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

Abstract

Fibroblast growth factors (FGFs) are important factors regulating osteogenesis. However, the early mechanisms and signaling pathways involved in FGF actions in osteoblasts are unknown. We investigated the effects of FGF-2 on cell-cell adhesion and cadherin expression and the underlying signaling pathways in immortalized human neonatal calvaria (IHNC) cells. These cells express E- and N-cadherins, as shown by immunocytochemical and Western blot analyses. rhFGF-2 increased cell-cell adhesion at 24-72 h, as measured in a cell aggregation assay, and this effect was blocked by specific neutralizing anti-N-cadherin, but not anti-E-cadherin antibodies. Accordingly, ELISA and 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 showed that rhFGF-2 transiently increased N-cadherin mRNA levels in IHNC cells. The RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole prevented the rhFGF-2-induced up-regulation of N-cadherin mRNA, suggesting that transcription is necessary for this effect. Analysis of signaling molecules showed evidence that PLCgamma-PKC, Src, Erk 1/2 and p38 MAPK pathways are activated by rhFGF-2 in IHNC cells. The selective PKC inhibitors calphostin C, Ro-31-8220, Gö6976 and Gö6983 abrogated the stimulatory effect of rhFGF-2 on N-cadherin mRNA levels. The src-family tyrosine kinase inhibitor PP1 also blocked rhFGF-2-promoted N-cadherin expression. In contrast, 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 identifies N-cadherin as a previously unrecognized target gene for FGF-2 signaling pathway that regulates cell-cell adhesion in human osteoblasts.

Tyrosine Phosphorylation of I-κB Kinase α/β by Protein Kinase C-Dependent c-Src Activation Is Involved in TNF-α-Induced Cyclooxygenase-2 Expression

“…Involvement of a tyrosine kinase upstream of IKK activation has also been reported in CD23 signaling in U937 cells (32) and in Ag receptor stimulation in B cell (33). A similar PKC-dependent c-Src activation pathway has been found in human osteoblasts (36), in which fibroblast growth factor-2 increases N-cadherin expression; in A7r5 vascular smooth muscle cells (37), in which TPA induces Rho-dependent actin reorganization; and in SH-SY5Y neuroblastoma cells (38), in which TPA induces CasCrk complex formation. Furthermore, the PKC/c-Src/IKK pathway, in this study shown to be involved in induction of COX-2 expression, might be a common signaling pathway for inducible gene expression, as TNF-␣-, IL-1␤-, or IFN-␥-induced ICAM-1 expression in human alveolar epithelial cells also involves PKCdependent activation of c-Src or Lyn (26, 39 -41).…”

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

“…Functional interactions may also occur between PKC␣ and the tyrosine kinase Src (65,66). In osteoblasts, PKC␣ is known to activate Src signaling (67)(68)(69). However, we previously showed that MT FGFR2 reduces Src activity which thereby contributes to promote osteoblast differentiation (39).…”

FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease

“…FGF2 and FGF18 stimulate ERK2 phosphorylation, which promotes mitogenesis (Hurley et al 1996;Chaudhary andAvioli 1997, 2000;Shimoaka et al 2001) and down-regulates procollagen gene expression in calvarial osteoblasts (Chaudhary andAvioli 1997, 2000). The protein kinase C (PKC) pathway is involved in the control of sodium-dependent phosphate transport (Suzuki et al 2000) and expression of N-cadherin in calvarial osteoblasts (Debiais et al 2001). Mutations in Fgfr2 that cause Apert syndrome induce constitutive activation of PKC in human calvarial osteoblasts (Fragale et al 1999;Lomri et al 2001).…”

“…In human osteoblasts, Apert syndrome mutations do not increase cell proliferation (Lomri et al 1998;Fragale et al 1999;Lemonnier et al 2000) but alternatively, increase the expression of type 1 collagen, osteocalcin, and osteopontin, and enhance osteogenesis (Lomri et al 1998;Lemonnier et al 2000). This premature osteogenic cell differentiation induced by Apert Fgfr2 mutations is associated with increased N-cadherin expression and cell-cell adhesion (Lemonnier et al 2001b), which is reproduced by application of FGF2 (Debiais et al 2001). The increased osteoblast differentiation and bone formation induced by activating mutations in Fgfr2 is consistent with the phenotype of stenotic sutures in human nonsyndromic craniosynostosis (De Pollak et al 1996;Shevde et al 2001).…”