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

The p53 gene undergoes rearrangement in a high percentage of osteosarcomas, resulting in loss of its expression. A p53-null murine osteosarcoma cell line F6 was transfected with either a wild-type or a mutant p53 gene. Stably transfected cell lines were obtained, and their differentiation capabilities were compared in vitro with the parental cell line. Alkaline phosphatase and osteocalcin expression were measured as early and late differentiation markers, respectively. Induction of alkaline phosphatase expression was not affected by the presence of either p53 gene, whereas osteocalcin expression was seen in cells containing the wild-type p53 gene but not in the parental p53-null or mutant-expressing cell lines. That the induction of osteocalcin was intrinsically dependent on the presence of wild-type p53 was also indicated by the use of a temperature-sensitive Val 135 p53 mutant at 32 degrees C; predominant expression of p53 in the wild-type conformation resulted in osteocalcin expression. While the wild-type p53 gene could suppress tumor formation in vivo, the tumors expressing the mutant p53 gene grew two to three times as large as the tumors that did not express p53. Therefore, the absence of end-point differentiation in bone due to p53 rearrangements may contribute to the maintenance of the tumorigenic phenotype in osteosarcomas.
Mol Carcinog 1993
PMID:Dependence of induction of osteocalcin gene expression on the presence of wild-type p53 in a murine osteosarcoma cell line. 828 Mar 78

Tumor necrosis factor (TNF) down-regulates the production of bone matrix proteins by osteoblasts, thereby inhibiting bone formation. Osteocalcin, the major noncollagenous protein in bone, is inhibited by TNF at the transcriptional level. Mapping studies were undertaken to characterize the TNF-responsive element (TNFRE) in the osteocalcin promoter. Deletion analysis localized the TNFRE to the -522/-511 region, which contains a 9-bp palindromic motif (AGGCTGCCT). Promoter segments containing this sequence down-regulated a heterologous simian virus 40 promoter. Site-specific mutagenesis of the TNFRE eliminated TNF down-regulation. Mobility shift assays demonstrated that a constitutively expressed nuclear factor bound to the TNFRE; this factor was tentatively identified as the p50 homodimer of NF-kappa B. TNF stimulation induced a second TNFRE-binding protein which displaced the constitutive factor. The TNF-induced protein was not inhibitable by the NF-kappa B consensus sequence and was unreactive with anti-NF-kappa B antiserum. DNase footprinting demonstrated that both factors protected the -522/-501 portion of the promoter, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that the generalized catabolic activities of TNF in infectious and malignant diseases may be regulated via this novel element.
Mol Cell Biol 1993 Jun
PMID:Characterization of a tumor necrosis factor-responsive element which down-regulates the human osteocalcin gene. 838 44

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

To study the direct effects of thyroid hormones on human osteoblasts we examined the effects of triiodothyronine (T3) on proliferation and differentiation of human osteoblast-like (hOB) cells in vitro. T3 increased 3H-thymidine incorporation in DNA of hOB cells (p < 0.05, n = 10). Half maximal effects obtained at a T3 concentration of 1-10 nM which lies within the physiological concentration of the hormone. In addition, T3 increased alkaline phosphatase production (p < 0.05, n = 13) and inhibited procollagen type I carboxyterminal propeptide (PICP) production (p < 0.05, n = 13). T3 interaction with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was also studied. 1,25-(OH)2D3 (10(-9)M) alone doubled AP production and induced osteocalcin expression by hOB cells. Concurrent addition of T3 and 1,25-(OH)2D3 did not further increase production of AP, PICP or osteocalcin by hOB cells. In conclusion, T3 exerts significant effects on osteoblast proliferation and differentiation, suggesting that human osteoblasts are targets for thyroid hormones.
Biochem Mol Biol Int 1993 Jul
PMID:Effects of triiodothyronine on DNA synthesis and differentiation markers of normal human osteoblast-like cells in vitro. 840 33

Osteocalcin is a major noncollagenous protein of bone regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and is believed to be expressed only by differentiated osteoblasts. We introduced a 3.9-kilobase human osteocalcin gene promoter (hOCP)-chloramphenicol acetyltransferase (CAT) fusion gene into the germ line of mice. Examination of tissue extracts from these transgenic mice demonstrated that the expression of CAT was restricted to bone-associated tissues and the brain. Immunohistochemical staining of femur tissue sections using CAT antibodies localized the production of CAT protein to osteoblasts and maturing chondrocytes. Previous studies via transient transfection into osteoblast-like cells have identified a vitamin D response element approximately 500 basepairs up-stream of the hOCP capable of mediating 1,25-(OH)2D3 induction. As a consequence, regulation of the transgene was examined in homozygous transgenic lines for sensitivity to 1,25-(OH)2D3. Hormonal deficiency was created using a low calcium diet supplemented with 0.8% SrCl2 for 7 days and was restored in experimental mice by injection of 25 ng 1,25-(OH)2D3/day, ip, for 3 days. The low vitamin D3 diet decreased CAT activity several-fold in extracts from calvaria, femur, and brain compared to that in mice maintained on a normal diet, while 1,25-(OH)2D3 supplementation restored and enhanced CAT activity over control values. These data demonstrate that hOCP is sufficient to direct osteoblast-specific 1,25-(OH)2D3-sensitive gene expression in mice in addition to the unexpected regulatable expression in brain tissue.
Mol Endocrinol 1993 Mar
PMID:The human osteocalcin promoter directs bone-specific vitamin D-regulatable gene expression in transgenic mice. 848 81

To identify osteoblast-specific cis-acting elements and trans-acting factors, we initiated an analysis of the promoter of a mouse osteocalcin gene, an osteoblast-specific gene. In this promoter, we identified two osteoblast-specific cis-acting elements (Ducy, P. and Karsenty, G. (1995) Mol. Cell. Biol. 15, 1858-1869). The sequence of one of these elements, OSE2, is identical to the DNA-binding site of the PEBP2 alpha/AML-1 transcription factors, the mammalian homologues of the Drosophila Runt protein. Here we show, using nuclear extracts, recombinant protein, and a specific antiserum against AML-1 proteins in DNA-binding assays, that one member of this family, AML-1B, binds specifically to OSE2 and is immunologically related to OSF2, the factor present in osteoblast nuclear extracts that binds to OSE2. By DNA cotransfection experiments, we also demonstrate that AML-1B can increase the activity of a short osteocalcin promoter through its binding to OSE2. Lastly, the different mobilities of osteoblast nuclear extract-DNA complexes compared with T-cell nuclear extract-DNA complexes, along with the inability of OSF2 to be upregulated by retinoic acid, unlike the other PEBP2 alpha factors, suggest that OSF2 is a new member of this family of transcription factors. Thus, this study demonstrates that AML-1B can increase gene expression of an osteoblast-specific gene through its binding to an osteoblast-specific cis-acting element and presents evidence that OSF2 is a member of the PEBP2 alpha/AML-1 family of transcription factors.
 ...
PMID:A PEBP2 alpha/AML-1-related factor increases osteocalcin promoter activity through its binding to an osteoblast-specific cis-acting element. 853 54

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

Unlike estrogen and progesterone receptors that operate as homodimers on response elements, retinoid X receptors (RXRs) and vitamin D receptors (VDRs) can function as heterodimers. Studies concerning the significance of heterodimeric partnerships are usually performed utilizing mammalian or insect cells. These cells express endogenous nuclear receptors, making it impossible to assign a role for one receptor subtype over another while studying the function of transfected receptor(s). Yeast lacks endogenous VDRs and RXRs and their ligands and provides a unique cellular context to study nuclear receptor function. We examined the interaction between human VDR and human RXR alpha, mouse RXR beta 2, and mouse RXR gamma to identify physiologically important receptor interactions. DNA binding studies on consensus, osteocalcin, or the rat 24-hydroxylase vitamin D response elements (VDREs) indicated that although RXR complexes can form on the consensus DNA elements, RXR:VDR heterodimers preferentially interact with the natural VDREs. The interaction is RXR isotype-specific and affected by ligands. Transactivation studies using the rat 24-hydroxylase VDREs indicated that VDR preferentially associated with RXR alpha or RXR gamma to stimulate transcription, and the activity was potentiated by ligand. Although RXR beta 2:VDR bound tightly to DNA, the resulting heterodimer transactivated poorly. The regulation of the 24-hydroxylase promoter observed in yeast is similar with respect to transactivation potential of specific VDRE and fold activation observed in osteosarcoma cells. Ligand binding to both receptors in a RXR:VDR complex is required for maximal transcriptional activity, indicating that the isotype-specific RXR partner significantly contributes to the ability of RXR:VDR heterodimers to transactivate from target response elements in yeast.
Mol Endocrinol 1996 Apr
PMID:Retinoid X receptor isotype identity directs human vitamin D receptor heterodimer transactivation from the 24-hydroxylase vitamin D response elements in yeast. 872 85

We have studied the effects of the complex Glycyl-L-Histidyl-L-Lysine:Cu (GHK:Cu), the GHK sequence present in the alpha 2 (I) chain of human collagen (Coll I), and bone matrix glycoproteins containing either RGD (fibronectin, FN), or RGD and GHK (Coll I), on the spreading, attachment and markers of the osteoblast phenotype in rat calvaria cells (RC), human trabecular osteoblastic cells (HT) and human marrow stromal cells (HM). Coll I (20 micrograms/ml) and FN (20 micrograms/ml) coating enhanced osteoblastic cell spreading, whereas free GHK:Cu and GHK coating (10(-10)-10(-8) M) had no effect. FN and Coll I, as well as GHK:Cu and GHK, increased the attachment of RC and HT cells. The attachment of both total number of cells and alkaline phosphatase (ALP)-positive osteoblastic cells was increased, showing no preferential effect on cells expressing this early marker of the osteoblast phenotype. In addition, immunocytochemical analysis showed that FN, Coll I and GHK:Cu coating increased both the total number of HM cells and the number of HM cells expressing Coll I or osteocalcin, indicating that GHK:Cu and RGD-containing proteins acted similarly on cells expressing different maturational stages. In contrast to its effect on cell attachment, GHK:Cu coating slightly inhibited the basal and 1,25(OH)2D-induced stimulation of ALP activity or osteocalcin production in rat and human osteoblastic cells. The finding that GHK promotes cell attachment and decreases the phenotype of normal rat and human osteoblastic cells suggests that osteoblasts may interact with free GHK or GHK-containing proteins in the bone matrix.
Cell Mol Biol (Noisy-le-grand) 1995 Dec
PMID:Effects of the tripeptide glycyl-L-histidyl-L-lysine copper complex on osteoblastic cell spreading, attachment and phenotype. 874 89


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