Local and systemic growth regulators control normal bone formation. Of these, transforming growth factor-beta (TGF-beta) appears to have a critical and complex role in bone health and disease. Specific changes in TGF-beta receptor (TbetaR) levels occur on bone cells in response to important stimulators and inhibitors of osteogenic cell activity in vitro. Moreover, TGF-beta independently stimulates and inhibits several aspects of osteoblast activity, often in phenotype-related ways. These observations predict that gain or loss of specific TbetaRs can determine whether and how osteoblasts respond to TGF-beta exposure. In order to avoid detrimental effects by inappropriate expression of TbetaRs and to focus TGF-beta activity more accurately on bone cells, mechanisms that control TbetaR synthesis and signaling must be resolved. Studies in this proposal will continue to define genomic and biochemical elements that regulate these events. TbetaR gene promoters will be dissected by reporter gene and gel shift analyses, and by deletions and mutations to define cis- and trans-acting elements that regulate TbetaR gene expression. TbetaR protein and mRNA levels will be measured by radioligand binding, Western blot and RNase protection analyses. Interactions among individual TbetaRs will be examined by immunoprecipitation and Western analyses. Downstream effects on TGF-beta activity will be assessed on cell replication and matrix protein synthesis, and by assays and forced expression of genes that relate to the expression or the activity of individual TbetaRs. These parameters will be studied in response to well-defined local and systemic bone growth regulators. Studies in Aim I will define regulatory elements in the TbetaRI gene promoter that repress its expression, with an emphasis on Runx-sensitive cis- and trans-acting elements. Results will be extended to other Runx-sensitive gene targets that may be repressed in a similar way. Studies in Aim 2 will concentrate on the TbetaRII gene promoter, in particular on E box binding proteins that appear to control TbetaRII expression during osteoblast differentiation. They will also assess the possibility of co-ordinate control with TbetaRIII, and extend to a limited number of E box sensitive genes with relevance to bone. Studies in Aim 3 will define functional aspects of TbetaRlll expression in osteoblasts. They will examine interactions between TbetaRIII and its PDZ-domain binding cofactor GIPC, and the effect of TbetaRIII glycosylation on native and hormone-induced changes in TbetaR multimer complex formation and stability. Due to the importance of TGF-beta in bone metabolism, studies like these that initiate with TbetaR expression on osteoblasts may continue to define new molecular targets that could be used to focus and enhance bone growth and repair.
