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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The differential expression of mRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed. Steady state mRNA levels were severalfold higher in ROS 17/2.8 cells exhibiting an osteoblast-like phenotype, compared with the less osteoblast-like ROS 25/1. Thymosin beta 4 transcripts were also detected in rat UMR 106 osteosarcoma cells and in intact neonatal and fetal rat calvaria. Sequence analysis of the cDNA indicated that thymosin beta 4 transcripts may arise by processing at a more distal polyadenylation signal. Treatment of ROS 17/2.8 cells with dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3 decreased thymosin beta 4 mRNA. The phenotype-dependent expression in the ROS cells and the response to steroid hormone suggest that thymosin beta 4 expression contributes to the osteoblast phenotype.
Mol Endocrinol 1990 Jan
PMID:Thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner. 232 69

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] regulates the synthesis of bone gamma-carboxyglutamic acid (Gla) protein (BGP) by osteoblastic cells. In this study we examined the effect of cAMP, alone and in combination with 1,25-(OH)2D3, on the regulation of BGP mRNA levels in ROS 17/2 rat osteosarcoma cells. Elevation of intracellular cAMP levels by cAMP analogs or by isobutylmethylxanthine (IBMX), forskolin, or PTH, resulted in increased BGP mRNA levels and BGP secretion after 1 day of treatment. The effects of these agents were additive with 1,25-(OH)2D3 in stimulating BGP gene expression. After 4 days of treatment, pertussis toxin (PT) and 1,25-(OH)2D3 were synergistic in stimulating BGP mRNA, and the effect of PT could be mimicked by (Bu)2cAMP, IBMX, forskolin, cholera toxin, and to a lesser extent by PTH. The effect of 1-day treatment with cAMP alone and the synergistic effect with 1,25-(OH)2D3 on the stimulation of BGP mRNA were dependent on cell density, while basal and 1,25-(OH)2D3-stimulated synthesis were not. Cyclic AMP inhibited ROS 17/2 cell growth after 1 day of treatment, an effect that was also dependent on initial cell density. After 4 days of treatment, 1,25-(OH)2D3, cAMP, and PT all demonstrated inhibition of cell growth. When cells were treated with actinomycin D, both 1,25-(OH)2D3 and cAMP stimulation of BGP mRNA were blocked. In addition, neither agent was effective in enhancing BGP mRNA stability when prestimulated cells were exposed to actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1989 Jan
PMID:Bone Gla protein messenger ribonucleic acid is regulated by both 1,25-dihydroxyvitamin D3 and 3',5'-cyclic adenosine monophosphate in rat osteosarcoma cells. 246 56

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates the alkaline phosphatase of rat and human osteoblast-like cells in culture. Here the mechanism of this effect was investigated using the rat osteogenic sarcoma cell line ROS 17/2-8. We found that 50% maximum alkaline phosphatase stimulation is elicited by 1,25(OH)2D3 at 7 X 10(-10) M. The concentration of serum in the culture medium influences inversely the effective 1,25(OH)2D3 concentration. Increased alkaline phosphatase appears after a lag period of cell exposure to 1,25(OH)2D3 which is between 8 and 24 h; during 96 h culture in the presence of 1,25(OH)2D3 the enzyme activity continues to rise. Cycloheximide (0.1-1 micrograms/ml) added in the cultures for 3 days or actinomycin-D (1-30 ng/ml) added for 24 h inhibit the 1,25(OH)2D3 effect on alkaline phosphatase in a dose-dependent fashion; withdrawal of cycloheximide restores the responsiveness of cells to 1,25(OH)2D3 completely, but withdrawal of actinomycin-D restores cell responsiveness only partially. These findings suggest that 1,25(OH)2D3-induced stimulation of alkaline phosphatase in the osteoblast-like cells involves genome activation and de novo protein synthesis.
Mol Cell Endocrinol 1983 Nov
PMID:Mechanism of action of 1,25-dihydroxyvitamin D3-induced stimulation of alkaline phosphatase in cultured osteoblast-like cells. 635 97

We demonstrated previously that vitamin D metabolites modulate the response of bone and cartilage cells to 17 beta-estradiol (E2) and dihydrotestosterone (DHT) both in cell cultures and in vivo. In the present study, we investigated to what extent pretreatment with 1,25(OH)2D3 or 24,25(OH)2D3 would reduce the minimal effective dose of E2, DHT or progesterone (P) required for stimulation of DNA synthesis and creatine kinase specific activity in cultured osteoblast-like ROS 17/2.8 cells and in rat embryo epiphyseal cartilage cells, and to what extent such pretreatment would increase the maximal response. We measured responses to sex steroids after pretreatment of the cells for 5 days with 0.02% ethanol vehicle or with the vitamin D metabolites 1,25(OH)2D3 (0.12 nM), or 24,25(OH)2D3 (1.2 nM) singly or in combination. Pretreatment of ROS 17/2.8 cells with 1,25(OH)2D3, but not 24,25(OH)2D3, increased synergistically their response to E2 but not to P, and did not affect their lack of response to DHT. Pretreatment of epiphyseal cartilage cells with either 1,25(OH)2D3 or 24,25(OH)2D3 increased synergistically their DNA synthetic response to all three steroids, but their CK response only to E2 or DHT. The minimal dose for causing a significant response to E2 in ROS 17/2.8 cells or to either E2 or DHT in epiphyseal cartilage cells was reduced 10-fold after pretreatment with vitamin D metabolites. After pretreatment, the maximal response was more than doubled in ROS 17/2.8 cells; epiphyseal cartilage cells showed a similar 10-fold decrease in the dose required for maximal response to E2 or DHT; the improvement in the response to P was significant only for DNA synthesis. We conclude that pretreatment with the appropriate vitamin D metabolite(s) both reduces by an order of magnitude, or more, the amount of sex steroids needed to stimulate skeletal derived cells and increases synergistically the maximal response of the cells.
J Steroid Biochem Mol Biol 1995 Nov
PMID:Pretreatment with 1,25(OH)2 vitamin D or 24,25(OH)2 vitamin D increases synergistically responsiveness to sex steroids in skeletal-derived cells. 749

Bleomycin-induced, 6-thioguanine-resistant, "non deletion" mutants pretreated with or without either TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic, were analyzed by polymerase chain reaction (PCR)-directed DNA sequencing in a Chinese hamster ovary (CHO) cell derivative, AS52. Among the 23 bleomycin-induced mutants, six have 3-bp 5'-TGA-3' deletions in the region of 366-371, five have single-base deletions, seven have base substitutions, three have insertions, and two have possible translocations. Among the 16 bleomycin-induced mutants pretreated with TRIEN, six have the 5'-TGA-3' deletion (366-371), two have single-base deletions, one has a 13-bp deletion, four have single-base substitutions, one has a double-base substitution, and two have insertions. Among the 17 bleomycin-induced mutants pretreated with TEMPOL, six have the same TGA deletions, two have single-base deletions, two have single-base insertions, four have single-base substitutions, one mutant has a 12-bp deletion, one has a 13-bp deletion, and one mutant shows no detectable change in its coding region in the DNA sequence. A possible shift from a ROS-mediated mutational spectrum to a spontaneous mutational spectrum by TRIEN further indicates that reactive oxygen species play an important role in bleomycin mutagenesis in mammalian cells.
Environ Mol Mutagen 1994
PMID:Polymerase chain reaction-directed DNA sequencing of bleomycin-induced "nondeletion"-type, 6-thioguanine-resistant mutants in Chinese hamster ovary cell derivative AS52: effects of an inhibitor and a mimic of superoxide dismutase. 751 29

The antigenicity of native DNA modified with reactive oxygen species was examined. Goats were immunized with the modified polymer and the antibody response was estimated by direct binding and competition ELISA. The induced antibodies bound ROS-DNA and showed considerable binding to native DNA as well. Specificity analysis of the purified antibodies revealed the recognition of native B-, A- and allied conformations presented by various synthetic polynucleotides. The contribution of lysine residues to the immunochemical binding of purified IgG was investigated by modifying the free amino groups of lysine residues. The modification of lysine residues paralleled loss in IgG binding to ROS-DNA to the extent of 50%, suggesting that such residues might be involved in the antigen binding site of immunoglobulin molecule.
Biochem Mol Biol Int 1995 Jan
PMID:Antigenic specificity of anti-ROS DNA antibodies: involvement of lysyl residues in antigen binding. 753 71

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.
Mol Cell Biol 1995 Jun
PMID:Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype. 776 Aug 23

We investigated the contribution of c-fos protooncogene in the mitogenic effect of transforming growth factor-beta (TGF beta) in serum-deprived, confluent rat calvaria osteoblastic cells. The TGF beta-induced growth in these cells was associated with an immediate and transient c-fos mRNA accumulation, similar to the inductive effect of fetal calf serum. To assess the role of c-fos in the response to TGF beta, we used a c-fos antisense (AS) oligonucleotide displaying duplex formation with rat c-fos mRNA. Studies of AS and sense (S) uptake by osteoblastic cells demonstrated that incorporation of labeled oligomers was maximal at 2 h, and the incorporated AS oligonucleotide remained intact for 24 h. Immunofluorescence analysis of c-Fos-labeled cells demonstrated that AS, but not S, oligonucleotide reduced c-Fos protein expression, suggesting specific efficient inhibition of c-fos translation by the AS oligomer. Proliferation assays showed that cell growth induced by fetal calf serum was inhibited by the AS, but not by the S oligonucleotide, in both normal rat osteoblasts and ROS 17/2.8 osteosarcoma cells, demonstrating efficient and specific blockage of cell growth by the AS oligomer. The mitogenic effect of TGF-beta was abolished in cells cultured in the presence of AS, whereas S had no effect, showing that c-fos is required for TGF beta-induced osteoblast cell growth. The results show that the induction of c-fos is implicated in the mitogenic effect of TGF beta in osteoblastic cells and provide a cellular mechanism involved in the response of these cells to TGF beta.
Mol Endocrinol 1995 Feb
PMID:c-fos protooncogene is involved in the mitogenic effect of transforming growth factor-beta in osteoblastic cells. 777 69

Endothelins (ETs) (ET-1, ET-2, and ET-3), a family of 21-amino acid peptides, mediate a host of biological responses by binding to specific cell surface receptors termed ETA and ETB. Because a role for ET in bone remodeling has been suggested, the present study was undertaken (a) to characterize ET receptors and their responses in the rat osteosarcoma cell line ROS 17/2.8 and (b) to study their regulation by 1,25-dihydroxy-vitamin D3. Binding studies using 125I-ET-1 (a nonselective agonist) and 125I-IRL-1620 (an ETB receptor-selective agonist) indicated that these cells display high affinity ETA and ETB receptors in the ratio of 3:1. Addition of ET-1 or sarafotoxin 6c to myo-[3H]inositol-labeled cells resulted in an increase in inositol phosphate accumulation as well as in intracellular Ca2+ release, suggesting that these receptors are coupled to phospholipase C. In addition, ET-1 but not sarafotoxin 6c induced a modest increase in the expression of osteocalcin protein that was completely blocked by BQ123 (an ETA receptor-selective antagonist), indicating that activation of ETA receptors plays a role in the induction of osteocalcin. Treatment of ROS osteoblasts with 10 nM 1,25-dihydroxy-vitamin D3 for 14 hr resulted in a significant (> 50%) decrease in 125I-ET-1 and 125I-IRL-1620 binding. This decrease in binding was shown to be due to a decrease in the number of ET receptors, with no change in affinity. Although both ETA and ETB receptors were down-regulated in response to 1,25-dihydroxy-vitamin D3, only ETA receptor mRNA levels were significantly decreased, with very little change in ETB mRNA levels. These data indicate that ROS osteoblasts display both ETA and ETB receptors that are functional. Induction of osteocalcin was primarily mediated by ETA receptors, and these receptors were also down-regulated at the mRNA level by 1,25-dihydroxy-vitamin D3.
Mol Pharmacol 1995 Feb
PMID:Identification and characterization of endothelin receptors on rat osteoblastic osteosarcoma cells: down-regulation by 1,25-dihydroxy-vitamin D3. 787 34

We recently defined an element (ACTAATTGG) within the rat osteocalcin (OC) promoter at -84 to -92 which provides approximately 70% of basal promoter activity in osteoblastic cell lines and binds a specific nuclear factor found in OC-producing ROS 17/2.8 osteosarcoma cells. Since this element closely resembles the recently described Msx-1 (Hox 7.1) homeodomain DNA binding cognate, we examined rodent osteoblastic cells lines for expression of Msx homeodomain-encoding messages. We have found and cloned a cDNA for rat Msx-2 (Hox 8.1) from a ROS 17/2.8 library and detect high levels of expression in various osteoblastic cell lines (ROS 17/2.8, RCT3, RCT1) as well as in culture passage 3 neonatal rat calvarial osteoblastic cells. Little to no expression was detected in phenotypically immature MC3T3E1 osteoblastic cells or in a variety of nonosteoblastic (ROS 25/1, C2C12, TRAB 11) mesenchymal cell lines. Dexamethasone (DEX) down-regulates Msx-2 message levels in both RCT3 and ROS 17/2.8 cells. Recombinant rat Msx-2 homeodomain expressed in Escherichia coli as a glutathione-S-transferase fusion protein binds to the rat OC promoter region -74 to -100 as determined by gel shift analysis. Recognition is dependent upon the intact ACTAATTGG motif at -84 to -92. In transient cotransfection assays using MC3T3E1 cells (which expresses very little or no endogenous Msx-2), Msx-2 suppresses the rat OC promoter 2- to 3-fold via the Msx-2 binding motif at -84 to -92. However, in ROS 17/2.8 cells, where a high level of endogenous Msx-2 mRNA is present, expression of exogenous Msx-2 does not suppress the rat OC promoter; surprisingly, Msx-2 further augments basal promoter activity by approximately 50-70%, again dependent upon the ACTAATTGG motif at -84 to -92. These data directly demonstrate that the Msx-2 homeodomain binds the rat OC promoter and that Msx-2 can act as a sequence-specific transcriptional regulator of the rat OC promoter in cultured osteoblastic cell lines. This activity is dependent upon the specific osteoblastic cellular context, similar to previous observations in nonosseous systems with other homeodomain transcription factors. These data suggest that Msx-2 may play a role in the transcriptional regulation of the osteoblast phenotype during development in the morphogenetic fields where it is expressed.
Mol Endocrinol 1994 Nov
PMID:Msx-2/Hox 8.1: a transcriptional regulator of the rat osteocalcin promoter. 787 17


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