The Ser252Trp fibroblast growth factor receptor-2 (FGFR-2) mutation induces PKC-independent downregulation of FGFR-2 associated with premature calvaria osteoblast differentiation

We recently showed that the Apert Ser252Trp fibroblast growth factor recept or-2 (FGFR-2) mutation causes premature osteoblast differentiation and incr eased subperiosteal calvaria bone matrix formation. To gain further insight into the cellular mechanisms involved in these effects, we examined the ef fects of the mutation on the expression of FGFRs in relation to cell prolif eration and differentiation markers in vivo and in vitro, and we analyzed t he underlying signaling pathways in mutant cells. Immunohistochemical analy sis of the Apert calvaria suture showed that the Ser252Trp FGFR-2 mutation increased type 1 collagen, osteocalcin, and osteopontin expression in preos teoblasts compared to normal, whereas cell growth was not affected. The pre mature osteoblast differentiation induced by the mutation was associated wi th lower than normal FGFR-2 immunolabeling, whereas FGFR-1 and FGFR-3 level s were not decreased. Immunocytochemical analysis in osteoblasts isolated f rom Apart coronal suture showed that the Ser252Trp mutation induced constit utive downregulation of FGFR-2 in mutant cells. Western blot analysis of FG FRs in immortalized mutant osteoblastic cells confirmed that the mutation i nduced FGFR-2 downregulation. FGFR-2 mRNA levels were not altered in mutant cells, indicating that FGFR-2 downregulation resulted from receptor intern alization rather than from changes in receptor mRNA The signaling pathway i nvolved in FGFR-2 downregulation was studied using specific inhibitors of F GF signaling molecules. The selective PKC inhibitor calphostin C markedly r educed FGFR-2 protein levels in mutant cells, in contrast to the p38 MAP ki nase inhibitor SE 203580 or the Erk 1,2 MAP kinase inhibitor PD-98059, show ing that PRC is involved in FGFR-2 regulation, but not in FGFR-2 downregula tion in mutant cells, The results indicate that the premature osteoblast di fferentiation induced by the FGFR-2 Ser252Trp mutation is associated with a PKC-independent downregulation of FGFR-2 in human calvaria cells. (C) 2000 Academic Press.