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

Abstract

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 over-expression 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/FGFR/ERKsignaling. 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 FGF receptor tyrosine kinase inhibitor SU5402 and the MAP kinase 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, Runx2, and osterix, and the osteocyte-related gene 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 byPD98059. 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.

Knockout of Nuclear High Molecular Weight FGF2 Isoforms in Mice Modulates Bone and Phosphate Homeostasis

“…However, this alone may not be responsible for the increased bone mass phenotype in the HMWKO because modulation of osteoblast-related genes could contribute to the increased bone mass phenotype. Specifically, in contrast to the HMWTg mice (10,11) in which a number of osteoblast differentiation promoting genes such as Runx2, osterix, and OC and pro-mineralization genes such as Phex and Dmp1 were decreased and inhibitory genes such as Mgp and Sost were increased, we observed increased expression of Runx2, osterix, OC, and Dmp1, normal expression of Mgp, and markedly reduced expression of Sost in the bones of HMWKO mice. The level of Col1a1 mRNA expression was significantly increased in the HMWKO mouse model, but it was significantly decreased in the HMWTg mice previously studied (10).…”

“…HMWFGF2 can function as intracrine signals through FGF receptors that translocate into the nucleus via importin-␤-mediated transport (37). When bound to FGF2 ligands, the complexes can function as transcriptional activators (37) to regulate Fgf23 (11). FGF23 produced by osteoblasts/osteocytes is released into the circulation to affect P i homeostasis via the kidney.…”

“…BMSCs Culture-BMSCs were extracted from excised tibial bones as described previously (11). BMSCs were plated in 6-well dishes at 2 million/well with 2 ml of minimum essential medium ␣ (Invitrogen) ϩ 10% FBS ϩ penicillin/streptomycin.…”

“…Mechanistically, these differences were shown to be due, in part, to modulation of the Fgf23/Klotho signaling pathway (10). In vitro bone formation studies using bone marrow stromal cells (BMSCs) from HMWTg mice demonstrated a significant decrease in pro-osteoblast differentiation and mineralization gene expressions such as osterix, osteocalcin, and * This work was supported, in whole or in part, by National Institutes of Health dentin matrix acidic phosphoprotein 1 (DMP1) as well as a significant increase in inhibitory matrix Gla protein (Mgp) and Fgf23 (11).…”

“…As an endocrine hormone, FGF23 is released into the bloodstream, increases P i wasting, and modulates vitamin D homeostasis via its effects on the kidneys (13). FGF23 signals in the kidney by interacting with Klotho (14), an anti-aging protein that is not found in bone, and FGF receptor(s) (15)(16)(17) to activate downstream signaling pathways, including the MAPK/ ERK pathway (11). This in turn inhibits the expression of the sodium-phosphate transporter 2a (Npt2a) (18).…”