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)

Previous in vivo and in vitro studies suggest that insulin-like growth factor (IGF-I) could be a regulator of the renal production of 1,25-(OH)2D3. In the present work, the local effect of low nanomolar concentrations of IGF-I on the 25-OH-D3-1 alpha-hydroxylase activity and the mechanism of its action have been investigated. To do so, an in vitro model of mouse proximal tubular cells in primary culture has been developed. These cells bear specific high affinity IGF-I binding sites (apparent Kd = 1.95 +/- 0.46 nM) and express the ability to convert [3H]25-(OH)D3 into [3H]1,25-(OH)2D3 (Km = 139 +/- 15.7 nM). Human recombinant IGF-I (10-100 ng/ml) stimulated both sodium-dependent phosphate uptake and 1,25-(OH)2D3 synthesis by these cells, in a time- and dose-dependent manner. IGF-I did not alter the apparent Michaelis constant but increased the maximum velocity of the 25-OH-D3-1 alpha-hydroxylase activity. This effect required protein synthesis. It was not affected by calphostin or GF109203X, two protein kinase C inhibitors, and was not mimicked by phorbol 12-myristate 13-acetate. In contrast, it was blocked by verapamil, a calcium channel blocker. Calcium depletion of the medium blunted the IGF-I effect but not that of human 1-34 parathyroid hormone 5 x 10(-8) M. IGF-I thus appears to be the first example of a physiological calcium-dependent regulator of the renal metabolism of vitamin D.
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PMID:Insulin-like growth factor I, a unique calcium-dependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells. 759 14

The regulation of synthesis and phosphorylation of osteopontin in relation to avian epiphyseal growth-plate chondrocyte differentiation was studied in situ and in culture. Osteopontin gene expression was evaluated in the tibia growth-plate of 3-week-old chickens by in situ hybridization. The gene was expressed mainly at the lower hypertrophic zone where cartilage matrix is calcified and endochondral bone formation is initiated. Within the hypertrophic region, a poorly labeled area separated the layer of osteopontin-positive hypertrophic chondrocytes from those associated with endochondral bone formation. In culture, proliferative chondrocytes show no alkaline phosphatase activity in contrast to ascorbic acid-treated chondrocytes which display the enzyme activity. Chondrocytes not treated with ascorbic acid, exhibited lower levels of osteopontin mRNA than the treated cells. The phorbol ester TPA--an activator of protein kinase C--and to a lesser extent FGF but not EGF, stimulated osteopontin gene expression. Chondrocytes secreted low levels of phosphorylated osteopontin to the medium. EGF treatment resulted in the appearance of phosphorylated osteopontin in the medium, without affecting the synthesis of other proteins. FGF and TGF beta, but not IGF-I or IGF-II, also caused phosphorylation of osteopontin. Ascorbic acid-treated chondrocytes secreted higher levels of phosphorylated osteopontin than the non-treated cells, but addition of FGF or TPA did not stimulate osteopontin phosphorylation any further. Parathyroid hormone caused a dose-dependent attenuation of osteopontin phosphorylation and inhibited the EGF-dependent osteopontin phosphorylation. The results suggest that osteopontin gene expression and phosphorylation in chondrocytes are regulated by separate mechanisms. The response to the various controlling agents varies with the state of differentiation. Both processes--the synthesis and phosphorylation of osteopontin--are under the control of local growth factors which are involved in bone growth and calcification.
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PMID:Synthesis and phosphorylation of osteopontin by avian epiphyseal growth-plate chondrocytes as affected by differentiation. 765 84

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G1 phase of the cell cycle instead of G0. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G0 phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G1, 12% were in S and 37% in G2 phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P < 0.01) [3H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P < 0.01) [3H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G1 phase of the cycle to enhance IGF-I effects in cell proliferation.
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PMID:cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle. 773 22

Previously we have shown that IGF-I protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). In the present study we investigated the ability of protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the protein kinase A activator 8-bromoadenosine 3'5'-cyclic monophosphate (Br-cAMP), and the enzyme inhibitor aurintricarboxylic acid (ATA) to protect MCF-7 cells against death, due to a continuous presence of CHX. Cell death was evaluated after 48 h of incubation by several techniques (trypan blue staining, release of lactic dehydrogenase, cellular ATP content, transmission electron microscopy, and DNA fragmentation). Apoptosis which terminates in necrosis, characterized this mode of cell death. TPA and ATA at optimal concentrations of 40 ng/ml and 100 micrograms/ml, respectively, reduced cell death to the control level (without CHX), while Br-cAMP at an optimal concentration of 650 micrograms/ml reduced cell death only partially. IGF-1, TPA, and ATA, which stimulated protein synthesis in the control MCF-7 cells, had no effect on protein synthesis in the CHX-treated cells, indicating that the survival effect is not due to new protein synthesis. The protein kinase C inhibitor staurosporine blocked the survival effect of TPA and IGF-1 in a dose-dependent manner, however did not affect the survival effect of ATA. The tyrosine kinase inhibitor genistein blocked the survival effect of IGF-1, but not that of TPA and ATA. Our results provide evidence for several distinctive pathways, the activation of which protects MCF-7 cells against death, due to protein synthesis inhibition.
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PMID:Multiple pathways are involved in protection of MCF-7 cells against death due to protein synthesis inhibition. 777 99

Aluminum (Al3+) stimulates de novo bone formation in dogs and is a potent stimulus for DNA synthesis in non-transformed osteoblasts in vitro. The recent identification of a G-protein coupled cation-sensing receptor (BoPCaR), which is activated by polyvalent agonists [e.g., gadolinium (Gd3+) > neomycin > calcium (Ca2+)], suggests that a similar physiologically important cation sensing receptor may be present in osteoblasts and pharmacologically activated by Al3+. To evaluate that possibility, we assessed whether known BoPCaR agonists stimulate DNA synthesis in MC3T3-E1 osteoblasts and examined the additive effects of Al3+ and BoPCaR agonists on DNA synthesis in MC3T3-E1 osteoblast-like cells. We found that Al3+, Gd3+, neomycin, and Ca2+ stimulated DNA synthesis in a dose-dependent fashion, achieving 50% effective extracellular concentrations (EC50) of 10 microM, 30 microM, 60 microM, and 2.5 mM, respectively. Al3+ displayed non-additive effects on DNA synthesis with the BoPCaR agonists as well as an unrelated G-protein coupled receptor agonist, PGF2 alpha, suggesting shared mechanisms of action. In contrast, the receptor tyrosine kinase agonist, IGF-I (10 eta g/ml), displayed additive proliferative effects when combined with AlCl3, indicating distinct signalling pathways. AlCl3 (25 microM) induced DAG levels 2-fold and the phosphorylation of the myristoylated alanine-rich C kinase (MARCKS) substrate 4-fold, but did not increase intracellular calcium concentrations. Down-regulation of PKC by pre-treatment with phorbol 12-myristate 13-acetate as well as PKC inhibition by H-7 and staurosporine blocked Al(3+)-induced DNA synthesis. Finally, Al3+, Gd3+, neomycin, and Ca2+ activated G-proteins in osteoblast membranes as evidenced by increased covalent binding of [32P]-GTP-azidoanilide to putative G alpha subunits. Our findings suggest that Al3+ stimulates DNA synthesis in osteoblasts through a cation sensing mechanism coupled to G-protein activation and signalling cascades involving DAG and PKC-dependent pathways.
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PMID:Aluminum-induced DNA synthesis in osteoblasts: mediation by a G-protein coupled cation sensing mechanism. 780 84

To investigate the responsiveness to phorbol ester-mediated malignant cell transformation of Balb/c 3T3 variant clones that were morphologically phorbol ester-sensitive and -resistant, we used an in vitro two-stage transformation assay in which cells were treated with 0.1 microgram/ml of MCA as an initiator and subsequently with 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. The morphologically TPA-sensitive variant, TR5, and the parent cells showed a relatively low sensitivity to TPA-induced cell transformation, whereas the morphologically TPA-resistant variant, TR4 cells, exhibited 50- to 100-fold higher sensitivity to phorbol ester-induced cell transformation than the parent or the TR5 cells. We investigated the effects of TPA on protein kinase C activity, 80 kDa PKC substrate phosphorylation, the organization of actin stress fibers, DNA synthesis, and anchorage-independent growth in the three cell clones. They showed similar responses to these biological and biochemical events, indicating that these PKC-mediated events may not be the causes of the differential responsiveness of the variant cells to TPA-induced cell transformation. We further examined their responsiveness to growth factor-mediated and spontaneous induction of membrane ruffling. When these cells were stimulated by PDGF in their growing phase, membrane ruffling was rapidly induced in the three cells. However, the PDGF-mediated membrane ruffling was completely suppressed in parent and TR5 but not TR4 cells in the confluent, contact-inhibited (steady-state) growth phase. Similar responses were observed by other growth factors such as insulin, IGF-I, acidic or basic FGF. In addition, when the cells were cultured beyond confluence in the presence of TPA, spontaneous membrane ruffling was induced continuously up to termination of culture in TR4 but not in parent and TR5 cells. These results suggest that the deficiency in cell contact-mediated inhibition of membrane ruffling may be responsible for hypersensitivity of TR4 cells to TPA-induced cell transformation.
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PMID:Differential susceptibility of morphologically TPA-resistant and -sensitive Balb/c 3T3 variants to TPA-induced cell transformation: relationship to induction of membrane ruffling. 782 Aug 74

The involvement of phospholipase D (PLD) in phosphatidylcholine hydrolysis by epidermal (EGF), insulin-like (IGF-I), and basic fibroblast (bFGF) growth factors was investigated in rat pancreatic acini. Acini were prelabeled with [3H]myristic acid which is mostly incorporated into phosphatidylcholine. EGF, IGF-I, and bFGF caused significant and dose-dependent increases in [3H]phosphatidic acid (PA) accumulation in the presence of propranolol, a phosphatidic acid phosphohydrolase inhibitor. The effects of EGF and IGF-I were significant after 5, 15, and 30 min of stimulation, whereas that of bFGF was evident only at 30 min. PA production in response to all three factors was dose dependent with maximal responses to EGF at 25 nM, to IGF-I at 16.5 nM, and to bFGF at 50 pM. Preincubation of acini with staurosporine, a protein kinase C and tyrosine kinase inhibitor, totally inhibited PA production by the three factors. Similarly, acini preincubation with genistein, a specific tyrosine kinase inhibitor, also neutralized the influence of the three factors on PA accumulation. In the presence of 1% ethanol, EGF, IGF-I, and bFGF caused significant phosphatidylethanol production after 20 min of incubation, thus confirming the involvement of PLD in PA production. These data present for the first time the description of a new signaling pathway through which EGF, IGF-I, and bFGF may operate to induce some of their specific effects on the pancreas in association with these growth factor receptors' tyrosine kinase activity.
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PMID:Activation of pancreatic acinar cell phospholipase D by epidermal, insulin-like, and basic fibroblast growth factors involves tyrosine kinase. 789 61

Enzymes involved in lipid metabolism exist within the nucleus and are responsive to external stimuli. In particular, the kinases which phosphorylate phosphatidylinositol and phosphatidylinositol-4-monophosphate have been demonstrated in nuclei of both undifferentiated and differentiated Friend cells and of quiescent Swiss 3T3 cells as well as of those exposed to insulin-like growth factor I. Besides the lipid kinases, also the phosphoinositidases C (PIC) are active inside the nucleus. In Swiss 3T3 cells the nuclear PIC beta 1 is activated and its activation by IGF-I temporally precedes the translocation to the nucleus of protein kinase C. In Friend cell nuclei, on the other hand, when erythroid differentiation is induced, the PIC beta 1 activity is reduced. Another aspect of the nuclear signalling transduction system which appears quite interesting is its actual localization at subcellular level. By using electron microscope immunogold labelling, the nuclear PIC isoforms (the beta 1 isoform in Swiss 3T3 cells, the beta 1 and gamma 1 in Friend cells) are localized mainly in the interchromatin domains. This localization has been further confirmed on in situ matrix preparations of 3T3 cells in which PIC beta 1 is associated with the inner nuclear matrix but not with the nuclear pore-lamina complex. Colocalization experiments indicate that nuclear PIC beta 1 is present in sites in which both nuclear phospholipids and PKC can be detected, while the cytoplasmic PIC gamma 1 can be identified in close association with cytoskeletal filaments identified by anti-actin antibodies. The precise localization of the different PIC isoforms strongly indicates that the signal transduction system operating at the nuclear level may be part of a cross-talk between the cytoplasm and the nucleus controlling either cell proliferation or differentiation.
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PMID:Lipid-dependent nuclear signalling: morphological and functional features. 794 70

To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent Swiss 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF-I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan-anti-PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub-cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the translocation process, we have tested nine isozyme-specific anti-PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of alpha, beta I, epsilon and zeta isoforms. In isolated nuclei from GF-exposed cells only the alpha isozyme was detected: immunostaining was very intense after IGF-I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC alpha and, because of the dramatic effect of IGF-I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF.
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PMID:Selective nuclear translocation of protein kinase C alpha in Swiss 3T3 cells treated with IGF-I, PDGF and EGF. 801 64

Based on the unique susceptibility of the neonatal pulmonary circulation to hypoxia-induced structural alteration in vivo, we hypothesized that pulmonary artery (PA) smooth muscle cells (SMC) from the neonate would demonstrate enhanced growth capacity in vitro compared to adult cells. To test this hypothesis, matched neonatal and adult bovine SMC were tested for differences in size, serum-stimulated proliferation, susceptibility to senescence, resistance to serum withdrawal, autocrine growth capacity, and responsiveness to a locally important growth factor (insulin-like growth factor I; IGF-I) and an activator of protein kinase C (PKC) (phorbol 12-myristate 13-acetate; PMA). Neonatal PA SMC were smaller, grew faster, reached a higher plateau density, and were less susceptible to senescence. They were more resistant to serum withdrawal, had spontaneous autocrine growth capacity, and were more responsive to IGF-I, PMA, and the combination. Acquisition of increased growth factor responsiveness occurred between d5 and d14 after birth. Increased neonatal growth to IGF-I was associated with reduced IGF-I binding activity, implicating a post-receptor mechanism in enhanced responsiveness. Increased membrane-bound PKC catalytic activity was found in serum-deprived neonatal SMC. This basal increase was equal to that stimulated by 1 nM PMA in adult SMC, a pretreatment that caused these cells to become as responsive to IGF-I as untreated neonatal ones. We conclude that neonatal bovine PA SMC have marked enhancement of growth capacity in vitro, the acquisition of which is dependent on time from birth and is associated with auto-activation of PKC, These increased growth properties detected in vitro may contribute to the striking hyperplasia of neonatal PA SMC found in vivo following hypoxic exposure.
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PMID:Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: dependence on cell size, time from birth, insulin-like growth factor I, and auto-activation of protein kinase C. 807 85


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