EC:2.7.11.13 - FACTA Search

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

12(S)-HETE, a lipoxygenase metabolite of arachidonic acid induced a nondestructive and reversible endothelial cell (EC) retraction. 12(S)-HETE induced EC retraction was inhibited by protein kinase C inhibitors calphostin C and staurosporine but not by the protein kinase A inhibitor H8. The role of EC integrins alpha v beta 3 and alpha 5 beta 1 in 12(S)-HETE induced EC retraction was investigated. In confluent EC cultures, alpha v beta 3 is localized to focal adhesions at both the cell body and cell-cell borders and is colocalized with vinculin-containing focal adhesions. In contrast, alpha 5 beta 1 is primarily enriched at the cell-cell borders, demonstrating codistribution with cell cortical microfilaments and extracellular fibronectin. Both receptors were functional in mediating cell-cell or cell-matrix interactions based on the observations that specific antibodies inhibited EC adhesion to intact subendothelial matrix and disrupted the monolayer integrity. 12(S)-HETE induced a multistep, temporally defined redistribution of the alpha v beta 3-containing focal adhesions, leading to an eventual decrease in alpha v beta 3 plaques in the retracted ECs. This effect of 12(S)-HETE was inhibited by calphostin C but not by H8. The alterations of alpha v beta 3-containing focal adhesions preceded the development of EC retraction. 12(S)-HETE also enhanced EC alpha v beta 3 surface expression as revealed by immunofluorescence, flow cytometry, and digitized image analysis. 12(S)-HETE-induced alpha v beta 3 rearrangement (i.e., decreased focal adhesion localization and enhanced surface expression) did not result from altered mRNA transcription (as revealed by semi-quantitative RT-PCR analysis) or protein translation (as revealed by Western blotting). In contrast to its effect on alpha v beta 3, 12(S)-HETE did not demonstrate a temporally related, well-defined effect on the distribution pattern and the surface expression of alpha 5 beta 1, although the cell-cell border staining pattern of alpha 5 beta 1 was disrupted due to EC retraction. It is concluded that 12(S)-HETE-induced decrease of alpha v beta 3 localization to focal adhesions may contribute to the development of EC retraction and that 12(S)-HETE induced increase in alpha v beta 3 surface expression may promote adhesion of inflammatory leukocytes as well as tumor cells to endothelium.
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PMID:Protein kinase C-dependent effects of 12(S)-HETE on endothelial cell vitronectin receptor and fibronectin receptor. 768 91

Initial arrest of tumor cells in the microvasculature and their attachment to the endothelium and subendothelial matrix (SEM) are essential prerequisites for metastasis to occur. Factors mediating these interactions are viewed as important determinants of the tumor-cell metastatic phenotype. In this work we have studied the effects of thrombin, its analogs and its precursors on the adhesive properties and metastatic potential of tumor cells. We show that alpha-thrombin, the native form of the key coagulation enzyme, is capable of enhancing tumor-cell adhesion to both the endothelium and SEM components represented by fibronectin. Subclotting, physiological concentrations of alpha-thrombin produced a 2- to 5-fold increase in tumor-cell adhesion. A bell-shaped dose-response curve was observed, with maximal effect at 0.1 U/ml. Maximum effect occurred when cells were exposed to the agonist for 15 min and exposure for up to 4 hr resulted in enhanced tumor-cell adhesion. Prolonged incubation with thrombin resulted in a decline in the thrombin-enhanced adhesion which reached unstimulated control levels by 24 hr. Thrombin precursors and active-site-inhibited thrombin analogs only had minimal adhesion-enhancing activity; nitro- and exosite-alpha-thrombin, which retain a functional active site, mimicked, although to a lesser degree, the action of alpha-thrombin. Tumor-cell incubation with thrombin resulted in an upregulated cell-surface expression of the alpha11b beta 3 integrin, a receptor mediating interactions between tumor cells and endothelial cells, and between tumor cells and SEM. Antibodies against alpha 11b beta 3 integrin effectively inhibited thrombin-enhanced tumor-cell adhesion. Thrombin effects on tumor cells involved the PKC signal transduction pathway as thrombin-enhanced adhesion was inhibited by pre-incubation with PKC inhibitors and a transient PKC translocation from cytosol to membrane was observed following thrombin challenge. In vivo, thrombin-treated tumor cells demonstrated a 2-fold increase in their lung-colonizing ability. In contrast to the adhesion results, the metastasis-enhancing effects of alpha-thrombin were mimicked by a thrombin precursor (prothrombin) and thrombin analogs.
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PMID:Thrombin increases the metastatic potential of tumor cells. 768 87

Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell behavior. We have investigated the role of protein kinase C in alpha 5 beta 1 integrin-mediated signal transduction in Chinese hamster ovary cells. Up-regulation of protein kinase C activity by phorbol esters was found to enhance cell adhesion, spreading, and migration on a fibronectin substrate, without affecting the cell surface expression or fibronectin binding of the alpha 5 beta 1 integrin, whereas inhibition of protein kinase C activity by calphostin C inhibited these functions as well in unstimulated cells. In addition, we observed that protein kinase C activity in the cell membrane fraction transiently increases preceding cell spreading on fibronectin, but not on polylysine, additionally implying a specific role of protein kinase C in alpha 5 beta 1 integrin-mediated spreading on fibronectin. Experiments with phorbol esters and calphostin C also suggested that protein kinase C activity is required for enhanced phosphorylation of the focal adhesion kinase, pp125fak, in cells plated on fibronectin. Protein kinase C-alpha was not, however, found to directly act on pp125fak, suggesting that other mechanisms are involved in this phenomenon.
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PMID:Activation of protein kinase C precedes alpha 5 beta 1 integrin-mediated cell spreading on fibronectin. 769 9

High concentrations of glucose in vitro inhibit cell proliferation and stimulate matrix protein synthesis. These studies sought to characterize the relationship between the effects of glucose on cell proliferation and matrix synthesis and to assess the mechanism(s) responsible for these cellular effects of glucose. The initial experiments showed that high glucose levels stimulate fibronectin (FN) synthesis by human mesangial cells (HMC) but only in those cultures in which cell proliferation was inhibited by glucose. To assess whether this relationship was due to an effect of glucose on the capacity of HMC to respond to cytokines, the responses of HMC to serum or cytokines were measured in the presence of different glucose concentrations. High concentrations of glucose inhibited (3H)thymidine incorporation in response to serum and platelet-derived growth factor. Under the conditions of these experiments, transforming growth factor-beta (TGF-beta) also stimulated thymidine incorporation by HMC, and high glucose concentrations inhibited thymidine incorporation in response to TGF-beta. In contrast, high concentrations of glucose did not inhibit the stimulation of FN synthesis caused by platelet-derived growth factor, serum, or TGF-beta. The antiproliferative effects of high glucose levels were first observed after 48 h of incubation and were reversible after the withdrawal of high glucose from the media. The following evidence suggest that the effects of glucose may be mediated via protein kinase C (PKC): (1) incubation with high glucose concentrations caused an increase in HMC PKC levels; (2) PKC activation with phorbol esters inhibited HMC proliferation; and (3) depletion or inhibition of PKC stimulated HMC proliferation and prevented the antiproliferative effects of glucose. In contrast to these findings, inhibitors of protein glycosylation and myo-inositol supplementation of culture media did not prevent the antiproliferative effects of glucose. In conclusion, high glucose concentrations acutely and reversibly inhibit HMC proliferation, perhaps by a PKC-dependent mechanism. Because PKC can also stimulate FN synthesis, glucose-induced changes in PKC may explain the relationship between the effects of high glucose concentrations on cell proliferation and FN synthesis.
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PMID:Effects of high glucose concentrations on human mesangial cell proliferation. 775 94

Transforming growth factor-beta (TGF-beta) is a multifunctional peptide that elicits a wide variety of responses in cells. TGF-beta binds to cell surface receptors that contain cytoplasmic serine/threonine kinase domains. Here we provide evidence that both phospholipase C and protein kinase C (PKC) are involved in the TGF-beta activation of transcription and luciferase expression from the p3TP-Lux plasmid. Down-regulation of PKC prevents TGF-beta 1 induction of luciferase expression. Staurosporin and Calphostin C, inhibitors of PKC, block the ability of TGF-beta 1 to initiate transcription of the luciferase gene. Further, D609, an inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), and secondarily PKC also blocks TGF-beta 1-induced transcription of the transgene in A549 cells while the phosphatidylinositol-PLC pathway inhibitor U73122 is without effect. TGF-beta elevates steady-state mRNA levels for the endogenous PAI-1 and fibronectin genes. Treatment of cells with calphostin C or D609 prevents the TGF-beta-induced increase in these mRNAs. Together, these results suggest that PC-PLC and PKC are in a TGF-beta signaling pathway that results in elevated gene expression.
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PMID:Evidence for involvement of phosphatidylcholine-phospholipase C and protein kinase C in transforming growth factor-beta signaling. 777 10

Low-density lipoprotein (LDL) cholesterol has been implicated in the pathogenesis of glomerulosclerosis in diabetes and other forms of glomerular injury. In the present study we evaluated the effect of LDL on fibronectin synthesis in cultured rat mesangial cells (MCs) and the roles of protein kinase C (PKC) and transforming growth factor-beta (TGF-beta) in mediating this LDL action. In MCs, 25 micrograms to 100 micrograms/ml LDL increased PKC activity within 15 minutes, as reflected by enhanced in situ phosphorylation of the 80 kd myristoylated alanine-rich C kinase substrate protein, a specific endogenous substrate of PKC in MC. The same concentrations of LDL subsequently (18 to 72 hours) enhanced fibronectin synthesis, as reflected by increased incorporation of labeled methionine into fibronectin. GF 109203X, a selective inhibitor of PKC, blocked increases in both PKC activity and fibronectin synthesis induced by LDL in MCs. Furthermore, prior downregulation of PKC to less than 1% of basal activity by exposure of MCs to 0.5 mumol/L phorbol myristate acetate (PMA) also prevented LDL stimulation of fibronectin synthesis. The activation of PKC by LDL seen after 15 minutes of exposure was transient and was not observed after 4 or 48 hours of exposure of MCs to LDL. However, exposure to LDL for 48 hours, but not for 15 minutes or 4 hours, increased both maximal PKC responses to phorbol dibutyrate (PDBu) and tritiated PDBu binding to MCs by 30%. These findings suggest that chronic exposure to LDL increases the total PKC content in MCs and thereby might modulate responses to other PKC agonists. Neither the cyclooxygenase inhibitor piroxicam nor the thromboxane/prostaglandin endoperoxide receptor blocker Sq-29548 altered LDL stimulation of fibronectin synthesis in MCs, suggesting that this action of LDL was not mediated by changes in MC eicosanoid generation. By contrast, antibody to TGF-beta blocked LDL stimulation of fibronectin synthesis in MCs. TGF-beta bioactivity, determined with the mink lung epithelial cell assay, was two to three times higher in the medium of MCs cultured with LDL for 24 to 48 hours as compared with corresponding control values. Total TGF-beta bioactivity examined after heat activation of latent TGF-beta was also two times higher in the medium of MCs exposed to LDL as compared with that of controls. Prior down-regulation of PKC by exposure of MCs to PMA blocked the increases in TGF-beta bioactivity induced by LDL.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Low-density lipoprotein stimulation of mesangial cell fibronectin synthesis: role of protein kinase C and transforming growth factor-beta. 782 50

Thromboxane (TX) has been implicated in the pathogenesis of glomerulosclerosis in several models of glomerular injury. In the present study, we examined the role of the protein kinase C (PKC) signalling system in expression of the action of the TXA2/PGH2 analogue U-46619 to stimulate fibronectin (Fn) synthesis in cultured rat mesangial cells (MC), and the influence of cGMP on this MC response. U-46619 activated PKC and enhanced Fn synthesis in MC in a time and concentration dependent fashion. Both responses to U-46619 were blocked by GF 109203X, a selective inhibitor of PKC activity, as well as by calphostin C and staurosporine, PKC inhibitors structurally distinct from GFX. Down-regulation of PKC by prior sustained exposure of MC to 0.5 microM phorbol myristate acetate similarly blocked increases in Fn synthesis induced by U-46619. The TXA2/PGH2 receptor antagonist Sq-29548 also prevented activation of PKC and stimulation of Fn synthesis by U-46619, consistent with transduction of these responses via specific high affinity TXA2/PGH2 receptors on MC. Addition of exogenous 8-Br-cGMP or stimulation of endogenous cGMP generation with atrial natriuretic peptide (ANP) suppressed both U-46619 activation of PKC and stimulation of Fn synthesis. cGMP did not alter TXA2/PGH2 receptor number of affinity in MC, but significantly suppressed phorbol ester activation of PKC. Thus, cGMP inhibition of U-46619 actions is expressed at steps distal to TX receptor binding and may involve effects at and proximal to activation of PKC. Interactions between the PKC and cGMP cellular signalling systems may be important determinants of MC matrix protein production in response to TX.
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PMID:Thromboxane stimulation of mesangial cell fibronectin synthesis is signalled by protein kinase C and modulated by cGMP. 786 1

Video microscopy and digital imaging were used to quantitatively analyze lymphocyte adhesion and formation of pseudopodia on the extracellular matrix protein fibronectin (FN). A morphology kinetics assay comparing pseudopodial extension values over a 24-h period showed that HPB-ALL T leukemic cells undergo a wave of morphologic change, returning to a round shape after 8 h. Using anti-alpha 4 and anti-alpha 5 mAbs and a panel of cell types that are single or double positive for expression of the alpha 4/beta 1 and alpha 5/beta 1 FN binding integrins, it was determined that cell adhesion to FN was influenced by both beta 1-integrins, whereas alpha 4/beta 1 was found to be the major FN receptor mediating pseudopodia extension. The protein kinase inhibitor staurosporine, the protein kinase C inhibitors calphostin C and chelerythrine, and the protein tyrosine kinase inhibitor herbimycin A blocked pseudopodial extension in HPB-ALL cells. In contrast, two cAMP-dependent protein kinase inhibitors H8 and H89 did not inhibit. Inhibitors of phospholipase A2, lipoxygenases, and cyclooxygenases could block formation of pseudopodia, yet had little or no effect on cell adhesion to FN. The preincubation of cells with arachidonic acid could prevent the inhibition mediated by the reversible phospholipase A2 inhibitor cibacron blue. We conclude that the formation of lymphocyte pseudopodia in response to FN can utilize the adhesive and signaling activities of the alpha 4/beta 1-integrin and the enzymatic activities of protein kinases and phospholipases.
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PMID:Regulation of lymphocyte pseudopodia formation by triggering the integrin alpha 4/beta 1. 786 87

We investigated the role of signal transduction systems in the attachment of human uveal melanoma cells to matrix proteins. Ocular melanoma cells established from primary tumours attached rapidly to all substrates examined. Preferred substrates of attachment were collagens type I, III and IV and fibronectin rather than laminin, gelatin, arginine-glycine-aspartine, vitronectin, poly-L-lysine or plastic. All cells showed rapid attachment to the preferred substrates (80% within 10 min). Manipulation of intracellular cyclic AMP or protein kinase C activity had relatively little effect on cell attachment. In contrast, attachment was significantly reduced by manipulating either intracellular calcium or calmodulin. After 15 min at 37 degrees C, the calcium ionophore ionomycin (5 microM) reduced attachment to 25%, and TMB8 (50 microM), which can reduce intracellular calcium, reduced attachment to 60%. The experimental calmodulin antagonist J8 (25 microM), a substituted naphthalene sulphonamide, reduced attachment to 40%. Similarly tamoxifen (25 microM), which has calmodulin antagonist activity in vitro, reduced attachment to 55%. Both J8 and tamoxifen inhibited cell attachment to a wide range of matrix proteins, suggesting that this effect on attachment is not dependent on the presence of specific adhesion receptors. Reduction of ocular melanoma tumour cell/matrix interactions through manipulation of intracellular calcium or calmodulin may therefore merit further investigation as a possible approach to reducing metastatic spread.
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PMID:Investigation of the role of signal transduction in attachment of ocular melanoma cells to matrix proteins: inhibition of attachment by calmodulin antagonists including tamoxifen. 792 90

It has been previously shown that rat glomerular mesangial cells synthesized increased amounts of fibronectin, laminin, and type IV collagen when grown in medium containing 30 mM glucose. High glucose exerted its effect at the mRNA level since transcripts for all three extracellular matrix (ECM) proteins were similarly elevated. High glucose appeared to exert its effect on ECM mRNA levels through protein kinase C activation. Using quantitative reverse transcription (RT) PCR, we now report that mRNA levels for c-fos and c-jun were increased approximately twofold after treatment with high glucose. The fos levels were elevated 15 minutes after addition of high glucose and were maintained elevated through 30 minutes; by one hour mRNA levels for fos returned to control levels. c-jun, on the other hand, was increased at two hours and remained elevated at 24 and 48 hours. Fibronectin mRNA levels were increased three- to fourfold at 24 and 48 hours. Immunofluorescence studies with polyclonal antibodies to c-fos and c-jun revealed that high glucose treatment for four hours increased nuclear staining intensity two- to threefold for both proteins. Nuclear staining for fos returned to control levels by 24 hours while staining for jun remained elevated. These determinations were made on images obtained on a confocal laser scanning microscope. Thus, high glucose may effect gene expression of ECM proteins by elevating the transcription factors c-fos and c-jun which complex with one another to form activator protein 1 (AP-1).
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PMID:High glucose elevates c-fos and c-jun transcripts and proteins in mesangial cell cultures. 793 27

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