Mechanism of conditional repression of human osteocalcin gene activity by glucocorticoids

Sammanfattning: Bone is one of the major target sites of steroid hormone action. Steroid hormones like cortisol, vitamin D and estradiol are responsible for principle events associated with bone formation and resorption. The action of these hormones in bone is reflected by the specific regulation of genes in bone cells, thereby affecting bone formation and resorption. Glucocorticoids exert a suppressive effect on bone formation. Increasing levels of glucocorticoids results in the development of osteoporosis in the long term. An example of a gene which is negatively regulated by glucocorticoids in bone is the gene for the major noncollagenous bone protein osteocalcin. In vitro analysis has identified a glucocorticoid receptor binding element in the human osteocalcin promoter that overlaps a functionally important basic transcription element, the TATA box. The project the function of this dual element and mechanistic aspects of the glucocorticoid dependent transrepression have been characterized in this thesis work. A specific mutation of rat GR, K461A, has previously been described to transform the indirect protein-protein interaction-dependent transrepressive effect of GR to an activating function. This mutation was shown to transform the transrepressive effect of wild-type GR, acting on reporter constructs containing a negative composite GRE or a negative tethering GRE, into an activating function. In contrast, the K461A mutation had no effect on the transrepressive effect of GR on the osteocalcin promoter. Thus, mutation of rat GR at position 461 differentiates between transrepressive functions of GR dependent on GR-DNA interaction (repression by occlusion) and GR-protein interaction (active repression). Furthermore we studied the function of the osteocalcin TATA site in respect to the negative glucocorticoid mediated effect on osteocalcin gene activity. We demonstrate that changing the osteocalcin TATAAAC box into a standard TATAAAA element blocks the repression by hormone activated glucocorticoid receptor. The mutation did not influence the specific GR binding indicating that this weak TATA box has an active function in the regulatory process. In additional studies we showed that the repressive effect of glucocorticoids on this promoter is the result of competitive binding to a basal transcriptional element and that it does not appear to require direct protein-protein interaction between the competitive factors. Our data strongly indicates that the occupation of the dual binding site prevents TBP binding to the TATA box and disrupts the assembly of an active preinitiation transcriptional complex. Finally, differences in the mechanism of GR mediated transrepression of the basal and vitamin D activated osteocalcin gene activity have been characterized. Our results support a model wherein the GR mediated transrepression of the activated osteocalcin gene activity is dependent on two distinct mechanisms.