Basic fibroblast growth factor (FGF-2), a potent mitogen for osteoblasts and marrow stromal cells, is synthesized by bone cells. FGF-2 null mice have marked decreased trabecular bone volume, decreased bone nodule formation in marrow cultures and reduce osteoclast formation in response to a variety of agonists. We hypothesize that FGF-2 regulates osteoblast replication and differentiation and that disruption of the FGF-2 gene results in impaired bone formation. In addition, osteoclast formation is reduced due to either insufficient support cells (osteoblasts), or reduced production of factors important for osteoclastogenesis.
Aim 1 : To elucidate the role of endogenous FGF-2 in bone formation. We will compare mice with no FGF-2 gene (FGF-2-/-) with wild type (FGF-2+/+) mice. Dynamic histomorphometry and bone densitometry will be performed on mice of different ages to determine the rate that decreased trabecular bone volume in FGF-2(-/-) mice develops. We will examine osteoblast differentiation in bone marrow stromal cell cultures and calvarial cell cultures from FGF-2 (-/-) and FGF-2 (+/+). We will determine the effect of loss of FGF-2 on gene markers of differentiation of early and mature osteoblasts including, CBFA1, type 1 collagen, alkaline phosphatase and osteocalcin. We will examine the expression of genes which support osteoblast differentiation which may be downstream of FGF-2 including BMP2 and TGFbeta. We will compare bone formation in calvariae by measuring DNA, thymidine labeling and proline incorporation into collagen and noncollagen proteins. We will determine whether in vivo administration of FGF-2 reverses bone loss in FGF-2 (-/-) mice.
Aim 2 : To determine whether osteoclast formation and bone resorption are altered in FGF-2 null mice. We will compare the ability of agonists to induce osteoclast formation using bone marrow, as well as coculture models of primary calvarial enriched osteoblasts and spleen cells from FGF-2 (-/-) and FGF-2 (+/+) mice. We will measure osteoclast precursor numbers in these animals by the CFU-GM assay. We will examine the expression of genes that are critical for osteoclast formation including osteoprotegerin ligand (OPGL), osteoprotegerin (OPG), receptor activator of NFKB (RANK) and macrophage colony stimulating factor. We will examine the expression of genes which are important in bone turnover which may be downstream of FGF-2 including PGHS-2, IGF-I, collagenase. To assess bone resorption, we will measure 45Ca release from prelabeled calvariae in the unstimulated and stimulated state. Analysis of bone formation and resorption in FGF-2 null mice is relevant not only to understanding the physiologic and pathologic role of this growth factor in bone remodeling, but also to facilitate the development of therapeutic usage of FGF-2 in bone disorders such as osteoporosis.
