Anabolic effect of aminoterminally truncated fibroblast growth factor 4 (FGF4) on bone

Fibroblast growth factor 4 (FGF4), a member of the FGF family, plays several important roles in bone development during embryogenesis. Systemic administration of FGF4 increases bone mass in rats, which suggests the potential therapeutic usefulness of this growth factor in treatment for osteopenia and in bone regeneration. We investigated the length of FGF4 required to exert its anabolic effects, because this information may be useful in developing new molecules to mimic the effects of FGF4. Because the active site of FGF family molecules is in the carboxylterminal region, we produced aminoterminally truncated recombinant human FGF4s (rhFGF4s) of different sizes. Human FGF4 cDNA containing almost the full length of the coding region (573 bp, 191 amino acid residues) was inserted into pUC18 vector and then deleted from the 5' end using the ExoIII system. Each of the deleted FGF4 cDNAs was subcloned into a pET29(+) expression vector. Differently sized recombinant proteins were expressed in the BL21(DE3)pLysS Escherichia coli strain and then purified. The growth-stimulative effects on NIH3T3 cells of each recombinant protein were examined by means of MTT colorimetric assay. Full-length and the shortened recombinant proteins, which stimulated NIH3T3 cell growth, were then subcutaneously administered into male ddY mice (6 weeks old) every day for 2 weeks. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). The rhFGF4 of 134 amino acid residues, the region homologous to other members of the FGF family, exerted a growth-stimulative effect on NIH3T3 cells comparable to the full-length version of FGF4; however, the shortest version, with 111 amino acid residues, showed a limited growth-stimulative effect. Systemic administration of the rhFGF4 of 134 amino acid residues increased the bone mineral density (BMD) of femurs at a dose of 0.1 mg/kg, which was comparable to that of the full-length rhFGF4. DEXA analysis, pQCT analysis, soft X-ray photos, and contact microradiographs revealed an increase in femoral trabecular bone in FGF4-treated animals; an increase in bone formation was also evident upon histomorphometric analysis. These results indicate that the region of FGF4 that is homologous to other FGF family members provides a sufficient anabolic effect in bone and that this recombinant protein is potentially useful as a therapeutic agent in bone.