The responsiveness of genes to steroid hormones is principally mediated by functional interactions between DNA-bound hormone receptors and components of the transcriptional initiation machinery, including TATA
Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.
The mouse MC3T3-E1 cell line is nontumorigenic and undergoes a typical program of osteoblast differentiation in vitro, producing a bone-like mineralized extracellular matrix. We report responses of these cells to dexamethasone (Dex) and 1,25-(OH)2D3 that are in contrast to findings from other osteoblast culture systems. First, chronic exposure of both early- and late-passaged MC3T3-E1 cells to 10(-7) M Dex, initiated during the proliferation period, blocked osteoblast differentiation, in contrast to the enhanced differentiation observed in cultures of fetal rat calvarial-derived cells. Secondly, 1,25-(OH)2D3 did not up-regulate expression (messenger RNA or protein synthesis) of the endogenous mouse osteocalcin (OC) gene. Several lines of evidence are presented that suggest this response is caused by sequence specific properties of the mouse OC vitamin D response element. We also observed both qualitative and quantitative differences in expression of cell growth (histone H2B) and phenotype-related genes (collagen, OC, osteopontin, glucocorticoid receptor, and 1, 25-(OH)2D3 receptor), between pre- and postmineralization stage osteoblasts, in response to 24 h steroid hormone treatment. Our findings in MC3T3-E1 cells are consistent with current concepts of selective influences of 1,25-(OH)2D3 and glucocorticoids as a function of osteoblast maturation. However, the inhibition of osteoblast differentiation by chronic Dex at 10(-7) M and the down-regulation of OC by 1,25-(OH)2D3 are novel observations relevant to species-specific responsiveness of mouse bone-expressed genes to steroid hormones during osteoblast differentiation.
Responsiveness of genes to steroid hormones is a complex process involving synergistic and/or antagonistic interactions between specific receptors and other nonreceptor transcription factors. Thus, DNA recognition elements for steroid hormone receptors are often located among binding sites for other trans-acting factors. The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, stimulates transcription of the tissue-specific osteocalcin (OC) gene in osteoblastic cells. The rat OC vitamin D response element contains an internal acitvating protein-1 (AP-1) site. Here, we report for the first time that this AP-1 site is critical for the transcriptional enhancement of rat osteocalcin gene expression mediated by vitamin D. Precise mutations were introduced either in the steroid half-elements or in the internal AP-1 sequences. One mutation within the internal AP-1 site retained vitamin D receptor/retinoid X receptor binding equivalent to that of the wild-type sequence, but resulted in complete loss of vitamin D inducibility of the OC promoter. These results suggest a functional interaction between the hormone receptor and nuclear oncoproteins at the rat OC vitamin D response element. This cooperation of activities may have important consequences in physiological regulation of osteocalcin transcription during osteoblast differentiation and bone tissue development in vivo.
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.
We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts.
We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts.
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