Normal bone formation is controlled by local and systemic growth regulators. Of these, transforming growth factor-beta (TGF-beta) appears to have a critical and complex role in bone health and disease. In vitro studies show significant changes in TGF-beta receptor (TGF- betaR) levels on bone cells in parallel with differentiation, and in response to important stimulators and inhibitors of osteogenic cell activity. Furthermore, gain or loss of specific TGF-betaRs can determine whether and how bone cells respond to TGF-beta exposure. In order to focus TGF-beta more accurately to appropriate bone cells, and to reverse detrimental effects by inappropriate expression of TGF- betaRs, the mechanisms that control TGF-betaR synthesis and stability must be resolved. Studies in this proposal will assess genomic and biochemical elements that regulate TGF-betaR levels on bone cells. TGF- betaR gene promoters will be examined by promoter/reporter analysis, gel shift analysis, and by deletions and mutations to define cis- and trans- acting elements that regulate TGF-betaR expression. Variations in steady state TGF-betaR mRNA and protein levels will be examined by radio-ligand binding, RNase protection and Western blot analyses. Flux of cell surface TGF-betaRs will be evaluated immunohistochemically. Interactions among individual TGF-betaRs will be examined by immunoprecipitation and Western analysis. These parameters will be assessed in response to well defined local and systemic bone growth regulators.
Each specific aim concentrates on unique studies related to individual TGF-betaRs, and includes studies to integrate common themes.
Specific aim 1 will address regulatory elements in the TGF- betaR type I (TGF-betaRI) gene promoter that may contribute to differential expression of TGF-betaRI by osteoblasts. TGF-betaRI flux and its specific interactions with TGF-betaRs type II and type III (TGF- betaRII and TGF-betaRIII) will be examined in response to heterologous and homologous receptor regulation.
Specific aim 2 will define control elements in the TGF-betaRIII promoter that we have just now cloned. It will also assess TGF-betaRIII flux, interactions with other TGF-betaRs, and effects by specific TGF-betaRIII regulators.
Specific aim 3 will further define TGF-betaRII promoter sequences to determine control elements that may be cryptic or absent in the TGF-betaRII promoter clones previously studied. Promoter activity, TGF-betaRII flux, interactions with other TGF-betaRs, and effects of TGF-betaRII regulators will also be examined. Due to the importance of TGF-beta in bone metabolism, our studies of TGF-betaR expression on osteoblasts may define new molecular targets that could be used to focus and enhance bone growth and repair.
