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)

To explore the role of glomerular endothelial cells (GEN) in the pathogenesis of glomerulonephritis, the in vitro production of monocyte chemoattractant protein-1 (MCP-1) by bovine GEN was determined by chemotaxis assay, and Western blot analysis, and immunocytochemistry. Monocyte chemotactic activity of GEN-conditioned media was detectable by a chemotaxis assay using human peripheral blood monocytes. Exposure to human recombinant interleukin-1 beta (IL-1 beta) and phorbol myristate acetate (PMA) significantly increased the chemotactic activity of GEN-conditioned media. A checkerboard analysis showed that the response of monocytes to GEN-conditioned media was truly chemotactic. Immunoadsorption with a monoclonal antibody to human MCP-1 reduced the chemotactic activity of GEN-conditioned media by 85%. Northern blot analysis revealed that MCP-1 mRNA was constitutively expressed by GEN and that IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) increased MCP-1 mRNA levels in a dose- and time-dependent manner. Furthermore, PMA induced an increase in MCP-1 mRNA levels, whereas dibutyryl cyclic AMP and forskolin had minimal effects. Inhibition study using protein kinase inhibitors revealed that MCP-1 mRNA expression induced by IL-1 beta and TNF-alpha was suppressed by the tyrosine kinase inhibitor genistein, not by the protein kinase C inhibitors staurosporine or H-7, or the protein kinase A inhibitor H-89, suggesting an important role of tyrosine kinase in the cytokine-induced MCP-1 gene expression. Dexamethasone had a small inhibitory effect on constitutive MCP-1 mRNA expression, but no effect on the induction by TNF-alpha. By immunoperoxidase staining and Western blot analysis using an anti-MCP-1 monoclonal antibody. MCP-1 protein was detected in untreated GEN and increased by exposure to TNF-alpha. These results demonstrate the cytokine-induced production of MCP-1 by GEN at gene and protein levels as well as bioactivity, and suggest that GEN may participate in the development of glomerulonephritis through the production of MCP-1.
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PMID:Production of monocyte chemoattractant protein-1 by bovine glomerular endothelial cells. 858 46

Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well-recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS-2 or cyclooxygenase-2, that is up-regulated in many cell systems by cytokines and growth factors and down-regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E2 (PGE2) after stimulation with either interleukin-1 beta (IL-1 beta) or the protein kinase C activator phorbol 12-myristate 13-acetate (TPA). This increase in PGE2 content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS-2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL-1 beta. Dexamethasone inhibited this induction of PGHS-2 mRNA by IL-1 beta. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor alpha and lipopolysaccharide, but not interleukin-6, also stimulated PGHS-2 mRNA expression. Relative to IL-1 beta, the greater increases in PGE2 production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS-2 mRNA. Furthermore NS-398, a specific inhibitor of cyclooxygenase-2, blocked > 80% of the cyclooxygenase activity in TPA-treated astrocytes. These findings indicate that increased expression of PGHS-2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors.
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PMID:Interleukin-1 beta induces prostaglandin G/H synthase-2 (cyclooxygenase-2) in primary murine astrocyte cultures. 863 79

Prostaglandin E2 (PGE2) levels are elevated in malignant human breast tissue. However, the cellular mechanisms regulating this arachidonate metabolism and the autocrine influence PGE2 production may have on breast cancer cell growth and function are unclear. In the present study, we have investigated the effects of 2 putative cyclo-oxygenase inducers, interleukin-1beta (IL-1beta) and the protein kinase C agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on PGE2 production, growth and aromatase activity in the MDA MB 231 breast cancer cell line. TPA stimulated a dose-dependent increase in PGE2 production, inhibited cell growth and stimulated aromatase activity. Although IL-1beta alone had no effect on any of these breast cancer cell functions, the cytokine greatly potentiated PGE2 production in the presence of TPA. Similarly, growth inhibition and aromatase stimulation in response to TPA were both further enhanced by IL-1beta treatment. Indomethacin and dexamethasone both prevented PGE2 production in response to IL-Ibeta and TPA but had no effect on the anti-proliferative action of the cytokine and phorbol ester. While indomethacin had no effect on induction of aromatase activity by IL-1beta and TPA, dexamethasone exhibited a temporally biphasic action. Dexamethasone alone stimulated aromatase activity and demonstrated a permissive action on aromatase stimulation by IL-1beta and TPA. However, pre-treatment of cells with dexamethasone prevented subsequent induction of aromatase activity by IL-1beta and TPA. Our study describes a novel synergistic interaction in response to protein kinase C activation and IL-1beta during the regulation of arachidonate metabolism, cell growth and aromatase activity in human breast cancer cells. We conclude that the cyclooxygenase pathway does not play a mediatory role during the inhibition of cell growth and the induction of aromatase activity by IL-1beta and TPA.
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PMID:Regulation of arachidonic acid metabolism, aromatase activity and growth in human breast cancer cells by interleukin-1beta and phorbol ester: dissociation of a mediatory role for prostaglandin E2 in the autocrine control of cell function. 878 59

The mouse adrenocortical Y-1 cell line expresses a high level of neuropeptide Y1 receptor (NPY-Y1). Moreover the receptor density can be up-regulated by dexamethasone or down-regulated by cAMP. To determine whether such regulation occurs at the level of gene expression, Y1 receptor mRNA was measured using a reverse transcriptase-competitive PCR method. Dexamethasone treatment increased Y1 mRNA in Y-1 cells, whereas the cAMP and ACTH decreased it. We also observed that the amount of Y1 receptor RNA was unaffected by phorbol 12-myristate 13-acetate, a protein kinase C stimulator, but was abolished in a cell line expressing apolipoprotein E (apoE). The results indicated that NPY-Y1 receptor mRNA in Y-1 cells is highly regulated by several intracellular messengers. The role of apoE in such regulation is of particular interest in view of evidence that the isoform of the molecule is highly correlated to the age of onset of Alzheimer's disease. The effect observed in the Y-1 cell line which expresses apoE may implicate a possible role of this protein in the process of neuronal death that occurred in the Alzheimer's disease.
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PMID:Neuropeptide Y receptor gene regulation in mouse adrenocortical Y-1 cells. 879 89

Tumor necrosis factor (TNF) may contribute to the pathogenesis of inflammatory airway disorders via the regulation of inflammatory and cellular immune responses. Shed cell surface TNF receptors can act as soluble TNF binding proteins and modulate TNF biological activity. We report that normal human airway epithelial cells, as well as two human airway epithelial cell lines, shed soluble type I TNF receptors (sTNF-RI) in a concentration-dependent fashion following protein kinase C (PKC) activation by PMA. Interleukin (IL)-1beta also induced concentration-dependent sTNF-RI shedding from NCI-H292 cells, which could be inhibited by the PKC inhibitor calphostin C. Since corticosteroids are commonly utilized as antiinflammatory agents in airway disorders, the effect of dexamethasone on sTNF-RI release was assessed. Dexamethasone inhibited constitutive, as well as PMA- and IL-1beta-mediated sTNF-RI release from NCI-H292 cells in a concentration-dependent fashion. Furthermore, dexamethasone increased while PMA decreased total cellular 55 kDa TNF-RI protein as detected by immunoblotting. These changes in total cellular 55kDa TNF-RI protein did not appear to be mediated at the mRNA level, as assessed by ribonuclease protection assays. This suggests that sTNF-RI shedding represents a mechanism by which airway epithelial cells can actively participate in local cytokine networks and modulate TNF-mediated inflammation. Furthermore, since corticosteroids inhibit sTNF-RI release and are known to downregulate TNF synthesis, this may represent a mechanism by which equilibrium between TNF ligand and soluble binding protein is maintained in the airway microenvironment.
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PMID:Protein kinase C, interleukin-1 beta, and corticosteroids regulate shedding of the type I, 55 kDa TNF receptor from human airway epithelial cells. 884 76

Interleukin-1 beta (IL-1 beta) stimulates osteoclast-like cell formation via prostaglandin E2 (PGE2) production. However, the regulatory mechanism for the production of PGE2 in bone cells is still unclear. Recently, it has been shown that two prostaglandin endoperoxide H synthase (PGHS) isozymes exist, termed PGSH-1 and PGHS-2. We report here that IL-1 beta induces PGE2 production in bone marrow culture induced by a PGHS-2-dependent mechanism. IL-1 beta stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) and the production of PGE2 in mouse bone marrow cultures. The dose response curves for the indomethacin inhibition of TRAP-positive MNC formation and PGE2 production were nearly identical. Cycloheximide (CHX) suppressed IL-1 beta-induced PGE2 production, suggesting that the production of PGE2 induced by IL-1 beta required de novo protein synthesis. Northern blot analysis determined that IL-1 beta induced PGHS-2 expression by 30 minutes and mRNA levels were maximal by 1-2 h. Cycloheximide potentiated the accumulation of PGHS-2 mRNA linearly up to 8 h. Dexamethasone, an inhibitor of the induction of PGHS-2, inhibited IL-1 beta-induced PGHS-2 mRNA expression and also suppressed IL-1 beta-stimulated formation of TRAP-positive MNC. Furthermore NS-398, as a selective PGHS-2 inhibitor, completely inhibited IL-1 beta-induced TRAP-positive MNC formation. Moreover, IL-1 beta-induced PGHS-2 mRNA expression and formation of TRAP-positive MNC were inhibited by calphostin C, a selective inhibitor of protein kinase C (PKC). These results indicate that IL-1 beta-induced formation of osteoclast-like cells requires PKC activation, induction of PGHS-2, and subsequent prostaglandin synthesis by this enzyme.
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PMID:Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast-like cell formation induced by interleukin-1 beta. 885 50

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E2 (PGE2) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE2 provoked a 3.9 +/- 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca++ channel blockers, verapamil and nifedipine, and the Ca++/calmodulin inhibitor, W-7. The Ca+2 ionophore, ionomycin, mimicked the effects of PGE2 as did the phorbol ester PMA, which activates Ca++/-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE2 did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE2 secretagogue, IL-1 beta, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 +/- 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE2 does not mediate the effect of its secretagogue and that IL-1 beta signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2 receptor signalling pathways. Taken together, our results suggest that PGE2 modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism.
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PMID:Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: role of calcium and protein kinase A and C. 891 83

The regulation of proenkephalin (proENK) mRNA levels by cAMP and protein kinase C (PKC) pathways was studied in cultured rat spinal cord cells in the present study. Spinal cord cells were cultured from 14 day (E 14) embryos of Sprague-Dawley rats. After 7 days in vitro, the spinal cord cells were incubated with either forskolin (5 microM) or phorbol-13-myristate acetate (PMA; 2.5 microM) for 1, 3, 6, 9, 12 or 24 h and total RNA and proteins were isolated for Northern and Western blot analyses. The proENK mRNA level began to increase within an hour, then reached and remained at a peak 3-12 h after stimulation by both forskolin and PMA. The increased proENK mRNA level in forskolin-treated cells was slightly decreased 24 h after the stimulation, whereas the level of proENK mRNA returned to basal levels in PMA-treated cells. A Western blot assay revealed that the intracellular level of proENK protein was not changed by treatment with either forskolin or PMA. Pretreatment of cells with cycloheximide (a protein synthesis inhibitor; 10 microM) did not affect the forskolin- or PMA-induced increase of proENK mRNA. However, pretreatment with nimodipine (an L-type Ca2+ channel blocker; 2 microM), omega-conotoxin (an N-type Ca2+ channel blocker; 1 microM), calmidazolium (a calmodulin antagonist; 1 microM) or KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor; 5 microM) attenuated the forskolin- or PMA-induced increase of proENK mRNA levels. Dexamethasone (1 microM) did not affect the forskolin-induced increase of proENK mRNA levels. Our results suggest that the elevation of proENK mRNA levels in the spinal cord is regulated by both cAMP and PKC pathways. Calcium influx through both L- and N-type calcium channels, calmodulin and Ca2+/calmodulin-dependent protein kinase II appear to be involved in the increase of proENK mRNA levels induced by either forskolin or PMA. Furthermore, ongoing protein synthesis is not required for forskolin- or PMA-induced alterations in proENK mRNA.
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PMID:Molecular mechanisms underlying the regulation of proenkephalin gene expression in cultured spinal cord cells. 892 15

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E2 (PGE2) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE2 provoked a 2.7 +/- 0.3- and 3.8 +/- 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca(++) channel blocker, verapamil, and the Ca(++)/calmodulin inhibitor, W-7. The Ca(++)ionophore, ionomycin, mimicked the effects of PGE2. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE2 secretagogue, interleukin-1 beta (IL-beta). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE2 synthesis, down-regulated control, constitutive levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca(++) and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE2 activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes.
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PMID:Prostaglandin E2 stimulates insulin-like growth factor binding protein-4 expression and synthesis in cultured human articular chondrocytes: possible mediation by Ca(++)-calmodulin regulated processes. 913 96

Exposure of mouse macrophages to either phorbol ester or certain bacteria was previously shown to cause increased phosphorylation of the cytosolic 85 kDa phospholipase A2 as well as a stable increase in its catalytic activity. We have now attempted to map the major phosphorylation sites on the enzyme in such cells. Phosphorylation occurred on serine residues without a detectable increase in either phosphothreonine or phosphotyrosine. After CNBr cleavage five fragments showed increased 32P labelling. Among those the most heavily labelled fragment was identified as the most C-terminal (residues 698-749), containing six serine residues. This was true whether phorbol ester or bacteria, causing protein kinase C-independent phospholipase A2 activation, was used as stimulus. The heavy phosphorylation of the most C-terminal fragment and an analysis of tryptic peptides derived from it suggested that more than one of the six serine residues became phosphorylated. Smaller increases also occurred in other CNBr-cleaved fragments from the C-terminal part of the protein, including that carrying Ser-505, a known target of the mitogen-activated protein kinase ERK-2 (extracellular-signal regulated kinase). Dexamethasone treatment (1-100 nM for 20 h), which was earlier shown to dose-dependently down-regulate the 85 kDa phospholipase A2 and its activation by phorbol ester and zymosan, was here shown also to counteract the protein kinase C-independent activation and arachidonate release elicited by bacteria. It remains to be determined whether all phosphorylation sites are equally affected under those conditions.
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PMID:Multiple C-terminal serine phosphorylation accompanies both protein kinase C-dependent and -independent activation of cytosolic 85 kDa phospholipase A2 in macrophages. 923 Jan 20


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