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)

Bone is a tissue that responds to mechanical load by changing its internal architecture. However, the mode of transmission of mechanical stimuli into biological signals and the effect of load at the cellular level are still not clear. An in vitro system, a Flexercell Strain Unit, was used to apply cyclic load to osteoblast-like cells in culture. In the first series of experiments, ROS 17/2.8 rat osteosarcoma cells, cultured on Flex I, flexible bottomed culture plates, were subjected to a 0.05 Hz, 0.24 STRAIN cyclic load regime for 3 and 7 days, in vitro. One group subjected to load received verapamil, a calcium channel blocker, throughout the experimental period. A second group was exposed to load but received no verapamil. A third group had no drug or load and a fourth group had no load but received verapamil. Cultures were incubated for 24 hours prior to collection with 10 microCi of 45CaCl in the medium, then well bottoms were divided to yield outer (maximum) and inner (minimum) load zones for assay of radioactivity. The effect of verapamil during a 7-day loading period was studied by adding the drug to individual cultures at daily intervals. Results indicated that mechanical loading stimulates calcium incorporation in ROS 17/2.8 cell cultures by day 7 but not by day 3. Only early verapamil addition decreased load-induced calcium incorporation when drug was added prior to day 4. If verapamil was added after 4 days, the channel blocker did not diminish load-induced calcium incorporation.
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PMID:Verapamil decreases cyclic load-induced calcium incorporation in ROS 17/2.8 osteosarcoma cell cultures. 128 12

A proinflammatory cytokine cascade, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8, is activated in response to infection or immunologic insult. Besides their immunologic effects, several of these mediators stimulate bone resorption and inhibit bone formation. Osteocalcin, the most abundant noncollagenous protein present in bone, is an osteoblast-specific product whose production closely correlates with bone formation, and which has also been implicated in control of bone resorption. IL-1 and TNF have previously been shown to down-regulate osteocalcin production in vitro and in vivo, although the mechanism of this inhibition is unknown. In the present studies, IL-1 beta and TNF-alpha both inhibited 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin protein and mRNA by ROS 17/2.8 osteosarcoma cells, whereas IL-6 had no effect on protein and only weakly inhibited mRNA. To determine if down-regulation was exerted at the transcriptional level, an osteocalcin promoter-chloramphenicol acetyltransferase (CAT) fusion gene was constructed (PHOC-CAT). After transient transfection of PHOC-CAT into ROS 17/2.8 osteosarcoma cells, reporter CAT activity was up-regulated by vitamin D at concentrations above 10(-12) M. In screening studies, TNF-alpha (-57%) and IL-6 (-37%) inhibited vitamin D-stimulated osteocalcin transcription, whereas IL-1 alpha, IL-1 beta, and IL-8 had no effect. Other immune cytokines and growth factors, including IL-2, IL-3, IL-7, and M-CSF, also failed to regulate osteocalcin transcription. Despite their lack of promoter regulation, IL-1 alpha and IL-1 beta also stimulated PGE2 production by ROS 17/2.8, further confirming the ability of the host cell to respond to these mediators. In dose-response experiments, down-regulation by TNF-alpha was significant at concentrations as low as 0.14 pM (0.1 U/ml), whereas approximately 10(4)-fold higher concentration of IL-6 was required to exert a similar effect. TNF-alpha-mediated down-regulation was unaffected by indomethacin. These data demonstrate that of these cytokines, TNF-alpha alone potently down-regulates osteocalcin promoter function, whereas IL-1 acts post-transcriptionally, possibly by reducing mRNA stability. Heterogeneity therefore exists among the proinflammatory cytokines with respect to the level at which control of osteocalcin expression is exerted.
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PMID:Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin gene promoter. 130 41

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.
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PMID:Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium. 131 66

The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.
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PMID:Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression. 132 35

The present study was designed to further understand the role of PTH on the secretion of the neutral metalloproteinases, collagenase and gelatinase, from the rat osteosarcoma clonal cell line, ROS 17/2.8. Semiconfluent cells were treated with bovine parathyroid hormone, b-PTH-(1-34) at 100 nM-0.01 nM for 24-96 hours and pooled, concentrated media were analyzed by functional assay for collagenase (3H-methyl collagen) and gelatinase (3H-methyl gelatin). Collagenase activity significantly decreased (P less than 0.01) in the PTH conditioned media in a dose-dependent manner before (98-64%) and after (91-39%) reduction and alkylation. SDS-PAGE and fluorography apparently showed the most degradation to alpha A chains in collagen with controls, whereas this substrate remained intact with PTH (100 nM). PTH (100 nM) media also showed neutral gelatinase activity approximately 2% compared to control before and after reduction and alkylation (P less than 0.01). Significant amounts of an inhibitor to collagenase and gelatinase might have been secreted at 1 nM and 0.01 nM PTH, since collagenase and gelatinase activities were greater after reduction and alkylation. Reduction and alkylation likely destroyed these significant amounts of inhibitor. Polymorphonuclear leukocyte collagenase activity was also inhibited 80% by PTH conditioned media, but not by control. However, upon reduction and alkylation which destroyed inhibitor, the PTH treated media showed only a 14% inhibition against polymorphonuclear leukocyte collagenase (P less than 0.01). PTH appeared to downregulate neutral metalloproteinase activities through its effects on an inhibitor. This downregulation may represent a specific phenotypic response to PTH in ROS 17/2.8 cells.
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PMID:Parathyroid hormone regulation of matrix degrading enzymes in rat osteoblastic osteosarcoma 17/2.8 cells. 132 16

The beta-adrenergic blocking agent propranolol was shown in previous studies to increase orthotopic bone formation in rats. To understand the cellular mechanisms underlying this observation, propranolol was tested for its effects on osteoblastic cells, which possess adenylate cyclase-coupled beta-adrenergic receptors. The ability of propranolol to modulate parathyroid hormone (PTH) and isoproterenol effects on adenylate cyclase activity and on alkaline phosphatase expression was studied in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. At concentrations between 0.1 and 10 microM, DL-propranolol specifically inhibited adenylate cyclase stimulation by the beta-adrenergic agonist isoproterenol, but did not alter either basal or PTH-stimulated activity. At these concentrations, propranolol also blunted the inhibition of alkaline phosphatase activity by isoproterenol but not PTH. Propranolol alone had minimal effects on ROS alkaline phosphatase activity at low concentrations (0.1-1 microM), but became inhibitory at high concentrations (10-100 microM). Thus, the direct effects of physiologically relevant propranolol concentrations on osteoblastic cells can be attributed principally to beta-adrenergic blockade. These findings further suggest that propranolol may enhance bone formation by preserving osteoblastic activity in the face of inhibition by beta-adrenergic agonists.
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PMID:Effects of beta-adrenergic blockade in an osteoblast-like cell line. 134 41

Local secretion of insulin-like growth factor binding proteins (IGFBPs) may modulate the effects of IGF-I and IGF-II on bone cell metabolism and proliferation. Several osteoblast-derived cell lines are currently used as interchangeable models to study IGFBP production, although it is unknown whether findings in one cell line can be extrapolated to another cell line or to normal human osteoblasts. In this study, we examined by Western ligand blotting both basal and regulated secretion of IGFBPs in vitro in 1) normal human osteoblast-like (hOB) cells cultured from explants of human trabecular bone; 2) an SV40-transformed hOB (HOBIT) cell line; and 3) several human (U-2, MG-63, TE-85) and rat (ROS 17/2.8 and UMR-106-01) osteosarcoma cell lines. Constitutively, hOB and HOBIT cells produced a similar pattern of IGFBPs, while all other cell lines produced their own unique pattern of IGFBPs. The two rat cell lines differed from the human cell lines as well as from each other. The response to hormonal stimulation also varied among the cell lines. Treatment of hOB and HOBIT cells with IGF-I resulted in a 2-fold increase in medium levels of IGFBP-3; IGF-I decreased levels of 24-kilodalton (kDa) IGFBP in hOB cell-conditioned medium. In addition, IGF-I markedly increased levels of the 29/32/34 kDa IGFBP triplet in U-2 cells, but had little or no effect in the other human and rat osteosarcoma cell lines. PTH increased a 29-kDa IGFBP apparent only in UMR 106-01 cell-conditioned medium, whereas GH had no direct effect on IGFBP secretion in any of the osteoblast-like cells tested. We conclude that basal and regulated secretion of IGFBPs from osteoblast-like cells is cell-line specific. Spontaneously transformed human or rat osteoblast-like cells provide unique model systems to study features of distinct IGFBPs and their regulation; however, hOB cells and their derivatives may be more appropriate models for understanding the regulation of IGFs in human bone.
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PMID:Basal and regulated secretion of insulin-like growth factor binding proteins in osteoblast-like cells is cell line specific. 137 4

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106-01, vasoactive intestinal polypeptide (VIP, 0.001-1 microM), calcitonin gene-related peptide (CGRP, 0.3-30 nM), and NE (0.1-300 microM), but not neuropeptide Y (NPY, 0.001-1 microM) or substance P (SP, 0.1-10 microM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1-3 microM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 microM) and VIP (0.3 microM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 microM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 microM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 microM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106-01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells. 138 76

Osteoblasts possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent ascorbate transporter located in the plasma membrane. The transporter is specific for ascorbate and stereoselective for L-ascorbate over D-isoascorbate. The present study examined the effects of ascorbate supplementation and deprivation on the activity of this transport system. L-ascorbate transport activity was determined by measuring uptake of the vitamin by ROS 17/2.8 osteosarcoma cells during 1 minute incubations with 5 microM L-[14C]ascorbate. The initial rate of L-[14C]ascorbate uptake by ROS 17/2.8 cells grown for 18 h in L-ascorbate-replete medium was 89 +/- 8 nmol/g protein per minute. Following removal of L-ascorbate from the growth medium, the initial rate of uptake increased within 6 h to 126 +/- 13 nmol/g protein per minute. Conversely, the initial rate of uptake by cells grown in ascorbate-free medium decreased following the addition of L-ascorbate, but not D-isoascorbate, to the medium. The effect of ascorbate pretreatment was specific for ascorbate transport in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-glucose. Kinetic analysis revealed that modulation of ascorbate transport arose from changes in the apparent maximum rate of transport (Vmax) without changes in the affinity of the transport system for L-ascorbate. These experiments are the first to show that ascorbate transport by osteoblastic cells responds to vitamin C deprivation and supplementation. Adaptation of transport activity to substrate availability may play an important role in the physiological regulation of intracellular ascorbate levels.
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PMID:Adaptive regulation of ascorbate transport in osteoblastic cells. 141 86

Using immunoprecipitation and tryptic peptide microsequencing we confirmed the identity of normal rat kidney (NRK) cell-secreted 69-kDa major phosphoprotein as osteopontin (OP). We then immunoselected a 1.4-kilobase pair (kb) OP cDNA from a lambda gt11 library prepared from Kirsten sarcoma virus-transformed NRK (KNRK) cellular mRNA, using rabbit anti-69-kDa OP serum. Sequence analysis of this cDNA revealed the presence of a 52-nucleotide-long insert in the 5'-noncoding region, which was absent in OP cDNA cloned from the cDNA library of ROS 17/2.8 rat osteosarcoma cells. The insert sequence is flanked by putative intron splice junctions and is located 15-nucleotide upstream of the translational initiation site. An insert-specific 30-mer oligonucleotide probe hybridized to a single 1.5-kb RNA species from both NRK and KNRK cells, but not from ROS 17/2.8 cells. However, Southern analysis showed the presence of this insert sequence in the genomic DNA of both NRK and ROS 17/2.8 cells. Furthermore, PCR amplification of the insert-containing region using genomic DNAs from both NRK and ROS 17/2.8 cells gave products of identical size and sequence. Since OP is a single copy gene, these data provide strong evidence for differential cell type-specific processing of OP transcripts. In addition, we demonstrate that, in contrast to most transformed cells, levels of OP expression are significantly reduced in KNRK cells as compared to NRK cells.
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PMID:Differential processing of osteopontin transcripts in rat kidney- and osteoblast-derived cell lines. 142 23


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