FGF2 transgenic mice were developed in which type I collagen regulatory sequences drive the nuclear high molecular weight FGF2 isoforms in osteoblasts (TgHMW). The phenotype of TgHMW mice included dwarfism, decreased bone mineral density (BMD), osteomalacia, and decreased serum phosphate (P i ). When TgHMW mice were fed a high P i diet, BMD was increased, and dwarfism was partially reversed. The TgHMW phenotype was similar to mice overexpressing FGF23. Serum FGF23 was increased in TgHMW mice. Fgf23 mRNA in bones and fibroblast growth factor receptors 1c and 3c and Klotho mRNAs in kidneys were increased in TgHMW mice, whereas the renal Na shown that FGF23 is the phosphaturic factor (5) responsible for autosomal dominant hypophosphatemic rickets (6) and tumorinduced osteomalacia (7). FGF23 also mediates phosphatewasting disorders, such as X-linked hypophosphatemic rickets/ osteomalacia, the most common cause of vitamin D-resistant rickets (5). Loss of function mutations in PHEX, a phosphateregulatory gene with homology to endopeptidases on the X-chromosome, have been identified in X-linked hypophosphatemic rickets/osteomalacia (8); however, the mechanism by which this elevates FGF23 levels remains unclear. Autosomal recessive hypophosphatemic rickets/osteomalacia, caused by loss of function mutations in DMP1 (dentin matrix protein), is also associated with increased FGF23 (9). Increased FGF23 may occur in McCune-Albright syndrome due to a somatic gain of function mutation in GNAS1 arising during embryogenesis, characterized by chimeric distribution of hyperpigmented skin lesions, fibrous dysplasia of bone, and, often, hypophosphatemia (5). Murine models of this group of disorders include the FGF23 transgenic mouse, a model of autosomal dominant hypophosphatemic rickets (10); the Hyp mouse, a homologue of X-linked hypophosphatemic rickets/osteomalacia with a Phex deletion (11); and the Dmp1 null mouse, a model of autosomal recessive hypophosphatemic rickets/osteomalacia (12). These murine homologues demonstrate many of the phenotypic changes of the human disorders, including elevated serum FGF23.Another FGF ligand, FGF2 (fibroblast growth factor-2) is widely expressed and is a mitogen for many cell types, including osteoblasts and chondrocytes (1,13,14). The FGF2 gene encodes multiple FGF2 high molecular weight (HMW) protein isoforms expressed from unique CUG alternative translation start sites located 5Ј to the classical AUG initiation codon for the 18-kDa low molecular weight (LMW) exported isoform (1,

Activation of the signal transducers and activators of transcription (STAT) pathway is important in fibroblast growth factor (FGF) modulation of chondrocyte proliferation and endochondral bone formation during embryogenesis. However, it is not known if the FGF/STAT signaling pathway is important for postnatal bone formation. To examine this, we have characterized a novel skeletal phenotype in Stat1؊/؊ mice in which we find a significant increase in bone mineral density, bone mineral content, and other parameters of bone growth. The data show that osteoblasts derived from Stat1؊/؊ mice have decreased expression of cell cycle inhibitor p21WAF/CIP and FGF receptor 3, a known negative regulator of chondrocyte proliferation. Interestingly, Stat1؊/؊ osteoblasts showed increased expression of FGF18 in vivo and increased responsiveness to FGF18 in vitro. These results suggest a mechanism for the regulation of the osteoblast in which Stat1 functions not only to directly regulate the cell cycle but also to modify the repertoire of FGF receptor expression from a potentially inhibitory receptor, FGFR3 to a stimulatory receptor such as FGFR1 or FGFR2.

Disruption of the fibroblast growth factor 2 (FGF-2) gene results in reduced bone mass in mice and impairs expression of bone morphogenic protein-2 (BMP-2) an important mediator of osteoblast and osteoclast differentiation. Since the relationship between FGF-2 and BMP-2 in bone remodeling has not been fully determined, in this study we examined whether endogenous FGF-2 was necessary for maximal effect of BMP-2 on periosteal bone formation in vivo and bone nodule formation and osteoclast formation in vitro in Fgf2-/- mice. We showed that BMP-2 significantly increased periosteal bone formation by 57% in Fgf2+/+ mice but the changes were not significant in Fgf2-/- littermates. In line with these results we found no significant increase in alkaline phosphatase positive (ALP) activity in calvarial osteoblasts or ALP mineralized colonies in stromal cultures from Fgf2-/- mice after BMP-2 treatment. Moreover, BMP-2 induced osteoclast formation was also impaired in marrow stromal cultures from Fgf2-/- mice. Interestingly, BMP-2 induced nuclear accumulation of the runt related transcription factor (Runx2) was markedly impaired in osteoblasts from Fgf2-/- mice. Examination of the effect of loss of FGF-2 on BMP-2 signaling pathway showed that BMP-2 caused a similar induction of phospho-Smad1/5/8 within 30 min in calvarial osteoblasts from both genotypes. In contrast BMP-2-induced p42/44 MAPK was reduced in Fgf2-/- mice. These findings strongly demonstrated that endogenous FGF-2 is important in the maximal responses of BMP-2 in bone and that this may be dependent on the p42/44 MAPK signaling pathway and downstream modulation of Runx2.

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Over-expression of human FGF-2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1-6 month-old wild (NTg) and TgFGF2 mice were studied. FGF-2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro-CT of trabecular bone of the distal femurs from 6-month-old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double-labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non-targeted over-expression of FGF-2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF-2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.