Fibroblast growth factor-2 (FGF-2) increases N-cadherin expression throughprotein kinase C and Src-kinase pathways in human calvaria osteoblasts

Fibroblast growth factors (FGFs) are important factors regulating osteogene sis. However, the early mechanisms and signaling pathways involved in FGF a ctions in osteoblasts are unknown. We investigated the effects of FGF-2 on cell-cell adhesion and cadherin expression and the underlying signaling pat hways in immortalized human neonatal calvaria (IHNC) cells. These cells exp ress E- and N-cadherins, as shown by immunocytochemical and Western blot an alyses, rhFGF-2 increased cell-cell adhesion at 24-72 h, as measured in a c ell aggregation assay, and this effect was blocked by specific neutralizing anti-N-cadherin, but not anti-E-cadherin antibodies. Accordingly, ELISA an d Western blot analyses showed that rhFGF-2 (10-100 ng/ml) dose dependently increased N-cadherin but not E-cadherin protein levels. RT-PCR analysis sh owed that rhFGF-2 transiently increased N-cadherin mRNA levels in IHNC cell s. The RNA polymerase II inhibitor 5,6-dichloro-1-beta -D-ribofuranosyl ben zimidazole prevented the rhFGF-2-induced upregulation of N-cadherin mRNA, s uggesting that transcription is necessary for this effect. Analysis of sign aling molecules showed evidence that PLC gamma -PKC, Src, Erk 1/2 and p38 M APK pathways are activated by rhFGF-2 in IHNC cells. The selective PKC inhi bitors calphostin C, Ro-31-8220, Go6976 and Go6983 abrogated the stimulator y effect of rhFGF-2 on N-cadherin mRNA levels. The src-family tyrosine kina se inhibitor PP1 also blocked rhFGF-2-promoted N-cadherin expression. In co ntrast, the p38 MAP kinase inhibitor SB 203580 or the MEK inhibitor PD98059 had no effect on rhFGF-2-induced N-cadherin mRNA levels. Our data indicate that FGF-2 increases N-cadherin expression and function in human calvaria osteoblasts via activation of PKC and src-kinase pathways. This study ident ifies N-cadherin as a previously unrecognized target gene for FGF-2 signali ng pathway that regulates cell-cell adhesion in human osteoblasts. (C) 2001 Wiley-Liss, Inc.