The goal of this work is to determine how local agents may be used to enhance bone growth and repair, emphasizing growth regulators within the transforming growth factor B (TGF-B) gene family. The specific focus concerns changes that may occur in select TGF-B receptors during osteoblast maturation or in response to the bone morphogenetic proteins (BMPs), relationships only now emerging in skeletal tissue. The TGF-B gene family comprises at least three closely related isoforms (TGF-B1, -B2, -B3) in mammals, and other highly homologous proteins, including activins and the BMPs themselves. These factors may control cell growth and differentiation in various tissues, but specific family members appear to be expressed differentially during skeletal development, and to accumulate in bone matrix and control bone cell activity in vivo and in vitro. Bone cells possess at least four distinct groups of TGF-B binding sites, once termed types I-IV receptors. Binding to type I (53 kD) and II (75 kD) sites appears to mediate signal transduction. Type III (>250 kD) sites are proteoglycans that do not seem to signal directly, but may facilitate extracellular TGF-B storage, activation, or binding to and signaling by other receptors. Sites previously termed type IV (55-75 kD) bind activins and TGF-Bs, and may comprise a group of related molecules that also bind other distant TGF-B gene family proteins. Several other low affinity sites of unknown function have also been described. but these have not yet been detected on normal bone cells. However, the investigators have shown that hormones, growth factors and various TGF-B family members induce complex changes in bone cell activity that may be associated in part with changes in binding to specific TGF-B receptors. Cloning of several TGF-B receptor species now permits a more detailed analysis at the mRNA level. The proposed studies will attempt to define more fully the expression of TGF-B receptors in bone cell cultures. Based on previous binding and activity profiles established in fetal rat bone cell cultures. these studies will specifically address the expression of members of each receptor class for which cDNA probes have been obtained. Studies will focus primarily on changes in TGF-B binding to select receptors as a function of osteoblast phenotype and in response to other bone growth regulators within the TGF-B family, the BMPs. Steady state mRNA levels will be examined by Northern and quantitative Sl nuclease analyses, and inhibitors of mRNA synthesis will be used to assess mRNA transcript turnover. Receptor protein levels will be examined using chemical cross-linking and resolving ligand receptor complexes on polyacrylamide gels. Where available, receptor specific antibody preparations will also be used in immunoprecipitation studies to assess specific receptor expression and turnover. The consequence of changes in receptor expression will be examined by well defined biological activities known to be induced by various TGF-B family members in these cultures. These changes will be challenged by transfection studies using intact and deficient receptor constructs, with the intent to increase, restore, or delete specific receptor binding and biological functions. Results from these studies could help to clarify relationships now emerging among various TGF-B gene family members in bone, and to assist development of interventions that selectively alter TGF-B function to enhance bone formation or repair.
