different between patients with CF and control subjects, CF ASM responded differently in the presence of the inflammatory mediator IL-13, showing impairment in β-adrenergic-induced relaxation. Indeed, the percentage of relaxation measured at maximal isoproterenol concentrations in the CF ASM was significantly lower after incubation with IL-13 (46.0% ± 6.7% relaxation) than without IL-13 (74.0% ± 7.7% relaxation, P = 0.018). It was also significantly lower than that observed in control ASM incubated with IL-13 (68.8% ± 4.9% relaxation, P = 0.048) and without IL-13 (82.4% ± 9.9%, P = 0.0035). CF ASM incubated with IL-13 also expressed greater levels of MLCK. Thus, our data suggest that the combination of an increase in ASM mass, increased MLCK expression, and inflammation-induced β-adrenergic hyporesponsiveness may contribute to airway dysfunction in CF.

MicroRNA-92a promotes vascular smooth muscle cell proliferation and migration through the ROCK/MLCK signalling pathway.

To identify the interaction between known regulators of atherosclerosis, microRNA‐92a (miR‐92a), Rho‐associated coiled‐coil‐forming kinase (ROCK) and myosin light chain kinase (MLCK), we examined their expressions during proliferation and migration of platelet‐derived growth factor‐BB (PDGF‐BB)‐regulated vascular smooth muscle cells (VSMCs), both in vivo and in vitro. During the formation of atherosclerosis plaque in mice, a parallel increase in expression levels of MLCK and miR‐92a was observed while miR‐92a expression was reduced in ML‐7 (an inhibitor of MLCK) treated mice and in MLCK‐deficient VSMCs. In vitro results indicated that both MLCK and miR‐92a shared the same signalling pathway. Transfection of miR‐92a mimic partially restored the effect of MLCK's deficiency and antagonized the effect of Y27632 (an inhibitor of ROCK) on the down‐regulation of VSMCs activities. ML‐7 increased the expression of Kruppel‐like factor 4 (KLF4, a target of miR‐92a), and siRNA‐KLF4 increased VSMCs' activity level. Consistently, inhibition of either MLCK or ROCK enhanced the KLF4 expression. Moreover, we observed that ROCK/MLCK up‐regulated miR‐92a expression in VSMCs through signal transducer and activator of transcription 3 (STAT3) activation. In conclusion, the activation of ROCK/STAT3 and/or MLCK/STAT3 may up‐regulate miR‐92a expression, which subsequently inhibits KLF4 expression and promotes PDGF‐BB‐mediated proliferation and migration of VSMCs. This new downstream node in the ROCK/MLCK signalling pathway may offer a potential intervention target for treatment of atherosclerosis.

Regulation of hypoxia inducible factor-1α on permeability of vascular endothelial cells and the mechanism

Regulation of hypoxia inducible factor-1α on permeability of vascular endothelial cells and the mechanism

Glabridin attenuates endothelial dysfunction and permeability, possibly via the MLCK/p-MLC signaling pathway.

Atherosclerosis is caused by various factors, and Glabridin may have protective effects on the cardiovascular system. The purpose of the present study was to evaluate the effects of Glabridin on atherosclerosis and evaluate whether Glabridin attenuates arteriosclerosis and endothelial permeability by suppressing the myosin light chain (MLC) kinase (MLCK)/phosphorylated (p)-MLC system via the mitogen activated protein kinase (MAPK) signaling pathway. Male New Zealand rabbits were randomly divided into 3 groups: The control group was administered an ordinary diet, whereas the high fat group and the Glabridin (2 mg/kg/d) intervention group were administered a high fat diet. Following 12 weeks, the blood lipid levels of rabbits, the morphological structure of the arterial wall, the arterial intimal permeability, the endothelial function and the mRNA levels of MLCK were measured. Western blot analysis was used to detect the levels of MLCK, p-c-Jun N-terminal kinase (JNK), p-extracellular signal regulated kinase (ERK), and p-p38. The high-fat diet group exhibited significantly increased total cholesterol and triglycerides, and endothelial dysfunction, which were attenuated by Glabridin treatment. Notably, the aortic endothelial permeability was increased in the high-fat diet group but was ameliorated in the Glabridin treatment group. Hyperlipidemia enhanced the expression of p-MLC and MLCK, which were associated with the increased phosphorylation of ERK, p38 and JNK. These changes were also ameliorated by Glabridin. In conclusion, the results of the present study suggested that atherosclerosis may be associated with upregulated MLCK expression and activity, which was downregulated by Glabridin. The mechanism of action of Glabridin was thought to proceed through modulating MAPK pathway signal transduction. However, further studies are required to adequately illuminate the exact regulatory mechanisms involved.

Angiotensin II Facilitates Matrix Metalloproteinase-9-Mediated Myosin Light Chain Kinase Degradation in Pressure Overload-Induced Cardiac Hypertrophy

Background/Aims: Angiotensin II (Ang II) has been shown to promote cardiac remodeling during the process of hypertrophy. Myosin light chain kinase (MLCK), a specific kinase for the phosphorylation of myosin light chain 2 (MLC2), plays an important role in regulating cardiac muscle contraction and hypertrophy. However, whether Ang II could facilitate cardiac hypertrophy by altering the expression of MLCK remains unclear. This study aimed to investigate this effect and the underlying mechanisms. Methods: Cardiac hypertrophy was induced via pressure overload in rats, which were then evaluated via histological and biochemical measurements and echocardiography. Angiotensin-converting enzyme inhibitor (ACEI) was used to inhibit Ang II. Neonatal rat cardiomyocytes were stimulated with Ang II to induce hypertrophy and were treated with a matrix metalloproteinase 9 (MMP9) inhibitor. Myocyte hypertrophy was evaluated using immunofluorescence and qRT-PCR. Degradation of recombinant human MLCK by recombinant human MMP9 was tested using a cleavage assay. The expression levels of MLCK, MLC2, phospho-myosin light chain 2 (p-MLC2), myosin phosphatase 2 (MYPT2), and calmodulin (CaM) were measured using western blotting. Results: ACEI improved cardiac function and remodeling and increased the levels of MLCK and p-MLC2 as well as reduced the expression of MMP9 in pressure overload-induced cardiac hypertrophy. Moreover, the MMP9 inhibitor alleviated myocyte hypertrophy and upregulated the levels of MLCK and p-MLC2 in Ang II-induced cardiomyocyte hypertrophy. Recombinant human MLCK was concentration- and time-dependently degraded by recombinant human MMP9 in vitro, and this process was prevented by the MMP9 inhibitor. Conclusion: Our results suggest that Ang II is involved in the degradation of MLCK in pressure overload-induced cardiac hypertrophy and that this process was mediated by MMP9.

Loss of MLCK leads to disruption of cell-cell adhesion and invasive behavior of breast epithelial cells via increased expression of EGFR and ERK/JNK signaling.

Myosin light chain kinase (MLCK) expression is downregulated in breast cancer, including invasive ductal carcinoma compared with ductal breast carcinoma in situ and metastatic breast tumors. However, little is known about how loss of MLCK expression contributes to tumor progression. MLCK is a component of the actin cytoskeleton and its known role is the phosphorylation of the regulatory light chain of myosin II. To gain insights into the role of MLCK in breast cancer, we perturbed its function using small interfering RNA (siRNA) or pharmacological inhibition in untransformed breast epithelial cells (MCF10A). Loss of MLCK by siRNAs led to increased cell migration and invasion, disruption of cell-cell adhesions and enhanced formation of focal adhesions at the leading edge of migratory cells. In addition, downregulation of MLCK cooperated with HER2 in MCF10A cells to promote cell migration and invasion and low levels of MLCK is associated with a poor prognosis in HER2-positive breast cancer patients. Associated with these altered migratory behaviors were increased expression of epidermal growth factor receptor and activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways in MLCK downregulated MCF10A cells. By contrast, inhibition of the kinase function of MLCK using pharmacological agents inhibited cell migration and invasion, and did not affect cellular adhesions. Our results show that loss of MLCK contributes to the migratory properties of epithelial cells resulting from changes in cell-cell and cell-matrix adhesions, and increased epidermal growth factor receptor signaling. These findings suggest that decreased expression of MLCK may have a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Melatonin inhibits the Migration of Colon Cancer RKO cells by Down-regulating Myosin Light Chain Kinase Expression through Cross-talk with p38 MAPK

Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.

Evaluation the effect of thiamin deficiency on intestinal immunity of young grass carp (Ctenopharyngodon idella).

Our study explored the effect of dietary thiamin on growth and immunity (intestine, head kidney, spleen and liver) of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing six graded levels of thiamin at 0.12-2.04 mg/kg diet for 8 weeks. The percentage weight gain (PWG), feed intake and feed efficiency were lower in fish fed the 0.12 mg/kg diet. Thiamin deficiency decreased complement 3 content, lysozyme (LA) and acid phosphatase activities, mRNA levels of hepcidin and interleukin (IL) 10, elevated mRNA levels of interferon γ2, tumor necrosis factor α, IL-1β and IL-8 in intestine, head kidney, spleen and liver. The mRNA levels of inhibitor protein-κBα, target of rapamycin (TOR) and NF-E2-related factor 2 (Nrf2), the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase were down-regulated, mRNA levels of myosin light-chain kinase (MLCK), IκB kinases (IKKβ and IKKγ), nuclear factor κB P65 (NF-κB P65) and Kelch-like-ECH-associated protein 1a (Keap1a) were up-regulated in the intestine of fish fed the thiamin-deficient diet. Additionally, thiamin deficiency decreased claudin b, c and 3, ZO-1 and occludin mRNA levels in each intestinal segment, increased claudin 12 and claudin 15a mRNA levels in distal intestine. In conclusion, thiamin deficiency decreased fish growth and immunity of intestine, head kidney, spleen and liver. The dietary thiamin requirement of young grass carp (242-742 g) based on intestinal LA activity or PWG were determined to be 1.15 or 0.90 mg/kg diet, respectively.

A novel all-trans retinoid acid derivative N-(3-trifluoromethyl- phenyl)- retinamide inhibits lung adenocarcinoma A549 cell migration through down-regulating expression of myosin light chain kinase.

To observe the effects of a novel all-trans retinoid acid (ATRA) derivative, N-(3-trifluoromethyl-phenyl)- retinamide (ATPR), on lung adenocarcinoma A549 cells and to explore the potential mechanism of ATPR inhibiting of A549 cell migration. The cytotoxicity of ATRA and ATPR on A549 cells was assessed using MTT assay. Wound healing assays were used to analyze the influences of ATRA, ATPR, ML-7 (a highly selective inhibitor of myosin light chain kinase (MLCK)), PMA (an activator of MAPKs) and PD98059 (a selective inhibitor of ERK1/2) on the migration of A549 cells. Expression of MLCK and phosphorylation of myosin light chain (MLC) were assessed by Western blotting. ATRA and ATPR inhibited the proliferation of A549 cells in a dose- and time-dependent manner, and the effect of ATPR was much more remarkable compared with ATRA. Relative migration rate and migration distance of A549 cells both decreased significantly after treatment with ATPR or ML-7. The effect on cell migration of PD98059 combining ATPR treatment was more notable than that of ATPR alone. Moreover, compared with control groups, the expression levels of MLCK and phosphorylated MLC in A549 cells were both clearly reduced in ATRA and ATPR groups. ATPR could suppress the migration and invasion of A549 cells, and the mechanism might be concerned with down- regulating the expression of MLCK in the ERK-MAPK signaling pathway, pointing to therapeutic prospects in lung cancer.

Dexamethasone Increases Cdc42 Expression in Human TM-1 Cells

Purpose: Changes in the cytoskeletal organization of the human trabecular meshwork (HTM) is thought to be responsible for primary open-angle glaucoma (POAG) pathologies. Cdc42 is a Rho GTPase; Rho GTPases are important modulatory agents of the cytoskeleton. This study aimed to investigate the effects of dexamethasone (DEX) on Cdc42 in a transformed HTM cell line, TM-1 to understand the molecular pathologies underlying POAG.Methods: TM-1 cells were cultured in vitro. The cultures were treated with DEX at 10−6 and 10−7 M for 1–4 days. Cdc42 was silenced using small interfering RNA (siRNA). The expression levels of Cdc42 in the TM-1 cells were measured using reverse transcription (RT)–PCR, western blotting analysis and immunofluorescence. Its downstream effectors, p21-activated kinase phosphorylation (phospho-PAK) and myosin light chain kinase (MLCK), were measured using western blotting analysis. In addition, the F-actin of TM-1 cells was stained using phalloidin.Results: The mRNA and protein levels of Cdc42 showed an increase in TM-1 cells with DEX treatment and a decrease in TM-1 cells transfected with Cdc42 siRNA. Moreover, phospho-PAK levels increased, whereas MLCK levels appeared to decrease, with DEX treatment. The F-actin of DEX-treated TM-1 cells displayed a rearrangement. Cdc42 siRNA decreased the expression of Cdc42 and its related proteins, resulting in an attenuation of the effects of DEX on Cdc42 and F-actin organization in TM-1 cells.Conclusions: DEX increases Cdc42 expression in TM-1. This may represent a potential mechanism of DEX-induced HTM cytoskeletal rearrangement.

Myosin Light Chain Kinase Is Involved in the Mechanism of Gastrointestinal Dysfunction in Diabetic Rats

It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca(2+) via myosin light chain phosphorylation, which is activated by myosin light chain kinase (MLCK). Recently, MLCK has been demonstrated to play an important role in smooth muscle contraction and normal gastrointestinal motility. The aim of our study is to investigate whether MLCK is involved in the mechanism of gastrointestinal dysfunction and the ameliorating effects of insulin on gastrointestinal dysfunction in diabetic rats. A diabetic rat model was established by an intravenous injection with streptozotocin. Rats were randomized into three groups: control group, diabetic group, and insulin-treated group. The gastrointestinal functions were assessed in terms of gastric emptying and intestinal transit. The expression of MLCK in the pylorus and ileum of the three groups was determined by real-time polymerase chain reaction (PCR) and Western blot methods. The diabetic group exhibited a significant delay in gastric emptying and intestinal transit than the control group. Insulin treatment significantly ameliorated the gastric emptying and intestinal transit in diabetic rats. The expression levels of MLCK in the pylorus and ileum of the diabetic group were both significantly decreased compared with the control group, and the changes of MLCK expression in these tissues of diabetic rats were partially reversed after treatment with insulin. Decreased expression of MLCK in gastrointestinal tissues could be a possible cause for gastrointestinal dysfunction. Insulin may partly ameliorate gastrointestinal dysfunction by restoring the expression of MLCK.

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.

Fast intestinal barrier recovery following ischemia/reperfusion damage in the human gut: myosin light chain mediated closure of the villus tip

In a recently developed human model of intestinal ischemia reperfusion (I‐IR) a rapid reversal of intestinal damage by shedding of injured enterocytes and villus tip closure was described. Here we investigated the putative involvement of myosin light chain kinase (MLCK) mediated actin contraction in villus tip closure.During surgery, an isolated part of the jejunum scheduled to be removed was subjected to 30 min ischemia and subsequent reperfusion. Immunohistochemistry was used to study the localization of MLCK, phosphorylated‐MLC (pMLC), filamentous actin and claudin‐3. Protein levels of MLCK, pMLC and claudin‐3 were semi‐quantitatively analyzed using western blot. Intestinal claudin‐3 production was determined by qPCR.After 30 min ischemia subepithelial spaces were noted as retractions of the basal membrane. A 30 min reperfusion period resulted in disintegration of epithelial lining at the villus tip, with epithelial cell damage and loss of claudin‐3. Both MLCK and pMLC increased following ischemia and were distributed around the disrupted villus tips after 30 min of reperfusion (MLCK; 918±430 versus 641±338 INT, P&lt;0.01) (fig 1). After 60 min reperfusion complete barrier recovery was observed.This study shows fast gut barrier recovery following I‐IR damage in the human gut after myosin light chain kinase mediated closure of the villus tip.Figure 1A) Before ischemiaB) After 30 min ischemiaC) After 30 min reperfusionD) After 60 min reperfusion

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.

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.