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

Fibroblast growth factor 4 (FGF4), a member of the FGF family, plays severa l important roles in bone development during embryogenesis. Systemic admini stration of FGF4 increases bone mass in rats, which suggests the potential therapeutic usefulness of this growth factor in treatment for osteopenia an d in bone regeneration. We investigated the length of FGF4 required to exer t its anabolic effects, because this information may be useful in developin g new molecules to mimic the effects of FGF4, Because the active site of FG F family molecules is in the carboxylterminal region, we produced aminoterm inally truncated recombinant human FGF4s (rhFGF4s) of different sizes. Huma m 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 f rom 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 t hen purified. The growth-stimulative effects on NIH3T3 cells of each recomb inant protein were examined by means of MTT colorimetric assay. Full-length and the shortened recombinant proteins, which stimulated NIH3T3 cell growt h, were then subcutaneously administered into male ddY mice (6 weeks old) e very day for 2 weeks. Bone mineral density (BMD) was measured using dual-en ergy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomog raphy (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 sh ortest version, with 111 amino acid residues, showed a limited growth-stimu lative effect. Systemic administration of the rhFGF4 of 134 amino acid resi dues 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 i n bone formation was also evident upon histomorphometric analysis. These re sults indicate that the region of FGF4 that is homologous to other FGF fami ly members provides a sufficient anabolic effect in bone and that this reco mbinant protein is potentially useful as a therapeutic agent in bone, (C) 1 999 by Elsevier Science Inc. an rights reserved.