Glucocorticoids (GC) are clinically important anti-inflammatory agents that affect cellular growth and differentiation, particularly in skeletal tissues. Long term GC treatment is associated with osteopenia and related to bone disorders. Little is known, however, regarding the mechanisms of GC mediated regulation of gene expression in osteoblasts during bone development. These molecular mechanisms can offer insight into actions of GC from both therapeutic and pathobiological perspectives. Recently, the bone specific osteocalcin gene has been characterized with respect to the representation and organization of regulatory sequences which resulted in the identification of multiple promoter elements, including at least two putative GREs. Our grant will address questions related to in vivo responsiveness of the osteocalcin gene to GCs in bone cells at various stages of osteoblast growth and differentiation to define the conditions and requirements of functionality of GC responsive promoter regulatory element. The focus of our studies will specifically be to: (1) determine the functionality of the putative GRE sequences in the promoter and establish specific nucleotides contributing to glucocorticoid-mediated transcription, (2) characterize the GC receptor complex protein-DNA interactions as a function of osteoblast phenotype development, and then 3) identify the accessory proteins of the GC receptor complexes during osteoblast growth and differentiation as well as 4) determine modifications in chromatin structure associated with GC binding to the osteocalcin gene. Developmentally regulated hormone responsiveness of the osteocalcin gene as a function of osteoblast growth and differentiation is reflected by: 1) the absence of transcription in proliferating osteoblasts, 2) induction of expression in the post-proliferative period and 3) further upregulation (100-fold) during mineralization of the bone matrix. These parameters of OC gene expression occur during fetal calvarial development and in cultured osteoblasts. A rat calvarial osteoblast culture system that produces an extracellular matrix with bone-like tissue organization provides a model together with the osteocalcin gene, for systematically examining GC receptor complexes during osteoblast phenotype development. These studies will now improve our understanding of osteoblast GR and associated transcription factors that contribute to selective activation and/or repression of transcription of a tissue-specific gene. Such knowledge can serve as a basis for selective utilization of GC and their analogues therapeutically.
