Abstract
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.
Highlights
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]; the mechanism by which this elevates FGF23 levels remains unclear
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]
Localized expression of HMW FGF2 isoforms from the transgene was confirmed by immunofluorescent staining (Fig. 1, C and D)
Summary
Generation and Identification of Mice Expressing HMW Isoforms of hFGF2—To elucidate the role of endogenous HMW FGF2 isoforms in a bone-specific manner, we generated an expression vector, called Col3.6-Fgf2 24-, 23-, and 22-kDa isoforms-IRES/GFP (Col3.6-HMW Fgf isoforms-IRES-GFPsaph or HMW). 400 g of nuclear protein from BMSC or 300 g of total protein from kidney were diluted with 1ϫ radioimmune precipitation buffer and incubated at 4 °C overnight with Protein A/G PLUS-agarose (20 l) (Santa Cruz Biotechnology) as well as FGF2 (Millipore), KLOTHO (R&D Systems), Pp44/42 (Cell Signaling Technology, Beverly, MA), or NPT2a (Alpha Diagnostic International Inc. San Antonio, TX) antibody. Infected 293GPG packaging cell lines were grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin, 2 g/ml puromycin, 0.1 g/ml tetracycline, and 0.4 mg/ml G418. Transduction of Mouse Primary Calvarial Osteoblasts—Primary calvarial osteoblasts from Fgf KO mice were plated in 6-well dishes and grown in Dulbecco’s modified Eagle’s medium (Sigma) containing 10% heat-inactivated fetal bovine serum plus 100 units/ml penicillin and 100 g/ml streptomycin. Differences between groups were analyzed using Student’s t test, and differences were considered significant at p values of less than 0.05
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