Myosin Light Chain Kinase Activity 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.

An Inulin-Type Fructan CP-A from Codonopsis pilosula Alleviated 5-Fluorouracil-Induced Intestinal Mucositis via the ERK/MLCK/MLC2 Pathway and Regulation of Gut Microbiota.

Intestinal mucositis (IM) is a common adverse effect of chemotherapy, limiting its clinical application. Codonopsis pilosula-derived CP-A (an inulin-type fructan) is an edible Chinese medicine with anti-inflammatory and gastrointestinal protective effects, which may be useful for treating IM. Here, we explored CP-A's role in ameliorating IM induced by 5-fluorouracil (5-FU) and investigated the underlying mechanism using in vitro experiments and rat models. Western blotting, immunohistochemistry (IHC), and real-time PCR (RT-PCR) analyses were used to assess protein expression related to the extracellular-regulated protein kinases (ERK)/myosin light chain kinase (MLCK)/myosin light chain 2 (MLC2) signaling pathway and tight junction proteins. Inflammatory factors were quantified using enzyme-linked immunosorbent assays (ELISAs), and 16S rRNA amplicon sequencing was employed for cecum content analysis. The results indicated that CP-A restored body weight and food intake and reversed histopathological changes in IM rats. Further, abnormal MLCK activation induced by 5-FU was attenuated by CP-A via the ERK/MLCK/MLC2 pathway. CP-A treatment improved tight junction protein levels and reduced inflammatory factor expression. Moreover, CP-A intervention regulated the intestinal microbiota community structure, increasing the abundance of Lactobacillus and decreasing the abundance of Shigella. In conclusion, CP-A mitigates 5-FU-induced IM by inhibiting the ERK/MLCK/MLC2 pathway, reducing the expression of inflammatory factors, improving the intestinal mucosal barrier, and regulating the intestinal microbial community. This study highlights CP-A's therapeutic potential in IM treatment and provides insights for future research.

Allergic Reaction to Aminophylline in a Case of Lower Respiratory Tract Infection with Measles: A Rare Case Report

Measles is an acute respiratory infectious disease caused by the measles virus. It leads to respiratory involvement with manifestations such as pneumonia, laryngobronchitis, pneumonitis, etc. It can also cause secondary bacterial and fungal infections. Aminophylline is a methylxanthine bronchodilator composed of theophylline and ethylenediamine. Airway blockage is reversed by bronchial smooth muscle relaxation, increased myosin light chain kinase activity, and decreased intracellular calcium concentration. It relaxes the smooth muscle of the bronchial airways and pulmonary blood vessels, reducing airway responsiveness to histamine, methacholine, adenosine, and allergens. Allergic skin reactions secondary to aminophylline administration have been rarely seen. Aminophylline can be given orally as well as intravenously. A seven-month-old male infant presented with a lower respiratory tract infection and a maculopapular rash involving the face and trunk. A history of contact with measles was present. Intravenous aminophylline was administered for persistent wheezing. The infant developed a generalised erythematous papular rash with a more widespread distribution than before within one hour of administration. The child was treated with antihistamines for the same. After a detailed physical examination, looking at the pattern of the rash and excluding other causes like environmental factors and drug history, the diagnosis of an allergic reaction to aminophylline was made. Early identification of allergic reactions is paramount, as prompt cessation of the offending agent and initiation of appropriate medical interventions can significantly mitigate the severity of the reaction and prevent potential life-threatening complications.

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.

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spatial transcriptomics approach to hypercontractile parenchymal arteries associated with ischemia-reperfusion

Background: Despite vessel recanalization, up to 67% of ischemic-stroke patients experience futile reperfusion increasing the risk of neurological morbidity and death. Arterial hypercontractility has been suggested to underlie the no-flow phenomenon, however, this remains to be fully elucidated. Parenchymal arteries in the area surrounding the ischemic core (penumbra) display an elevated vascular tone due to increased intracellular Ca 2+ sensitivity. Ischemia-reperfusion (IR) reduces Na + , K + -ATPase activity and is inversely proportional to Src activation, a tyrosine kinase, which is known to sensitize the contractile machinery to Ca 2+ via myosin light chain kinase (MLCK) activation. We hypothesize that IR reduces Na + , K + -ATPase activity and increases Src with consequent Ca 2+ sensitization and hypercontractility of parenchymal arteries. Materials and Methods: Five 3-month-old C57BL/6J male mice were exposed to one-hour transient middle cerebral artery occlusion by a filament followed by 24-hour reperfusion. Mice were anesthetized with isoflurane (3% induction, 1.5–2% maintenance) and intraperitoneally injected with Temgesic (0.1 mg/kg) 0.5- and 6 hours post-operative. Nissl staining was used to detect infarct areas. Spatial transcriptomics (ST) of coronal slices was performed. Infarct size was corrected for edema and calculated as the percentage of the whole hemisphere. ST was cell-predicted, and the cortex region was specified using gene markers from a single-cell RNA sequencing dataset. Pearson’s correlation coefficient was used to predict cell-cell correlation in the ST spots. Cells of interest were used for average gene expression analysis of scaled data. The expression levels were compared between the two hemispheres using t-tests. Data are presented as mean ± SD. Results: The ischemic core area was 0.67 ± 0.34 cm 2 corresponding to 18.11 ± 8.28% of the whole hemisphere. An interrelationship between cell types from the cerebral blood vessels and astrocytes was found, and these cell types were pooled. When comparing the average gene expressions between the contralateral and ipsilateral hemisphere, the Na + , K + -ATPase α 1 and α 2 isoforms were downregulated in the ipsilateral cortex area (p=2.7e -6 and p=3.3e -6 , respectively). Src (p=2.3e -4 ) and MLCK were downregulated in the ipsilateral cortex (4.8e -2 ). All genes for the myosin light chain phosphatase (MLCP) were downregulated. Conclusion: The data suggest that potentiated hypercontractility of parenchymal arteries is not due to Na + , K + -ATPase, and Src kinase-associated activation of the Ca 2+ -dependent MLCK. However, hypercontractility of parenchymal arteries may be due to a downregulated MLCP. Funding: This study was funded by the Lundbeck Foundation (R344-2020-952). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transepithelial barrier dysfunction drives microbiota dysbiosis to initiate epithelial clock-driven inflammation.

Factors that contribute to inflammatory bowel disease (IBD) pathogenesis include genetic polymorphisms, barrier loss, and microbial dysbiosis. A major knowledge gap exists in the origins of colitogenic microbiome and its relationship with barrier impairment. Epithelial myosin light chain kinase (MLCK) is a critical regulator of the paracellular barrier, but the effects of MLCK activation on intraepithelial bacteria (IEB) and dysbiosis are incompletely understood. We hypothesize that MLCK-dependent bacterial endocytosis promotes pathobiont conversion and shapes a colitogenic microbiome. To explore this, transgenic (Tg) mice with barrier loss induced by intestinal epithelium-specific expression of a constitutively active MLCK were compared to wild-type (WT) mice. When progeny of homozygous MLCK-Tg mice were separated postweaning by genotype (Tg/Tg, Tg/WT, WT/WT), increased IEB numbers associated with dysbiosis and more severe colitis were present in Tg/Tg and Tg/WT mice, relative to WT/WT mice. Cohousing with MLCK-Tg mice induced dysbiosis, increased IEB abundance and exacerbated colitis in WT mice. Conversely, MLCK-Tg mice colonized with WT microbiota at birth displayed increased Escherichia abundance and greater colitis severity by 6 weeks of age. Microarray analysis revealed circadian rhythm disruption in WT mice co-housed with MLCK-Tg mice relative to WT mice housed only with WT mice. This circadian disruption required Rac1/STAT3-dependent microbial invasion but not MLCK activity, and resulted in increased proinflammatory cytokines and glucocorticoid downregulation. In summary, the data demonstrate that barrier dysfunction induces dysbiosis and expansion of invasive microbes that lead to circadian disruption and mucosal inflammation. These results suggest that barrier-protective or bacterium-targeted precision medicine approaches may be of benefit to IBD patients.

Chemo-mechanical modeling of smooth muscle cell activation for the simulation of arterial walls under changing blood pressure

In this paper, a novel chemo-mechanical model is proposed for the description of the stretch-dependent chemical processes known as Bayliss effect and their impact on the active contraction in vascular smooth muscle. These processes are responsible for the adaptive reaction of arterial walls to changing blood pressure by which the blood vessels actively support the heart in providing sufficient blood supply for varying demands in the supplied tissues. The model is designed to describe two different stretch-dependent mechanisms observed in smooth muscle cells (SMCs): a calcium-dependent and a calcium-independent contraction. For the first one, stretch of the SMCs leads to an inlet of calcium ions which activates the myosin light chain kinase (MLCK). The increased activity of MLCK triggers the contractile units of the cells resulting in the contraction on a comparatively short time scale. For the calcium-independent contraction mechanism, stretch-dependent receptors of the cell membrane stimulate an intracellular reaction leading to an inhibition of the antagonist of MLCK, the myosin light chain phosphatase resulting in a contraction on a comparatively long time scale. An algorithmic framework for the implementation of the model in finite element programs is derived. Based thereon, it is shown that the proposed approach agrees well with experimental data. Furthermore, the individual aspects of the model are analyzed in numerical simulations of idealized arteries subject to internal pressure waves with changing intensities. The simulations show that the proposed model is able to describe the experimentally observed contraction of the artery as a reaction to increased internal pressure, which can be considered a crucial aspect of the regulatory mechanism of muscular arteries.

Abstract B021: Piezo1 and ROCK2 promote fast amoeboid migration in confined environments

Abstract Cell migration through confined environments may induce a phenotypic transition to fast amoeboid (leader bleb-based) migration. However, the molecular mechanism(s) controlling this phenotypic transition remain poorly understood. Here, we show that regulation of intracellular calcium levels by the plasma membrane tension sensor, Piezo1, promotes the Leader Bleb-Based Migration (LBBM) of melanoma cells. Using a ratiometric assay, intracellular calcium is shown to rise with increasing levels of confinement. Chelation of extracellular and intracellular calcium by BAPTA and BAPTA-AM, respectively, inhibits LBBM. Moreover, in highly motile cells, we found intracellular calcium levels to be dramatically increased at the cell rear. Using the Piezo1 inhibitor, GsMTx4, and RNAi, we can inhibit the phenotypic transition to fast amoeboid (leader bleb-based) migration. Therefore, we wondered if Piezo1 through calcium/calmodulin activates Myosin Light Chain Kinase (MLCK) to promote actomyosin contractility and amoeboid migration. Using a microchannel based assay, we find that ROCK2 and not MLCK, promotes amoeboid migration. Altogether, our work reveals an unanticipated collaboration between Piezo1 and ROCK2 in amoeboid migrating melanoma cells. Citation Format: Alexa Caruso, Neelakshi Kar, Jeremy Logue. Piezo1 and ROCK2 promote fast amoeboid migration in confined environments [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B021.

Abstract 15402: P90 Ribosomal S6 Kinase Rescues Contractility of Myosin Light Chain Kinase Null Smooth Muscle

Introduction: Smooth muscle (SM) contraction is regulated by an increase in [Ca 2+ ] , activation of myosin light chain kinase (MLCK) and phosphorylation of myosin regulatory light chain (RLC 20 ) activating myosin crossbridge cycling. MLCK null mice die at birth, but SM retains the ability to contract suggesting the presence of another kinase(s). Based on studies with WT and p90 ribosomal S-6 kinase (RSK2) null mice, we reported that RSK2 activity accounts for ~ 25% of maximal contractile force in wild type (WT) resistance arteries. Hypothesis: RSK2 confers contractility via RLC 20 activation in smooth muscle cells of MLCK null mice. Methods: Tension was measured on E18 aorta, bladder, and umbilical artery from WT and MLCK -/- embryos, in response to phenylephrine, carbachol, high potassium, SM myosin phosphatase, calyculinA (10 uM), as well as Ca 2+ following permeabilization with α-toxin. VSMCs were cultured from E18 MLCK -/- aortae and from WT and RSK2 -/- mouse mesenteric arteries. Serum starved cells were stimulated with serum and LPA (0.5 uM), in the presence and absence of RSK2 inhibitor, LJH685 (10 μM). Protein-protein interactions were assessed by in-situ PLA assay and immunoprecipitation. On-going in-vitro studies using purified myosin and kinases address RSK2 direct regulation of MLCK activity. Results: Strips from bladder SM and umbilical artery from WT and MLCK -/- embryos (E18), contracted in response to agonists and high [K]. pCa-tension curves showed the same sensitivity to Ca 2+ in MLCK -/- and WT. Maximal force induced by calyculinA was also the same indicating an intact normal contractile apparatus in the MLCK -/- . Significant phosphorylation of RLC 20 at Ser19 occurred in LPA- and serum-stimulated MLCK -/- SM cells, which was significantly inhibited by the RSK2 inhibitor, LJH685 (n=3). PLA assay showed co-localization of RSK2 and MLCK (<40nm) (n=3). RSK2 is immunoprecipitated along with its activators ERK1/2, PDK1, suggestive of cross talk between RSK2 and MLCK on the actin filament (n=5). Conclusions: RSK2 signaling accounts for the contractile activity in the absence of MLCK. Furthermore, RSK2 and MLCK form a signaling complex on the actin filament, optimally positioning them for interaction with adjacent myosin heads.

Platelet-activating factor and microvesicle particles as potential mediators for the toxicity associated with intoxicated thermal burn injury.

Thermal burn injuries (TBIs) in patients who are alcohol-intoxicated result in greater morbidity and mortality. The systemic toxicity found in human patients, which includes both immediate systemic cytokine generation with multiple organ failure and a delayed systemic immunosuppression, has previously been replicated in mouse models combining ethanol and localized TBI. Though considerable insights have been provided with these models, the exact mechanisms for these pathologic effects are unclear. In this review, we highlight the roles of the lipid mediator platelet-activating factor (PAF) and subcellular microvesicle particle (MVP) release in response to intoxicated thermal burn injury (ITBI) as effectors in the pathology. Particularly, MVP is released from keratinocytes in response to PAF receptor (PAFR) activation due to excess PAF produced by ITBI. These subcellular particles carry and thus protect the metabolically labile PAF which enable binding of this potent lipid mediator to several key sites. We hypothesize that PAF carried by MVP can bind to PAFR within the gut, activating myosin light chain kinase (MLCK). The subsequent gut barrier dysfunction in response to MLCK activation then allows bacteria to invade the lymphatic system and, eventually, the bloodstream, resulting in sepsis and resultant dysregulated inflammation in multiple organs. PAF in MVP also activate the skin mast cell PAFR resulting in migration of this key effector cell to the lymph nodes to induce immunosuppression. This review thus provides a mechanism and potential therapeutic approaches for the increased toxicity and immunosuppressive outcomes of TBI in the presence of acute ethanol exposure.

Streptococcal autolysin promotes dysfunction of swine tracheal epithelium by interacting with vimentin.

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium.

Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells.

Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains—Lacticaseibacillus paracasei BL23 and Lacticaseibacillus rhamnosus GG—in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.

Aftiphilin Regulation of Myosin Light Chain Kinase Activity Promotes Actin Dynamics and Intestinal Epithelial Barrier Function

The expression levels of aftiphilin (AFTPH) are significantly lower in inflamed colonic tissues from patients with ulcerative colitis (UC) and mice with experimental colitis. During colonic inflammation, the selective permeability of the colonic epithelium is compromised largely due to dysregulation of proteins associated with either the tight junction (TJ) complex and actomyosin contraction rings. Here, we hypothesized that inflammation-associated reduction in AFTPH levels might cause an increase in the selective permeability of the colonic epithelium. In this study, we measured the transepithelial electric resistance (TEER), sodium (Na+) ion flux and dextran permeability in polarized colonic epithelial cells after manipulation of AFTPH. Silencing of AFTPH reduced TEER, increased Na+ ion flow and dextran permeability. Examination of mRNA and protein levels of multiple TJ proteins and Na+ ion transporters suggested that AFTPH deficiency did not significantly change expression of most of these transmembrane proteins. While the gross structure of the TJs in AFTPH gene-silenced cells appeared normal, elevated levels of junctional Occludin were observed. Most notably we observed that AFTPH co-localized with myosin light chain kinase (MLCK) and attenuated cellular MLCK activity as observed by phospho- myosin light chain 2 (pMLC2) western blots. Importantly, inhibition of MLCK activity reversed the reduction of TEER in AFTPH-deficient monolayers. Lastly, examination of microvilli by transmission electron microscopy and immunofluorescence imaging of actin filament arrangement demonstrated that AFTPH deficiency also affected filament arrangement in colonic epithelial cells. Taken together, these results suggest that AFTPH regulates intestinal epithelial permeability and actin polymerization in colonic epithelium through interfering with MLCK/MLC interactions.

Brain-derived neurotrophic factor regulates LYN kinase–mediated myosin light chain kinase activation to modulate nonmuscle myosin II activity in hippocampal neurons

Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system.

Study on the mechanism of Mycoplasma gallisepticum infection on chicken tracheal mucosa injury

ABSTRACT Mycoplasma gallisepticum (MG) is a pathogenic microorganism that causes serious harm to the poultry industry. It is mainly adsorbed on the cilia and mucosa of respiratory epithelial cells, causing tracheal mucosal damage or cilia loss, causing chronic respiratory disease (CRD). In order to study the effect of MG infection on chicken tracheal mucosa injury and explore its possible mechanism, specific-pathogen-free (SPF) chickens were challenged with Mycoplasma gallisepticum wild-type strain MG-HY. Then, transcriptome sequencing analysis was performed to study the mechanism of MG tracheal mucosal damage. During infection, MG localizes and proliferates in the chicken trachea, and induces mucosal damage. A total of 3112 significantly (P < 0.01) differentially expressed genes (DEGs) were selected by RNA-seq, including 1646 up-regulated genes and 1466 down-regulated genes. Functional analysis showed increased expression levels of genes involved in immune defense response and mechanical barrier of tracheal mucosa in infected chicks. The expression level of pro-inflammatory cytokines (TNF-α) increased, activating the upstream protein Ras of the ERK-MLCK signaling pathway, Ras causing ERK phosphorylation levels to rise and MLCK activation, thus causing phosphationalization of MLC, and further regulating the expression and mucous distribution of tight junction protein (TJ), leading to tracheal mucosal injury in chicks. The results of qRT-PCR assay and immunohistochemical analysis were consistent with the results of transcriptome analysis. Overall, Our findings provide a basis for further research on the underlying mechanism of chick tracheal mucosal damage caused by MG infection, and help to understand how MG induces respiratory immune damage in birds.

Molecular mechanism of the TGF‑β/Smad7 signaling pathway in ulcerative colitis.

Aberrant TGF-β/Smad7 signaling has been reported to be an important mechanism underlying the pathogenesis of ulcerative colitis. Therefore, the present study aimed to investigate the effects of a number of potential anti-colitis agents on intestinal epithelial permeability and the TGF-β/Smad7 signaling pathway in an experimental model of colitis. A mouse model of colitis was first established before anti-TNF-α and 5-aminosalicyclic acid (5-ASA) were administered intraperitoneally and orally, respectively. Myeloperoxidase (MPO) activity, histological index (HI) of the colon and the disease activity index (DAI) scores were then detected in each mouse. Transmission electron microscopy (TEM), immunohistochemical and functional tests, including Evans blue (EB) and FITC-dextran (FD-4) staining, were used to evaluate intestinal mucosal permeability. The expression of epithelial phenotype markers E-cadherin, occludin, zona occludens (ZO-1), TGF-β and Smad7 were measured. In addition, epithelial myosin light chain kinase (MLCK) expression and activity were measured. Anti-TNF-α and 5-ASA treatments was both found to effectively reduce the DAI score and HI, whilst decreasing colonic MPO activity, plasma levels of FD-4 and EB permeation of the intestine. Furthermore, anti-TNF-α and 5-ASA treatments decreased MLCK expression and activity, reduced the expression of Smad7 in the small intestine epithelium, but increased the expression of TGF-β. In mice with colitis, TEM revealed partial epithelial injury in the ileum, where the number of intercellular tight junctions and the expression levels of E-cadherin, ZO-1 and occludin were decreased, all of which were alleviated by anti-TNF-α and 5-ASA treatment. In conclusion, anti-TNF-α and 5-ASA both exerted protective effects on intestinal epithelial permeability in an experimental mouse model of colitis. The underlying mechanism may be mediated at least in part by the increase in TGF-β expression and/or the reduction in Smad7 expression, which can inhibit epithelial MLCK activity and in turn reduce mucosal permeability during the pathogenesis of ulcerative colitis.

Angiotensin II-Induced Vasoconstriction via Rho Kinase Activation in Pressure-Overloaded Rat Thoracic Aortas.

Angiotensin II (Ang II) induces vasoconstriction through myosin light chain (MLC) kinase activation and MLC phosphatase inactivation via phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) by Rho kinase. However, the detailed mechanism underlying Rho kinase activation by Ang II is still unknown. We investigated the mechanism of Ang II-induced vasoconstriction mediated by Rho kinase in pressure-overloaded rat thoracic aortas. Pressure-overloaded rats were produced by coarctation of the suprarenal abdominal aorta in four-week-old male Wistar rats. The contractile response to Ang II was significantly enhanced in the pressure-overloaded rats. Ang II-induced vasoconstriction was attenuated by inhibitors of Rho kinase, extracellular signal-regulated kinase 1 and 2 (Erk1/2), and epidermal growth factor receptor (EGFR) in both the sham-operated and pressure-overloaded rats. The Ang II-induced vasoconstriction was attenuated by a Janus kinase 2 (JAK2) inhibitor in only the pressure-overloaded rats. The protein levels of MYPT1 and JAK2 increased only in the pressure-overloaded rat thoracic aortas. These results suggested that Ang II-induced contraction is mediated by Rho kinase activation via EGFR, Erk1/2, and JAK2 in pressure-overloaded rat thoracic aortas. Moreover, Ang II-induced contraction was enhanced in pressure-overloaded rats probably because the protein levels of MYPT1 and JAK2 increased in the thoracic aortas.

TMEM16A inhibits angiotensin II-induced basilar artery smooth muscle cell migration in a WNK1-dependent manner

Vascular smooth muscle cell (VSMC) migration plays a critical role in the pathogenesis of many cardiovascular diseases. We recently showed that TMEM16A is involved in hypertension-induced cerebrovascular remodeling. However, it is unclear whether this effect is related to the regulation of VSMC migration. Here, we investigated whether and how TMEM16A contributes to migration in basilar artery smooth muscle cells (BASMCs). We observed that AngII increased the migration of cultured BASMCs, which was markedly inhibited by overexpression of TMEM16A. TMEM16A overexpression inhibited AngII-induced RhoA/ROCK2 activation, and myosin light chain phosphatase (MLCP) and myosin light chain (MLC20) phosphorylation. But AngII-induced myosin light chain kinase (MLCK) activation was not affected by TMEM16A. Furthermore, a suppressed activation of integrinβ3/FAK pathway, determined by reduced integrinβ3 expression, FAK phosphorylation and F-actin rearrangement, was observed in TMEM16A-overexpressing BASMCs upon AngII stimulation. Contrary to the results of TMEM16A overexpression, silencing of TMEM16A showed the opposite effects. These in vitro results were further demonstrated in vivo in basilar arteries from VSMC-specific TMEM16A transgenic mice during AngII-induced hypertension. Moreover, we observed that the inhibitory effect of TMEM16A on BASMC migration was mediated by decreasing the activation of WNK1, a Cl−-sensitive serine/threonine kinase. In conclusion, this study demonstrated that TMEM16A suppressed AngII-induced BASMC migration, thus contributing to the protection against cerebrovascular remodeling during AngII-infused hypertension. TMEM16A may exert this effect by suppressing the RhoA/ROCK2/MLCP/MLC20 and integrinβ3/FAK signaling pathways via inhibiting WNK1. Our results suggest that TMEM16A may serve as a novel therapeutic target for VSMC migration-related diseases, such as vascular remodeling.

Matrix Metalloproteinase-9 (MMP-9) induced disruption of intestinal epithelial tight junction barrier is mediated by NF-κB activation.

BackgroundMatrix Metalloproteinase-9 (MMP-9) has been shown to play a key role in mediating inflammation and tissue damage in inflammatory bowel disease (IBD). In patients with IBD, the intestinal tight junction (TJ) barrier is compromised as characterized by an increase in intestinal permeability. MMP-9 is elevated in intestinal tissue, serum and stool of patients with IBD. Previous studies from our laboratory showed that MMP-9 causes an increase in intestinal epithelial TJ permeability and that the MMP-9 induced increase in intestinal permeability is an important pathogenic factor contributing to the development of intestinal inflammation in IBD. However, the intracellular mechanisms that mediate the MMP-9 modulation of intestinal barrier function remain unclear.AimsThe main aim of this study was to further elucidate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ permeability using Caco-2 monolayers as an in-vitro model system.ResultsMMP-9 induced increase in Caco-2 TJ permeability was associated with activation and cytoplasmic-to-nuclear translocation of NF-κB p65. Knocking-down NF-κB p65 by siRNA transfection prevented the MMP-9 induced expression of the NF-κB target gene IL-8, myosin light chain kinase (MLCK) protein expression, and subsequently prevented the increase in Caco-2 TJ permeability. In addition, the effect of MMP-9 on Caco-2 intestinal epithelial TJ barrier function was not mediated by apoptosis or necrosis.ConclusionOur data show that the MMP-9 induced disruption of Caco-2 intestinal epithelial TJ barrier function is regulated by NF-κB pathway activation of MLCK.