Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:APRD00369 (ROS)
 19,271 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study the involvement of protein kinase-C (PKC) in the regulation of the vitamin D receptor (VDR) and interaction of PKC with cAMP-induced up-regulation of VDR in osteoblast-like cells were examined. Activation of PKC by incubation for 4 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a comparable dose-dependent decrease in 1,25-dihydroxyvitamin D3 binding in the osteoblast-like cell lines UMR 106 and ROS 17/2.8, with a maximum inhibition at 100 nM and an IC50 at 5 nM PMA. Time-course studies revealed that in both UMR 106 and ROS 17/2.8 cells, 24-h incubation with PMA caused an increase in 1,25-dihydroxyvitamin D3 binding. This can be related to down-regulation of PKC. Scatchard analysis demonstrated that activation of PKC resulted not in a change in receptor affinity, but, rather, in an increase in VDR number. This is supported by Northern blot analysis, which shows at 2 h a decrease and at 24 h an increase in VDR mRNA. At 4 h, when activation of the cAMP pathway results in an increase in VDR, activation of PKC results in a decrease in VDR. Coincubation for 4 h with PMA caused a decrease in PTH- and forskolin-induced up-regulation of VDR. This inhibition is not due to a reduction in cAMP production, as PTH-stimulated cAMP production was potentiated by PMA. The effect of activation of PKC on VDR is not a general effect, as PMA does not affect basal ornithine decarboxylase activity and potentiates PTH-induced ornithine decarboxylase activity. The present study demonstrates that PKC is involved in the regulation of VDR in UMR 106 and ROS 17/2.8 and that PKC interacts with cAMP in the regulation of VDR. The current data point to a negative controlling role for PKC in the regulation of VDR. Moreover, two different cAMP-regulated actions in UMR 106 cells (VDR up-regulation and ornithine decarboxylase activity) are differently modulated by PKC. Although the precise mechanism by which PKC represses and stimulates gene expression is not yet clear, this study demonstrates the important regulatory role for PKC in two osteoblast-like sarcoma cell lines.
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PMID:Bidirectional regulation of the 1,25-dihydroxyvitamin D3 receptor by phorbol ester-activated protein kinase-C in osteoblast-like cells: interaction with adenosine 3',5'-monophosphate-induced up-regulation of the 1,25-dihydroxyvitamin D3 receptor. 131 52

PTH receptors on osteoblasts and calcitonin receptors on osteoclasts are coupled to adenylate cyclase. Despite similar transduction mechanisms, these hormones have opposing physiological actions. We investigated the consequences of persistent protein phosphorylation on bone resorption in neonatal mouse calvariae using okadaic acid (OA) and calyculin-A, two inhibitors of protein phosphatase-1 and -2A. These two inhibitors caused different responses in bone at picomolar and low nanomolar concentrations. OA inhibited, in a dose-dependent manner, bone resorption stimulated by PTH, 1,25-Dihydroxyvitamin D3, phorbol ester, and prostaglandin E2 (PGE2). OA did not inhibit the generation of the second messengers cAMP or PGs and did not have nonspecific toxic effects, as measured by protein and RNA synthesis. Thus, OA appeared to mimic the global inhibitory action of calcitonin on bone resorption. Unlike OA, calyculin-A elicited a biphasic dose response. At concentrations of 3.3 nM and greater, calyculin-A inhibited, in a dose-dependent manner, stimulated bone resorption. However, calyculin-A alone, at 0.625 and 2.5 nM, stimulated bone resorption via a PG-independent pathway. In calvariae, OA and calyculin-A increased phosphorylation of a 58- to 60-kilodalton protein. A protein of similar molecular mass was hyperphosphorylated in OA-treated ROS 17/2.8 osteoblast-like cells. We conclude that in addition to hormonal regulation of protein kinase activity, protein dephosphorylation plays a functionally important role in the modulation of bone resorption.
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PMID:Protein phosphatase inhibitors and bone resorption: inhibition by okadaic acid and biphasic actions of calyculin-A. 137 1

The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormone-binding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for casein kinase-II or a related protein kinase.
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PMID:Vitamin D receptor phosphorylation in transfected ROS 17/2.8 cells is localized to the N-terminal region of the hormone-binding domain. 165 37

The PTH activates both adenylate cyclase and a mechanism that increases membrane-associated protein kinase-C (PKC) activity. To define the hormone's PKC activation domain we have used a panel of PTH fragments and ROS 17/2 rat osteosarcoma cells as the target cells. PTH equally and maximally increased PKC activity in ROS 17/2 cell membranes at physiological concentrations between 1-50 pM and 5-50 nM, but not at intermediate concentrations or concentrations above 50 nM. The PKC-stimulating picomolar concentrations of PTH did not stimulate adenylate cyclase in ROS 17/2 cells, while the PKC-stimulating nanomolar concentrations of the hormone did stimulate adenylate cyclase, with an EC50 of 1-2 nM. Very high concentrations of PTH, such as 100 nM, that did not increase membrane PKC activity were still able to maximally stimulate adenylate cyclase. PTH fragments lacking the N-terminal amino acids needed for adenylate cyclase activation increased membrane PKC activity, and the PKC activation domain was found to lie within the 28-34 region of the PTH molecule. This was confirmed by showing that optimally effective picomolar concentrations of the human PTH-(28-34) fragment itself were able to increase membrane-associated PKC activity to the same extent as the optimally effective picomolar concentrations of the intact PTH-(1-84) or the larger PTH-(1-34) or PTH-(3-34) fragments.
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PMID:The protein kinase-C activation domain of the parathyroid hormone. 172 20

Phosphoproteins appear to be involved in several ways in the regulation of the orderly deposition and crystal growth of mineral within the performed collagenous matrix of bone and dentine. The phosphorylation of these proteins is not yet understood. Potential protein kinases were extracted from an osteoblast-like cell line, ROS 17/2.8. The ROS 17/2.8 line was shown to produce a full complement of known kinases. However, neither bone phosphoproteins (BPP) nor dentine phosphophoryn (DPP) could be phosphorylated by the messenger dependent kinases. DPP and dephosphorylated BPP (dBPP) were substrates for a unique messenger independent kinase distinct from casein kinase II, and dDPP was a still better substrate. Thus, BPP and DPP are phosphorylated by a unique kinase or set of kinases which are messenger independent and have very specific substrate sequence requirements.
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PMID:Phosphorylation of extracellular bone and dentine matrix proteins. 216 64

Glucocorticoids increase and 1,25-dihydoxyvitamin D3 [1,25-(OH)2D3] decreases the activity of PTH-responsive adenylate cyclase, altering intracellular cAMP in a rat osteoblast-like cell line (ROS 17/2.8). This study was undertaken to measure the subsequent activation of the cAMP-dependent protein kinase (PKA). Pretreatment of ROS cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM), shifted the dose-response curve for PKA activation by PTH upward compared to the control value. Basal PKA activity was enhanced 50% by TRM, and the PTH concentration required for maximal activation of PKA decreased from 1.0 to 0.05 ng/ml. At the lowest effective PTH concentration (0.05 ng/ml) the mean PKA activity ratio increased to 0.73 in TRM-treated cells compared with 0.45 in untreated cells. Pretreatment with 1,25-(OH)2D3 had opposite effects, shifting the dose-response curve for PKA activation by PTH downward and to the right, decreasing the basal activity ratio from 0.26 to 0.16, and increasing the PTH concentration required for maximal activation to 10 ng/ml. 1,25-(OH)2D3-treated cells stimulated with 0.5-1 ng/ml PTH consistently had lower PKA activity ratios than untreated cells. Simultaneous treatment with 1,25-(OH)2D3 reversed the effect of TRM. There were no differences in total PKA activity (2.57 +/- 0.09 pmol 32P/min.micrograms protein) between treatment groups, suggesting that TRM and 1,25-(OH)2D3 do not alter the cellular PKA concentration. In control experiments exogenous PKA was added to sonication buffer of PTH-stimulated cells to verify that the TRM and 1,25-(OH)2D3 shifts in PKA activation at low PTH doses occur before sonication. cAMP-dependent protein kinase activation was also studied by measuring the progressive occupation of regulatory subunit-binding sites by hormonally stimulated endogenous cAMP. [3H] cAMP binding was expressed as the percent change in bound [3H]cAMP per microgram protein compared to that in unstimulated cells not steroid treated. [3H]cAMP binding to all cytosol fractions decreased as PTH increased over the concentration range predicted by our PKA activation experiments. TRM treatment shifted the curve for [3H]cAMP binding to regulatory subunit downward and to the left, and 1,25-(OH)2D3 treatment shifted it upward and to the right. In cells treated with both TRM and 1,25-(OH)2D3, the curve was similar to control curve. Sonicating unstimulated cells in buffer containing comparable concentrations of added cAMP did not alter [3H]cAMP binding. These and the previous controls suggest that changes in PKA activation at low doses of PKA reflect cellular events occurring before cell disruption.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Glucocorticoids and 1,25-dihydroxyvitamin D3 regulate parathyroid hormone stimulation of adenosine 3',5'-monophosphate-dependent protein kinase in rat osteosarcoma cells. 245 15

Glucocorticoid increases and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases PTH activation of adenylate cyclase and cAMP-dependent protein kinase in rat osteosarcoma cells (ROS 17/2.8). Since selective cAMP-dependent protein kinase isoenzyme activation may account for specific physiological hormonal responses, we investigated steroid effects on activation of isoenzymes I and II in response to PTH using a new ion exchange separation procedure. Pretreatment of cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM) or 1,25-(OH)2D3 altered the degree of cAMP-dependent protein kinase isoenzyme activation by PTH in accordance with their modulation of intracellular cAMP accumulation, but did not alter the amount of each isoenzyme present or the order in which isoenzymes I and II were activated. In all treatment groups isoenzyme I was preferentially activated by low doses of PTH, while high concentrations activated both isoenzymes, as predicted by the relative affinities of each isoenzyme for cAMP. Glucocorticoid reduced the concentration of bovine PTH-(1-34) required for maximal activation of isoenzyme I from 1 to 0.05 ng/ml and that required for activation of isoenzyme II from 10 to 1 ng/ml. This effect was abolished by simultaneous treatment of cells with 1,25-(OH)2D3. At doses of PTH that caused partial activation (0.05-0.1 ng/ml for isoenzyme I; 1 ng/ml for isoenzyme II), 1,25-(OH)2D3 treatment attenuated this activation. In all groups both isoenzymes were fully activated by 100 ng/ml PTH. Control experiments demonstrated that isoenzyme activation is not a result of cell disruption over the range of PTH doses that regulation by steroid hormone was observed. These results extend our studies on modulation of the cAMP pathway by steroid hormones and make it feasible to correlate selective isoenzyme activation with specific responses to PTH.
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PMID:Glucocorticoid and 1,25-dihydroxyvitamin D modulate the degree of adenosine 3',5'-monophosphate-dependent protein kinase isoenzyme I and II activation by parathyroid hormone in rat osteosarcoma cells. 255 28

Late passage cultures of a clonal osteogenic sarcoma line (ROS 17/2.8) failed to respond to PTH with activation of cAMP-dependent protein kinase isoenzymes despite showing a sensitive and dose-dependent increase in cAMP after treatment with the hormone. When cells were treated with hydrocortisone or dexamethasone, protein kinase responsiveness to PTH was readily demonstrated; such treatment also resulted in enhanced cAMP production. Forskolin preincubation resulted in a cAMP response to PTH of similar magnitude to that seen with hydrocortisone but no activation of cAMP-dependent protein kinase occurred. Thus, the effect of glucocorticoid cannot be explained merely by the increased amplitude and sensitivity of the cAMP response which developed with glucocorticoid treatment in these cells. The data indicate that cellular activation of cAMP-dependent protein kinase does not automatically follow cAMP generation and that information transfer can be restored by pharmacological means.
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PMID:Glucocorticoid treatment facilitates cyclic adenosine 3',5'-monophosphate-dependent protein kinase response in parathyroid hormone-responsive osteogenic sarcoma cells. 300 48

Teleost rod photoreceptors elongate in the light and shorten in darkness. We are investigating the role of cAMP-dependent protein kinase (PKA), phosphatases and target phosphoproteins in the regulation of photoreceptor cell shape. Preparations of rod fragments, consisting of the motile inner segment with attached photosensory outer segment (RIS-ROS), undergo light-stimulated elongation in culture. The PKA-selective inhibitor, H89, enhanced RIS-ROS elongation in both light and darkness, suggesting that elongation is associated with dephosphorylation of PKA substrates. Okadaic acid and calyculin A, inhibitors of type 1 and 2A phosphatases, blocked light-dependent and light-independent elongation with relative potencies suggesting that elongation requires dephosphorylation by type 1 phosphatase in light and type 2A phosphatase in darkness. To identify targets of PKA and phosphatases, RIS-ROS were isolated from retinas prelabeled with 32P-orthophosphate, and then incubated in the presence of kinase inhibitors or phosphatase inhibitors. Two phosphoproteins, PP33 and PP35, were phosphorylated by PKA and dephosphorylated by type 1 or 2A phosphatases in light- and dark-cultured RIS-ROS. PP35 (but not PP33) was immunoprecipitated by an antibody to phosducin, a PKA-regulated modulator of phototransduction (Lee et al., 1992); PP35 was also phosphorylated in vitro by a Ca2+ calmodulin-activated kinase. PP33 further differed from PP35 in its phosphopeptide maps and phosphorylation by PKC. We conclude that RIS-ROS elongation is correlated with the dephosphorylation of PKA substrates by type 1 or 2A phosphatases. Candidate mediator proteins include PP35, a fish phosducin homolog, and PP33, a newly described photoreceptor phosphoprotein.
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PMID:Phosducin and PP33 are in vivo targets of PKA and type 1 or 2A phosphatases, regulators of cell elongation in teleost rod inner-outer segments. 747 10

We report here that osteoblasts and osteoblast-like osteosarcoma cells express PMCA1b, an alternatively spliced transcript of plasma membrane Ca(2+)-ATPase. Synthetic oligonucleotide pairs were designed based upon unique regions of the cDNA encoding known PMCA isoforms (PMCA1-3) and used as primers in PCR-mediated amplification of cDNA synthesized from ROS 17/2.8 osteosarcoma cell RNA. A product was observed only when PMCA1-specific primers were present; no products were seen with PMCA2 or PMCA3 primers unless cDNA synthesized from rat brain RNA was present. Examination of the cDNA encoding the C terminus of PMCA1 from ROS 17/2.8 cells revealed that the mRNA is spliced to yield the PMCA1b isoform, a Ca(2+)-ATPase containing a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. PMCA1b was also detected in UMR-106-01 osteosarcoma cells and unpassaged primary rat calvarial osteoblasts. These results suggest that the regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca(2+)-ATPase.
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PMID:Osteoblasts express the PMCA1b isoform of the plasma membrane Ca(2+)-ATPase. 750 68


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