Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein-coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase- and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction.
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PMID:GIT1 mediates thrombin signaling in endothelial cells: role in turnover of RhoA-type focal adhesions. 1501 33

Lung epithelial and endothelial barrier dysfunction is critical to the physiologic derangement observed in acute lung injury, but remains poorly understood. We utilized human alveolar epithelial (A549) and endothelial cells (EC) to study cytoskeletal remodeling, myosin light chain (MLC) phosphorylation and barrier regulation evoked by the edemagenic agent, thrombin. Thrombin-challenged human EC monolayers demonstrated increased MLC phosphorylation, actin stress fiber formation and loss of barrier integrity reflected by decreased transmonolayer electrical resistance (TER). In contrast, thrombin produced prominent circumferential localization of actin fibers, increased MLC phosphorylation and increased TER across epithelial monolayers, consistent with barrier protection. Reductions in MLC phosphorylation induced by cell pretreatment with pharmacological inhibitors of MLC kinase (ML-7) and Rho kinase (Y-27632) significantly attenuated thrombin-mediated TER changes and MLC phosphorylation in both lung cell types. Thrombin-produced, time-dependent activation of Rho GTPase in both epithelial and EC, whereas Rac GTPase activation was observed only in A549 cells. Molecular inhibition of Rac activity by adenoviral transfer of dominant-negative Rac mutant abolished thrombin-induced TER increases in alveolar epithelial cells. Finally, A549 cells, but not endothelium, demonstrated increased levels of tight junction proteins (ZO-1 and occludin) after thrombin at the cell-cell interface areas linked to thrombin-elicited barrier protection. These results demonstrate differential pulmonary endothelial and alveolar epithelial barrier regulation via unique actomyosin remodeling and cytoskeletal interactions with tight junction complexes, which confer selective barrier responses to edemagenic stimuli.
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PMID:Differential regulation of human lung epithelial and endothelial barrier function by thrombin. 1528 75

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is associated with the development of vascular tumors including renal cell carcinoma. Aside from the role played by the VHL protein (pVHL) in negative regulation of hypoxia-inducible factor, 41F-1alpha, pVHL also takes part in cytoskeletal organization. Thrombin is a serine protease involved in angiogenesis and in cancer progression and its action is mediated by the protease-activated receptors (PARs). In several cell types, thrombin induces reorganization of the cytoskeleton along with RhoA activation. Thus, we conducted an investigation on the capacity of thrombin to regulate pVHL expression. Our results demonstrated that VHL mRNA and protein levels were increased by thrombin in cultured renal cancer cells. Cytoplasmic pVHL was redistributed to perinuclear regions and membrane fractions following thrombin treatments. Stimulation of Caki-1 cells with PAR1, PAR2 and PAR4 agonist peptides demonstrated that PAR1 was the receptor involved in thrombin-induced pVHL expression. Western blot analysis confirmed that these cells express PAR1 and that its expression was increased by thrombin. PAR1 activation by both thrombin and an agonist peptide stimulated renal cancer cell invasion through Matrigel. Interestingly, the upregulation of pVHL was dependent on RhoA because C3 exotoxin abolished pVHL induction. However, the pharmacological Rho kinase inhibitor, Y27632, did not influence pVHL expression in the presence of thrombin, suggesting that other RhoA effectors were involved in the process. Together, these results demonstrate that thrombin induces both pVHL expression via PAR1/RhoA activation as well as the stimulation of renal cancer cell invasion suggesting a role for thrombin in tumor invasion.
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PMID:von Hippel-Lindau tumor suppressor protein stimulation by thrombin involves RhoA activation. 1538 85

The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G(i)-mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCbeta activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCbeta. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by G-protein-coupled receptor agonists.
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PMID:Two different pathways link G-protein-coupled receptors with tyrosine kinases for the modulation of growth and survival in human hematopoietic progenitor cells. 1560 23

Protein kinase C-potentiated phosphatase inhibitor of 17 kDa (CPI-17) mediates some agonist-induced smooth muscle contraction by suppressing the myosin phosphatase in a phosphorylation-dependent manner. The physiologically relevant kinases that phosphorylate CPI-17 remain to be identified. Several previous studies have shown that some agonist-induced CPI-17 phosphorylation in smooth muscle tissues was attenuated by the Rho kinase (ROCK) inhibitor Y-27632, suggesting that ROCK is involved in agonist-induced CPI-17 phosphorylation. However, Y-27632 has recently been found to inhibit protein kinase C (PKC)-delta, a well-recognized CPI-17 kinase. Thus the role of ROCK in agonist-induced CPI-17 phosphorylation remains uncertain. The present study was designed to address this important issue. We selectively activated the RhoA pathway using inducible adenovirus-mediated expression of a constitutively active mutant RhoA (V14RhoA) in primary cultured rabbit aortic vascular smooth muscle cells (VSMCs). V14RhoA caused expression level-dependent CPI-17 phosphorylation at Thr38 as well as myosin phosphatase phosphorylation at Thr853. Importantly, we have shown that V14RhoA-induced CPI-17 phosphorylation was not affected by the PKC inhibitor GF109203X but was abolished by Y-27632, suggesting that ROCK but not PKC was involved. Furthermore, we have shown that the contractile agonists thrombin and U-46619 induced CPI-17 phosphorylation in VSMCs. Similarly to V14RhoA-induced CPI-17 phosphorylation, thrombin-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X, but it was blocked by inhibition of RhoA with adenovirus-mediated expression of exoenzyme C3 as well as by Y-27632. Taken together, our present data provide the first clear evidence indicating that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs.
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PMID:RhoA-Rho kinase pathway mediates thrombin- and U-46619-induced phosphorylation of a myosin phosphatase inhibitor, CPI-17, in vascular smooth muscle cells. 1581 90

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.
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PMID:Activation of protease-activated receptor-1 triggers astrogliosis after brain injury. 1585 58

Thrombin activates protease-activated receptor-1 (PAR-1) by cleavage of the amino terminus to unmask a tethered ligand. Although peptide analogs can activate PAR-1, we show that the functional responses mediated via PAR-1 differ between the agonists. Thrombin caused endothelial monolayer permeability and mobilized intracellular calcium with EC(50) values of 0.1 and 1.7 nm, respectively. The opposite order of activation was observed for agonist peptide (SFLLRN-CONH(2) or TFLLRNKPDK) activation. The addition of inactivated thrombin did not affect agonist peptide signaling, suggesting that the differences in activation mechanisms are intramolecular in origin. Although activation of PAR-1 or PAR-2 by agonist peptides induced calcium mobilization, only PAR-1 activation affected barrier function. Induced barrier permeability is likely to be Galpha(12/13)-mediated as chelation of Galpha(q)-mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of Galpha(i/o), or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the Galpha(12/13)-mediated Rho kinase abrogated the response. Similarly, calcium mobilization is Galpha(q)-mediated and independent of Galpha(i/o) and Galpha(12/13) because pertussis toxin Y-27632 and had no effect, whereas U-73122 inhibition of phospholipase C-beta blocked the response. It is therefore likely that changes in permeability reflect Galpha(12/13) activation, and changes in calcium reflect Galpha(q) activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate Galpha(12/13) or Galpha(q). This functional selectivity was characterized quantitatively by a mathematical model describing each step leading to Rho activation and/or calcium mobilization. This model provides an estimate that peptide activation alters receptor/G protein binding to favor Galpha(q) activation over Galpha(12/13) by approximately 800-fold.
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PMID:Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1. 1587 70

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.
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PMID:LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells. 1589 90

Increased contractility of the actin cytoskeleton by phosphorylation of the regulatory myosin light chain (MLC) results in a loss of barrier integrity in corneal endothelial cells. This study has investigated the effect of extracellular ATP, which may influence both Ca2+ and cAMP signalling, on MLC phosphorylation and barrier integrity in cultured bovine corneal endothelial cells (BCEC) known to express A2B and P2Y purinergic receptors, and ecto-nucleotidases. Extracellular ATP (100 microM) promoted MLC dephosphorylation (pMLC=61.8% at 18 min; n=9). Pre-exposure to ARL-67156, an ecto-nucleotidase inhibitor, prevented ATP-induced dephosphorylation. Other P2Y agonists, UTP and ATPgammaS, also induced MLC dephosphorylation but to a lesser degree compared to ATP. Thrombin (2 U/ml), which activate Rho kinase through PAR-1 receptors in the endothelium, induced MLC phosphorylation (pMLC=129.2%; n=14). This phosphorylation was completely abolished by concomitant exposure to ATP. When cells were pretreated with adenosine (100 microM; A2B agonist) or forskolin (10 microM), thrombin-induced phosphorylation was suppressed. ATP also led to a significant increase in cAMP (> 3-fold compared to 10 microM adenosine). Thrombin-induced increase in trans-endothelial flux of horseradish peroxidase (44 kDa) and disruption of the cortical actin were suppressed by ATP. These findings indicate that in BCEC (1) ATP induces elevated cAMP through its metabolite adenosine leading to MLC dephosphorylation, (2) Stimulation of P2Y2 receptors also leads to activation of MLCP since UTP- and ATPgammaS caused MLC dephosphorylation, and (3) ATP is antagonistic to thrombin since the latter inhibits MLCP through increased activity of Rho kinase. These findings further emphasize the role of contractility of the actin cytoskeleton in regulating the barrier integrity of corneal endothelium.
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PMID:Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells. 1608 Sep 12

We recently reported that proteinase-activated receptors type I (PAR-1) are coupled to both negative and positive invasion pathways in colonic and kidney cancer cells cultured on collagen type I gels. Here, we found that treatments with the cell-permeant analog 8-Br-cGMP and the soluble guanylate cyclase activator BAY41-2272, and Rho kinase (ROK) inhibition by Y27632 or a dominant negative form of ROK lead to PAR-1-mediated invasion through differential Rac1 and Cdc42 signaling. Hypoxia or the counteradhesive matricellular protein SPARC/BM-40 (SPARC: secreted protein acidic rich in cysteine) overexpressed during cancer progression also commutated PAR-1 to cellular invasion through the cGMP/protein kinase G (PKG) cascade, RhoA inactivation, and Rac1-dependent or -independent signaling. Cultured primary cancer cells isolated from peritoneal and pleural effusions from patients with colon cancer or other malignant tumors harbored PAR-1, as shown by RT-PCR and FACS analyses. These malignant effusions also contained high levels of activated thrombin and fibrin, and induced a proinvasive response in HCT8/S11 human colorectal cancer cells. Our data underline the essential role of the tumor microenvironment and of several commutators targeting cGMP/PKG signaling and the RhoA-ROK axis in the control of PAR-1 proinvasive activity and metastatic potential of cancer cells in distant organs and peritoneal or pleural cavities. We also add new insights into the mechanisms linking the coagulation mediators thrombin and PAR-1 in the context of blood coagulation disorders and venous thrombosis often observed in cancer patients, as described in 1865 by Armand Trousseau.
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PMID:Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho-Rho kinase axis and tumor microenvironment. 1609 33


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