Myosin Light Chain Phosphorylation Research Articles

Chlorogenic Acid Relaxes Rat Ileal Smooth Muscles by Mechanisms Independent of Rho Kinase Mediated Effects on Myosin Light Chain Phosphorylation

Chlorogenic acid (CGA) is a polyphenolic compound found in fruits and vegetables and generally known to have antioxidant, anti-inflammatory and antithrombotic effects. Studies have shown that CGA induces nitric oxide-sGC-PKG pathway to cause relaxation of the vascular smooth muscles. But the effects of CGA on ileal smooth muscles are still unknown. In our previous experiments, we have shown that CGA increases basal tone with no effect on relaxation of rat ileal smooth muscles when they are depolarized by 140 mM potassium kreb's buffer. The present study is designed to evaluate if CGA acts through the rho-kinase mediated pathway in relaxing rat ileal smooth muscles when challenged with acetylcholine. Effects of CGA (10 μM) on basal tension and maximum contractile tension induced by acetylcholine (0.5μM) in isolated rat ileal rings (3mm) were determined using tissue bath apparatus. CGA effects on myosin light chain phosphorylation at maximum contractile tension in response to acetylcholine (0.5 μM) were also measured by freezing the ileal rings and extracting the protein and running Western blots. The role of rho-kinase in regulating smooth muscle relaxation is tested by incubating ileal rings with rho-kinase inhibitor Y-27632 (1 μM) and measuring the basal tension, maximum contractile tension and myosin light chain phosphorylation as described above. The results show that basal tension increases from 100% (control group) to 130.8 ± 19.0% and 121.7 ± 30.6 % when treated with CGA alone and the combination of CGA and Y-27632 respectively. The peak contractile tension in response acetylcholine changes from 100% (control group) to 50.4 ± 12.13 % and 55 ± 4.05 % when treated with CGA alone and the combination of CGA and Y-27632 respectively. The results from Western blots show that myosin light chain phosphorylation didn't change significantly between CGA (41.8 ± 5.5%) and the combination of CGA and Y-27632 (37.7 ± 3.7%) treatments. These results suggest that CGA alone and in combination with rho-kinase inhibitor is involved in increasing the basal tone of ileal smooth muscles when compared with the control group. In contrast, CGA significantly decreased the acetylcholine induced peak contractile tension both in presence and absence of rho-kinase inhibitor with out any corresponding changes in myosin light chain phosphorylation. Therefore, we conclude that CGA relaxes ileal smooth muscles by mechanisms independent of rho-kinase mediated effects on myosin light chain phosphorylation. Further studies on effects of CGA on activity of soluble guanylate cyclase and phosphodiesterase activity may provide some insights on mechanism of action of CGA on smooth muscles.

SARS‐Cov‐2 Spike Protein Causes Human Lung Endothelial Inflammation

Patients with COVID‐19 often present with pulmonary inflammation and blood coagulopathy, with severe cases advancing to acute respiratory distress syndrome (ARDS) and multiple organ failure. Although the pathogenesis is not completely understood, endothelial inflammation is suspected to be a major causative factor for the systemic manifestations of COVID‐19. In this study, we investigated the direct effects of SARS‐Cov‐2 spike protein on pulmonary microvascular endothelial cells (ECs) isolated from the small airway region of viable human lungs. We verified the expression of angiotensin‐converting enzyme 2 (Ace‐2), the receptor for SARS‐Cov‐2 spike protein, in pulmonary ECs using flow cytometry analysis and immunofluorescence labeling. Treating ECs with recombinant spike protein caused increased F‐actin stress fibers, enhanced phosphorylation of myosin light chain 2, and disruption of adherens junction continuity. In addition to the junction and cytoskeletal changes, spike protein‐treated ECs showed increased expression of intercellular adhesion molecule 1 and enhanced adherence for monocytic cells. Interestingly, there was a significant accumulation of von Willebrand factor long strands on the cell surface in ECs isolated from COVID‐19 positive human lungs, as well as on ECs from normal human lungs following spike protein treatment. In conclusion, we demonstrate that the SARS‐Cov‐2 spike protein is capable of directly activating human lung ECs and promoting inflammation/coagulopathy.

IL‐13/IL4Rα Signaling Increases Tension in Human Circular and Longitudinal Esophageal Smooth Muscle Through Distinct Molecular Pathways: Potential Contribution to Reduced Esophageal Distensibility in EoE

Introduction Reduced esophageal distensibility (ED) in EoE has been attributed largely to fibrosis, but inflammatory cell infiltration and increased tension in esophageal smooth muscle (ESM) also might contribute to reduced ED. EoE is a type 2 immune disease in which type 2 cytokines IL-4 and IL-13 signal through the IL-4 receptor alpha (IL4Rα), activating Janus kinases (JAKs) that in turn activate STAT6. Dupilumab, a monoclonal antibody against IL4Rα, can improve ED in EoE patients, but it is unclear whether this is due to effects on fibrosis, inflammatory cells, or ESM tension. To explore how IL4Rα signaling might affect ESM tension, we treated ESM cells with IL-13 and selective inhibitors of STAT6 (AS1517499, 600 nM) or JAK1/2 (ruxolitinib, 100 ng/ml). Since ESM tension is induced by phosphorylation of myosin light chains through PKC and ROCK pathways, we also studied effects of selective inhibitors of PKC (calphostin C, 500 nM) and ROCK (Y-27632, 10 µM) on ESM tension. Methods Using 1 circular (C) and 1 longitudinal (L) telomerase-expressing ESM cell lines generated from muscularis propria of a human organ donor, cell contraction was assessed in the attached gel contraction model. Gels containing equal numbers of ESM cells were cultured for 48 hours with and without IL-13 (50 ng/ml), during which ESM tension developed; contraction was initiated by releasing the gel from the sides of the culture plate. Histamine (100 µM), a contraction mediator, was added immediately prior to release in some studies. Contraction was assessed by measuring gel surface area over 24 hours using Image J. Gel contraction studies were repeated in the presence of pathway inhibitors. Results In non-treated cells, gel sizes of CESM were significant smaller at all time points than those of LESM (p<0.001). Compared to non-treated controls, IL-13 significantly reduced gel size in both CESM and LESM (Figures 1&2). Also compared to non-treated controls, IL-13-treatment resulted in smaller gel sizes at all time points following histamine stimulation in both CESM and LESM (p<0.05). In LESM, IL-13-induced reductions in gel size were significantly inhibited by ruxolitinib, calphostin C, and Y-27632, but not by AS1517499 (Figure 2). In CESM, these IL-13-induced reductions were significantly inhibited by ruxolitinib and calphostin C, but not by Y-27632 or AS1517499 (Figure 1). Conclusions CESM generates more baseline tension that LESM. IL-13 increases tension and enhances the contractile response to histamine in both CESM and LESM. In LESM, both the PKC and ROCK pathways contribute to IL-13-mediated increases in tension whereas in CESM, only the PKC pathway contributes to this effect. These findings demonstrate distinct molecular pathways whereby IL-13 contributes to LESM and CESM tension, and suggest that IL-13-induced ESM tension may contribute to the reduced ED of EoE.

Identification of PDZ‐RhoGEF (PRG) as a Novel Gα i Target

Cell-migration and adhesion are fundamental biological processes regulated by chemokine G-protein-coupled receptors that couple to PTX-sensitive Gi proteins. The role of Gβγ subunits released from Gi in chemokine signaling has been well characterized. In contrast, Gαi has been thought to have no direct signaling function. Our laboratory recently uncovered a role for Gαi-GTP in regulating cell adhesion. However, a direct effector of Gαi responsible was not identified. To discover novel targets of active Gαi, we used BioID2 proximity-based labeling coupled to TMT quantitative proteomic mass spectrometry. We subsequently compared the abundance ratios of proteins in Gαi1-QL-BioID2 (constitutively active) to Gαi1-WT-BioID2 (inactive) samples. BioID2-CaaX was used as a membrane-targeted control. Experiments were done in HT1080 fibrosarcoma cells where we previously demonstrated Gαi regulation of adhesion. Known binding partners for Gαi subunits including, GPCRs, Gβ, Gγ, adenylate cyclase, and Ric8A were identified by mass spectrometry further validating the method. A candidate Gαi effector identified was PDZ-RhoGEF (PRG), a Rho guanine nucleotide exchange factor that regulates cell adhesion in neutrophils. To determine if PRG can be activated by Gαi; we co-transfected PRG with Gαi1 subunit and an SRE luciferase Rho reporter in A293 cells. Gαi1-QL but not Gαi1-WT strongly activated PRG dependent SRE luciferase, which was confirmed in a rhotekin assay for active RhoA. PRG activation was specific to the Gαi subfamily and required the PRG-RGS domain. A proximal interaction was confirmed in aproximity ligation assay in A293 cells transfected with PRG and Gαi1 cDNAs. Purified Cy5-labeled myr-His-Gαi bound specifically to GST-PRG in a TR-FRET assay. This binding was independent of the nucleotide state of Gαi1 but was inhibited by the addition of Gβγ subunits. In neutrophils, activation of the formyl peptide receptor (FPR) by fMLP, and subsequent PRG activation leads to Rho-dependent accumulation of phosphorylated-myosin light chain (P-MLC) at the trailing edge and has been proposed to result from FPR-dependent Gα13 activation. In our preliminary experiments, stimulation of primary human neutrophils with 10 nM fMLP promoted polarized P-MLC accumulation that was inhibited by PTX. This supports a model where at physiological concentrations of chemoattractant, activation of PRG/Rho is through Gαi-GTP rather than Gα13 in primary human neutrophils. Overall, we identified relatively ubiquitously expressed PRG as a novel Gαi-GTP target. Identification of a novel signaling pathway regulated by Gi-coupled GPCRs will have a significant impact on our understanding of the biology regulated by these ubiquitous and pharmacologically important receptors.

Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells.

Single-cell motility is spatially heterogeneous and driven by metabolic energy. Directly linking cell motility to cell metabolism is technically challenging but biologically important. Here, we use single-cell metabolic imaging to measure glycolysis in individual endothelial cells with genetically encoded biosensors capable of deciphering metabolic heterogeneity at subcellular resolution. We show that cellular glycolysis fuels endothelial activation, migration and contraction and that sites of high lactate production colocalize with active cytoskeletal remodelling within an endothelial cell. Mechanistically, RhoA induces endothelial glycolysis for the phosphorylation of cofilin and myosin light chain in order to reorganize the cytoskeleton and thus control cell motility; RhoA activation triggers a glycolytic burst through the translocation of the glucose transporter SLC2A3/GLUT3 to fuel the cellular contractile machinery, as demonstrated across multiple endothelial cell types. Our data indicate that Rho-GTPase signalling coordinates energy metabolism with cytoskeleton remodelling to regulate endothelial cell motility.

Nongenomic Thyroxine‐Induced Relaxation of Rat Skeletal Muscle Arteries Is Mediated by Integrin αvβ3, Integrin‐Linked Kinase, ERK1/2 and ROCK

Thyroid hormones (TH) can decrease total peripheral resistance by changing vascular tone. They induce genomic effects which are necessarily associated with the change in gene expression and nongenomic effects which could involve a wide spectrum of molecular pathways resulting in relatively rapid vasodilation (within several minutes). The mechanisms behind the nongenomic vasorelaxation are poorly understood and may differ among the vascular beds. Skeletal muscles resistance arteries are important contributors to the total peripheral resistance. Therefore, in this study, we tested the hypothesis that both TH (T3 and T4) induce rapid nongenomic dilation of rat skeletal muscle arteries. Gastrocnemius feed (sural) arteries from Wistar rats (m=300-450 g) were mounted in wire myograph to record their responses in isometric mode. We discovered that T4 (10 microM) induced more prominent relaxation than T3 (10 microM) in arteries preconstricted by methoxamine (α1-adrenoceptor agonist): to 37.7±6.6% and 67.9±11.8 % from contraction level in control, respectively. Preincubation with T4 (10 microM) was also more effective in depressing contractile responses to methoxamine than preincubation with T3 (10 microM). T3-induced relaxation disappeared after endothelium removal, while T4-induced relaxation did not depend on the presence of endothelium. Further, we studied the mechanisms of the more prominent nongenomic effect of T4 in details. The effect of T4 in endothelium-denuded arteries was abolished by tetrac (3 microM), the competitive inhibitor of integrin αvβ3. Integrin αvβ3 may mediate the extracellular matrix signalling by activation of associated kinases like Src-kinase, integrin-linked kinase (ILK) or ERK1/2. Src-kinase inhibitor PP2 (10 microM) did not affect T4-induced relaxation. However, in the presence of ILK inhibitor compound 22 (10 microM) or ERK1/2 inhibitor U0126 (10 microM), the effect of T4 was absent. One of the downstream targets of ILK could be RhoA-activated kinase (ROCK) which potentiates smooth muscle contraction by inhibiting myosin light chain phosphatase through MYPT1 phosphorylation. ROCK inhibitor Y27632 (3 microM) decreased the effect of T4. The results of functional studies were supported by Western blot data. Preincubation with T4 (10 microM) reduced the contents of phosphorylated MYPT1 (Thr850) and MLC2 (Ser19) in endothelium-denuded arterial samples, the effect of T4 was not observed in the presence of ILK inhibitor compound 22. To conclude, T3 induces endothelium-dependent rapid nongenomic relaxation of rat sural arteries. The rapid relaxing effect of T4 is more powerful and, unlike T3, endothelium-independent. The molecular pathway of T4-induced relaxation of smooth muscle includes suppression of extracellular matrix signalling via integrin αvβ3 and downstream kinases ILK, ERK 1/2 and ROCK with subsequent decrease of MYPT1 (Thr850) and MLC2 (Ser19) phosphorylation.

SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

SummaryHepatic stellate cells (HSCs) are crucial for liver injury repair and cirrhosis. However, the mechanism of chemotactic recruitment of HSCs into injury loci is still largely unknown. Here, we demonstrate that serum amyloid A1 (SAA1) acts as a chemokine recruiting HSCs toward injury loci signaling via TLR2, a finding proven by gene manipulation studies in cell and mice models. The mechanistic investigations revealed that SAA1/TLR2 axis stimulates the Rac GTPases through PI3K-dependent pathways and induces phosphorylation of MLC (pSer19). Genetic deletion of TLR2 and pharmacological inhibition of PI3K diminished the phosphorylation of MLCpSer19 and migration of HSCs. In brief, SAA1 serves as a hepatic endogenous chemokine for the TLR2 receptor on HSCs, thereby initiating PI3K-dependent signaling and its effector, Rac GTPases, which consequently regulates actin filament remodeling and cell directional migration. Our findings provide novel targets for anti-fibrosis drug development.

Dynamic cytosolic Ca2+ and force responses to muscarinic stimulation in airway smooth muscle.

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+]cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 μM). Both maximum [Ca2+]cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+]cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+]cyt, the extent of regulatory myosin light-chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation.

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.

Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100nM. Some LECs were pretreated with 1μM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100nM MBG or cinobufotalin (CINO). Expression of β-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure β-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1nM) and CINO (≥ 10nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1μM L-NAME attenuated (p < 0.05) the MPLEC. The β-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain.

Activated ezrin-radixin-moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologues LOK and SLK. CRISPR/Cas9 was used to knock out all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM-activating phosphorylation. The apical domains of cells lacking LOK/SLK or ERMs are strikingly similar and selectively altered, with loss of microvilli and with junctional actin replaced by ectopic myosin-II-containing apical contractile structures. Constitutively active ezrin can reverse the phenotypes of either ERM or LOK/SLK knockouts, indicating that a central function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.

NOX5 AND VASCULAR SIGNALLING IN HUMAN HYPERTENSION

Objective: Nox5, a key ROS-generating oxidase in human vessels, regulates vascular contraction and is implicated in vascular damage. However molecular mechanisms regulating vascular Nox5 are elusive. We questioned the role of Nox5 in vascular smooth muscle cell signalling in hypertension. Design and method: Intact small arteries and vascular smooth muscle cells (VSMC) isolated from small arteries from normotensive (NT) and hypertensive (HT) subjects were studied. Nox5 expression and phosphorylation (western blot, immunoprecipitation); ROS generation (chemiluminescence); protein oxidation (oxidised peroxiredoxin/DJ-1), activation of signalling pathways (Src, PKC, MLC phosphorylation; immunoblotting), and actin cytoskeletal organization (phalloidin staining) were assessed in the absence and presence of Nox5 inhibition (mellitin, 100 nM). To exclude Nox1–4 effects, we used p22phox silenced VSMCs (siRNA). Results: Nox5 expression was increased in intact arteries from HT subjects (70 + 26.79% vs NT, p &lt; 0.05). In VSMC, ROS levels (139 ± 27% vs NT, p &lt; 0.05), and oxidation of peroxiredoxin (201.8 + 40.34% vs NT, p &lt; 0.05) and DJ-1 (71.7 + 16.51% vs NT, p &lt; 0.05) were increased in HT. Nox5 expression (103 ± 23% vs NT, p &lt; 0.05) and phosphorylation (77 + 17.93% vs NT, p &lt; 0.05) were increased in the HT group. Nox5 inhibition reduced Ang II-induced ROS generation in both groups (p &lt; 0.05 vs Ctl). In contrast, p22phox silencing increased ROS in both groups, an effect blocked by mellitin (p &lt; 0.05 vs Ctl). Nox5 associates with c-Src. Treatment with a Src inhibitor (PP2, 10 μM) did not reduce Nox5 phosphorylation. Furthermore, PP2 failed to reduce AngII-induced ROS generation in p22phox-silenced VSMCs. Nox5 inhibition reduced Ang II-induced c-Src activation (p &lt; 0.05 vs Ctl), suggesting that Nox5 is upstream of c-Src. Next, we investigated the role of Nox5 in vascular signaling. In cells from hypertensive subjects, p22phox silencing was associated with sustained Ang II-induced PKC and MLC20 phosphorylation, an effect blocked by mellitin and PP2 (p &lt; 0.05 vs Ctl). VSMC from hypertensive subjects had an increased number of stress fibres, which was attenuated by mellitin and PP2. Conclusions: Our findings demonstrate that in human hypertension Nox5 is upregulated. This is associated with activation of c-Src, increased redox-sensitive signalling and VSMC cytoskeletal reorganisation. We define a novel Nox5-ROS-c-Src signalling pathway that may play a role in vascular remodelling in hypertension.

Depletion of Arg/Abl2 improves endothelial cell adhesion and prevents vascular leak during inflammation

Endothelial barrier disruption and vascular leak importantly contribute to organ dysfunction and mortality during inflammatory conditions like sepsis and acute respiratory distress syndrome. We identified the kinase Arg/Abl2 as a mediator of endothelial barrier disruption, but the role of Arg in endothelial monolayer regulation and its relevance in vivo remain poorly understood. Here we show that depletion of Arg in endothelial cells results in the activation of both RhoA and Rac1, increased cell spreading and elongation, redistribution of integrin-dependent cell-matrix adhesions to the cell periphery, and improved adhesion to the extracellular matrix. We further show that Arg is activated in the endothelium during inflammation, both in murine lungs exposed to barrier-disruptive agents, and in pulmonary microvessels of septic patients. Importantly, Arg-depleted endothelial cells were less sensitive to barrier-disruptive agents. Despite the formation of F-actin stress fibers and myosin light chain phosphorylation, Arg depletion diminished adherens junction disruption and intercellular gap formation, by reducing the disassembly of cell-matrix adhesions and cell retraction. In vivo, genetic deletion of Arg diminished vascular leak in the skin and lungs, in the presence of a normal immune response. Together, our data indicate that Arg is a central and non-redundant regulator of endothelial barrier integrity, which contributes to cell retraction and gap formation by increasing the dynamics of adherens junctions and cell-matrix adhesions in a Rho GTPase-dependent fashion. Therapeutic inhibition of Arg may provide a suitable strategy for the treatment of a variety of clinical conditions characterized by vascular leak.

Pterostilbene Ameliorates DSS-Induced Intestinal Epithelial Barrier Loss in Mice via Suppression of the NF-κB-Mediated MLCK-MLC Signaling Pathway.

The integrity of the intestinal barrier is critical for homeostasis. In this study, we investigated the protective effect of pterostilbene (PTE) on the intestinal epithelium barrier. In vitro results of transepithelial electrical resistance (TEER) in Caco-2 cells indicated that PTE counteracted tumor necrosis factor α (TNFα)-induced barrier damage. In vivo PTE pretreatment markedly ameliorated intestinal barrier dysfunction induced by dextran sulfate sodium (DSS). Notably, intestinal epithelial tight junction (TJ) molecules were restored by PTE in mice exposed to DSS. The mechanism study revealed that PTE prevented myosin light-chain kinase (MLCK) from driving phosphorylation of MLC (p-MLC), which is crucial for maintaining intestinal TJ stability. Furthermore, PTE blunted translocation of NF-κB subunit p65 into the nucleus to downregulate MLCK expression and then to safeguard TJs and barrier integrity. These findings suggest that PTE protected the intestinal epithelial barrier through the NF-κB- MLCK/p-MLC signal pathway.

Mechanism of α1-Adrenergic Receptor-Induced Increased Contraction of Rat Mesenteric Artery in Aging Hypertension Rats

Introduction: Vasoconstriction is triggered by an increase in intracellular-free calcium concentration. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase (ROCK), protein kinase C (PKC), and so on. In this study, we studied the changes of vascular reactivity as well as the underlying signaling pathways in aging spontaneously hypertensive rats (SHRs). Methods: The artery tension induced by α1-adrenergic receptor activator (α1-AR) phenylephrine (PE) was measured in the absence or presence of myosin light chain kinase (MLCK), PKC, and ROCK inhibitors. The α1-AR, PKC, ROCK, phosphorylation of myosin light chain (MLC), and PKC-potentiated phosphatase inhibitors of 17 kDa (CPI-17) of rat mesenteric arteries were analyzed at the mRNA level or protein level. Results: The vascular tension measurements showed that there was a significant increase in the mesenteric artery contraction induced by PE in old SHR. MLCK inhibitor ML-7 can similarly inhibit PE-induced vasoconstriction. PKC inhibitor GF109203X has the weakest inhibitory effect on PE-induced contraction in old SHR. At the presence of ROCK inhibitor H1152, PE-induced contraction was significantly reduced in young Wistar-Kyoto (WKY) rats, but this phenomenon disappeared in other rats. Furthermore, in old SHR the protein expression of α1-AR decreased and phosphorylation of MLC and CPI-17 were upregulated and MLC phosphatase (MLCP) activity was significantly lower. The expressions of PKC were upregulated in SHR and old rats. In addition, the expression of ROCK-1 was decreased and ROCK-2 was significantly upregulated with age in SHR. Conclusion: In aging hypertension, the expression/activity of PKC or ROCK-2/CPI-17 excessively increased, MLCP activity decreased and MLC phosphorylation enhanced, leading to increased α1-AR-induced vasoconstriction.

Protective effects of curcumin against rat intestinal inflammation‑related motility disorders.

Intestinal inflammation frequently occurs alongside dysmotility, which is characterized by altered myosin light chain phosphorylation levels. Curcumin, an active component from the ginger family, is reported to confer anti-inflammatory effects. However, the effects of curcumin on both diarrhea and constipation associated inflammation remains to be elucidated. The present study was designed to investigate the effects of curcumin on diarrhea and constipation and to determine the related mechanisms. Sprague-Dawley rats were used to establish diarrhea and constipation models via intracolonic acetic acid (4%) instillation or cold water gavage for 2 weeks, respectively. Blood samples were collected to measure the serum levels of the cytokines TNF-α and IL-1β using ELISA kits. Western blotting was performed to measure NF-κB, RhoA, Rho-related kinase 2, phosphorylated MLC20, phosphorylated myosin phosphorylated target subunit 1, 130k Da-MLC kinase (MLCK), c-kit tyrosine kinase protein expression, and reverse transcription-quantitative PCR was conducted to measure MLCK expression levels. The results indicated that curcumin reversed the elevations in the pro-inflammatory cytokines IL-1β and TNF-α by inhibiting the NF-κB pathway in rats with diarrhea and constipation. The results also indicated that myosin light chain (MLC) phosphorylation in intestinal smooth muscle was positively and negatively associated with the motility of inflammation-related diarrhea and constipation in rats, respectively. Curcumin significantly reversed the increased MLC phosphorylation in the jejunum of the rats with diarrhea, significantly enhanced the reductions in inflammatory mediators, including TNF-α and IL-1β, of rats with constipation and significantly ameliorated the related hyper-motility and hypo-motility in rats with both diarrhea and constipation. In conclusion, the potential roles of the MLC kinase, c-kit tyrosine and Rho A/Rho-associated kinase 2 pathways, which are involved in curcumin-induced amelioration of inflammation-related diarrhea and constipation, were explored in the present study. Results from the present study suggested that curcumin has potential therapeutic value for treating intestinal inflammation and inflammation-related motility disorders.

IGF1R controls mechanosignaling in myofibroblasts required for pulmonary alveologenesis.

Ventilation throughout life is dependent on the formation of pulmonary alveoli, which create an extensive surface area in which the close apposition of respiratory epithelium and endothelial cells of the pulmonary microvascular enables efficient gas exchange. Morphogenesis of the alveoli initiates at late gestation in humans and the early postnatal period in the mouse. Alveolar septation is directed by complex signaling interactions among multiple cell types. Here, we demonstrate that IGF1 receptor gene (Igf1r) expression by a subset of pulmonary fibroblasts is required for normal alveologenesis in mice. Postnatal deletion of Igf1r caused alveolar simplification, disrupting alveolar elastin networks and extracellular matrix without altering myofibroblast differentiation or proliferation. Moreover, loss of Igf1r impaired contractile properties of lung myofibroblasts and inhibited myosin light chain (MLC) phosphorylation and mechanotransductive nuclear YAP activity. Activation of p-AKT, p-MLC, and nuclear YAP in myofibroblasts was dependent on Igf1r. Pharmacologic activation of AKT enhanced MLC phosphorylation, increased YAP activation, and ameliorated alveolar simplification in vivo. IGF1R controls mechanosignaling in myofibroblasts required for lung alveologenesis.

Phosphorylated‑myosin light chain mediates thedestruction of small intestinal epithelial tight junctions in mice with acute liver failure.

Tight junction dysregulation and epithelial damage contribute to intestinal barrier loss in patients with acute liver failure (ALF); however, the regulatory mechanisms of these processes remain poorly understood. The aim of the present study was to investigate the changes of intestinal tight junction and intestinal mucosa in mice with ALF and their mechanisms. In the present study, ALF was induced in mice through an intraperitoneal injection of D-galactosamine and lipopolysaccharide (D-GalN/LPS), and the morphological changes of the liver or small intestine were analyzed using hematoxylin and eosin staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The intestinal tissues and isolated serum were analyzed using western blotting, immunofluorescence staining and ELISA. D-GalN/LPS-induced mice exhibited signs of hepatocyte necrosis, alongside inflammatory cell infiltration into the liver tissue and partial microvilli detachment in the small intestinal mucosa. TEM demonstrated that the intestinal epithelial tight junctions were impaired, whereas SEM micrographs revealed the presence of abnormal microvilli in D-GalN/LPS-induced mice. In addition, the expression levels of phosphorylated (p)-myosin light chain (MLC), MLC kinase (MLCK) and Rho-associated kinase (ROCK) were significantly increased in the D-GalN/LPS-induced mice compared with those in the control mice, whereas the subsequent inhibition of MLCK or ROCK significantly reduced p-MLC expression levels. Conversely, the expression levels of occludin and zonula occludens-1 (ZO-1) were significantly decreased in the D-GalN/LPS-induced mice, and the inhibition of MLCK or ROCK significantly increased occludin and ZO-1 protein expression levels compared with those in the control group. Changes in the serum levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were similar to the trend observed in p-MLC expression levels. In conclusion, the findings of the present study suggested that in a D-GalN/LPS-induced ALF model, TNF-α and IL-6 signaling may increase MLCK and ROCK expression levels, further mediate phosphorylation of MLC, which may result in tight junction dysregulation and intestinal barrier dysfunction.

Therapeutic Hypothermia Mitigates the Sepsis-Increased Permeability in EA. hy926 Cells by Preserving Rap1 Expression.

To determine the effect and potential mechanisms of therapeutic hypothermia (TH) on the permeability of septic cells. Human EA. hy926 cells were transfected with, or without, control or ras-proximate-1 (Rap1)-specific siRNA and treated with 2 μg/mL of lipopolysaccharide (LPS). The cells were cultured in normal temperature (NT) or a temporary TH for 10 hours. The cellular permeability of each group of cells was determined by transwell permeability assay. The relative levels of Rap1, RhoA (a small GTP enzyme of the Rho family), VE-cadherin expression, and myosin light chain (MLC) phosphorylation were quantified by Western blot and immunofluorescent assays. Compared with the control group, LPS stimulation increased cellular permeability in EA. hy926 cells under an NT condition, but significantly mitigated by TH. The effect of TH decreased after Rap1 silencing. Furthermore, LPS upregulated RhoA expression and MLC phosphorylation, but reduced Rap1 and VE-cadherin expression, which were also enhanced by Rap1 silencing, but significantly mitigated by TH. Immunofluorescent analyses indicated that LPS significantly increased phosphorylated MLC, but decreased VE-cadherin expression, which were further deteriorated by Rap1 silencing, but significantly mitigated by TH in EA. hy926 cells. TH significantly mitigated the sepsis-increased permeability of EA. hy926 cells by enhancing the Rap1 expression to attenuate the RhoA/MLC signaling.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.