Basic fibroblast growth factor(bFGF) is a potent mitogen for osteoblasts and has both stimulatory and inhibitory effects on collagen synthesis in bone. However, the role of this growth factor in normal bone growth and remodeling is unknown. Recently, bFGF mRNA and protein were identified in cultured bovine bone explants. In preliminary studies we have demonstrated that osteoblastic MC3T3-E1 cells express bFGF mRNA and bFGF protein. The fact that bFGF is made by bone cells and that exogenous bFGF affects collagen production and cell replication in bone suggests that local bFGF production may play a role in bone cell function. 1.) The first goal of this study is to assess bFGF expression in vitro and in vivo using osteoblastic MC3T3-E1 cells, primary mouse calvarial cells and organ cultures of mouse calvaria. bFGF mRNA will be quantitated by Northern blot hybridization assays. bFGF protein will be determined by Western blot analysis. Immunohistochemical localization and in situ hybridization will be used to identify the cells within bone which produce bFGF in vivo. 2.) The second goal is to determine the role of endogenous bFGF on osteoblast proliferation and differentiation in bone cell cultures and bone organ cultures and the role of endogenous bFGF in unstimulated bone resorption in mouse calvariae. We will utilize antisense bFGF oligodeoxynucleotides which block translation of bFGF mRNA and antibodies to bFGF protein in these studies. We will determine by Northern blot analysis whether there are changes in bFGF mRNA and by Western blot analysis changes in bFGF protein production. We will measure DNA synthesis and collagen synthesis in the presence of bFGF antisense oligodeoxynucleotides. If there are changes in cell replication in the presence of antisense bFGF oligodeoxynucleotides, we will determine by Northern blot analysis whether these changes are associated with modulation of the mRNA for the early genes c-fos and c-jun since the mitogenic signalling pathway of bFGF involves activation of these early genes. If there are changes in collagen synthesis, we will determine whether alpha1(I) collagen mRNA is altered. 3.) We have found that TGFbeta increases bFGF mRNA in MC3T3-E1 cells. Hence the third goal of this proposal is to extend our studies of TGFbeta regulation of bFGF expression in bone. MC3T3-E1 cells and mouse calvariae will be treated with TGFbeta. bFGF mRNA levels will be determined by Northern blot analysis and bFGF protein by Western blot analysis in the presence and absence of TGFbeta. We will determine the effects of TGFbeta on bFGF mRNA and protein expression in the presence of antisense bFGF oligodeoxynucleotides. Immunohistochemical localization and in situ hybridization will be used to determine whether TGFbeta regulates bFGF production in bone. Understanding the mechanism (s) regulating bFGF expression in bone, may be important in delineating the role of this growth factor in both physiologic and pathologic states of bone metabolism.
