Fibroblast Growth Factor 2 High Molecular Weight Isoforms in Dentoalveolar Mineralization.

Transgenic mice overexpressing human high molecular weight fibroblast growth factor 2 (HMWFGF2) isoforms in osteoblast and odontoblast lineages (HMWTg) exhibit decreased dentin and alveolar bone mineralization, enlarged pulp chamber, and increased fibroblast growth factor 23 (FGF23). We examined if the alveolar bone and dentin mineralization defects in HMWTg mice resulted from increased FGF23 expression and whether an FGF23 neutralizing antibody could rescue the hypomineralization phenotype. HMWTg and VectorTg control mice were given subcutaneous injections of FGF23 neutralizing antibody twice/week starting at postnatal day 21 for 6 weeks. Since Calcitriol (1,25D) have direct effects in promoting bone mineralization, we also determined if 1,25D protects against the defective dentin and alveolar bone mineralization. Therefore, HMWTg mice were given subcutaneous injections of 1,25D daily or concomitantly with FGF23 neutralizing antibody for 6 weeks. Our results showed that HMWTg mice displayed thickened predentin, alveolar bone hypomineralization, and enlarged pulp chambers. FGF23 neutralizing antibody and 1,25D monotherapy partially rescued the dentin mineralization defects and the enlarged pulp chamber phenotype in HMWTg mice. 1,25D alone was not sufficient to rescue the alveolar bone hypomineralization. Interestingly, HMWTg mice treated with both FGF23 neutralizing antibody and 1.25D further rescued the enlarged pulp chamber size, and dentin and alveolar bone mineralization defects. We conclude that the dentin and alveolar bone mineralization defects in HMWTg mice might result from increased FGF23 expression. Our results show a novel role of HMWFGF2 on dentoalveolar mineralization.

Ablation of low-molecular-weight FGF2 isoform accelerates murine osteoarthritis while loss of high-molecular-weight FGF2 isoforms offers protection.

FGF2 is an essential growth factor implicated in osteoarthritis (OA), and deletion of full-length FGF2 (Fgf2ALLKO ) leads to murine OA. However, the FGF2 gene encodes both high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms, and the effects of selectively ablating individual isoforms, as opposed to total FGF2, has not been investigated in the context of OA. We undertook this study to examine whether mice lacking HMW FGF2 (Fgf2HMWKO ) or LMW FGF2 (Fgf2LMWKO ) develop OA and to further characterize the observed OA phenotype in Fgf2ALLKO mice. Fgf2HMWKO mice never developed OA, but 6- and 9-month-old Fgf2LMWKO and Fgf2ALLKO mice displayed signs of OA, including eroded articular cartilage, altered subchondral bone and trabecular architecture, and increased OA marker enzyme levels. Even with mechanical induction of OA, Fgf2HMWKO mice were protected against OA, whereas Fgf2LMWKO and Fgf2ALLKO displayed OA-like changes of the subchondral bone. Before exhibiting OA symptoms, Fgf2LMWKO or Fgf2ALLKO joints displayed differential expression of genes encoding key regulatory proteins, including interleukin-1ß, insulin-like growth factor 1, bone morphogenetic protein 4, hypoxia-inducible factor 1, B-cell lymphoma 2, Bcl2-associated X protein, a disintegrin and metalloproteinase with thrombospondin motifs 5, ETS domain-containing protein, and sex-determining region Y box 9. Moreover, Fgf2LMWKO OA cartilage exhibited increased FGF2, FGF23, and FGFR1 expression, whereas Fgf2HMWKO cartilage had increased levels of FGFR3, which promotes anabolism in cartilage. These results demonstrate that loss of LMW FGF2 results in catabolic activity in joint cartilage, whereas absence of HMW FGF2 with only the presence of LMW FGF2 offers protection from OA.

FGFR Inhibitor Ameliorates Hypophosphatemia and Impaired Engrailed-1/Wnt Signaling in FGF2 High Molecular Weight Isoform Transgenic Mice.

High molecular weight FGF2 transgenic (HMWTg) mouse phenocopies the Hyp mouse, homolog of human X-linked hypophosphatemic rickets with hypophosphatemis, and abnormal FGF23, FGFR, Klotho signaling in kidney. Since abnormal Wnt signaling was reported in Hyp mice we assessed whether Wnt signaling was impaired in HMWTg kidneys and the effect of blocking FGF receptor (FGFR) signaling. Bone mineral density and bone mineral content in female HMWTg mice were significantly reduced. HMWTg mice were gavaged with FGFR inhibitor NVP-BGJ398, or vehicle and were euthanized 24 h post treatment. Serum phosphate was significantly reduced and urine phosphate was significantly increased in HMWTg and was rescued by NVP-BGJ398. Analysis of kidneys revealed a significant reduction in Npt2a mRNA in HMWTg that was significantly increased by NVP-BGJ398. Increased FGFR1, KLOTHO, P-ERK1/2, and decreased NPT2a protein in HMWTg were rescued by NVP-BGJ398. Wnt inhibitor Engrailed-1 mRNA and protein was increased in HMWTg and was decreased by BGJ398. Akt mRNA and protein was decreased in HMWTg and was increased by NVP-BGJ398. The active form of glycogen synthase 3 beta (pGSK3-ß) and phosphor-ß-catenin were increased in HMWTg and were both decreased by NVP-BGJ398 while decreased active-ß-catenin in HMWTg was increased by NVP-BGJ398. We conclude that FGFR blockade rescued hypophosphatemia by regulating FGF and WNT signaling in HMWTg kidneys. J. Cell. Biochem. 117: 1991-2000, 2016. © 2016 Wiley Periodicals, Inc.

Age-Related Changes in FGF-2, Fibroblast Growth Factor Receptors and ß-Catenin Expression in Human Mesenchyme-Derived Progenitor Cells.

FGF-2 stimulates preosteoblast replication, and knockout of the FGF-2 gene in mice resulted in osteopenia with age, associated with decreased Wnt-ß-Catenin signaling. In addition, targeted expression of FGF-2 in osteoblast progenitors increased bone mass in mice via Wnt-ß-Catenin signaling. We posited that diminution of the intrinsic proliferative capacity of human mesenchyme-derived progenitor cells (HMDPCs) with age is due in part to reduction in FGF-2. To test this hypothesis HMDPCs from young (27-38), middle aged (47-56), and old (65-76) female human subjects were isolated from bone discarded after orthopedic procedures. HMDPCs cultures were mostly homogeneous with greater than 90% mesenchymal progenitor cells, determined by fluorescence-activated cell sorting. There was a progressive decrease in FGF-2 and FGFR1 mRNA and protein in HMDPCs with age. Since FGF-2 activates ß-catenin, which can enhance bone formation, we also assessed its age-related expression in HMDPCs. An age-related decrease in total-ß-Catenin mRNA and protein expression was observed. However there were increased levels of p-ß-Catenin and decreased levels of activated-ß-Catenin in old HMDSCs. FGF-2 treatment increased FGFR1 and ß-Catenin protein, reduced the level of p-ß-Catenin and increased activated-ß-Catenin in aged HMDPCs. In conclusion, reduction in FGF-2 expression could contribute to age-related impaired function of HMDPCs via modulation of Wnt-ß-catenin signaling.

Accelerated fracture healing in transgenic mice overexpressing an anabolic isoform of fibroblast growth factor 2.

The effect of targeted expression of an anabolic isoform of basic fibroblast growth factor (FGF2) in osteoblastic lineage on tibial fracture healing was assessed in mice. Closed fracture of the tibiae was performed in Col3.6-18 kDaFgf2-IRES-GFPsaph mice in which a 3.6 kb fragment of type I collagen promoter (Col3.6) drives the expression of only the 18 kD isoform of FGF2 (18 kDaFgf2/LMW) with green fluorescent protein-sapphire (GFPsaph) as well as Vector mice (Col3.6-IRES-GFPsaph, Vector) that did not harbor the FGF2 transgene. Radiographic, micro-CT, DEXA, and histologic analysis of fracture healing of tibiae harvested at 3, 10 and 20 days showed a smaller fracture callus but accelerated fracture healing in LMWTg compared with Vector mice. At post fracture day 3, FGF receptor 3 and Sox 9 mRNA were significantly increased in LMWTg compared with Vector. Accelerated fracture healing was associated with higher FGF receptor 1, platelet derived growth factors B, C, and D, type X collagen, vascular endothelial cell growth factor, matrix metalloproteinase 9, tartrate resistant acid phosphatase, cathepsin K, runt-related transcription factor-2, Osterix and Osteocalcin and lower Sox9, and type II collagen expression at 10 days post fracture. We postulate that overexpression of LMW FGF2 accelerated the fracture healing process due to its effects on factors that are important in chondrocyte and osteoblast differentiation and vascular invasion.

Knockout of nuclear high molecular weight FGF2 isoforms in mice modulates bone and phosphate homeostasis.

We previously reported that targeted overexpression of the fibroblast growth factor 2 (FGF2) high molecular weight (HMW) isoforms in osteoblastic lineage cells in mice resulted in phenotypic changes, including dwarfism, rickets, osteomalacia, hypophosphatemia, increased serum parathyroid hormone, and increased levels of the phosphatonin FGF23 in serum and bone. This study examined the effects of genetically knocking out the FGF2HMW isoforms (HMWKO) on bone and phosphate homeostasis. HMWKO mice were not dwarfed and had significantly increased bone mineral density and bone mineral content in femurs and lumbar vertebrae when compared with the wild-type (WT) littermates. Micro-computed tomography analysis of femurs revealed increased trabecular bone volume, thickness, number, and connective tissue density with decreased trabecular spacing compared with WT. In addition, there was significantly decreased cortical porosity and increased cortical thickness and sub-periosteal area in femurs of HMWKO. Histomorphometric analysis demonstrated increased osteoblast activity and diminished osteoclast activity in the HMWKO. In vitro bone marrow stromal cell cultures showed there was a significant increase in alkaline phosphatase-positive colony number at 1 week in HMWKO. At 3 weeks of culture, the mineralized area was also significantly increased. There was increased expression of osteoblast differentiation marker genes and reduced expression of genes associated with impaired mineralization, including a significant reduction in Fgf23 and Sost mRNA. Normal serum phosphate and parathyroid hormone were observed in HMWKO mice. This study demonstrates a significant negative impact of HMWFGF2 on biological functions in bone and phosphate homeostasis in mice.

Effects of GBT1118, a voxelotor analog, on bone disease in sickle cell disease mice.

We assessed the effect of GBT1118, a sickle hemoglobin polymerization inhibitor on bone loss in humanized sickle cell disease (SCD) mice. Healthy control (Ctrl) 4-months-old female and male mice were fed Vehicle-chow for 2-months, while SCD mice were fed Vehicle-chow or GBT1118-chow. By micro-CT, GBT1118 significantly increased femur metaphyseal trabecular thickness (Tb.Th) and tissue mineral density (TMD), and significantly decreased trabecular spacing in female SCD mice. In SCD male mice, there was significant reduction in epiphyseal trabecular bone volume fraction (BV/TV), Tb.Th and TMD and GBT1118 significantly increased BV/TV and TMD but not Tb.Th. A significant decrease in cortical area fraction in SCD female mice was rescued by GBT1118 but not SCD males. Markedly decreased mineralized femur trabeculae in SCD females and males was partially rescued by GBT1118. Bone histomorphometry of femurs demonstrated significantly decreased bone formation parameters and increased bone resorption parameters in SCD mice of both sex that were rescued by GBT1118. Significant alteration in bone and hypoxia related genes of SCD mice of both sexes were differentially modulated by GBT1118. We conclude that "a sickle hemoglobin polymerization inhibitor" might be efficacious in improving some parameters of SCD bone loss.

Prostaglandin F2α: a bone remodeling mediator.

Prostaglandin F2α (PGF2α) plays multiple roles on bone metabolism by regulating a wide range of signaling pathways. PGF2α, via activation of PKC, stimulates Na-dependent inorganic phosphate (Pi) transport system in osteoblasts; up-regulates interleukin (IL)-6 synthesis; increases vascular endothelial growth factor (VEGF). In addition, PGF2α acts as a strong mitogenic and survival agent on osteoblasts, and these effects are, at least in part, mediated by the binding of fibroblast growth factor-2 (FGF-2) to the specific receptor FGFR1. The understanding of PGF2α intracellular network, albeit complex to clarify, provides molecular bases useful to identify the players of osteoblast proliferation, apoptosis, and the associated angiogenic processes. Indeed, the molecular mechanism that underline PGF2α-regulated bone metabolism may be a promising platform for the development of novel targeted therapies in the treatment of bone disorders and disease.

FGF-2 enhances Runx-2/Smads nuclear localization in BMP-2 canonical signaling in osteoblasts.

Bone morphogenetic protein 2 (BMP-2) is one of the most potent regulators of osteoblast differentiation and bone formation. R-Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they are translocated in the nucleus regulating transcription target genes by interacting with various transcription factors. Runx-2 proteins have been shown to interact through their C-terminal segment with Smads and this interaction is required for in vivo osteogenesis. In particular, recruitment of Smads to intranuclear sites is Runx-2 dependent, and Runx-2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites. Previously, we have shown, by in vitro and in vivo experiments, that BMP-2 up-regulated FGF-2 which is important for the maximal responses of BMP-2 in bone. In this study, we found that endogenous FGF2 is necessary for BMP-2 induced nuclear accumulation and co-localization of Runx-2 and phospho-Smads1/5/8, while Runx/Smads nuclear accumulation and co-localization was reduced in Fgf2-/- osteoblasts. Based on these novel data, we conclude that the impaired nuclear accumulation of Runx-2 in Fgf2-/- osteoblasts reduces R-Smads sub-nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice.

Fibroblast Growth Factor 23 Neutralizing Antibody Ameliorates Abnormal Renal Phosphate Handling in Sickle Cell Disease Mice.

We assessed the involvement of fibroblast growth factor 23 (FGF23) in phosphaturia in sickle cell disease (SCD) mice. Control and SCD mice were treated with FGF23 neutralizing antibody (FGF23Ab) for 24 hours. Serum ferritin was significantly increased in SCD mice and was significantly reduced in female but not male SCD mice by FGF23Ab. FGF23Ab significantly reduced increased erythropoietin in SCD kidneys. Serum intact FGF23 was significantly increased in SCD female mice and was markedly increased in SCD male mice; however, FGF23Ab significantly reduced serum intact FGF23 in both genotypes and sexes. Serum carboxy-terminal-fragment FGF23 (cFGF23) was significantly reduced in SCD IgG male mice and was markedly but not significantly reduced in SCD IgG female mice. FGF23Ab significantly increased cFGF23 in both sexes and genotypes. Serum 1,25-dihydroxyvitamin D3 was significantly increased in SCD IgG and was further significantly increased by FGF23Ab in both sexes and genotypes. Significantly increased blood urea nitrogen in SCD was not reduced by FGF23Ab. The urine phosphate (Pi)/creatinine ratio was significantly increased in SCD in both sexes and was significantly reduced by FGF23Ab. Increased SCD kidney damage marker kidney injury molecule 1 was rescued, but sclerotic glomeruli, increased macrophages, and lymphocytes were not rescued by short-term FGF23Ab. FGF23Ab significantly reduced increased phospho-fibroblast growth factor receptor 1, αKlotho, phosphorylated extracellular signal-regulated kinase, phosphorylated serum/glucocorticoid-regulated kinase 1, phosphorylated sodium-hydrogen exchanger regulatory factor-1, phosphorylated janus kinase 3, and phosphorylated transducer and activator of transcription-3 in SCD kidneys. The type II sodium Pi cotransporter (NPT2a) and sodium-dependent Pi transporter PiT-2 proteins were significantly reduced in SCD kidneys and were increased by FGF23Ab. We conclude that increased FGF23/FGF receptor 1/αKlotho signaling promotes Pi wasting in SCD by downregulating NPT2a and PIT2 via modulation of multiple signaling pathways that could be rescued by FGF23Ab.

Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway.

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/ß-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and ß-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and ß-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3ß, a negative regulator of Wnt/ß-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted ß-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.

Role of fibroblast growth factor 2 and Wnt signaling in anabolic effects of parathyroid hormone on bone formation.

Osteoporosis poses enormous health and economic burden worldwide. One of the very few anabolic agents for osteoporosis is parathyroid hormone (PTH). Although great progress has been made since the FDA approved PTH in 2002, the detailed mechanisms of the bone anabolic effects of intermittent PTH treatment is still not well understood. PTH bone anabolic effect is regulated by extracellular factors. Maximal bone anabolic effect of PTH requires fibroblast growth factor 2 (FGF2) signaling, which might be mediated by transcription factor activating transcription factor 4 (ATF4). Maximal bone anabolic effect of PTH also requires Wnt signaling. Particularly, Wnt antagonists such as sclerostin, dickkopf 1 (DKK1) and secreted frizzled related protein 1 (sFRP1) are promising targets to increase bone formation. Interestingly, FGF2 signaling modulates Wnt/ß-Catenin signaling pathway in bone. Therefore, multiple signaling pathways utilized by PTH are cross talking and working together to promote bone formation. Extensive studies on the mechanisms of action of PTH will help to identify new pathways that regulate bone formation, to improve available agents to stimulate bone formation, and to identify potential new anabolic agents for osteoporosis.

FGF receptor inhibitor BGJ398 partially rescues osteoarthritis-like phenotype in older high molecular weight FGF2 transgenic mice via multiple mechanisms.

We have used Basic Fibroblast Growth Factor (FGF2) transgenic mice as experimental models for human X-linked hypophosphatemia (XLH)-related degenerative osteoarthritis (OA) to investigate the pathogenesis of the disease and to test potential pharmacotherapies for treatment. This study tested the efficacy of BJG398, a small molecule fibroblast growth factor receptor tyrosine kinase (FGFRTK) inhibitor, to rescue the knee joint osteoarthritis phenotype in High Molecular Weight fibroblast growth factor 2 transgenic (HMWTgFGF2) mice. BJG398 was administered in vivo to 8-month-old female HMWTgFGF2 mice for six weeks. Histomorphometry, immunohistochemistry and micro-CT were used to examine the knee joints in BGJ398-treated and control mice. We assessed: Fibroblast Growth Factor 23 (FGF23) expression and FGFR1 activity; Matrix metalloproteinase 13 (MMP13) and Aggrecanase2 (ADAMTS5) expression; then signaling by SMAD1/5/8-pSMAD6, pERK1/2 and Runt-related transcription factor 2 (RUNX2). Using PrimePCR arrays, we identified a contributing role for major target genes in the TGFB/BMP2 signaling pathway that were regulated by BGJ398. BGJ398 inhibited HMWFGF2/FGF23-induced increase in bone morphogenic protein receptor-1, bone morphogenic protein-2 and 4 and Serine peptidase inhibitor, clade E, member 1. The results from Micro-CT and histology show BGJ398 treatment rescued the OA changes in subchondral bone and knee articular cartilage of HMWTgFGF2 mice. The gene expression and signal transduction results provide convincing evidence that HMWFGF2 generates OA through FGFRTK with characteristic downstream signaling that defines OA, namely: increased FGF23-FGFR1 activity with BMP-BMPR, activation of pSMAD1/5/8-RUNX2 and pERK signaling pathways, then upregulation of MMP13 and ADAMTS5 to degrade matrix. BGJ398 treatment effectively reversed these OA molecular phenotypes, providing further evidence that the OA generated by HMWFGF2 in the transgenic mice is FGFR-mediated and phenocopies the OA found in the Hyp mouse homolog of XLH with a spontaneous mutation in the Phex (phosphate regulating endopeptidase on the X chromosome) gene and human XLH-OA. Overall, the results obtained here explain how the pleotropic effects of FGF2 emanate from the different functions of HMW protein isoforms for cartilage and bone homeostasis, and the pathogenesis of XLH-degenerative osteoarthropathy. BGJ398 inhibits HMWFGF2-induced osteoarthritis via multiple mechanisms. These results provided important scientific evidence for the potential application of BGJ398 as a therapeutic agent for osteoarthritis in XLH.

Nuclear isoforms of fibroblast growth factor 2 are novel inducers of hypophosphatemia via modulation of FGF23 and KLOTHO.

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(+)/P(i) co-transporter Npt2a mRNA was decreased. Immunohistochemistry and Western blot analyses of TgHMW kidneys showed increased KLOTHO and decreased NPT2a protein. The results suggest that overexpression of HMW FGF2 increases FGF23/FGFR/KLOTHO signaling to down-regulate NPT2a, causing P(i) wasting, osteomalacia, and decreased BMD. We assessed whether HMW FGF2 expression was altered in the Hyp mouse, a mouse homolog of the human disease X-linked hypophosphatemic rickets/osteomalacia. Fgf2 mRNA was increased in bones, and Western blots showed increased FGF2 protein in nuclear fractions from osteoblasts of Hyp mice. In addition, immunohistochemistry demonstrated co-localization of FGF23 and HMW FGF2 protein in osteoblasts and osteocytes from Hyp mice. This study reveals a novel mechanism of regulation of the FGF23-P(i) homeostatic axis.

The impaired bone anabolic effect of PTH in the absence of endogenous FGF2 is partially due to reduced ATF4 expression.

Parathyroid hormone (PTH) is currently the only approved anabolic agent for osteoporosis pharmacotherapy in the USA. However, the molecular and cellular mechanisms underlying which intermittent PTH stimulates bone formation are not fully established. Activating transcription factor 4 (ATF4) was recently identified to be a downstream target of PTH signaling in osteoblasts and FGF2 is able to rapidly increase ATF4 mRNA and protein expression in osteoblasts. Furthermore, ATF4 expression is markedly reduced in Fgf2(-/-) osteoblasts. In addition, FGF2 is required for the anabolic action of PTH on bone formation. Therefore, we hypothesize that the impaired anabolic effect of PTH in Fgf2(-/-) mice is partially due to reduced ATF4 expression. To test this hypothesis, we examined the ability of PTH to increase ATF4 expression in vitro and in vivo. In vitro data showed that PTH induced a significant increase in ATF4 mRNA expression as early as 15 min in Fgf2(+/+) primary bone marrow stromal cells (BMSCs) but not in Fgf2(-/-) BMSCs. In vivo data showed that treatment with PTH (1-34) (40 µg/kg/d) treatment for 2 weeks in 21-23 months female mice increased lumbar vertebrae bone mineral density in Fgf2(+/+) (13.8% increase). In contrast there was a 2.1% decrease in Fgf2(-/-) mice. Interestingly, basal ATF4 mRNA expression in tibiae was significantly lower in Fgf2(-/-) mice (46% decrease) compared to Fgf2(+/+) mice. PTH treatment increased ATF4 mRNA by 97% (p<0.05) in Fgf2(+/+) compared to 8% (p=0.57) in Fgf2(-/-) mice. Immunohistochemistry of vertebrae showed less ATF4 staining in Fgf2(-/-) tissue, and treatment with PTH increased ATF4 staining in Fgf2(+/+) but the increase was attenuated in Fgf2(-/-) tissue. In summary, reduced ATF4 expression may result in decreased osteoblast differentiation, and possibly contribute to the impaired stimulation of PTH on bone formation in Fgf2(-/-) mice.

Gait disturbances and muscle dysfunction in fibroblast growth factor 2 knockout mice.

Fibroblast growth factor 2 (FGF2) is important in musculoskeletal homeostasis, therefore the impact of reduction or Fgf2 knockout on skeletal muscle function and phenotype was determined. Gait analysis as well as muscle strength testing in young and old WT and Fgf2KO demonstrated age-related gait disturbances and reduction in muscle strength that were exacerbated in the KO condition. Fgf2 mRNA and protein were significantly decreased in skeletal muscle of old WT compared with young WT. Muscle fiber cross-sectional area was significantly reduced with increased fibrosis and inflammatory infiltrates in old WT and Fgf2KO vs. young WT. Inflammatory cells were further significantly increased in old Fgf2KO compared with old WT. Lipid-related genes and intramuscular fat was increased in old WT and old Fgf2KO with a further increase in fibro-adipocytes in old Fgf2KO compared with old WT. Impaired FGF signaling including Increased ß-Klotho, Fgf21 mRNA, FGF21 protein, phosphorylated FGF receptors 1 and 3, was observed in old WT and old Fgf2KO. MAPK/ ERK1/2 was significantly increased in young and old Fgf2KO. We conclude that Fgf2KO, age-related decreased FGF2 in WT mice, and increased FGF21 in the setting of impaired Fgf2 expression likely contribute to impaired skeletal muscle function and sarcopenia in mice.

Endogenous FGF-2 levels impact FGF-2/BMP-2 growth factor delivery dosing in aged murine calvarial bone defects.

Bone repair in elderly mice has been shown to be improved or negatively impacted by supplementing the highly osteogenic bone morphogenetic protein-2 (BMP-2) with fibroblast growth factor-2 (FGF-2). To better predict the outcome of FGF-2 supplementation, we investigated whether endogenous levels of FGF-2 play a role in optimal dosing of FGF-2 for augmenting BMP-2 activity in elderly mice. In vivo calvarial bone defect studies in Fgf2 knockout mice with wildtype controls were conducted with the growth factors delivered in a highly localized manner from a biomimetic calcium phosphate/polyelectrolyte multilayer coating applied to a bone graft substitute. Endogenous FGF-2 levels were measured in old mice versus young and found to decrease with age. Optimal dosing for improving bone defect repair correlated with levels of endogenous FGF-2, with a larger dose of FGF-2 required to have a positive effect on bone healing in the Fgf2 knockout mice. The same dose in wildtype old mice, with higher levels of FGF-2, promoted chondrogenesis and increased osteoclast activity. The results suggest a personalized medicine approach, based on a knowledge of endogenous levels of FGF-2, should guide FGF-2 supplementation in order to avoid provoking excessive bone resorption and cartilage formation, both of which inhibited calvarial bone repair.

Signaling pathways implicated in PGF2alpha effects on Fgf2+/+ and Fgf2-/- osteoblasts.

Prostaglandin F2alpha (PGF2alpha) regulates fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor (FGFR) expression in osteoblasts. Here, the role of FGF-2 in PGF2alpha-induced proliferation and the signaling pathway involved, were determined in calvarial osteoblasts (COBs) from Fgf2+/+ and Fgf2-/- mice. The involvement of the exported FGF-2 isoform, was determined using the FGF-2 neutralizing antibody to alter its binding to FGFR1. PGF2alpha increased activity of Ras, and MAP-kinase cascade as well as Bcl-2 and c-Myc levels in Fgf2+/+ but not in Fgf2-/- COBs. Moreover, in Fgf2+/+ COBs, PGF2alpha-enhanced nuclear accumulation and co-localization of Bcl-2/c-Myc. Although up-regulation of multiple proliferative and survival signals were induced by PGF2alpha in Fgf2+/+ COBs, phospho-p53 was unmodified while p53 was increased. Increased phospho-p53 was, instead, found in Fgf2-/- COBs without up-regulation of oncogenic proteins. The lack of p53 activation in wild type osteoblasts could be due in part to the overexpression of MDM2 caused by PGF2alpha via FGF-2. PGF2alpha, also, increased cyclins D and E in Fgf2+/+ COBs and induced an expansion of Fgf2+/+ osteoblasts in G(2)/M phase. These data clearly show that PGF2alpha induces proliferation via endogenous FGF-2 and the exported isoform mediates PGF2alpha effects by acting in autocrine manner. Furthermore, silencing of FGFR1 in Fgf2+/+ COBs blocked PGF2alpha induced increase of phospho-MDM2 and cyclins.

Fibroblast growth factor 2 positively regulates expression of activating transcription factor 4 in osteoblasts.

Our previous studies showed that basic fibroblast growth factor 2 (FGF2) null mice display markedly reduced bone mass and bone formation. However, the mechanism by which FGF2 regulates bone mass or bone formation is not fully defined. Activating transcription factor 4 (ATF4), one member of activating transcription factor/cAMP response element binding family, is a transcription factor required for osteoblast terminal differentiation. Here we investigate the ability of FGF2 to increase expression of ATF4 in bone marrow stromal cells (BMSCs) and examine ATF4 expression in Fgf2(-/-) BMSCs. We found that FGF2 stimulated ATF4 mRNA expression as early as 20 min and increased ATF4 protein expression after three hours of treatment. BMSCs from Fgf2(+/+) and Fgf2(-/-) mice were cultured in osteogenesis medium. We observed reduced alkaline phosphatase staining, decreased mineralized nodules and reduced osteocalcin expression, and reduced expression of ATF4 in Fgf2(-/-) BMSC cultures compared to Fgf2(+/+) BMSCs. This study is the first demonstration that ATF4 expression can be stimulated by FGF2 in osteoblasts and that ATF4 expression is significantly reduced in differentiated Fgf2(-/-) BMSCs. These results suggest that impaired bone mass and bone formation in Fgf2 null mice may be due in part to reduced ATF4 expression.

Inhibition of FGFR Signaling Partially Rescues Osteoarthritis in Mice Overexpressing High Molecular Weight FGF2 Isoforms.

Fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptors (FGFRs) are key regulatory factors in osteoarthritis (OA). HMWTg mice overexpress the high molecular weight FGF2 isoforms (HMWFGF2) in osteoblast lineage and phenocopy both Hyp mice (which overexpress the HMWFGF2 isoforms in osteoblasts and osteocytes) and humans with X-linked hypophosphatemia (XLH). We previously reported that, similar to Hyp mice and XLH subjects who develop OA, HMWTg mice also develop an OA phenotype associated with increased degradative enzymes and increased FGFR1 compared with VectorTg mice. Therefore, in this study, we examined whether in vivo treatment with the FGFR tyrosine kinase inhibitor NVP-BGJ398 (BGJ) would modulate development of the OA phenotype in knee joints of HMWTg mice. VectorTg and HMWTg mice (21 days of age) were treated with vehicle or BGJ for 13 weeks. Micro-computed tomography images revealed irregular shape and thinning of the subchondral bone with decreased trabecular number and thickness within the epiphyses of vehicle-treated HMWTg knees, which was partially rescued following BGJ treatment. Articular cartilage thickness was decreased in vehicle-treated HMWTg mice, and was restored to the cartilage thickness of VectorTg mice in the BGJ-treated HMWTg group. Increased OA degradative enzymes present in HMWTg vehicle-treated joints decreased after BGJ treatment. OA in HMWTg mice was associated with increased Wnt signaling that was rescued by BGJ treatment. This study demonstrates that overexpression of the HMWFGF2 isoforms in preosteoblasts results in osteoarthropathy that can be partially rescued by FGFR inhibitor via reduction in activated Wnt signaling.