Basic fibroblast growth factor (FGF-2), an important modulator of cartilage and bone growth and differentiation, is expressed and regulated in osteoblastic cells. To investigate the role of FGF-2 in bone, we examined mice with a disruption of the Fgf2 gene. Measurement of trabecular bone architecture of the femoral metaphysis of Fgf2 +/+ and Fgf2 -/-adult mice by micro-CT revealed that the platelike trabecular structures were markedly reduced and many of the connecting rods of trabecular bone were lost in the Fgf2 -/-mice. Dynamic histomorphometry confirmed a significant decrease in trabecular bone volume, mineral apposition, and bone formation rates. In addition, there was a profound decreased mineralization of bone marrow stromal cultures from Fgf2 -/-mice. This study provides strong evidence that FGF-2 helps determine bone mass as well as bone formation.

Human and mouse genetic and in vitro evidence has shown that canonical Wnt signaling promotes bone formation, but we found that mice lacking the canonical Wnt antagonist Dickkopf2 (Dkk2) were osteopenic. We reaffirmed the finding that canonical Wnt signaling stimulates osteogenesis, including the differentiation from preosteoblasts to osteoblasts, in cultured osteoblast differentiation models, but we also found that canonical Wnts upregulated the expression of Dkk2 in osteoblasts. Although exogenous overexpression of Dkk before the expression of endogenous canonical Wnt (Wnt7b) suppressed osteogenesis in cultures, its expression after peak Wnt7b expression induced a phenotype resembling terminal osteoblast differentiation leading to mineralization. In addition, osteoblasts from Dkk2-null mice were poorly mineralized upon osteogenic induction in cultures, and Dkk2 deficiency led to attenuation of the expression of osteogenic markers, which could be partially reversed by exogenous expression of Dkk2. Taken together with the finding that Dkk2-null mice have increased numbers of osteoids, these data indicate that Dkk2 has a role in late stages of osteoblast differentiation into mineralized matrices. Because expression of another Wnt antagonist, FRP3, differs from Dkk2 expression in rescuing Dkk2 deficiency and regulating osteoblast differentiation, the effects of Dkk2 on terminal osteoblast differentiation may not be entirely mediated by its Wnt signaling antagonistic activity.

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,

In serum-free mouse osteoblastic MC3T3-E1 cells, basic fibroblastic growth factor (bFGF) induced mRNA and protein for prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in arachidonic acid (AA) conversion to prostaglandins. mRNA accumulation peaked at 1 h with bFGF 1 nM. In cells stably transfected with a 371-bp PGHS-2 promoterluciferase reporter, bFGF stimulated luciferase activity, which peaked at 2-3 h with bFGF 1-10 AM. In the presence of exogenous AA, bFGF stimulated PGE2 production, which paralleled luciferase activity. In serum-free neonatal mouse calvarial cultures, bFGF stimulated PGE2 production in the absence of exogenous AA. bFGF stimulated PGHS-2 mRNA accumulation, which peaked at 2-4 h and then decreased; there were later mRNA elevations at 48 and 96 h that were inhibited by indomethacin. In both MC3T3-E1 cells and neonatal calvariae, bFGF produced smaller and slower increases in PGHS-1 mRNA levels than for PGHS-2. bFGF stimulated bone resorption in mouse calvariae with a maximal increase of 80% at 1 nM. Stimulation was partially inhibited by nonsteroidal anti-inflammatory drugs. We conclude that bFGF rapidly stimulates PGE2 production in osteoblasts, largely through transcriptional regulation of PGHS-2, and that prostaglandins mediate some of bFGF 's effects on bone resorption. (J. Clin. Invest. 1995. 96:923-930.)

Here we determine the Fibroblast Growth Factor-2 (FGF2) dependency of the time course of changes in bone mass in female mice. This study extends our earlier reports that knockout of the FGF2 gene (Fgf2) caused low turnover bone loss in Fgf2 −/− male mice by examining bone loss with age in Fgf2 −/− female mice, and by assessing whether reduced bone formation is associated with differentiation of bone marrow stromal cells (BMSCs) towards the adipocyte lineage. Bone mineral density (BMD) was similar in 3 month old female Fgf2 +/+ and Fgf2 −/− mice but was significantly reduced as early as 5 months of age in Fgf2 −/− mice. In vivo studies showed that there was a greater accumulation of marrow fat in long bones of 14 and 20 month old Fgf2 −/− mice compared with Fgf2 +/+ littermates. To study the effect of disruption of FGF2 on osteoblastogenesis and adipogenesis, BMSCs from both genotypes were cultured in osteogenic or adipogenic media. Reduced alkaline phosphatase positive (ALP), mineralized colonies and a marked increase in adipocytes were observed in Fgf2 −/− BMSC cultures. These cultures also showed an increase in the mRNA of the adipogenic transcription factor PPARγ2 as well as the downstream target genes aP2 and adiponectin. Treatment with exogenous FGF2 blocked adipocyte formation and increased ALP colony formation and ALP activity in BMSC cultures of both genotypes. These results support an important role for endogenous FGF2 in osteoblast (OB) lineage determination. Alteration in FGF2 signaling may contribute to impaired OB bone formation capacity and to increased bone marrow fat accumulation both of which are characteristics of aged bone.