Inhibition Of Myosin Light Chain Phosphorylation Research Articles

NLRX1 Mediates the Disruption of Intestinal Mucosal Function Caused by Porcine Astrovirus Infection via the Extracellular Regulated Protein Kinases/Myosin Light-Chain Kinase (ERK/MLCK) Pathway.

Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill-defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD-like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV-4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV-4 infection significantly up-regulated NLRX1 and mitophagy in Caco-2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3-MA inhibited PAstV-4 replication. Additionally, PAstV-4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight-junction proteins (occludin and ZO-1) as well as MUC-2 expression. The silencing of NLRX1 or the treatment of 3-MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO-1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV-4 infection. Yet, adding PD98059 or ML-7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV-4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy.

Esrp1-Regulated Splicing of Arhgef11 Isoforms Is Required for Epithelial Tight Junction Integrity

SUMMARYThe epithelial-specific splicing regulators Esrp1 and Esrp2 are required for mammalian development, including establishment of epidermal barrier functions. However, the mechanisms by which Esrp ablation causes defects in epithelial barriers remain undefined. We determined that the ablation of Esrp1 and Esrp2 impairs epithelial tight junction (TJ) integrity through loss of the epithelial isoform of Rho GTP exchange factor Arhgef11. Arhgef11 is required for the maintenance of TJs via RhoA activation and myosin light chain (MLC) phosphorylation. Ablation or depletion of Esrp1/2 or Arhgef11 inhibits MLC phosphorylation and only the epithelial Arhgef11 isoform rescues MLC phosphorylation in Arhgef11 KO epithelial cells. Mesenchymal Arhgef11 transcripts contain a C-terminal exon that binds to PAK4 and inhibits RhoA activation byArhgef11. Deletion of the mesenchymal-specific Arhgef11 exon in Esrp1/2 KO epithelial cells using CRISPR/Cas9 restored TJ function, illustrating how splicing alterations can be mechanistically linked to disease phenotypes that result from impaired functions of splicing regulators.

Alpha-Isocubebene modulates vascular tone by inhibiting myosin light chain phosphorylation in murine thoracic aorta

Alpha-Isocubebene modulates vascular tone by inhibiting myosin light chain phosphorylation in murine thoracic aorta

Adipose-Derived Stem Cells of Blind Mole Rat Spalax Exhibit Reduced Homing Ability: Molecular Mechanisms and Potential Role in Cancer Suppression.

Adipose-derived stem cells (ADSCs) are recruited by cancer cells from the adjacent tissue, and they become an integral part of the tumor microenvironment. Here, we report that ADSCs from the long-living, tumor-resistant blind mole rat, Spalax, have a low ability to migrate toward cancer cells compared with cells from its Rattus counterpart. Tracking 5-ethynyl-2'-deoxyuridine (EdU)-labeled ADSCs, introduced to tumor-bearing nude mice, toward the xenografts, we found that rat ADSCs intensively migrated and penetrated the tumors, whereas only a few Spalax ADSCs reached the tumors. Moreover, rat ADSCs, but not Spalax ADSCs, acquired endothelial-like phenotype and incorporated in the intratumoral reticular structure resembling a vasculature. Likewise, endothelial-like cells differentiated from Spalax and rat ADSCs could form capillary-like structures; however, the tube densities were higher in rat-derived cells. Using time-lapse microscopy, in vitro wound-healing, and transwell migration assays, we demonstrated the impaired motility and low polarization ability of Spalax ADSCs. To assess whether the phosphorylated status of myosin light chain (MLC) is involved in the decreased motility of Spalax ADSCs, we inhibited MLC phosphorylation by blocking of Rho-kinase (ROCK). Inhibition of ROCK resulted in the suppression of MLC phosphorylation, acquisition of actin polarization, and activation of motility and migration of Spalax ADSCs. We propose that reduced ADSCs migration to cancer and poor intratumoral angiogenesis play a role in Spalax's cancer resistance. Learning more about the molecular strategy of noncancerous cells in Spalax to resist oncogenic stimuli and maintain a nonpermissive tumor milieu may lead us to developing new cancer-preventive strategy in humans. Stem Cells 2018;36:1630-1642.

α-Isocubebene modulates vascular tone by inhibiting myosin light chain phosphorylation in murine thoracic aorta.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms, but the mechanism responsible for this activity has not been determined. To determine the role played by ICB on the regulation of vascular tone, we investigated the inhibitory effects of ICB on vascular contractile responses by adrenergic α-receptor agonists. In addition, we investigated the role on myosin light chain (MLC) phosphorylation and cytosolic calcium concentration in vascular smooth muscle cells (VSMC). In aortic rings isolated from C57BL/6J mice, ICB significantly attenuated the contraction induced by phenylephrine (PE) and norepinephrine (NE), whereas ICB had no effects on KCl (60 mM)-induced contraction. In vasculatures precontracted with PE, ICB caused marked relaxation of aortic rings with or without endothelium, suggesting a direct effect on VSMC. In cultured rat VSMC, PE or NE increased MLC phosphorylation and increased cytosolic calcium levels. Both of these effects were significantly suppressed by ICB. In conclusion, our results showed that ICB regulated vascular tone by inhibiting MLC phosphorylation and calcium flux into VSMC, and suggest that ICB has anti-hypertensive properties and therapeutic potential for cardiovascular disorders related to vascular hypertension.

Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction

Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction.

Abstract 5904: Stiffness of extracellular matrix regulates breast cancer progression by stimulating mesenchymal stem cells

Abstract The tumor microenvironment contains cancer cells, non-cancerous cells, and extracellular components such as extracellular matrix (ECM). Previous studies have demonstrated that interactions between the cells and the microenvironment contribute to cancer progression via chemical stimuli, such as growth factors, and mechanical stimuli, such as stiffness of the ECM. Recently, it has been reported that mesenchymal stem cells (MSCs) differentiate into cancer associated fibroblasts (CAFs) in response to chemical stimuli from cancer cells and thereby promote cancer progression. However, the contribution of mechanical stimuli to MSCs in cancer is poorly understood. In this study, we revealed that MSCs showed CAF phenotypes and promote mammary cancer progression in response to mechanical stimuli. On a stiff substrate, MSCs treated with conditioned media from cancer cell culture expressed increased levels of alpha smooth muscle actin (alpha-SMA), a marker of CAFs, compared to the MSCs cultured on a soft substrate. MSCs grown on a stiff substrate displayed higher expression and activity of YAP and increased phosphorylation of myosin light chain (MLC) compared to MSCs grown on a soft substrate. In addition, knockdown of YAP by shRNA decreased the expression of alpha-SMA and phosphorylation of MLC in MSCs on a stiff substrate. Pharmacological inhibition of MLC phosphorylation by H1152 treatment also reduced expression of alpha-SMA and activity of YAP in MSCs. Cell-cell communication between MSCs and carcinoma cells was bi-directional, as conditioned medium from MSCs cultured on a stiff substrate, but not a soft substrate, increased growth of mammary carcinoma cells. The soluble factor prosaposin was highly secreted by the MSCs on a stiff substrate, and the addition of recombinant prosaposin increased proliferation and survival of mammary carcinoma cells. Furthermore, secretion of prosaposin was promoted in YAP-overexpressed MSCs on a soft substrate. Mammary carcinoma cells treated with prosaposin showed increased level of phosphorylation in Akt at T308. In addition, inhibition of phosphorylation of Akt at T308 prevented proliferation and survival in mammary carcinoma cells. These results suggest that increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSCs to CAFs via YAP and actomyosin contractility, secretion of prosaposion from MSCs, and as a result, trigger progression of mammary cancer via phosphorylation of Akt at T308. Citation Format: Seiichiro Ishihara, David R. Inman, Wan-Ju Li, Suzanne M. Ponik, Patricia J. Keely. Stiffness of extracellular matrix regulates breast cancer progression by stimulating mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5904. doi:10.1158/1538-7445.AM2017-5904

PP.LB02.02

Objective: Alpha-isocubebene (α-ICB) is a compound isolated from Schisandra chinensis fruit which is a well-known as a useful material in the preventive or treatment of cardiovascular symptoms in Korean. Design and method: In the present study, the vasoactivities of α-ICB were evaluated in the isolated thoracic aorta of mice (8-week-old male C57/BL6). Results: In isolated rings of aorta (about 2 mm in width), phenylephrine (0.1–3.0 μM), adrenergic α-receptor agonist, or norepinephrine (10–300 nM) induced vasocontraction in dose-dependent manners, which were markedly attenuated by α-ICB (1–10 μg/ml) in a concentration-dependent manner. However, α-ICB had no effects on vasodilation induced by acetylcholine (10–300 nM). These results suggested that α-ICB regulated vasocontraction via regulation of contractile machine including myosin light chain (MLC) on the vasculature. In the immunoblot analysis, α-ICB inhibited phosphorylation of MLC at the sites of Thr18 and Thr18/Ser19. The effects on Thr18 phosphorylation were more prominent than that at The18/Ser19 sites, suggesting a pivotal involvement of MLC phosphatase in the modulation of vasocontraction by α-ICB. Conclusions: Collectively, α-ICB attenuated vasocontraction induced by various agonists through inhibition of MLC phosphorylation via an increased activity of MLC phosphatase.

P21‐activated Kinase, a Novel Drug Target for the Treatment of Postoperative Ileus

Ileus, a common postoperative complication, has a significant impact on patient outcomes; however, there are no safe, effective pharmacological therapies for treating postoperative ileus (POI). We have shown that p21-activated kinase (PAK1) inhibits myosin light chain phosphorylation in human intestinal smooth muscle cells and PAK1 expression is increased in intestinal smooth muscle in an ileus model. The objective of this study was to determine the feasibility of targeting PAK1 for the treatment of POI. POI was induced via gut manipulation in wildtype and PAK1 knockout mice and basal and agonist-induced intestinal contractility and markers of inflammation, including a cytokine panel and edema development in intestinal smooth muscle, were determined. Gut manipulation caused a significant decrease in ileal contraction amplitude in wildtype mice but did not affect contraction amplitude in PAK1 knockouts. Carbachol response was increased and EC50 for carbachol was decreased in PAK1 knockout mice after gut manipulation compared to wildtype mice. Edema development was significantly increased after gut manipulation in both wildtype and PAK1 knockout mice. Inflammatory cytokines were differentially regulated in response to gut manipulation in PAK1 knockout mice compared to wildtype. While the inflammatory response appeared to be partially attenuated in PAK1 knockout mice, several markers of inflammation including monocyte chemotactic protein 1 (MCP-1) and edema were increased in both wildtype and PAK1 knockout mice. Thus, while inflammation still developed in intestinal smooth muscle of PAK1 knockout mice, POI was prevented. We conclude that PAK1 is a feasible target for the treatment of POI.

Disruption of endothelial adherens junctions by high glucose is mediated by protein kinase C-β-dependent vascular endothelial cadherin tyrosine phosphorylation.

BackgroundHyperglycemia has been recognized as a primary factor in endothelial barrier dysfunction and in the development of micro- and macrovascular diseases associated with diabetes, but the underlying biochemical mechanisms remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) leads to the disruption of endothelial adherens junctions and increases the transendothelial migration (TEM) of leukocytes.MethodsVE-cad tyrosine phosphorylation, adherens junction integrity and TEM of monocytes in human umbilical vein endothelial cells (HUVECs) treated with high-concentration glucose were evaluated. The role of protein kinase C (PKC) in induction of endothelial cells adherence junction disruption by exposure of HUVECs to high concentration of glucose was explored.ResultsThe treatment of HUVEC with high-concentration glucose increased VE-cad tyrosine phosphorylation, whereas mannitol or 3-O-methyl-D-glucose had no effect. In addition, high-concentration glucose increased the dissociation of the VE-cad–β-catenin complex, activation of the Wnt/β-catenin pathway, and the TEM of monocytes. These alterations were accompanied by the activation of endothelial PKC and increased phosphorylation of ERK and myosin light chain (MLC). High-concentration glucose-induced tyrosine phosphorylation of VE-cad was attenuated by: 1- the inhibition of PKC-β by overexpression of dominant-negative PKC-β 2- inhibition of MLC phosphorylation by overexpression of a nonphosphorylatable dominant-negative form of MLC, 3- the inhibition of actin polymerization by cytochalasin D and 4- the treatment of HUVECs with forskolin (an activator of adenylate cyclase).ConclusionsOur findings show that the high-concentration glucose-induced disruption of endothelial adherens junctions is mediated by tyrosine phosphorylation of VE-cad through PKC-β and MLC phosphorylation.

Schisandrin B suppresses TGFβ1-induced stress fiber formation by inhibiting myosin light chain phosphorylation

Ethnopharmacological relevanceSchisandra chinensis fruit extract (SCE) has been used as a traditional oriental medicine for treating vascular diseases. However, the pharmacologic effects and mechanisms of SCE on vascular fibrosis are still largely unknown. Transforming growth factor β1 (TGFβ1)-mediated cellular changes are closely associated with the pathogenesis of vascular fibrotic diseases. Particularly, TGFβ1 induces actin stress fiber formation that is a crucial mechanism underlying vascular smooth muscle cell (VSMC) migration in response to vascular injury. In this study, we investigated the effect of SCE and its active ingredients on TGFβ1-induced stress fiber assembly in A7r5 VSMCs. Materials and methodsTo investigate pharmacological actions of SCE and its ingredients on TGFβ1-treated VSMCs, we have employed molecular and cell biological technologies, such as confocal microscopy, fluorescence resonance energy transfer, western blotting, and radiometric enzyme analyses. ResultsWe found that SCE inhibited TGFβ1-induced stress fiber formation and cell migration. Schisandrin B (SchB) showed the most prominent effect among the active ingredients of SCE tested. SchB reduced TGFβ1-mediated phosphorylation of myosin light chain, and this effect was independent of RhoA/Rho-associated kinase pathway. Fluorescence resonance energy transfer and radiometric enzyme assays confirmed that SchB inhibited myosin light chain kinase activity. We also showed that SchB decreased TGFβ1-mediated induction of α-smooth muscle actin by inhibiting Smad signaling. ConclusionsThe present study demonstrates that SCE and its active ingredient SchB suppressed TGFβ1-induced stress fiber formation at the molecular level. Therefore, our findings may help future investigations to develop multi-targeted therapeutic strategies that attenuate VSMC migration and vascular fibrosis.

Disruption of endothelial adherens junctions by high glucose is mediated by protein kinase C-β–dependent vascular endothelial cadherin tyrosine phosphorylation

BackgroundHyperglycemia has been recognized as a primary factor in endothelial barrier dysfunction and in the development of micro- and macrovascular diseases associated with diabetes, but the underlying biochemical mechanisms remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) leads to the disruption of endothelial adherens junctions and increases the transendothelial migration (TEM) of leukocytes.MethodsVE-cad tyrosine phosphorylation, adherens junction integrity and TEM of monocytes in human umbilical vein endothelial cells (HUVECs) treated with high-concentration glucose were evaluated. The role of protein kinase C (PKC) in induction of endothelial cells adherence junction disruption by exposure of HUVECs to high concentration of glucose was explored.ResultsThe treatment of HUVEC with high-concentration glucose increased VE-cad tyrosine phosphorylation, whereas mannitol or 3-O-methyl-D-glucose had no effect. In addition, high-concentration glucose increased the dissociation of the VE-cad–β-catenin complex, activation of the Wnt/β-catenin pathway, and the TEM of monocytes. These alterations were accompanied by the activation of endothelial PKC and increased phosphorylation of ERK and myosin light chain (MLC). High-concentration glucose-induced tyrosine phosphorylation of VE-cad was attenuated by: 1- the inhibition of PKC-β by overexpression of dominant-negative PKC-β 2- inhibition of MLC phosphorylation by overexpression of a nonphosphorylatable dominant-negative form of MLC, 3- the inhibition of actin polymerization by cytochalasin D and 4- the treatment of HUVECs with forskolin (an activator of adenylate cyclase).ConclusionsOur findings show that the high-concentration glucose-induced disruption of endothelial adherens junctions is mediated by tyrosine phosphorylation of VE-cad through PKC-β and MLC phosphorylation.

P21‐activated kinase inhibits vascular reactivity via inhibition of MLCK

Changes in vascular reactivity contribute to the pathology of a number of different diseases including heart failure, hypertension, severe trauma, and sepsis. P21‐activated kinase (PAK) can alter smooth muscle contractile activity and has been implicated in altering vascular reactivity. The objective of this study was to determine the mechanism by which PAK regulates MLC phosphorylation in vascular smooth muscle to alter vascular reactivity. The effects of PAK activation and inhibition on basal and agonist‐induced aortic contraction in aortic rings and on myosin light chain (MLC) and myosin targeting subunit of MLC phosphatase (MYPT1) phosphorylation and MLC kinase (MLCK) activity in primary aortic smooth muscle cells (ASMC) were determined. Finally, the effects of increased mechanical stimulation on PAK activation were determined in ASMC. PAK activation inhibited both basal tone and agonist induced contraction of rat aortic rings. Conversely, PAK inhibition increased agonist induced contraction and basal tone. In ASMC, PAK activation inhibited MLC phosphorylation and PAK inhibition increased MLC phosphorylation. However, PAK activation increased MYPT1 phosphorylation in ASMC. Increased mechanical stimulation of ASMC increased PAK activation and decreased MLCK activation. These data suggest that PAK inhibits vascular reactivity via decreased inhibition of MLCK.

The MLK-related Kinase (MRK) Is a Novel RhoC Effector That Mediates Lysophosphatidic Acid (LPA)-stimulated Tumor Cell Invasion*

The small GTPase RhoC is overexpressed in many invasive tumors and is essential for metastasis. Despite its high structural homology to RhoA, RhoC appears to perform functions that are different from those controlled by RhoA. The identity of the signaling components that are differentially regulated by these two GTPases is only beginning to emerge. Here, we show that the MAP3K protein MRK directly binds to the GTP-bound forms of both RhoA and RhoC in vitro. However, siRNA-mediated depletion of MRK in cells phenocopies depletion of RhoC, rather than that of RhoA. MRK depletion, like that of RhoC, inhibits LPA-stimulated cell invasion, while depletion of RhoA increases invasion. We also show that active MRK enhances LPA-stimulated invasion, further supporting a role for MRK in the regulation of invasion. Depletion of either RhoC or MRK causes sustained myosin light chain phosphorylation after LPA stimulation. In addition, activation of MRK causes a reduction in myosin light chain phosphorylation. In contrast, as expected, depletion of RhoA inhibits myosin light chain phosphorylation. We also present evidence that both RhoC and MRK are required for LPA-induced stimulation of the p38 and ERK MAP kinases. In conclusion, we have identified MRK as a novel RhoC effector that controls LPA-stimulated cell invasion at least in part by regulating myosin dynamics, ERK and p38.

Loss of CaMKKβ attenuates endotoxin induced hypotension

Septic shock is a syndrome defined by persistently reduced blood pressure despite fluid resuscitation leading to multiple organ failure resulting from overwhelming infection. It is a major cause of intensive care admission and is associated with a high mortality rate. The phosphorylation state of myosin light chain (MLC) in smooth muscle cells directly controls the contractility of blood vessels and the maintenance of blood pressure. Activation of AMP-activated protein kinase (AMPK) by calcium/calmodulin dependent protein kinase kinase beta (CaMKKβ) has previously been reported to inhibit MLC phosphorylation, and induce vasodilation in mice.We investigated the involvement of CaMKKβ in blood pressure control using a model of endotoxin induced septic shock. Using implantable telemetry probes, we recorded aortic blood pressure in age-matched male wild-type (WT) and CaMKKβ knockout (CaMKK KO) mice under both basal conditions and during endotoxin induced sepsis. We showed that in mice with global deletion of CaMKKβ, basal blood pressure is increased compared with their wild-type littermates. In addition, when treated with intraperitoneally injected bacterial endotoxin, the lack of CaMKKβ appeared to confer protection against hypotension, with significantly higher blood pressure seen in CaMKK KO mice, as well as improved mobility and general physiological state, compared with WT mice. We found no indication of altered inflammatory response between CaMKK KO and WT mice after intraperitoneal endotoxin injection. We are addressing the role of CaMKKβ in vascular smooth muscle MLC phosphorylation and the potential for AMPK involvement in the signalling pathways.Our findings suggest that loss of CaMKKβ in mice protects against septic shock through the maintenance of blood pressure. Inhibition of CaMKKβ could be an alternative strategy to assist with the maintenance of blood pressure during sepsis. FundingUK Medical Research Council, Imperial College London, and National Institute for Health Research Biomedical Research Centre at Imperial College NHS Trust.

Biphasic Regulation of Myosin Light Chain Phosphorylation by p21-activated Kinase Modulates Intestinal Smooth Muscle Contractility

Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

Inhibition of MLC Phosphorylation Restricts Replication of Influenza Virus—A Mechanism of Action for Anti-Influenza Agents

Influenza A viruses are a severe threat worldwide, causing large epidemics that kill thousands every year. Prevention of influenza infection is complicated by continuous viral antigenic changes. Newer anti-influenza agents include MEK/ERK and protein kinase C inhibitors; however, the downstream effectors of these pathways have not been determined. In this study, we identified a common mechanism for the inhibitory effects of a significant group of anti-influenza agents. Our studies showed that influenza infection activates a series of signaling pathways that converge to induce myosin light chain (MLC) phosphorylation and remodeling of the actin cytoskeleton. Inhibiting MLC phosphorylation by blocking RhoA/Rho kinase, phospholipase C/protein kinase C, and HRas/Raf/MEK/ERK pathways with the use of genetic or chemical manipulation leads to the inhibition of influenza proliferation. In contrast, the induction of MLC phosphorylation enhances influenza proliferation, as does activation of the HRas/Raf/MEK/ERK signaling pathway. This effect is attenuated by inhibiting MLC phosphorylation. Additionally, in intracellular trafficking studies, we found that the nuclear export of influenza ribonucleoprotein depends on MLC phosphorylation. Our studies provide evidence that modulation of MLC phosphorylation is an underlying mechanism for the inhibitory effects of many anti-influenza compounds.

Rho Kinase Inhibitors as Potential Therapeutic Agents for Oncogenic KIT, FLT3, and BCR-ABL Induced Leukemogenesis.

Abstract 3909Poster Board III-845Myeloproliferative disorder (MPD) and acute myeloid leukemia (AML) are heterogeneous hematologic malignancies which share the common characteristics of myeloid cell overproduction and aberrant differentiation. With the exception of chronic myelogenous leukemia (CML), therapeutic intervention for these diseases is largely ineffective. There are increasing examples of mutations in tyrosine kinases (TKs) that contribute to MPD as well as AML. Key examples are the well characterized BCR-ABL transgene causing constitutively activated Abl tyrosine kinase function in CML. FLT3 internal tandem duplications (ITDs) induce ligand-independent autophosphorylation in AML, and activating KIT mutations seen in AML and in over 90% cases of systemic mastocytosis (SM). Although hyperactivation of several signaling molecules downstream from these tyrosine kinases have been reported; little is known about the identity of signaling molecules that are shared among these mutations. Rho kinases or Rho-associated coiled coil-containing protein kinases (ROCK1 and ROCK2) are protein serine/threonine kinases that control a wide variety of cellular functions, including cell growth, survival and motility. Rho kinases have been shown to be hyperactive in several cancers. Importantly, in preclinical models, ROCK inhibitors have demonstrated significant efficacy in repressing aspects of tumorigenesis. However, the role of ROCK kinases in MPDs/Leukemogenesis has not been investigated. We show that expression of wild-type (WT) KIT receptor or vector alone in 32D murine myeloid cells or WT FLT3 receptor in BaF3 murine lymphoid cells does not result in ligand independent activation of ROCK kinases as evident by lack of phosphorylation of its substrate MYPT1. In contrast, expression of oncogenic KITD816V, FLT3N51, and BCR-ABL in these cells results in constitutive activation of Rho kinases, which is completely inhibited in the presence of Rho kinase inhibitor dimethyl-fasudil. Importantly, treatment of oncogene bearing cells with dimethy-fasudil did not affect the activation of PKA, PKC, AKT, STAT3 or ERK MAPkinase. Furthermore, treatment of oncogenic KITD816V, FLT3N51 and BCR-ABL bearing cells with additional Rho kinase inhibitors (Y27632 and fasudil) also resulted in a dose dependent repression in ligand independent growth in a thymidine incorporation assay. In contrast, WT KIT, FLT3, or vector alone containing cells demonstrated little to no repression of growth in the presence of cytokines and Rho kinase inhibitors. A human mast cell line (HMC1.2) derived from a patient with mastocytosis bearing the activation loop mutation D816V in the KIT receptor, also showed a dose dependent suppression in ligand independent growth upon treatment with Rho kinase inhibitors. Similar inhibition of growth was observed when primary hematopoietic stem and progenitor cells (HSC/Ps) bearing the oncogenic KITD814V, FLT3N51 and BCR-ABL mutations were treated with Rho kinase inhibitors. In an effort to identify the mechanism behind reduced growth, we examined opoptosis in oncogene bearing cells treated with Rho kinase inhibitors. We found increased apoptosis in these cells compared to WT KIT receptor, FLT3 receptor, or vector alone expressing cells. Apoptosis was observed as early as two hours after Rho kinase inhibitor treatment in oncogene bearing cells. To determine the mechanism of such rapid apoptosis in oncogene bearing cells, we examined the activation of several known downstream substrates of ROCK kinases. We found that cells bearing the oncogenic KITD814V, FLT3N51 and BCR-ABL receptors demonstrated enhanced and constitutive phosphorylation of myosin light chain (MLC). Since inhibition of MLC phosphorylation in breast cancer lines has been associated with apoptosis, we next measured the activation of MLC in the presence of ROCK inhibitors. We found that MLC was dephosphorylated within minutes of treating oncogene bearing cells with Rho kinase inhibitors but not in wildtype receptor bearing cells. Taken together, these results suggest that dephosphorylation of a major downstream Rho kinase substrate (i.e. MLC) plays an essential role in inducing cell death in oncogene bearing cells but not in normal cells. These studies indicate Rho kinases as possible therapeutic targets for treating diseases such as AML and MPD. Disclosures:No relevant conflicts of interest to declare.

Volatile Anesthetics Inhibit Angiotensin II-Induced Vascular Contraction by Modulating Myosin Light Chain Phosphatase Inhibiting Protein, CPI-17 and Regulatory Subunit, MYPT1 Phosphorylation

Vascular contraction is regulated by myosin light chain (MLC) phosphorylation. Inhibition of MLC phosphatase (MLCP) increases MLC phosphorylation for a given Ca(2+) concentration, and results in promoting myofilament Ca(2+) sensitivity. MLCP activity is mainly determined by protein kinase C (PKC) and Rho kinase through the phosphorylation of both PKC-potentiated inhibitory protein (CPI-17) and myosin phosphatase target subunit (MYPT1). We have previously demonstrated that sevoflurane inhibits PKC phosphorylation and membrane translocation of Rho kinase. This study was designed to investigate the effects of sevoflurane and isoflurane on CPI-17, MYPT1, and MLC phosphorylation in response to angiotensin II (Ang II) in rat aortic smooth muscle. The effects of sevoflurane or isoflurane (1-3 minimum alveolar concentration) on the vasoconstriction and phosphorylation of MLC, CPI-17, MYPT1 at Thr853 and MYPT1 at Thr696 in response to Ang II were investigated using isometric force transducer and Western blotting, respectively. Ang II (10(-7) M) elicited a transient contraction of rat aortic smooth muscle that was inhibited by both sevoflurane and isoflurane in a concentration-dependent manner. Ang II also induced an increase in the phosphorylation of MLC, CPI-17, MYPT1/Thr853 and MYPT1/Thr696. Sevoflurane inhibited the phosphorylation of MLC, CPI-17, and MYPT1/Thr853 in response to Ang II in a concentration-dependent manner. Isoflurane also inhibited MLC phosphorylation in response to Ang II, which was associated with decreases in MYPT1/Thr853, but not in CPI-17. Neither sevoflurane nor isoflurane affected the Ang II-induced phosphorylation of MYPT1/Thr696. Although both volatile anesthetics inhibited Ang II-induced vasoconstriction and MLC phosphorylation to similar extent, the mechanisms behind the inhibitory effects of each anesthetic on MLCP activity appear to differ.

Molecular mechanisms affecting LPS‐induced endothelial cells (EC) hyperpermeability

LPS decreases transendothelial electrical resistance (TER), increases endothelium permeability for large molecules, and leads to sizeable gap formation in cells grown on glass coverslips. Forskolin suppresses LPS‐induced drop in TER and gap formation in EC monolayer, concomitant with the decrease in LPS‐induced myosin light chain (MLC) phosphorylation. Both protein kinase A activator 6‐Bnz‐cAMP and Epac activator O‐Me‐cAMP have similar effect on LPS‐induced TER changes and gap formation; however, only 6‐Bnz‐cAMP decreases LPS‐induced MLC phosphorylation and increases phosphorylation of VASP. Inhibition of MLC phosphorylation with Rho kinase inhibitor Y27632 significantly suppresses LPS‐induced gap formation. VASP depletion with siRNA exacerbates LPS‐induced changes in TER. These data suggest that MLC‐dependent actomyosin contraction contributes to LPS‐induced barrier dysfunction, whereas VASP‐dependent organization of actin near cell membrane plays barrier‐protective role. We next studied the role of p38 pathway in LPS‐induced pulmonary edema. p38 inhibitor SB203580 significantly suppresses LPS‐induced gap formation and permeability for large molecules, indicative of barrier‐disruptive role of p38 pathway. HSP27 depletion by siRNA exacerbates LPS‐induced changes in TER and gap formation, suggesting barrier‐protective role of this protein. This work was supported by HL‐067307.