Liping Xiao

Disruption of the Fgf2 gene activates the adipogenic and suppresses the osteogenic program in mesenchymal marrow stromal stem cells

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 PPARgamma2 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.

Over-expression of fibroblast growth factor-2 causes defective bone mineralization and osteopenia in transgenic mice.

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.

Reduced expression and function of bone morphogenetic protein-2 in bones of Fgf2 null mice.

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. J. Cell. Biochem. 103: 1975–1988, 2008.

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/β-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 β-catenin was decreased significantly in Fgf2−/− bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2−/− mice. Furthermore, Fgf2−/− osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-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.

Endogenous FGF-2 is critically important in PTH anabolic effects on bone.

Parathyroid hormone (PTH) increases fibroblast growth factor receptor‐1 (FGFR1) and fibroblast growth factor‐2 (FGF‐2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2−/− mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2−/− osteoblasts. PTH increased Runx‐2 protein expression in Fgf2+/+ but not Fgf2−/− osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx‐2 only in Fgf2+/+ osteoblasts. PTH and FGF‐2 regulate Runx‐2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho‐CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2−/− osteoblasts. Silencing of FGF‐2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx‐2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1‐cdk4/6 protein in Fgf2+/+ but not Fgf2−/− osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2−/− osteoblasts. PTH increased Bcl‐2/Bax protein ratio in Fgf2+/+ but not Fgf2−/− osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2−/− osteoblasts. These data suggest that endogenous FGF‐2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2−/− mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF‐2 expression. J. Cell. Physiol. 219: 143–151, 2009.

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 (Pi). When TgHMW mice were fed a high Pi 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+/Pi 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 Pi 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-Pi homeostatic axis.

Exported 18-kDa Isoform of Fibroblast Growth Factor-2 Is a Critical Determinant of Bone Mass in Mice

The role of the 18-kDa isoform of fibroblast growth factor-2 (FGF2) in the maintenance of bone mass was examined in Col3.6-18-kDa FGF2-IRES-GFPsaph transgenic (18-kDa TgFGF2) mice in which a 3.6-kb fragment of the type I collagen 5′-regulatory region (Col3.6) drives the expression of only the 18-kDa isoform of FGF2 with green fluorescent protein-sapphire (GFPsaph). Vector only transgenic mice (Col3.6-IRES-GFPsaph, VTg) were also developed as a control, and mice specifically deficient in 18-kDa FGF2 (FGF2lmw-/-) were also examined. Bone mineral density, femoral bone volume, trabecular thickness, and cortical bone area and thickness were significantly increased in 18-kDa TgFGF2 mice compared with VTg. Bone marrow cultures (BMSC) from 18-kDa TgFGF2 mice produced more mineralized nodules than VTg. Increased bone formation was associated with reduced expression of the Wnt antagonist secreted frizzled receptor 1 (sFRP-1). In contrast to 18-kDa TgFGF2 mice, FGF2lmw-/- mice have significantly reduced bone mineral density and fewer mineralized nodules, coincident with increased expression of sFRP-1 in bones and BMSC. Moreover, silencing of sFRP-1 in BMSC from FGF2lmw-/- mice reversed the decrease in β-catenin and Runx2 mRNA. Assay of Wnt/β-catenin-mediated transcription showed increased and decreased TCF-luciferase activity in BMSC from 18-kDa TgFGF2 and FGF2lmw-/- mice, respectively. Collectively, these results demonstrate that the 18-kDa FGF2 isoform is a critical determinant of bone mass in mice by modulation of the Wnt signaling pathway.

Nuclear fibroblast growth factor 2 (FGF2) isoforms inhibit bone marrow stromal cell mineralization through FGF23/FGFR/MAPK in vitro

Fibroblast growth factor 23 (FGF23) is responsible for phosphate wasting and the phenotypic changes observed in human diseases such as X‐linked hypophosphatemia (XLH). Targeted overexpression of nuclear high–molecular weight fibroblast growth factor 2 isoforms (HMW isoforms) in osteoblasts resulted in a transgenic mouse with phenotypic changes similar to XLH, including increased FGF23, hypophosphatemia, and rickets/osteomalacia. The goal of this study was to assess whether HMW isoforms also reduced mineralized bone formation via phosphate‐independent effects in bone marrow stromal cells (BMSCs) by modulating FGF23/FGF receptor (FGFR)/extracellular signal‐regulated kinase (ERK) signaling. To determine if decreased bone formation in BMSC cultures from HMW transgenic mice could be rescued by blocking this pathway, an FGF23 neutralizing antibody, the FGFR tyrosine kinase inhibitor SU5402 and the mitogen‐activated protein kinase (MAPK) inhibitor PD98059 were used. FGF23 levels in the conditioned medium of HMW BMSC cultures were dramatically increased compared to BMSC from control (Vector) mice. Mineralized nodule formation was significantly decreased in HMW BMSC cultures compared with control cultures. The decreased nodule formation in HMW cultures was partially rescued by the FGF23 neutralizing antibody, SU5402 and PD98059. mRNA levels for the osteoblast‐related genes, osteocalcin, Runt‐related transcription factor 2 (Runx2), and osterix, and the osteocyte‐related gene dentin matrix acidic phosphoprotein 1 (Dmp1) were significantly decreased in HMW cultures compared with control cultures, and the decreases were partially rescued by SU5402 or PD98059 treatment. Matrix‐gla‐protein (Mgp) mRNA was significantly higher in HMW cultures compared with control cultures, reduced by SU5402, but further increased by PD98059. Our results suggest that phosphate‐independent effects of HMW isoforms in vitro may be directly mediated in part via FGF23 and that HMW isoforms signal via FGF23/FGFR/MAPK to inhibit bone formation in vitro.

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. J. Cell. Physiol. 228: 2149–2158, 2013.

Signaling pathways implicated in PGF2α effects on Fgf2+/+ and Fgf2−/− osteoblasts

Prostaglandin F2α (PGF2α) regulates fibroblast growth factor‐2 (FGF‐2) and fibroblast growth factor receptor (FGFR) expression in osteoblasts. Here, the role of FGF‐2 in PGF2α‐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. PGF2α 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, PGF2α‐enhanced nuclear accumulation and co‐localization of Bcl‐2/c‐Myc. Although up‐regulation of multiple proliferative and survival signals were induced by PGF2α 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 PGF2α via FGF‐2. PGF2α, also, increased cyclins D and E in Fgf2+/+ COBs and induced an expansion of Fgf2+/+ osteoblasts in G2/M phase. These data clearly show that PGF2α induces proliferation via endogenous FGF‐2 and the exported isoform mediates PGF2α effects by acting in autocrine manner. Furthermore, silencing of FGFR1 in Fgf2+/+ COBs blocked PGF2α induced increase of phospho‐MDM2 and cyclins. J. Cell. Physiol. 224: 465–474, 2010.