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

A detailed analysis of the transcriptional machinery responsible for osteoblast-specific gene expression should provide tools useful for understanding osteoblast commitment and differentiation. We have defined three cis-elements important for basal activity of the rat osteocalcin (OC) promoter, located at about -200 to -180, -170 to -138, and -121 to -64 relative to the transcription initiation site. A motif (TCTGATTGTGT) present in the region between -200 and -170 that binds a multisubunit CP1/NFY/CBF-like CAAT factor complex contributes significantly to high level basal activity and presumably functions as the CAAT box for the rat OC promoter. We show that the region -121 to 32 is sufficient to confer osteoblastic cell type specificity in transient transfection assays of cultured cell lines using luciferase as a reporter. The basal promoter is active in rodent osteoblastic cell lines, but not in rodent fibroblastic or muscle cell lines. Although the rat OC box (-100 to -74) contains a CAAT motif, we could not detect CP1-like CAAT factor binding to this region. In fact, we demonstrate that a Msx-1 (Hox 7.1) homeodomain binding motif (ACTAATTG; bottom strand) in the 3'-end of the rat OC box is necessary for high level activity of the rat OC basal promoter in osteoblastic cells. A nuclear factor that recognizes this motif appears to be present in osteoblastic ROS 17/2.8 cells, which produce OC, but not in fibroblastic ROS 25/1 cells, which fail to express OC. This ROS 17/2.8 nuclear factor also recognizes the A/T-rich DNA cognates of the homeodomain-containing POU family of transcription factors. Taken together, these data suggest that a ubiquitous CP1-like CAAT factor and a cell type-restricted homeodomain containing (Msx or POU family) transcription factor interact with the proximal rat OC promoter to direct appropriate basal OC transcription in osteoblastic cells.
Mol Endocrinol 1994 May
PMID:Activity of the rat osteocalcin basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs. 791 73

The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected with the mouse ER in order to more easily evaluate the physiological role of estrogens in bone cell homeostasis. These transfected ROS.SMER 14 cells are highly responsive to estrogenic stimulation at subconfluence, but become refractory to estrogenic stimulation when postconfluency is reached. The purpose of these studies was to determine the mechanisms underlying this loss of responsiveness in these ER stably transfected cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the subconfluent cells were actively dividing, whereas none of the postconfluent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cells was low and often undetectable. Steady state ER mRNA levels were not significantly modified by postconfluency. ER protein levels were also unaffected by confluency status. Since protein kinase-C (PKC) has been reported to influence cell proliferation and steroid hormone receptor binding, PKC activity was measured in sub- and postconfluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no differences were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were treated with PKC inhibitors (H-7 or staurosporine) or with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulation of the PKC activity in postconfluent cells enhanced ER-binding capacity in a dose-dependent manner and estrogen responsiveness of an exogenous reporter gene and of the endogenous alkaline phosphatase, representing an endogenous estrogen-stimulated gene. These data indicate that there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the proliferative and/or differentiative state of the cells, resulting in modulation of hormone responsiveness.
Mol Endocrinol 1993 Sep
PMID:Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels. 824 15

A regulatory mechanism for the vitamin D receptor (VDR) in rat osteosarcoma cells (ROS 17/2.8) is stabilization of the receptor through binding of its ligand, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Increased transcription of the gene encoding VDR does not occur upon treatment of these osteoblast-like cells with 1,25-(OH)2D3. When 10 nM 1,25-(OH)2D3 was administered to confluent cultures of ROS 17/2.8 cells, no change in receptor mRNA was detected, as measured by a ribonuclease protection assay. VDR abundance was measured using an immunoradiometric assay at varying time points within a 24-h period after 1,25-(OH)2D3 treatment. Receptor protein levels increased rapidly and continued to rise over 24 h. By 2 h, the level of receptor increased 2.5-fold, achieving a maximum level of 8-fold above the baseline at 18 h. The half-life of the receptor protein is 2 h in the absence of hormone, as determined by blockage of translation in cycloheximide-treated cells. In the presence of hormone, however, receptor levels were unchanged for at least 6 h. The administration of 1,25-(OH)2D3 stabilizes the receptor, thereby resulting in its accumulation in ROS 17/2.8 cells.
Mol Endocrinol 1993 Oct
PMID:Stabilization of the vitamin D receptor in rat osteosarcoma cells through the action of 1,25-dihydroxyvitamin D3. 826 62

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.
Mol Cell Biol 1993 Sep
PMID:Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene. 839 17

Although numerous studies have shown potent antiproliferative and differentiation-inducing effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and its analogs on cells not directly related to bone metabolism, only few reports focussed on the effects of these analogs on bone. We compared the action of several recently developed analogs with that of 1,25-(OH)2D3 on human (MG-63) and rat (ROS 17/2.8) osteoblast-like cells and on in vitro bone resorption. In MG-63 cells the analogs EB1089 and KH1060 were about 166,000 and 14,000 times more potent than 1,25-(OH)2D3 in stimulating type I procollagen and 100 and 6,000 times more potent in stimulating osteocalcin production, respectively. Also in ROS 17/2.8 cells EB1089 and KH1060 were most potent in inducing osteocalcin synthesis. In vitro bone resorption was 2.3 and 17.5 times more potently stimulated by EB1089 and KH1060, respectively. In MG-63 cells, 1,25-(OH)2D3 and the analogs inhibited cell proliferation, whereas both 1,25-(OH)2D3 and the analogs stimulated the growth of ROS 17/2.8 cells. Differences in potency could neither be explained by affinity for the vitamin D receptor nor by a differential involvement of protein kinase C in the action of the analogs. Together, these data show that also in bone the analogs EB1089 and KH1060 are more potent than 1,25-(OH)2D3 but that the potency of the analogs compared to 1,25-(OH)2D3 is dependent on the biological response. On the basis of these observations it can be concluded that the reported reduced calcemic effect in vivo is not the result of a decreased responsiveness of bone to these analogs. Lastly, in view of eventual clinical application of 1,25-(OH)2D3-analogs, the observed stimulation of in vitro bone resorption and growth of an osteosarcoma cell line warrant in vivo studies to further examine these effects.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Differential effects of 1,25-dihydroxyvitamin D3-analogs on osteoblast-like cells and on in vitro bone resorption. 854 Dec 30

Previous studies identified several glucocorticoid response elements (GREs) in the 5'-promoter region of the rat osteocalcin (OC) gene by purified receptor binding. The present study addresses functionality of the GRE sequences in the proximal promoter at nucleotide (nt) -16 to -1 downstream of the TATA element together with the GRE half-element in the OC box at nt -86 to -81. This was done by assaying glucocorticoid responsiveness [at 10(-6) M dexamethasone (DEX)], and in combination with 10(-8) M 1,25-dihydroxyvitamin D3, of a series of deleted and mutated OC promoter reporter constructs (OCCAT) in osteoblast-like cells, the ROS 17/2.8 rat osteosarcoma line. Promoter deletion analysis revealed an additional GRE in the distal promoter at nt -697 to -683 that functions to suppress OC transcription. In the absence of this upstream negative GRE (nGRE), the -531 OCCAT construct exhibited enhanced promoter activity in response to DEX (1.8-fold DEX/Control), but further deletion (-348 and -108 OCCAT constructs) restored DEX suppression to OC promoter activity (0.6- and 0.8-fold DEX/Control, respectively). Mutations introduced in both the proximal GRE (nt -16 to -1) and the half-GRE in the OC box, or in the proximal GRE alone, nearly abrogated DEX responsiveness of OC promoter activity. Both distal and proximal GREs specifically bound glucocorticoid receptor present in ROS 17/2.8 nuclear extracts as shown by competition with wild type and mutated oligonucleotides and antibody inhibition of binding. Furthermore, both GREs, independently, conferred DEX-responsive transcriptional repression to the heterologous thymidine kinase basal promoter. We also report that glucocorticoid suppression of 1,25-dihydroxyvitamin D3-stimulated transcription occurs independently of distal or proximal GREs. Taken together, these results demonstrate that in vivo responsiveness of OC to DEX involves the integrative activities of several functional promoter elements.
Mol Endocrinol 1995 Jun
PMID:Contributions of distal and proximal promoter elements to glucocorticoid regulation of osteocalcin gene transcription. 859 14

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.
Mol Cell Endocrinol 1996 Feb 05
PMID:Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells. 864 14

Expression of the gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is down-regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Deletions of the 5'-flanking region of the rat PTHrP gene, when fused to the chloramphenicol acetyl-transferase gene and transfected into ROS 17/2.8 (rat osteosarcoma) cells, showed that the 1,25-(OH)2D3 responsive region is located between -1.05 and -0.71 kb upstream of the transcription start site. Further mapping of this region revealed that a 123-bp fragment is able to confer 1,25-(OH)2D3 responsiveness to a heterologous (SV40) promoter. This region contains two potential vitamin D response elements (VDREs). One of these motifs resembles the negative VDRE (nVDRE) from the PTH gene, which is also down-regulated by vitamin D3. The other element resembles the canonical VDRE (two hexanucleotide motifs separated by three nucleotides), which has been characterized in a number of genes whose expression is modulated by vitamin D3. Electrophoretic mobility shift assays using nuclear extracts from ROS 17/2,8 cells and from vitamin D receptor. (VDR)-enriched COS 1 cells revealed that both elements interact with the VDR. This protein-DNA interaction is disrupted by an anti-VDR antibody. Therefore, modulation of PTHrP gene transcription by 1,25-(OH)2D3 is mediated by the VDR interacting with one or both of the identified motifs in the 5'-flanking sequence of the gene.
Mol Endocrinol 1996 Jun
PMID:DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression. 877 27

An important physiological control of PTH gene expression is its transcriptional repression by 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. The mechanism of this 1,25-(OH)(2)D(3)-mediated transcriptional repression is poorly understood. Previous investigations have identified a DNA sequence in the 5'-regulatory region of the human PTH (hPTH) gene that binds the vitamin D receptor (VDR) and mediates transcription repression in response to 1,25(OH)(2)D(3) in GH4CI cells. The hPTH gene sequence does not mediate transcriptional repression in ROS 17/2.8 cells, even though up-regulatory vitamin D response elements (VDREs) are active in these cells. The hPTH DNA sequence differs from the upregulatory VDREs in that it contains a single copy of a hexameric motif (AGGUC) homologous to those repeated in the up-regulatory VDREs. The protein-DNA interactions of this sequence were examined using nuclear extracts from bovine parathyroid, GH4CI, and ROS 17/2.8 cells. In bovine parathyroid nuclear extracts, the VDR binds the down-regulatory hPTH DNA sequence independently of the retinoid X receptor (RXR). In GH4C1 nuclear extracts, two VDR-containing complexes are observed: one lacking RXR and one containing RXR. In ROS 17/2.8 nuclear extracts, a single VDRdependent complex containing RXR is observed. When the up-regulatory rat osteocalcin VDRE is used as a probe, only VDR-RXR-containing complexes are generated using nuclear extracts from all three cell types. These results demonstrate that the sequence that mediates transcriptional repression in response to 1 ,25-(OH)(2)D(3) differs from the up-regulatory response elements both in sequence composition and in its ability to bind VDR independently of RXR.
Mol Endocrinol 1996 Mar
PMID:Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor. 883 58

The 1 alpha,25-dihydroxyvitamin D3 (VD3)-dependent stimulation of osteocalcin (OC) and osteopontin (OP) gene transcription in bone tissue is mediated by interactions of trans-activating factors with distinct VD3-responsive elements (VDREs). Sequence variation between the OC- and OP-VDRE steroid hormone half-elements provides the potential for recognition by distinct hormone receptor homo- and heterodimers. However, the exact composition of endogenous VD3- induced complexes recognizing the OC- and OP-VDREs in osteoblasts has not been definitively established. To determine the identity of these complexes, we performed gel shift immunoassays with nuclear proteins from ROS 17/ 2.8 osteoblastic cells using a panel of monoclonal antibodies. We show that VD3- inducible complexes interacting with the OC- and OP-VDREs represent two distinct heterodimeric complexes, each composed of the vitamin D receptor (VDR) and the retinoid X receptor-alpha (RXR). The OC- and OP-VDR/RXR alpha heterodimers are immunoreactive with RXR antibodies and several antibodies directed against the ligand-binding domain of the VDR. However, while the OC-VDRE complex is also efficiently recognized by specific monoclonal antibodies contacting epitopes in or near the VDR DNA-binding domain (DBD) (between amino acids 57-164), the OP-VDRE complex is not efficiently recognized by these antibodies. By systematically introducing a series of point-mutations in the OC-VDRE, we find that two internal nucleotides of the proximal OC-VDRE half-site (nucleotide -449 and -448; 5'-AGGACA) determine differences in VDR immunoreactivity. These results are consistent with the well established polarity of RXR heterodimer binding to bipartite hormone response elements, with the VDR recognizing the 3'-half-element. Furthermore, our data suggest that the DBD of the VDR adopts different protein conformations when contacting distinct VDREs. Distinctions between the OC- and OP-VDR/RXR alpha complexes may reflect specialized requirements for VD3 regulation of OC and OP gene expression in response to physiological cues mediating osteoblast differentiation.
Mol Endocrinol 1996 Nov
PMID:Distinct conformations of vitamin D receptor/retinoid X receptor-alpha heterodimers are specified by dinucleotide differences in the vitamin D-responsive elements of the osteocalcin and osteopontin genes. 892 69


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