Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activation of protease-activated receptor-1 (PAR-1) produces a dual action, apamin-sensitive relaxation followed by contraction, in the rat duodenal smooth muscle, which is partially dependent on activation of L-type Ca2+ channels, protein kinase C (PKC) or tyrosine kinase (TK), and resistant to tetrodotoxin. The present study further characterized the PAR-1-mediated duodenal responses. Removal of extracellular Ca2+ as well as SK&F96365 reduced the contraction due to the PAR-1 agonist TFLLR-NH2 (TFp-NH2) by 60-80% that was similar to the extent of the inhibition by nifedipine. Lowering of the extracellular Na+ concentration, but not IAA-94, a Cl- channel inhibitor, reduced both the PAR-1-mediated contraction and relaxation by about 50%. U73122, a phospholipase C (PLC) inhibitor, or wortmannin, a phosphatidyl inositol 3'-kinase (PI3K) inhibitor, significantly reduced the PAR-1-mediated contraction, but not the relaxation, by itself, as the PKC inhibitor GF109203X and the TK inhibitor genistein did. U73122 or wortmannin, like GF109203X, when applied in combination with genistein, significantly reduced the PAR-1-mediated relaxation. The relaxation was resistant to antagonists of PACAP receptors, VIP receptors and P2 purinoceptors. Thus, the PAR-1-mediated contraction is considered to be dependent on intracellular and extracellular Ca2+, the influx of the latter being induced through activation of L-type Ca2+ channels triggered by the enhanced Na+ permeability, and that PLC and PI3K, in addition to PKC and TK, are involved in the PAR-1-mediated dual responses. Furthermore, non-adrenergic, non-cholinergic nerve neurotransmitter candidates that may modulate K+ channels do not appear to contribute to the relaxation by PAR-1 activation.
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PMID:Characterization of the protease-activated receptor-1-mediated contraction and relaxation in the rat duodenal smooth muscle. 1106 74

Human glioblastoma cell line A172 expressed protease-activated receptor-1 and -2 (PAR-1 and PAR-2). We investigated the effects of the stimulation of these receptors by receptor-activating agonist peptides on the Ca2+ signaling, protein kinase C translocation, cell morphology and cell proliferation in A172. Both PAR-1 agonist SFLLRN and PAR-2 agonist SLIGKV induced an increase in [Ca2+]i. The prior treatment of A172 with PAR-2 agonist SLIGKV did not influence the [Ca2+]i response to PAR-1 agonist SFLLRN or thrombin, however, the prior treatment with PAR-1 agonist SFLLRN or thrombin completely abolished the second response to PAR-2 agonist SLIGKV. Treatment with each agonist peptide produced thinner and fewer processes in A172. The PAR-2 agonist inhibited the proliferation of A172 significantly while PAR-1 agonist did not. PKC-alpha and gamma were translocated from cytosol to membrane with either PAR-1 or PAR-2 stimulation, however, L was specifically translocated with SFLLRN, and lambda with SLIGKV, respectively. These results indicated that PAR-1 and PAR-2 stimulation produced a similar [Ca2+]i response and morphological changes in A172 glioblastoma while the effects on the cell proliferation and activation of PKC isozymes were distinct, suggesting that different signal transduction pathways were activated by these receptors. The uni-directional cross desensitization implies a functional linkage between PAR-1 and PAR-2 receptors.
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PMID:The effects of stimulating protease-activated receptor-1 and -2 in A172 human glioblastoma. 1131 68

Thrombin activates mast cells to release inflammatory mediators through a mechanism involving protease-activated receptor-1 (PAR-1). We hypothesized that PAR-1 activation would induce mast cell adhesion to fibronectin (FN). Fluorescent adhesion assay was performed in 96-well plates coated with FN (20 microg/ml). Murine bone marrow cultured mast cells (BMCMC) were used after 3-5 wk of culture (>98% mast cells by flow cytometry for c-Kit expression). Thrombin induced beta-hexosaminidase, IL-6, and matrix metalloproteinase-9 release from BMCMC. Thrombin and the PAR-1-activating peptide AparafluoroFRCyclohexylACitY-NH(2) (cit) induced BMCMC adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive peptide FSLLRY-NH(2) had no effect. Thrombin and cit induced also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was inhibited by anti-alpha(5) integrin Ab (51.1 +/- 6.7%; n = 5). The combination of anti-alpha(5) and anti-alpha(4) Abs induced higher inhibition (65.7 +/- 7.1%; n = 5). Unlike what is known for FcepsilonRI-mediated adhesion, PAR-1-mediated adhesion to FN did not increase mediator release. We then explored the signaling pathways involved in PAR-1-mediated mast cell adhesion. Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 +/- 7.3% (n = 4), indicating that G(i) proteins are involved. Wortmannin and calphostin almost completely inhibited PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and protein kinase C are involved. Adhesion was partially inhibited by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5 +/- 3.3%; n = 3) and the p38 inhibitor SB203580 (25.1 +/- 10.4%; n = 3). The two inhibitors had additive effects. Therefore, thrombin mediates mast cell adhesion through the activation of G(i) proteins, phosphoinositol 3-kinase, protein kinase C, and mitogen-activated protein kinase pathways.
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PMID:Thrombin induces mast cell adhesion to fibronectin: evidence for involvement of protease-activated receptor-1. 1237 Mar 92

We have recently demonstrated that multiple signalling pathways are involved in thrombin-induced proliferation in rat astrocytes. Thrombin acts by protease-activated receptor-1 (PAR-1) via mitogen-activated protein kinase activity. Signalling includes both Gi/(betagamma subunits)-phosphatidylinositol 3-kinase and a Gq-phospholipase C/Ca2+/protein kinase C (PKC) pathway. In the present study, we investigated the possible protein tyrosine kinases which might be involved in thrombin signalling cascades. We found that, in astrocytes, thrombin can evoke phosphorylation of proline-rich tyrosine kinase (Pyk2) via PAR-1. This process is dependent on the increase in intracellular Ca2+ and PKC activity. Moreover, in response to thrombin stimulation Pyk2 formed a complex with Src tyrosine kinase and adapter protein growth factor receptor-bound protein 2 (Grb2), which could be coprecipitated. Furthermore, both thrombin-induced Pyk2 phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation can be attenuated by Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. From these data we conclude that PAR-1 uses Ca2+- and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation, which predominantly recruits Grb2 in rat astrocytes.
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PMID:The role of the Ca2+-sensitive tyrosine kinase Pyk2 and Src in thrombin signalling in rat astrocytes. 1261 35

Recently, we reported that thrombin specifically stimulates protease-activated receptor-1 (PAR-1) signaling in RPE entailing inhibition of Sp1 dependent HCMV replication. We now studied whether thrombin modulates the expression of the proinflammatory cytokine/chemokines IL-6 and IL-8 in mock- and cytomegalovirus-infected human retinal pigment epithelial cells (RPE). Our data show that thrombin/PAR-1 stimulates IL-6 and IL-8 gene transcription and protein secretion in both mock- and HCMV-infected RPE. Thrombin/PAR-1-mediated signaling stimulated PKC and NF-kappaB-dependent IL-6 and IL-8 gene expression via phosphoinositide 3-kinase and further downstream via p42/44 and p38 MAPKs. Thus, thrombin/PAR-1-mediated IL-6/IL-8 gene expression is uncoupled from Sp1 inhibition and may support proinflammatory pathomechanisms probably involved in hemorrhage/HCMV retinitis progression.
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PMID:Thrombin stimulates IL-6 and IL-8 expression in cytomegalovirus-infected human retinal pigment epithelial cells. 1471 42

G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinase-dependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNFalpha protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.
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PMID:Regulated shedding of PAR1 N-terminal exodomain from endothelial cells. 1498 36

Signaling pathways elicited by protease-activated receptor-1 (PAR-1) agonists, thrombin receptor-activating peptide (TRAP) and thrombin, are markedly different. Here we show that TRAP-induced disaggregation of platelets is a function of extracellular calcium. Chelation of calcium with EGTA after the onset of aggregation precluded subsequent destabilization of the aggregates in TRAP-stimulated platelets, whereas disaggregation was not observed in the platelets stimulated with thrombin. TRAP-induced disaggregation was independent of the activity of the calcium-dependent thiol protease, calpain. Inhibition of phosphoinositide 3-kinase activity provoked further destabilization of the platelet aggregates in the presence of calcium; however, EGTA attenuated this effect. Activation of protein kinase C (PKC) by phorbol ester prevented disaggregation of the TRAP-stimulated platelets independent of the extracellular calcium. Two proteins of relative mobilities 67 and 75 kD were found to be significantly dephosphorylated on tyrosine in calcium-pretreated platelets as compared to the EGTA-treated platelets following continued stimulation with either TRAP or thrombin for 15 min. Inhibition of phosphoinositide 3-kinase by two pharmacologically independent inhibitors also caused dephosphorylation of p67, which was completely abrogated by chelation of extracellular calcium. Platelet activation by phorbol ester was not associated with disaggregation, although dephosphorylation of p67 was induced under this condition. SHP-1, an abundant tyrosine phosphatase in platelets, co-migrated with the p67 protein and co-localized to the actin-based cytoskeleton of aggregated platelets; however, its identity with p67 was ruled out from immunoprecipitation studies.
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PMID:Sustained stimulation of platelet thrombin receptor is associated with tyrosine dephosphorylation of a novel p67 peptide in a manner regulated by extracellular calcium. 1531 16

Using a cDNA microarray screening approach, we have identified seven novel thrombin-responsive genes in human umbilical vein endothelial cells that were verifiable by Northern blot analysis. Among them CL-100, a dual-specificity phosphatase also known as MAP kinase phosphatase-1 (MKP-1), showed greatest induction by thrombin. Steady-state levels of CL-100 mRNA induction by thrombin peaked at 1 h and declined rapidly (t1/2 approximately 45 min). Induction by thrombin was protease-activated receptor-1-mediated, protein synthesis-independent, and transcriptionally regulated. Metabolic labeling followed by immunoprecipitation verified that the thrombin-induced CL-100 mRNA was translated into protein. We found that both Src-kinase and p42/p44 ERK activity are critical for thrombin-induced CL-100 expression, whereas phosphatidylinositol 3-kinase and protein kinase C activity were not required. Antisense-mediated inhibition of CL-100 was shown to prolong thrombin-induced ERK activity in endothelial cells, concomitant with an inhibition in thrombin-induced PDGF-A (platelet-derived growth factor A) and PDGF-B gene expression and an up-regulation in thrombin-induced VCAM-1 and E-selectin gene expression. Inhibition of ERK activation by PD98059 in endothelial cells was shown to potentiate thrombin-induced expression of PDGF-B (approximately 3-fold) while inhibiting thrombin-induced VCAM-1 and E-selectin gene expression by 60 and 70%, respectively. These results suggested that induced expression of the CL-100 phosphatase and its subsequent regulation of ERK activity play a key regulatory role in the thrombin signaling pathway and in the transcriptional regulation of pathologically important "endothelial cell activation genes."
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PMID:Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation. 1533 8

Thrombospondin-1 (THBS1) is a large extracellular matrix glycoprotein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. Increases in THBS1 expression have been liked to disease states including tumor progression, atherosclerosis, and arthritis. The present study focuses on the effects of thrombin activation of the G-protein-coupled, protease-activated receptor-1 (PAR-1) on THBS1 gene expression in the microvascular endothelium. Thrombin-induced changes in gene expression were characterized by microarray analysis of approximately 11,000 different human genes in human microvascular endothelial cells (HMEC-1). Thrombin induced the expression of a set of at least 65 genes including THBS1. Changes in THBS1 mRNA correlated with an increase in the extracellular THBS1 protein concentration. The PAR-1-specific agonist peptide (TFLLRNK-PDK) mimicked thrombin stimulation of THBS1 expression, suggesting that thrombin signaling is through PAR-1. Further studies showed THBS1 expression was sensitive to pertussis toxin and protein kinase C inhibition indicating G(i/o)- and G(q)-mediated pathways. THBS1 up-regulation was also confirmed in human umbilical vein endothelial cells stimulated with thrombin. Analysis of the promoter region of THBS1 and other genes of similar expression profile identified from the microarray predicted an EBOX/EGRF transcription model. Expression of members of each family, MYC and EGR1, respectively, correlated with THBS1 expression. These results suggest thrombin formed at sites of vascular injury increases THBS1 expression into the extracellular matrix via activation of a PAR-1, G(i/o), G(q), EBOX/EGRF-signaling cascade, elucidating regulatory points that may play a role in increased THBS1 expression in disease states.
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PMID:Thrombin modulates the expression of a set of genes including thrombospondin-1 in human microvascular endothelial cells. 1581 47

Thrombin, TNF-alpha, and LPS have each been implicated in endothelial cell and vascular smooth muscle cell (VSMC) activation. We wanted to test the hypothesis that these three agonists display mediator and/or cell type-specific properties. The addition of thrombin to human pulmonary artery endothelial cells resulted in an upregulation of PDGF-A, tissue factor (TF), ICAM-1, and urokinase-type plasminogen activator (u-PA), whereas TNF-alpha and LPS failed to induce PDGF-A. These effects were mimicked by protease-activated receptor-1 activation. In VSMC, thrombin induced expression of TF and PDGF-A but failed to consistently induce ICAM-1 or u-PA expression. In contrast, TNF-alpha and LPS increased expression of all four genes in this cell type. Inhibitor studies in endothelial cells demonstrated a critical role for PKC in mediating thrombin, TNF-alpha, and LPS induction of ICAM-1, TF, and u-PA and for p38 MAPK in mediating thrombin, TNF-alpha, and LPS induction of TF. Taken together, these results suggest that inflammatory mediators engage distinct signaling pathways and expression profiles in endothelial cells and VSMC. The data support the notion that endothelial cell activation is not an all-or-nothing phenomenon but rather is dependent on the nature of the extracellular mediator.
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PMID:Thrombin, TNF-alpha, and LPS exert overlapping but nonidentical effects on gene expression in endothelial cells and vascular smooth muscle cells. 1583


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