STRUCTURAL AND FUNCTIONAL ANALYSIS OF GLUCOCORTICOID RESPONSIVE ELEMENTS IN THE OSTEOCALCIN GENE PROMOTER

The Osteocalcin (OC) gene encodes a 10 Kda hone-specific protein which is expressed with the onset of mineralization during the differentiation of normal diploid osteoblasts. OC levels in the serum correlate with bone turnover. The tissue-specific expression of OC gene is regulated by a modular promoter composed of a complex series of regulatory elements. The transcriptional activity of the OC gene is controlled by several physiological and hormonal regulators that independently and synergistically or antagonistically modulate OC expression (e.g. vitamin D3 glucocorticoids. retinoic acid. TGF-β).\ud \ud Complexity of steroid responsiveness of OC gene during osteoblast development has been under observation. The understanding of molecular mechanisms mediating glucocorticoid responsiveness in osseous cells is limited. Glucocorticoids are physiological mediators of osteoblast proliferation and differentiation, as well as perturbants of bone formation and resorption in vivo. The main goal of the present study has been focused on functional characterization of glucocorticoid-mediated regulation of OC gene as a model system. Previous studies in the laboratory identified glucocorticoid-receptor binding sequences in the proximal part of rat OC gene promoter using partially purified receptor. Here, using a series of OC promoter deletion and mutation constructs, the functionality of the GR-binding sequences in the proximal promoter at nucleotide (nt) -16 to -1 downstream of TAT A element together with the glucocorticoid receptor element (GRE) half element in the OC box. at nt-86to-81 was assayed. This was done by assaying glucocorticoid responsiveness at 10-6 M Dexamethasone (DEX). and in combination with 10-8M 1.25(OH)2D3 (D3), with deleted and mutated OC promoter-reporter constructs (OCCAT) in osteoblast-like cells. the 17/2.8 rat osteosarcoma (ROS) cell line. Promoter deletion analysis revealed an additional GRE in the distal promoter at nt.-697to-683 that functions to suppress OC transcription. In transient transfection assays, in the absence of distal negative GRE (nGRE), the -531 OCCA T construct exhibited enhanced promoter activity in response to DEX (1.8 fold DEX/control), but further deletions (-348 and -108 OCCA T constructs) restored D EX suppression to OC promoter activity (0.6- and 0.8-fold DEX/control, respectively).Both distal and proximal GREs specifically bound glucocorticoid receptor present in ROS 17/2.8 and ROB cells as evidenced by competition with wild type and mutated oligonucleotides and antibody inhibition of binding. ROS 17/2.8 cell specific glucocorticoid-receptor complex was detected in nuclear matrix fractions binding to both proximal and distal GREs. Furthermore, both,GREs, independently, conferred DEX-responsive transcriptional repression to the heterologous thymidine kinase basal promoter. Mutations were introduced in the proximal GRE, half GRE in OC box and distal GRE either independently or in combination in context of the full OCCA T and in shorter OCCA T constructs. These mutants were functionally evaluated for ability to confer OC transcriptional activity in response to DEX and vitamin D3 In the absence of distal GRE, mutations in proximal GREs nearly ahn1gated DEX responsiveness of OC promoter activity in shorter OCCAT constructs. In context of full promoter. mutations introduced in the proximal and distal GREs and half GRE in OC box. abrogated DEX responsiveness of OC transcription and also significant abrogation of vitamin D3-mediated enhanced OC transcription was observed. The effect of DEX on OC promoter activity was much more pronounced when -1097 OCCAT construct was stably transfected into ROS 17/2.8 cells (3-4 fold repression compared to 0.7 fold repression in transient transfection assays),the same fold repression was observed for two shorter constructs. Furthermore, this effect required the -531 to -348 domain. which was not implicated in DEX-mediated repression in the transient transfection assays. These results suggest the higher order nuclear structure (e.g. nucleosomal organization, interactions with the nuclear matrix, which is better represented in the stable transfection assays), plays a role in glucocorticoid-mediated regulation of OC transcription. The results presented in this dissertation show that:\ud \ud (i) In vivo responsiveness of osteocalcin gene to DEX involves the integrated activities of several functional promoter elements.\ud \ud (ii) Glucocorticoid repression of vitamin D3-stimulated OC transcription occurs independently of distal or proximal GREs.\ud \ud (iii) The difference observed for deleted OCCA T promoter constructs in response to DEX, in transient compare to stable transfections, functionally demonstrate the contribution of chromatin structure to transcriptional control.\ud \ud (iv) Several distinct promoter segments were identified that exert either positive or negative effects on transcription in response to DEX. But also coma in regulatory determinants that influence basal and vitamin 03 enhanced OC transcription