Myosin Light Chain Phosphorylation Research Articles

Caldesmon enhances motility inhibition by myosin dephosphorylation.

Some studies have suggested that smooth muscle caldesmon (hCaD) participates in force maintenance at low myosin light chain (LC20) phosphorylation. To elucidate the effects of hCaD on the molecular mechanics of dephosphorylating smooth muscle myosin, we injected myosin light chain phosphatase (MLCP) during in vitro motility measurements performed in the presence of hCaD (400 nM) or its absence. Because hCaD increases the binding force of unphosphorylated smooth muscle myosin to actin, we hypothesized that, upon LC20 dephosphorylation, the fraction (fmot) and the average velocity (vavg) of moving actin filaments would decrease at a greater rate in the presence of hCaD. We fitted sigmoidal curves to fmot and vavg and estimated the rate constant (k) and the midpoint (t0) of the decrease. k was larger in the presence of hCaD (3.6×10−1±6.4×10−2 s−1) than without (1.8×10−1±1.4×10−2 s−1), p = 0.039, n = 5. Additionally, t0 was smaller in the presence of hCaD (32±3 s) than without (44±2 s), p = 0.008, n = 5. These results can be explained either by an increased dephosphorylation rate in the presence of hCaD or by an increase of the reattachment of dephosphorylated myosin to actin. In line with previous experiments that demonstrated that hCaD inhibits the actomyosin ATPase, our sigmoidal fits also show a lower vavg in the presence of hCaD (0.45±0.04 µm/s) than without (0.63±0.06 µm/s), p=0.04, before the addition of MLCP. However, hCaD had no effect on k or t0 of vavg during MLCP injection. The lack of concordance between the fmot and vavg data may be due to the fact that the vavg data are noisier after dephosphorylation. Our results agree with previous data obtained at the whole muscle level that showed that hCaD increases the rate of relaxation of arterial smooth muscle and accelerates the decay in tension of skinned smooth muscle strips.

Y-27632 Impairs Angiogenesis on Extra-Embryonic Vasculature in Post-Gastrulation Chick Embryos

Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in various embryonic developmental processes, including angiogenesis, by controlling actin cytoskeleton assembly and cell contractility. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos, leading to ventral body wall defects (VBWDs). Impaired angiogenesis has been hypothesized to contribute to VBWDs. ROCK is essential in transmitting signals downstream of vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis induces gene expressions and alterations of the actin cytoskeleton upon binding to VEGF receptors (VEGFRs). The aim of this study was to investigate effects of Y-27632 on angiogenesis in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Y-27632-treated embryos showed reduced extra-embryonic blood vessel formation with impaired circulation of the yolk sac, confirmed by fractal analysis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated myosin light chain. Interestingly, RT-PCR demonstrated increased gene expression of VEGF and VEGFR-2 1 h post-treatment. Protein levels of VEGF were higher in Y-27632-treated embryos at 8 h following treatment, whereas no difference was seen in membranes. We hypothesize that administration of Y-27632 impairs vessel formation during angiogenesis, which may contribute to failure of VWB closure, causing VBWDs.

Myosin light chain phosphorylation exhibits a gradient across the wall of cerebellar arteries under sustained ex vivo vascular tone

Small blood vessel diseases are often associated with impaired regulation of vascular tone. The current understanding of resistance arteries often focuses on how a level of vascular tone is achieved in the acute phase, while less emphasis is placed on mechanisms that maintain vascular tone. In this study, cannulated rat superior cerebellar arteries (SCA) developed spontaneous myogenic tone and showed a marked and sustained constriction in the presence of diluted serum (10%), a stimulus relevant to cerebrovascular disease. Both phosphorylated myosin light chain (MLC-p) and smooth muscle alpha actin (SM-α-actin) aligned with phalloidin-stained actin filaments in the vessel wall, while exhibiting a ‘high to low’ gradient across the layers of vascular smooth muscle cells (VSMC), peaking in the outer layer. The MLC-p distribution profile shifted towards the adventitia in serum treated vessels, while removal of the serum reversed it. Furthermore, a positive correlation between the MLC-p signal and vessel wall tension was also evident. The gradients of phosphorylated MLC and SM-α-actin are consistent with a spatial regulation of the myosin-actin apparatus in the vessel wall during the maintenance of vascular tone. Further, the changing profiles of MLC-p and SM-α-actin are consistent with SCA vasoconstriction being accompanied by VSMC cytoskeletal reorganization.

S-45-3: PARP INHIBITION AMELIORATES VEGF INHIBITOR-ASSOCIATED VASCULAR DYSFUNCTION VIA TRPM2

Hypertension is a common unwanted effect of VEGF inhibitors (VEGFi) commonly used as anti-angiogenic drugs in cancer patients. The combination of VEGFi with olaparib, a PARP inhibitor (PARPi), has been shown to reduce VEGFi-induced hypertension. However underlying molecular mechanisms are unknown. PARP plays major role in the activation of TRPM2, a redox-sensitive calcium channel, which is associated with hypertension-induced vascular dysfunction. Objective: to elucidate whether PARP/TRPM2 axis is involved in the protective mechanisms of the combination VEGFi/PARPi in vascular cells. Design and Methods: Human vascular smooth muscle cells (VSMC), aortic endothelial cells (HAEC), and mice mesenteric arteries were used. Cells/arteries were exposed to axitinib (VEGFi) alone and in combination with olaparib (PARPi). Wire myography assessed vascular function. Reactive oxygen species (ROS) production, Ca2+ influx, protein/gene analysis, PARP activity, and TRPM2 siRNA were assessed in VSMC, and nitric oxide (NO) levels in HAEC. Results: Axitinib increased ROS production in hVSMC (RUL: 0.8 ± 0.2 [Ct] vs. 1.1 ± 0.09 [Axi]), which was followed by an increase in PARP activity (a.u.: 0.09 ± 0.03 [Ct] vs. 0.14 ± 0.004 [Axi]). Axitinib reduced ACh-induced vasodilation (% relaxation: 70.5 [Ct] vs. 34.8 [Axi]), an effect blocked by olaparib. U46619- and ET-1-induced vasoconstriction (% KCl-U4: 101.2 [Ct] vs. 141.4 [Axi]; ET-1: 122.6 [Ct] vs. 152.5 [Axi]) were increased by axitinib, which not observed with the combination axitinib plus olaparib. TRPM2 channel blocker (8-Br-cADPR) attenuated the hypercontractile effects and endothelial dysfunction induced by axitinib in mesenteric arteries. PARP and TRPM2 blockage also overturned the increase in VSMC Ca2+ influx induced by axitinib (AUC: 17541 ± 4708 [Ct] vs. 22249 ± 1438 [Axi]). This was also confirmed by TRPM2 siRNA. Phosphorylation of MLC20 in VSMC (a.u.: 0.028 ± 0.02 [Ct] vs. 0.04 ± 0.01 [Axi]), and eNOS (Thr495) in HAEC (a.u.: 0.99 ± 0.35 [Ct] vs. 1.35 ± 0.10 [Axi]) were enhanced by axitinib and prevented by olaparib and TRPM2 siRNA. HAEC exposed to the combination olaparib and axitinib showed NO levels similar to VEGF-stimulated cells. Pro-inflammatory markers (IL-6, MCP-1 and IL-1B) were also upregulated in axitinib-stimulated VSMC (p < 0.01), which was reduced by ROS and PARP-TRPM2 inhibition. Conclusions: We identify that PARP inhibition, by reducing TRPM2 activation, attenuates the vascular deleterious effects of axitinib. Our study defines novel mechanisms whereby the combination VEGFi/PARPi may reduce vascular dysfunction in VEGFi-treated cancer patients.

The lysophosphatidic acid-regulated signal transduction network in ovarian cancer cells and its role in actomyosin dynamics, cell migration and entosis.

Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca2+ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference. Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis. Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression.

S-12-2: ENHANCING MAS1:ETBR INTERACTION AS A POTENTIAL VASOPROTECTIVE STRATEGY IN STROKE-PRONE SHR

We previously demonstrated that Ang-(1–7) has anti-inflammatory effects in endothelial cells by inducing interaction between Mas1 and ETBR. Using HTS of > 20,000 druggable compounds, we identified several compounds that enhance Mas1:ETBR interaction as a novel strategy to promote vasoprotection. The objective of this study was to evaluate effects of 2 candidate compounds, termed enhancers 3 and 4 (Enh3 and Enh4), on functional responses in vascular smooth muscle cells (VSMCs) and their therapeutic potential in hypertensive (SHRSP) rats. VSMCs from normotensive (WKY) and SHRSP rats were exposed to Enh3 and Enh4(10–5 M; 5–30 mins). Expression of pro-contractile and mitogenic molecules was assessed by immunoblotting. Ca2+ influx was evaluated using fluorescence microscopy. ROCK2 activity was assessed using a colorimetric immunoassay which detected MYPT1 phosphorylation at Thr696. Male SHRSP rats (18–21weeks) were treated subcutaneously with Enh4 (10 mg/kg/day) for 7 days. Changes in blood pressure and vascular function were assessed via radiotelemetry and wire myography respectively. In WKY VSMCs, Enh3 reduced basal ERK1/2 phosphorylation (26.8 ± 5.9% vs veh,), an effect absent in SHRSP VSMCs, while Enh4 had no effect. Only in SHRSP VSMCs, Enh3, but not Enh4, reduced basal AKT phosphorylation (63.5 ± 8.9% vs veh). Enh4, but not Enh3, reduced basal MLC20 phosphorylation (54.9 ± 7.5% vs veh) in WKY but not in SHRSP VSMCs. ET-1-induced Ca2+ influx in WKY and SHRSP VSMCs was unaffected by Enh3 and Enh4. Assessment of Ca2+ -independent contractile mechanisms in VSMCs showed that ROCK2 activity is increased in SHRSP compared to WKY VSMCs (WKY: 0.27 ± 0.05; SHRSP:4.78 ± 0.34) and Enh4, but not Enh3, reduced ROCK2 activity in SHRSP VSMCs (61.5 ± 0.43% vs veh). Enh4 treatment in SHRSP rats reduced daytime systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) (SBP:4.26 ± 1.98; DBP:1.48 ± 0.92; MAP:2.07 ± 0.99 vs veh) compared to baseline. Furthermore, Enh4 treatment reduced U46619 induced contraction in SHRSP rat vessels (Emax(Mn): Veh- 13.16 ± 1.81; Enh4- 9.48 ± 1.31). Taken together, enhancing Mas1:ETBR interaction attenuates mitogenic and contractile signaling pathways in WKY and SHRSP VSMCs, and does not increase Ca2+ signaling, important in VSMC contraction. Enhancing Mas1:ETBR interaction reduces blood pressure and improves vascular function in SHRSP rats. Hence, Mas1:ETBR enhancers may be of potential therapeutic use as a new vasoprotective strategy to improve vascular function and to reduce blood pressure in hypertension.

The First EGF Domain of Coagulation Factor IX Increases PAR1 Distributionin Lipid Rafts and Modulates the Response to Thrombin in Endothelial Cells

The first epidermal growth factor 1 (EGF-1) domain of coagulation factor IX (FIX) contains a CXDXXXXYXCXC consensus amino acid sequence, which reportedly induces endocytosis and clustering of lipid rafts in adhesive cells. Endocytosis and lipid rafts regulate signaling via receptors on the plasma membrane. In this study, weinvestigated the effect of FIX EGF-1 domains on intracellular signaling by thrombin (FIIa) in endothelial cells.Recombinant EGF-1 protein of FIX (EGF-FIX) alone did not induce ROCK-dependent phosphorylation ofmyosin light chain (MLC) in human umbilical vein endothelial cells (HUVECs). On the other hand, a high concentration (100 ng/ml) of FIIa increased phosphorylated MLC, and EGF-FIX enhanced it by 1.3-fold. Interestingly, low concentrations (0.3 ng/ml) of FIIa suppressed the phosphorylation of MLC, and the combination oflow-concentration FIIa and EGF-FIX promoted MLC phosphorylation. Methyl-beta-cyclodextrin (MβCD), whichinhibits the formation of lipid rafts, suppressed the phosphorylation of MLC regardless of the FIIa concentration,whereas EGF-FIX attenuated the effects of MβCD. EGF-FIX increased the distribution of the FIIa receptor(PAR1) in the lipid raft fraction. Active FIX could modulate intracellular signaling by other molecules in endothelial cells via its EGF domain.

Involvement of Aquaporin 1 in the Motility and in the Production of Fibrillin 1 and Type I Collagen of Cultured Human Dermal Fibroblasts

Aminocarbonyl proteins increase with age in the dermal layer. Gene Chip analysis of mRNA expression in human dermal fibroblasts cultured on collagen gels treated with glyceraldehyde as an aminocarbonyl protein and on untreated collagen gels showed a decrease in the amount of aquaporin 1 (AQP1) mRNA. In this study, we clarified the involvement of AQP1 in collagen gel contraction and the production of fibrillin 1 and type I collagen in cultured human dermal fibroblasts. In the experiment, AQP1 siRNA was transfected into cultured human dermal fibroblasts to deplete AQP1, and the cell motility and contractile activity of the collagen gel were assessed. The production of fibrillin 1 and type I collagen was also examined by RT–qPCR and antibody staining. AQP1 depletion decreased the collagen gel contractile activity, and both the amount of mRNA and the antibody staining of fibrillin 1 and type I collagen decreased. Furthermore, the depletion of AQP1 reduced the levels of F-actin and phosphorylated myosin light chain 2, suggesting their involvement in reductions of the motility and collagen gel contractile activity of fibroblasts. These findings suggest that AQP1 is an important biomolecule for cell motility in human dermal fibroblasts and that decreased motility results in decreased expression of extracellular matrix proteins such as fibrillin 1 and type I collagen.

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.

Abstract 13258: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Introduction: 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. PPP1R12CPP1c 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 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.

Morphological and Functional Colonic Defects Caused by a Mutated Thyroid Hormone Receptor α.

Background: Mutations of thyroid hormone receptor α (TRα1) result in resistance to thyroid hormone (RTHα), exhibiting symptoms of retarded growth, delayed bone maturation, anemia, and severe constipation. Using a mouse model of RTHα (Thra1PV/+ mouse), we aimed at understanding the molecular basis underlying the severe constipation observed in patients. Methods: The Thra1PV/+ mouse expresses a strong dominant negative mutant, PV, which has lost T3 binding and transcription activity. Thra1PV/+ mouse faithfully reproduces growth abnormalities and anemia as shown in RTHα patients and therefore is a valid model to examine causes of severe constipation in patients. We used histopathological analysis, confocal fluorescence imaging, transmission electron microscopy (TEM), and gene expression profiles to comprehensively analyze the colonic abnormalities of Thra1PV/+ mouse. Results: We found a significant increase in colonic transit time and decrease stool water content in Thra1PV/+ mouse, mimicking constipation as found in patients. Histopathological analysis showed expanded lamina propria filled with interstitium fluid between crypt columns, enlarged muscularis mucosa, and increased content of collagen in expanded submucosa. The TEM analysis revealed shorter muscle fibers with wider gap junctions between muscle cells, fewer caveolae, and hypoplastic interstitial cells of Cajal (ICC) in the rectal smooth muscles of Thra1PV/+ mice. These abnormal histological manifestations suggested defective intercellular transfer of small molecules, electrolytes, and signals for communication among muscles cells, validated by Lucifer Yellow transferring assays. Expression of key smooth muscle contractility regulators, such as calmodulin, myosin light-chain kinase, and phosphorylated myosin light chain, was markedly lower, and c-KIT signaling in ICC was attenuated, resulting in decreased contractility of the rectal smooth muscles of Thra1PV/+ mice. Collectively, these abnormal histopathological alterations and diminished contractility regulators led to the constipation exhibited in patients. Conclusions: This is the first demonstration that TRα1 mutants could act to cause abnormal rectum smooth muscle organization, defects in intercellular exchange of small molecules, and decreased expression of contractility regulators to weaken the contractility of rectal smooth muscles. These findings provide new insights into the molecular basis underlying constipation found in RTHα patients.

Berberine Depresses Inflammation and Adjusts Smooth Muscle to Ameliorate Ulcerative Colitis of Cats by Regulating Gut Microbiota.

Intestinal microbiota dysbiosis is a well established characteristic of ulcerative colitis (UC). Regulating the gut microbiota is an effective UC treatment strategy. Berberine (BBR), an alkaloid extracted from several Chinese herbs, is a common traditional Chinese medicine. To establish the efficacy and mechanism of action of BBR, we constructed a UC model using healthy adult shorthair cats to conduct a systematic study of colonic tissue pathology, inflammatory factor expression, and gut microbiota structure. We investigated the therapeutic capacity of BBR for regulating the gut microbiota and thus work against UC in cats using 16S rRNA genes amplicon sequencing technology. Our results revealed that dextran sulfate sodium (DSS)-induced cat models of UC showed weight loss, diarrhea accompanied by mucous and blood, histological abnormalities, and shortening of the colon, all of which were significantly alleviated by supplementation with BBR. A 16S rRNA gene-based microbiota analysis demonstrated that BBR could significantly benefit gut microbiota. Western blot, quantitative PCR, and enzyme-linked immunosorbent assays (ELISAs) showed that in DSS-induced cat models, the expression of the inflammatory factors was increased, activating the JAK2/STAT3 signaling pathway, and treatment with BBR reversed this effect. The myosin light chain (MLC) phosphorylation in the smooth muscle of the intestines is associated with motility of inflammation-related diarrhea in cats. This study used gut flora analyses to demonstrate the anti-UC effects of BBR and its potential therapeutic mechanisms and offers novel insights into the prevention of inflammatory diseases using natural products. IMPORTANCE Ulcerative colitis (UC) is common in clinics. Intestinal microbiota disorder is correlated with ulcerative colitis. Although there are many studies on ulcerative colitis in rats, there are few studies on colitis in cats. Therefore, this study explored the possibility of the use of BBR as a safe and efficient treatment for colitis in cats. The results demonstrated the therapeutic effects of BBR on UC based on the state of the intestinal flora. The study found BBR supplementation to be effective against dextran sulfate sodium (DSS)-induced colitis, smooth muscle damage, and gut microbiota dysbiosis.

NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart

To adapt to changing hemodynamic demands, regulatory mechanisms modulate actin-myosin-kinetics by calcium-dependent and -independent mechanisms. We investigate the posttranslational modification of human essential myosin light chain (ELC) and identify NIMA-related kinase 9 (NEK9) to interact with ELC. NEK9 is highly expressed in the heart and the interaction with ELC is calcium-dependent. Silencing of NEK9 results in blunting of calcium-dependent ELC-phosphorylation. CRISPR/Cas9-mediated disruption of NEK9 leads to cardiomyopathy in zebrafish. Binding to ELC is mediated via the protein kinase domain of NEK9. A causal relationship between NEK9 activity and ELC-phosphorylation is demonstrated by genetic sensitizing in-vivo. Finally, we observe significantly upregulated ELC-phosphorylation in dilated cardiomyopathy patients and provide a unique map of human ELC-phosphorylation-sites. In summary, NEK9-mediated ELC-phosphorylation is a calcium-dependent regulatory system mediating cardiac contraction and inotropy.

Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin localization in Ciona.

Ventral tail bending, which is transient but pronounced, is found in many chordate embryos and constitutes an interesting model of how tissue interactions control embryo shape. Here, we identify one key upstream regulator of ventral tail bending in embryos of the ascidian Ciona. We show that during the early tailbud stages, ventral epidermal cells exhibit a boat-shaped morphology (boat cell) with a narrow apical surface where phosphorylated myosin light chain (pMLC) accumulates. We further show that interfering with the function of the BMP ligand Admp led to pMLC localizing to the basal instead of the apical side of ventral epidermal cells and a reduced number of boat cells. Finally, we show that cutting ventral epidermal midline cells at their apex using an ultraviolet laser relaxed ventral tail bending. Based on these results, we propose a previously unreported function for Admp in localizing pMLC to the apical side of ventral epidermal cells, which causes the tail to bend ventrally by resisting antero-posterior notochord extension at the ventral side of the tail.

CapG promoted nasopharyngeal carcinoma cell motility involving Rho motility pathway independent of ROCK

BackgroundGelsolin-like capping actin protein (CapG) modulates actin dynamics and actin-based motility with a debatable role in tumorigenic progression. The motility-associated functions and potential molecular mechanisms of CapG in nasopharyngeal carcinoma (NPC) remain unclear.MethodsCapG expression was detected by immunohistochemistry in a cohort of NPC tissue specimens and by Western blotting assay in a variety of NPC cell lines. Loss of function and gain of function of CapG in scratch wound-healing and transwell assays were performed. Inactivation of Rac1 and ROCK with the specific small molecular inhibitors was applied to evaluate CapG’s role in NPC cell motility. GTP-bound Rac1 and phosphorylated-myosin light chain 2 (p-MLC2) were measured in the ectopic CapG overexpressing cells. Finally, CapG-related gene set enrichment analysis was conducted to figure out the significant CapG-associated pathways in NPC.ResultsCapG disclosed increased level in the poorly differentiated NPC tissues and highly metastatic cells. Knockdown of CapG reduced NPC cell migration and invasion in vitro, while ectopic CapG overexpression showed the opposite effect. Ectopic overexpression of CapG compensated for the cell motility loss caused by simultaneous inactivation of ROCK and Rac1 or inactivation of ROCK alone. GTP-bound Rac1 weakened, and p-MLC2 increased in the CapG overexpressing cells. Bioinformatics analysis validated a positive correlation of CapG with Rho motility signaling, while Rac1 motility pathway showed no significant relationship.ConclusionsThe present findings highlight the contribution of CapG to NPC cell motility independent of ROCK and Rac1. CapG promotes NPC cell motility at least partly through MLC2 phosphorylation and contradicts with Rac1 activation.

Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells.

Zearalenone (ZEN) is a widespread contaminant of cereals and agricultural products which causes food safety issues. Ingesting food or feed contaminated with ZEN can disrupt the intestinal epithelial barrier function. The RhoA/ROCK signaling pathway plays a key role in regulating the epithelial barrier function, but studies on such roles have rarely focused on the intestine. The aim of this experiment was to investigate the exact mechanism of ZEN-induced intestinal barrier damage and whether the RhoA/ROCK signaling pathway is involved. The results showed that ZEN significantly induced alkaline phosphatase (AP) activity and FITC–dextran (4 kDa) passage across the epithelial barrier, which significantly reduced the transepithelial resistance (TEER). Meanwhile, ZEN could induce the significantly down-regulated mRNA expression of tight junction proteins (occludin, claudin-1, ZO-1, and claudin-3) and redistribution of ZO-1 immunofluorescence. Further studies demonstrated that ZEN exposure activated the RhoA/ROCK signaling pathway, significantly up-regulated the mRNA expression of ROCK1, the main effector of the signaling pathway, the protein expression of phosphorylated myosin light chain (MLC) and myosin light chain kinase (MLCK), and relatively increased the activity of ATP in cells, simultaneously remodeling the cytoskeleton (F-actin). Overall, our study indicated that ZEN induced intestinal barrier dysfunction by activating the RhoA/ROCK signaling pathway.

Starving a Cell Promotes Airway Smooth Muscle Relaxation: Inhibition of Glycolysis Attenuates Excitation-Contraction Coupling.

Bronchomotor tone modulated by airway smooth muscle shortening represents a key mechanism that increases airway resistance in asthma. Altered glucose metabolism in inflammatory and airway structural cells is associated with asthma. Although these observations suggest a causal link between glucose metabolism and airway hyperresponsiveness, the mechanisms are unclear. We hypothesized that glycolysis modulates excitation-contraction coupling in human airway smooth muscle (HASM) cells. Cultured HASM cells from human lung donors were subject to metabolic screenings using Seahorse XF cell assay. HASM cell monolayers were treated with vehicle or PFK15 (1-(Pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one), an inhibitor of PFKFB3 (PFK-1,6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3) that generates an allosteric activator for glycolysis rate-limiting enzyme PFK1 (phosphofructokinase 1), for 5-240 minutes, and baseline and agonist-induced phosphorylation of MLC (myosin light chain), MYPT1 (myosin phosphatase regulatory subunit 1), Akt, RhoA, and cytosolic Ca2+ were determined. PFK15 effects on metabolic activity and contractile agonist-induced bronchoconstriction were determined in human precision-cut lung slices. Inhibition of glycolysis attenuated carbachol-induced excitation-contraction coupling in HASM cells. ATP production and bronchodilator-induced cAMP concentrations were also attenuated by glycolysis inhibition in HASM cells. In human small airways, glycolysis inhibition decreased mitochondrial respiration and ATP production and attenuated carbachol-induced bronchoconstriction. The findings suggest that energy depletion resulting from glycolysis inhibition is a novel strategy for ameliorating HASM cell shortening and bronchoprotection of human small airways.

Oculocerebrorenal syndrome of Lowe protein controls cytoskeletal reorganisation during human platelet spreading.

Lowe syndrome (LS) is a rare, X-linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5-phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-4-monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL-inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co-localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL-inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS.

The Effects of Cannabidiol on Aqueous Humor Outflow and Trabecular Meshwork Cell Signaling.

Intraocular pressure (IOP) is regulated primarily through aqueous humor production by ciliary body and drainage through uveoscleral and trabecular meshwork (TM) tissues. The goal of this study was to measure the effect of non-psychotropic cannabidiol (CBD) on aqueous humor outflow through TM and assess the effect of CBD on the TM cell signaling pathways that are important for regulating outflow. Perfused porcine eye anterior segment explants were used to investigate the effects of CBD on aqueous humor outflow. Cultured porcine TM cells were used to study the effects of CBD on TM cell contractility, myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation, and RhoA activation. In the anterior segment perfusion experiments, aqueous humor outflow was increased significantly within 1 h after adding 1 µM CBD and the effect was sustained over the 5 h of measurement. Treatment of TM cells with 1 µM CBD significantly decreased TM cell-mediated collagen contraction, inhibited phosphorylation of MLC and MYPT1, and reduced RhoA activation. Our data demonstrate, for the first time, that as a potential therapeutic agent for lowering intraocular pressure, CBD can enhance aqueous humor outflow and modify TM cell signaling.

TLR4 activation induces inflammatory vascular permeability via Dock1 targeting and NOX4 upregulation

The loss of vascular integrity is a cardinal feature of acute inflammatory responses evoked by activation of the TLR4 inflammatory cascade. Utilizing in vitro and in vivo models of inflammatory lung injury, we explored TLR4-mediated dysregulated signaling that results in the loss of endothelial cell (EC) barrier integrity and vascular permeability, focusing on Dock1 and Elmo1 complexes that are intimately involved in regulation of Rac1 GTPase activity, a well recognized modulator of vascular integrity. Marked reductions in Dock1 and Elmo1 expression was observed in lung tissues (porcine, rat, mouse) exposed to TLR4 ligand-mediated acute inflammatory lung injury (LPS, eNAMPT) in combination with injurious mechanical ventilation. Lung tissue levels of Dock1 and Elmo1 were preserved in animals receiving an eNAMPT-neutralizing mAb in conjunction with highly significant decreases in alveolar edema and lung injury severity, consistent with Dock1/Elmo1 as pathologic TLR4 targets directly involved in inflammation-mediated loss of vascular barrier integrity. In vitro studies determined that pharmacologic inhibition of Dock1-mediated activation of Rac1 (TBOPP) significantly exacerbated TLR4 agonist-induced EC barrier dysfunction (LPS, eNAMPT) and attenuated increases in EC barrier integrity elicited by barrier-enhancing ligands of the S1P1 receptor (sphingosine-1-phosphate, Tysiponate). The EC barrier-disrupting influence of Dock1 inhibition on S1PR1 barrier regulation occurred in concert with: 1) suppressed formation of EC barrier-enhancing lamellipodia, 2) altered nmMLCK-mediated MLC2 phosphorylation, and 3) upregulation of NOX4 expression and increased ROS. These studies indicate that Dock1 is essential for maintaining EC junctional integrity and is a critical target in TLR4-mediated inflammatory lung injury.