Thrombin-induced Phosphorylation Research Articles

Sesamin suppresses activation of microglia and p44/42 MAPK pathway, which confers neuroprotection in rat intracerebral hemorrhage

Thrombin plays important roles in the pathology of intracerebral hemorrhage (ICH). The recruitment of activated microglia, accompanied by thrombin-induced phosphorylation of the mitogen-activated protein kinase (MAPK) family, contributes to ICH-associated neuron loss. Here we investigated the possibility that sesamin, a lignan of sesame seed oil, is a natural candidate as an inhibitor of microglial activation and MAPK pathways under ICH insults. Sesamin (30–100μM) suppressed thrombin-induced nitric oxide (NO) production by primary-cultured rat microglia via inhibition of inducible NO synthase (iNOS) protein expression, independently of the antioxidative effect. Sesamin selectively inhibited p44/42 MAPK phosphorylation in the MAPK family (p38 and p44/42) involved in iNOS protein expression in primary-cultured rat microglia. An in vivo rat ICH model was prepared by intrastriatal injection of 0.20U collagenase type IV unilaterally. ICH evoked the phosphorylation of p44/42 MAPK, microglial proliferation with morphological change into the activated ameboid form, and neuron loss. The phosphorylation of p44/42 MAPK was inhibited by intracerebroventricular administration of 30-nmol sesamin. Sesamin prevented ICH-induced increase of microglial cells in the perihematomal area. Notably, ramified microglia, the resting morphology, were observed in brain sections of the animals administrated sesamin. Sesamin furthermore achieved neuroprotection in the perihematomal area but not in the hematomal center. These results suggest that sesamin is a promising natural product as a novel therapeutic strategy based on the regulation of microglial activities accompanied by the activated p44/42 MAPK pathway in ICH.

Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression of IL6 mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. The IL6 mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

Nox1 transactivation of epidermal growth factor receptor promotes N-cadherin shedding and smooth muscle cell migration

In atherosclerosis and restenosis, vascular smooth muscle cells (SMCs) migrate into the subendothelial space and proliferate, contributing to neointimal formation. The goal of this study was to define the signalling pathway by which Nox1 NAPDH oxidase mediates SMC migration. SMCs were cultured from thoracic aorta from Nox1(-/y) (Nox1 knockout, KO) and wild-type (WT) mice. In response to thrombin, WT but not Nox1 KO SMCs generated increased levels of reactive oxygen species (ROS). Deficiency of Nox1 prevented thrombin-induced phosphorylation of Src and the subsequent transactivation of the epidermal growth factor receptor (EGFR) at multiple tyrosine residues. Next, activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and matrix metalloproteinase-9 (MMP-9) by thrombin was inhibited by the EGFR inhibitor AG1478 and in Nox1 KO SMCs. Thrombin-induced shedding of N-cadherin from the plasma membrane was dependent on the presence of Nox1 and was blocked by AG1478 and an inhibitor of metalloproteinases. Migration of SMCs to thrombin was impaired in the Nox1 KO SMCs and was restored by expression of Nox1. Finally, treatment of WT SMCs with AG1478 abrogated Nox1-dependent SMC migration. The Nox1 NADPH oxidase signals through EGFR to activate MMP-9 and promote the shedding of N-cadherin, thereby contributing to SMC migration.

An important role for Akt3 in platelet activation and thrombosis

AbstractThe Akt family of serine/threonine kinases includes Akt1, Akt2, and Akt3 isoforms. Prior studies have reported that Akt1 and Akt2, but not Akt3, are expressed in platelets. Here, we show that Akt3 is expressed in substantial amounts in platelets. Akt3−/− mouse platelets selectively exhibit impaired platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A2 (TXA2), but not collagen or VWF. In contrast, platelets from Akt1−/− or Akt2−/− mice are defective in platelet activation induced by thrombin, TXA2, and VWF, but only Akt1−/− platelets show significant defects in response to collagen, indicating differences among Akt isoforms. Akt3−/− platelets exhibit a significant reduction in thrombin-induced phosphorylation of glycogen synthase kinase 3β (GSK-3β) at Ser9, which is known to inhibit GSK-3β function. Thus, Akt3 is important in inhibiting GSK-3β. Accordingly, treatment of Akt3−/− platelets with a GSK-3β inhibitor rescued the defect of Akt3−/− platelets in thrombin-induced aggregation, suggesting that negatively regulating GSK-3β may be a mechanism by which Akt3 promotes platelet activation. Importantly, Akt3−/− mice showed retardation in FeCl3-induced carotid artery thrombosis in vivo. Thus, Akt3 plays an important and distinct role in platelet activation and in thrombosis.

Metabolomics-Driven Nutraceutical Evaluation of Diverse Green Tea Cultivars

BackgroundGreen tea has various health promotion effects. Although there are numerous tea cultivars, little is known about the differences in their nutraceutical properties. Metabolic profiling techniques can provide information on the relationship between the metabolome and factors such as phenotype or quality. Here, we performed metabolomic analyses to explore the relationship between the metabolome and health-promoting attributes (bioactivity) of diverse Japanese green tea cultivars.Methodology/Principal FindingsWe investigated the ability of leaf extracts from 43 Japanese green tea cultivars to inhibit thrombin-induced phosphorylation of myosin regulatory light chain (MRLC) in human umbilical vein endothelial cells (HUVECs). This thrombin-induced phosphorylation is a potential hallmark of vascular endothelial dysfunction. Among the tested cultivars, Cha Chuukanbohon Nou-6 (Nou-6) and Sunrouge (SR) strongly inhibited MRLC phosphorylation. To evaluate the bioactivity of green tea cultivars using a metabolomics approach, the metabolite profiles of all tea extracts were determined by high-performance liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses, principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA), revealed differences among green tea cultivars with respect to their ability to inhibit MRLC phosphorylation. In the SR cultivar, polyphenols were associated with its unique metabolic profile and its bioactivity. In addition, using partial least-squares (PLS) regression analysis, we succeeded in constructing a reliable bioactivity-prediction model to predict the inhibitory effect of tea cultivars based on their metabolome. This model was based on certain identified metabolites that were associated with bioactivity. When added to an extract from the non-bioactive cultivar Yabukita, several metabolites enriched in SR were able to transform the extract into a bioactive extract.Conclusions/SignificanceOur findings suggest that metabolic profiling is a useful approach for nutraceutical evaluation of the health promotion effects of diverse tea cultivars. This may propose a novel strategy for functional food design.

Rho-kinase regulates thrombin-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

We have previously reported that thrombin stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of thrombin in the thrombin-stimulated IL-6 synthesis and the involvement of Rho-kinase in MC3T3-E1 cells. Thrombin time-dependently induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and myosin phosphatase targeting subunit-1 (MYPT-1), a Rho-kinase substrate. While SP600125, an inhibitor of SAPK/JNK, failed to reduce IL-6 synthesis, PD98059, a specific inhibitor of MEK, and SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by thrombin. Y27632, a specific Rho-kinase inhibitor, significantly reduced thrombin-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed thrombin-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect thrombin-induced phosphorylation of p44/p42 MAP kinase. Y27632 as well as fasudil attenuated thrombin-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates thrombin-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

Protease‐activated receptor ‐3 signals independently to phosphorylate endothelial nitric oxide synthase in human endothelial cells

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites. Failure of the eNOS/NO pathway leads to endothelial dysfunction, a precursor to cellular induced hypertension and other cardiovascular diseases. Using human umbilical vein endothelial cells (HUVECs) as a model for human endothelium, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide, SFNGGP‐NH2. Therefore, we hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS. Immunoblot analysis using eNOS specific antibodies showed that 25uM of SFNGGP lead to the phosphorylation of the positive site of eNOS at ‐Ser1177 and the negative site ‐Thr495 in a time‐dependent manner similar to that observed with thrombin. After HUVECs were pretreated with 10uM of SCH79797, a selective PAR‐1 inhibitor, then stimulated with SFNGGP for 5 minutes, we observed an increase in phosphorylation at both eNOS regulatory sites. Thrombin‐induced phosphorylation of these sites was inhibited in the presence of SCH79797. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site specific phosphorylation that is independent of the activation of PAR‐1. Cardiovascular disease is the leading cause of death among Americans, where hypertension can be a silent aggressor. Studying the signaling pathways responsible for eNOS‐induced NO production will lead to a better understanding of endothelial dysfunction in cardiovascular diseases such as hypertension.NIH‐NIGMS‐SCORE and NIH‐NHLBI‐T32

Indirubin-3′-Monoxime Blocks Vascular Smooth Muscle Cell Proliferation by Inhibition of Signal Transducer and Activator of Transcription 3 Signaling and Reduces Neointima Formation In Vivo

Our goal was to examine the influence of indirubin-3'-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2'-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-γ-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [(14)C]l-arginine/[(14)C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.

CAMP/PKA antagonizes thrombin-induced inactivation of endothelial myosin light chain phosphatase: role of CPI-17

Activation of cAMP signalling abrogates thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, one of the major determinants of endothelial barrier function, mainly via the activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied. Endothelial contractile machinery and barrier function were analysed in cultured human umbilical vein endothelial cells (HUVEC). The cAMP analogues 8-CPT-cAMP and 6-Bnz-cAMP were used to activate Epac and PKA, respectively, and forskolin (FSK) was used to activate adenylyl cyclase. The cells were challenged by thrombin to inhibit MLCP via the RhoA/Rock pathway. Activation of either PKA or Epac partially blocked thrombin-induced hyperpermeability. Simultaneous activation of PKA and Epac had additive effects that were comparable to that of FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, partly via inhibition of the RhoA/Rock pathway. FSK activated the MLCP catalytic subunit PP1 via dephosphorylation and dissociation of the PP1 inhibitory protein CPI-17. FSK blunted thrombin-induced CPI-17 phosphorylation, CPI-17/PP1 complex formation, and PP1 inactivation. Down-regulation of CPI-17 attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect. cAMP/PKA regulates the endothelial barrier via inhibition of the contractile machinery, mainly by the activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability-lowering effect of the cAMP/Epac pathway is independent of CPI-17.

Thrombin induces endothelial arginase through AP-1 activation

Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction.

Protein Kinase C-δ and Phosphatidylinositol 3-Kinase/Akt Activate Mammalian Target of Rapamycin to Modulate NF-κB Activation and Intercellular Adhesion Molecule-1 (ICAM-1) Expression in Endothelial Cells

We have shown that the mammalian target of rapamycin (mTOR) down-regulates thrombin-induced ICAM-1 expression in endothelial cells by suppressing the activation of NF-kappaB. However, the mechanisms by which mTOR is activated to modulate these responses remain to be addressed. Here, we show that thrombin engages protein kinase C (PKC)-delta and phosphattidylinositol 3-kinase (PI3K)/Akt pathways to activate mTOR and thereby dampens NF-kappaB activation and intercellular adhesion molecule 1 (ICAM-1) expression. Stimulation of human vascular endothelial cells with thrombin induced the phosphorylation of mTOR and its downstream target p70 S6 kinase in a PKC-delta- and PI3K/Akt-dependent manner. Consistent with this, thrombin-induced phosphorylation of p70 S6 kinase was defective in embryonic fibroblasts from mice with targeted disruption of PKC-delta (Pkc-delta(-)(/)(-)), p85alpha and p85beta subunits of the PI3K (p85alpha(-)(/)(-)beta(-)(/)(-)), or Akt1 and Akt2 (Akt1(-)(/)(-)2(-)(/)(-)). Furthermore, we observed that expression of the constitutively active form of PKC-delta or Akt was sufficient to induce NF-kappaB activation and ICAM-1 expression, and that co-expression of mTOR suppressed these responses. In reciprocal experiments, inhibition/depletion of mTOR augmented NF-kappaB activation and ICAM-1 expression induced by PKC-delta or Akt. In control experiments, increasing or impairing mTOR signaling by the above approaches produced similar effects on NF-kappaB activation and ICAM-1 expression induced by thrombin. Thus, these data reveal an important role of PKC-delta and PI3K/Akt pathways in activating mTOR as an endogenous modulator to ensure a tight regulation of NF-kappaB signaling of ICAM-1 expression in endothelial cells.

PKC isoenzymes differentially modulate the effect of thrombin on MAPK-dependent RPE proliferation

Thrombin signalling through PAR (protease-activated receptor)-1 is involved in cellular processes, such as proliferation, differentiation and cell survival. Following traumatic injury to the eye, thrombin signalling may participate in disorders, such as PVR (proliferative vitreoretinopathy), a human eye disease characterized by the uncontrolled proliferation, transdifferentiation and migration of otherwise quiescent RPE (retinal pigment epithelium) cells. PARs activate the Ras/Raf/MEK/ERK MAPK pathway (where ERK is extracellular-signal-regulated kinase, MAPK is mitogen-activated protein kinase and MEK is MAPK/ERK kinase) through the activation of G(alpha) and G(betagamma) heterotrimeric G-proteins, and the downstream stimulation of the PLC (phospholipase C)-beta/PKC (protein kinase C) and PI3K (phosphoinositide 3-kinase) signalling axis. In the present study, we examined the molecular signalling involved in thrombin-induced RPE cell proliferation, using rat RPE cells in culture as a model system for PVR pathogenesis. Our results showed that thrombin activation of PAR-1 induces RPE cell proliferation through Ras-independent activation of the Raf/MEK/ERK1/2 MAPK signalling cascade. Pharmacological analysis revealed that the activation of 'conventional' PKC isoforms is essential for proliferation, although thrombin-induced phosphorylation of ERK1/2 requires the activation of atypical PKCzeta by PI3K. Consistently, thrombin-induced ERK1/2 activation and RPE cell proliferation were prevented completely by PI3K or PKCzeta inhibition. These results suggest that thrombin induces RPE cell proliferation by joint activation of PLC-dependent and atypical PKC isoforms and the Ras-independent downstream stimulation of the Raf/MEK/ERK1/2 MAPK cascade. The present study is the first report demonstrating directly thrombin-induced ERK phosphorylation in the RPE, and the involvement of atypical PKCzeta in this process.

TIMAP is a positive regulator of pulmonary endothelial barrier function

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin‐induced morphological changes in 1321N1 human astrocytoma cells

Activated astrocytes show various patterns of Ca(2+) mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca(2+) dynamics through investigation of thrombin-induced unique Ca(2+) entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03-3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca(2+) concentration ([Ca(2+)](i)) showed that thrombin induced heterogeneous Ca(2+) responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca(2+) release from intracellular stores, followed by refilling of Ca(2+) from the extracellular region without a direct [Ca(2+)](i) increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca(2+) mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca(2+) entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca(2+) responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca(2+) influx. Moreover, TRPC3-mediated Ca(2+) dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca(2+) dynamics, including Ca(2+) entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca(2+) channel and might modulate astrocytic activation in response to brain hemorrhaging.

Cocoa procyanidins inhibit expression and activation of MMP-2 in vascular smooth muscle cells by direct inhibition of MEK and MT1-MMP activities

Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 microg/mL and 5 microM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.

Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which is involved in various physiological functions of the cardiovascular system. eNOS is activated by dephosphorylation at Thr495 and phosphorylation at Ser1177. Inhibition of Rho-kinase, an effector of the small GTPase RhoA, leads to activation of Akt/PKB, which phosphorylates eNOS at Ser1177 and thereby promotes NO production. However, little is known about the effects of Rho-kinase on phosphorylation of Thr495. We here found that the constitutively active form of Rho-kinase phosphorylated eNOS at Thr495 in vitro. Expression of the constitutively active form of RhoA or Rho-kinase increased this phosphorylation in COS-7 cells. Addition of thrombin to cultured human umbilical vein endothelial cells induced phosphorylation of eNOS at Thr495. Treatment with Y27632, a Rho-kinase inhibitor, suppressed thrombin-induced phosphorylation at Thr495. These results indicate that Rho-kinase can directly phosphorylate eNOS at Thr495 to suppress NO production in endothelium.

Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

Thrombin has been shown to activate endothelial NO synthase (eNOS) leading to endothelium-dependent vasorelaxation. In addition to its activation by Ca2+/calmodulin, eNOS has several regulatory sites. Ser1179 phosphorylation of eNOS by the phosphatidylinositol 3-kinase-dependent Akt stimulates its catalytic activity. In this study, we have elucidated the signaling mechanism of thrombin-induced phosphorylation of eNOS in the regulation of NO production. Immunoblot analysis showed that thrombin rapidly phosphorylates eNOS at Ser1179 in cultured bovine aortic endothelial cells. Also, thrombin was unable to stimulate eNOS if the Ser1179 was mutated to Ala. Akt is phosphorylated in response to thrombin at Ser473 at a later time point than eNOS. In this regard, a phosphatidylinositol 3-kinase inhibitor, LY294002, blocked Akt phosphorylation without affecting eNOS phosphorylation and cGMP production by thrombin. The Ca2+ ionophore A23187 stimulated eNOS phosphorylation, as well as cGMP production, and pretreatment with intracellular or extracellular Ca2+ chelators inhibited thrombin-induced eNOS phosphorylation and cGMP production. Moreover, infection of bovine aortic endothelial cell with adenovirus encoding dominant-negative mutants of protein kinase C (PKC) and PKC or pretreatment of bovine aortic endothelial cells with PKC inhibitors revealed that PKC is indispensable for thrombin-induced eNOS phosphorylation and activation. From these data, we concluded that thrombin induces the Ser1179 phosphorylation-dependent eNOS activation through a Ca2+-dependent, PKC-sensitive, but phosphatidylinositol 3-kinase/Akt-independent pathway.

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain.

Outside-in signaling of β3 integrins induces and requires phosphorylation at tyrosine-747 (Y747) and tyrosine-759 (Y759) of the β3 subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of β3 cytoplasmic domain. Here we show that tyrosine phosphorylation in the synthetic β3 cytoplasmic domain peptide inhibits calpain cleavage. In platelets, tyrosine phosphates inhibitor, sodium vanadate, enhances thrombin-induced phosphorylation at Y747 and Y759, which is associated with the reduced integrin cleavage by calpain. The effects of sodium vanadate is unlikely to be caused by its effects on calpain activity but is likely to be caused by the susceptibility of integrin cytoplasmic domain, because sodium vanadate did not affect the calpain cleavage of another substrate, fodrin, in platelets. To further support the protective effect of tyrosine phosphorylation against calpain cleavage, we show that mouse β3 (DiYF) with both Y759 and Y747 mutated to phenylalanine is more susceptible to calpain cleavage than wild type during thrombin-induced platelet aggregation. Furthermore, phosphorylation at Y747 and Y759 of β3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Y759 is associated with calpain cleavage at Y759. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the β3 cytoplasmic domain.

Rho Kinase Differentially Regulates Phosphorylation of Nonmuscle Myosin II Isoforms A and B during Cell Rounding and Migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.