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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Osteoblast-like cells, such as UMR 106 osteosarcoma cells, are known to be growth stimulated by growth factors such as EGF. In contrast, factors such as PTH and prostaglandin E2 inhibit their growth. The exact signal transduction mechanisms by which these latter factors act remain to be elucidated. Here we show that simultaneous treatment of UMR 106 cells with EGF and PTH-(1-34) resulted in a level of DNA synthesis intermediate between the levels of treatment with epidermal growth factor (EGF) and PTH alone. This correlated with the interference of PTH-(1-34) early in an EGF receptor-linked signal transduction pathway, i.e. the EGF-induced activation of p42 mitogen-activated protein (MAP) kinase. This effect was also found for prostaglandin E2, and could be potentiated by the phosphodiesterase inhibitor isobutyl-methylxanthine and mimicked by forskolin and 8-bromo-cAMP. There was a strict correlation between the lowest concentration of PTH-(1-34) required to enhance protein kinase A (PKA) activity and that required to inhibit MAP kinase activation, whereas saturating amounts of PTH-(3-34), a PTH analog unable to elevate PKA activity, had no effect. Lysophosphatidic acid- and 12-O-tetracanoylphorbol-13-acetate-induced MAP kinase activation were also inhibited by PTH-(1-34) and forskolin in these cells. Similar effects were seen on basic fibroblast growth factor-mediated MAP kinase activation in ROS 17/2.8 cells, indicating that this mechanism is a general feature of PTH in osteosarcoma cells. The inhibition of this mitogenic pathway through activation of PKA might play an important role in PTH-induced changes in proliferation and differentiation of osteoblasts.
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PMID:Parathyroid hormone inhibits mitogen-activated protein kinase activation in osteosarcoma cells via a protein kinase A-dependent pathway. 762 68

The objectives of the present study were to determine whether the G protein-coupled receptor for PTH and PTH-related protein (PTHrP) is subject to agonist-specific phosphorylation and to characterize the relevant kinase(s). The opossum kidney PTH/PTHrP receptor stably expressed in human embryonic kidney 293 cells was coupled to adenylyl cyclase, with half-maximal activation occurring in the presence of 0.1 nM bovine (b) PTH-(1-34). Immunoprecipitation of extracts of 32P-labeled cells using a monoclonal antibody to the PTH/PTHrP receptor revealed the presence of a major 32P-labeled protein of approximately 85 kilodaltons that was not evident in untransfected 293 cells. bPTH-(1-34) treatment produced a rapid dose-dependent increase in phosphorylation of the 85-kilodalton receptor, with a maximal effect that was 3.5 +/- 0.7-fold (n = 4) over basal. Half-maximal phosphorylation occurred with 10 nM bPTH-(1-34), similar to the hormone concentration required for 50% receptor occupancy. Activation of protein kinase A or protein kinase C with forskolin or phorbol 12-myristate 13-acetate also increased PTH/PTHrP receptor phosphorylation, but to a lesser degree than PTH. Neither of these kinases mediated the effect of PTH, as blockade of the protein kinase A pathway (with H-89) or the protein kinase C pathway (with the bisindolylmaleimide GF 109203X) did not inhibit bPTH-(1-34)-induced PTH/PTHrP receptor phosphorylation. These results suggest that agonist-stimulated PTH/PTHrP receptor phosphorylation may involve a nonsecond messenger-activated kinase, such as a member of the G protein-coupled receptor kinase family.
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PMID:Agonist-stimulated phosphorylation of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein. 766 44

We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FlCRhR. FlCRhR was microinjected into single cells, and the 500-530/> 560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10(-7) M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 +/- 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10(-7) M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10(-6) M) stimulation was able to increase the FlCRhR ratio (154 +/- 27%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10(-7) M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.
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PMID:Single-cell analysis of cyclic AMP response to parathyroid hormone in osteoblastic cells. 781 24

Osteoclast-mediated bone resorption plays a crucial role in osseous remodeling. Osteoblasts are important regulators of this activity, in part through their ability to produce osteoclast-regulating soluble factors such as interleukin-6 (IL-6). IL-11 is a newly appreciated pleotropic cytokine whose spectrum of biological activities overlaps with that of IL-6. As a result, we hypothesized that osteoblasts are an important skeletal source of this cytokine. To test this hypothesis, we characterized the IL-11 production of unstimulated and stimulated SaOS-2 human osteosarcoma cells. Unstimulated cells produced modest amounts of IL-11. The osteotropic agents recombinant IL-1 (0.25-5 ng/ml), transforming growth factor-beta 1 (0.1-10 ng/ml), PTH (10(-8)-10(-11) M), and PTH-related peptide ((10(-8)-10-11 M) further increased SaOS-2 cell IL-11 protein production and messenger RNA accumulation. These stimulatory effects were dose and time dependent, and the IL-11 that was produced was bioactive, as demonstrated by its ability to stimulate the proliferation of T10D plasmacytoma cells. The protein kinase-C activator, 12-O-Tetra-decanoylphorbol 13-acetate, and a variety of cAMP agonists [forskolin, prostaglandin E1, prostaglandin E2, and (Bu)2AMP] also stimulated osteoblast IL-11 protein production and messenger RNA accumulation. In contrast, recombinant IL-4, recombinant interferon-gamma, and endotoxin did not stimulate SaOS-2 cells in a similar fashion. Importantly, the ability to produce IL-11 was not a unique property of SaOS-2 cells, because primary human trabecular bone osteoblasts also produced significant amounts of bioactive IL-11 when stimulated with transforming growth factor-beta 1. These studies demonstrate that appropriately stimulated human osteoblasts and osteoblast-like cells are potent producers of IL-11 and suggest that osteoblast-derived IL-11 may be an important component of the cytokine network mediating osteoblast-osteoclast communication in normal and pathological bone remodeling.
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PMID:Cytokine and hormonal stimulation of human osteosarcoma interleukin-11 production. 783 81

The regulation of vitamin D receptor (VDR) abundance in MC3T3-E1 mouse osteoblasts and UMR 106-01 rat osteosarcoma cells by rat PTH 1-34, human PTH-related protein 1-34, and agents that activate specific signal transduction pathways was studied. Treatment of these cells with forskolin (FSK) caused up-regulation of VDR, whereas treatment with phorbol esters suppressed VDR levels. PTH or PTH-related protein treatment induced a 2- to 3-fold increase in VDR, which was equivalent to that elicited by FSK in UMR 106-01 cells but less than the FSK-induced increase (approximately 8-fold) in MC3T3-E1 cells. PTH treatment of MC3T3-E1 cells resulted in an approximately 3-fold increase in VDR levels with maximum stimulation occurring at 10(-9) M PTH after 4 h of treatment. In UMR 4-7 cells, a subclone of UMR 106-01 cells that express cAMP resistance due to regulated expression of a mutant form of the type 1 regulatory subunit of the cAMP-dependent protein kinase A (PKA), the up-regulation of VDR abundance due to FSK and PTH treatment was mostly prevented. Pretreatment of MC3T3-E1 cells with staurosporine, an inhibitor of PKC, resulted in an approximately 3-fold increase in basal VDR levels but did not enhance the PTH-mediated up-regulation of VDR. Collectively, these data suggest that the increase in VDR abundance observed in these target cells is mainly due to the activation of the PKA signal transduction pathway. Treatment of UMR 106-01 cells with PTH for 4 h before exposure of the cells to 1,25-dihydroxyvitamin D3 resulted in a 2-fold increase in the induction of 25-hydroxyvitamin D3-24 hydroxylase messenger RNA. Thus, exposure of target cells to PTH augments their response to 1,25-dihydroxyvitamin D3 due to up-regulation of VDR abundance.
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PMID:Regulation of 1,25-dihydroxyvitamin D3 receptors by parathyroid hormone in osteoblastic cells: role of second messenger pathways. 783 3

PTH-induced phosphaturia is exerted in part by cAMP added to the renal tubular lumen under the influence of the hormone. Modulation of renal phosphate transport by luminal cAMP requires degradation of the nucleotide into adenosine by brush-border membrane ectoenzymes, among them ecto-5'-nucleotidase (5'-NU). Hormonal modulation of 5'-NU activity was evaluated in cultured opossum kidney cells. PTH (1-100 nM) stimulated 5'-NU in a time-, concentration-, and protein synthesis-dependent manner. The effect of PTH-(1-34) was mimicked by PTH-(3-34), which does not activates adenylate cyclase, and by phorbol 12-myristate 13-acetate (PMA), but not by forskolin or (Bu)2cAMP. Down-regulation or pharmacological inhibition of protein kinase-C (PKC) abolished the effect of PTH fragments and PMA. PTH fragments increased intracellular Ca2+ and translocated PKC activity to the membrane. PTH or PMA did not affect 5'-NU messenger RNA content. Inhibition of sodium-phosphate cotransport by extracellular cAMP was decreased by 5'-NU inhibition and was magnified by PTH. These results indicate that 1) PTH stimulates 5'-NU activity in renal proximal tubular cells in a manner involving PKC activation and de novo protein synthesis; and 2) this effect participates in PTH modulation of renal phosphate transport.
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PMID:Parathyroid hormone stimulates ecto-5'-nucleotidase activity in renal epithelial cells: role of protein kinase-C. 786 81

Both PGE2 and PTH (1-34) caused a time- and concentration-dependent stimulation of proliferation by embryonic chick periosteal cells. Cells were exposed to the agents for different periods of time, the medium was replaced with fresh medium, and 3H-TdR incorporation was measured after 16 hours. Challenge with 10(-6) M prostaglandin E2 (PGE2) or 10(-7) M parathyroid hormone (1-34) (PTH) for 5 minutes produced 4- and 5.5-fold increases in 3H-TdR incorporation, respectively. Longer exposures, however, produced diminishing responses and after 45 minutes, only minimal effects or slight inhibitions were seen. These time-dependent effects were also seen with forskolin and dibutyryl-cAMP; TPA on the other hand stimulated DNA synthesis after both short- and long-term exposure. Both PGE2 and PTH (1-34) stimulated cAMP synthesis in periosteal cells but neither could be shown to stimulate protein kinase-C (PKC) at concentrations required for stimulation of proliferation, and dibutyryl-cyclic AMP (cAMP) effectively inhibited endogenous PKC activity. It is possible that the stimulation of proliferation by short-term exposure to PGE2 and PTH (1-34) is mediated by cAMP and that the time dependency possibly stems from the inhibition of endogenous PKC activity by increased intracellular cAMP levels.
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PMID:Time-dependent effects of parathyroid hormone and prostaglandin E2 on DNA synthesis by periosteal cells from embryonic chick calvaria. 798 35

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is believed to act as a local vasorelaxant by activating either the classical PTH or a unique PTHrP receptor. We used a newly cloned complementary DNA encoding the rat PTH/PTHrP receptor to study the expression of its messenger RNA (mRNA) in primary aortic vascular smooth muscle cells (VSMC) and in UMR-106 osteoblast-like cells under basal conditions and in response to treatment with agonists. Both cell types expressed a 2.4-kilobase PTH/PTHrP receptor mRNA transcript and exhibited hormone-induced desensitization of PTHrP-(1-34)NH2-stimulated cAMP. In VSMC, angiotensin-II, which induces PTHrP expression, also rapidly (30 min) desensitized the cAMP response and down-regulated (75-90%) receptor mRNA within 1 h. Treatment of cells with phorbol 12-myristate 13-acetate (0.1 microM) mimicked these effects, whereas neither PTHrP-(1-34)NH2, forskolin, nor (Bu)2cAMP altered receptor mRNA expression. By contrast, in UMR-106 cells, PTHrP-(1-34)NH2 induced time- and dose-dependent decreases in receptor mRNA that were preceded by pronounced desensitization (cAMP and ligand binding) of cell surface receptors. These effects were mimicked by (Bu)2cAMP and forskolin, but not by phorbol 12-myristate 13-acetate, suggesting that both receptor mRNA down-regulation and receptor desensitization in UMR cells were mediated through a protein kinase-A pathway. We suggest that VSMC and UMR cells express a common receptor, which is subject to cell-specific regulation. Such diversity in the PTH/PTHrP receptor regulatory mechanisms provides a means for restricting the length and duration of the cellular response to hormone in a cell/tissue-specific manner.
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PMID:Parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor and its messenger ribonucleic acid in rat aortic vascular smooth muscle cells and UMR osteoblast-like cells: cell-specific regulation by angiotensin-II and PTHrP. 807 Mar 51

Our recent study demonstrated the direct involvement of cAMP-dependent protein kinase (PKA) in the regulation of DNA synthesis by PTH-related peptide (PTHrP) in osteoblastic osteosarcoma cells, UMR-106. Since PTHrP has been reported to possess dual signal transduction systems [PKA and calcium/protein kinase C (Ca/PKC]), present study was performed to characterize the involvement of Ca/PKC signal transduction system in the regulation of DNA synthesis by PTHrP in these cells. Human PTHrP-(1-34) (10(-7) M) caused a rapid increase in intracellular Ca ([Ca2+]i), followed by return to the basal level within 1 min. Pretreatment with 10(-4) M TMB-8 or 10(-5) M dantrolene, inhibitors of calcium release from intracellular calcium store, significantly blocked the PTHrP-induced increase in [Ca2+]i, but did not affect the PTHrP-induced inhibition of DNA synthesis. Pretreatment with 50 uM H-7 or 1 nM staurosporine, inhibitors of PKC, significantly blocked the PTHrP (10(-9) to 10(-7) M)-induced inhibition of DNA synthesis. Pretreatment with 10(-6) M phorbol 12-myristate 13-acetate, which downregulated PKC, significantly blocked the inhibition of DNA synthesis by PTHrP. Present study indicates that in addition to PKA activation, PKC activation is coupled to the regulation of DNA synthesis by PTHrP in osteoblasts.
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PMID:Role of calcium/protein kinase C in the regulation of DNA synthesis by parathyroid hormone-related peptide in osteoblastic osteosarcoma cells. 811 63

Homologous down-regulation of PTH/PTH-related peptide (PTHrP) receptor expression occurs in several PTH-responsive osteoblastic cell lines, but the mechanisms responsible are not well understood. We have used wild-type SaOS-2 human osteoblastic cells, in which homologous PTH/PTHrP receptor down-regulation occurs within 4 h, and a mutant cAMP-resistant subclone (Ca4A strain), to investigate the mechanisms by which PTH/PTHrP receptor mRNA is regulated. SaOS-2 cells expressed a single 2.2- to 2.5-kilobase transcript of PTH/PTHrP receptor mRNA, as assessed by Northern blot analysis of total RNA with a cDNA probe encoding the human PTH/PTHrP receptor. Homologous down-regulation of this PTH/PTHrP receptor mRNA first became significant when SaOS-2 cells had been treated with human (h) PTH-(1-34) (10(-7) M) for 8-12 h. By 24 h, steady state levels of PTH/PTHrP receptor mRNA were reduced by about 50%. This effect was mimicked by both (Bu)2cAMP (DBcAMP; 0.5 mM) and forskolin (Fsk; 10(-5) M). In contrast, down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34), DBcAMP or Fsk was almost completely blocked in cAMP-resistant Ca4A cells. Short term (4-6 h) treatment with hPTH-(1-34), DBcAMP, or Fsk did not reduce steady state levels of PTH/PTHrP receptor mRNA in either SaOS-2 or Ca4A cells, although down-regulation was induced by 4-6 h of treatment with active phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (200 nM) or phorbol-12,13-didecanoate (200 nM). Neither thapsigargin (1 microM) nor ionomycin (200 nM), both of which stimulate calcium transients in these cells, altered PTH/PTHrP receptor mRNA expression. Treatment with hPTH-(39-84) and hPTH-(53-84), which do not activate either cAMP-dependent protein kinase or protein kinase-C, but do stimulate 45Ca2+ uptake in these cells, did not alter PTH/PTHrP receptor mRNA expression. In the presence of actinomycin-D (1 microgram/ml), down-regulation of PTH/PTHrP receptor mRNA by hPTH-(1-34) was not observed. Cycloheximide (10 micrograms/ml) did not block down-regulation of PTH/PTHrP receptor mRNA induced by hPTH-(1-34). We conclude that homologous down-regulation of PTH/PTHrP receptor mRNA in SaOS-2 cells occurs later than the decline in functional surface receptors via a mechanism that does not involve enhanced mRNA degradation or new protein synthesis, but is dependent upon cAMP/cAMP-dependent protein kinase.
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PMID:Role of protein kinase-A in homologous down-regulation of parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in human osteoblast-like SaOS-2 cells. 813 52


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