Protein kinase C-dependent upregulation of N-cadherin expression by phorbol ester in human calvaria osteoblasts

Cell-cell adhesion mediated by cadherins is believed to play an essential r ole in the control of cell differentiation and tissue formation. Our recent studies indicate that N-cadherin is involved in human osteoblast different iation. However, the signalling molecules that regulate cadherins in osteob lasts are not known. We tested the possibility that N-cadherin expression a nd function may be regulated by direct activation of protein kinase C (PKC) in human osteoblasts. Treatment of immortalized human neonatal calvaria (I HNC) cells with phorbol 12,13-dibutyrate (100 nM) transiently increased PKC activity. RT-PCR analysis showed that transient treatment with phorbol est er transiently increased N-cadherin mRNA levels at 4-12 h. Western blot ana lysis showed that N-cadherin protein levels were increased by phorbol ester at 24-48 h, and this was confirmed by immunocytochemical analysis. In cont rast, E-cadherin expression was not affected. Transient treatment of IHNC c ells with phorbol ester increased cell-cell aggregation, which was suppress ed by neutralizing N-cadherin antibody, showing that the increased N-cadher in induced by phorbol ester was functional. Finally, phorbol ester dose-dep endently increased alkaline. phosphatase activity, an early marker of osteo blast differentiation. This effect was comparable to the promoting effect o f BMP-2, a potent activator of osteoblast differentiation. These data show that direct activation of PKC by phorbol ester increases N-cadherin express ion and function, and promotes ALP activity in human calvaria osteoblasts, which provides a signaling mechanism by which N-cadherin is regulated and s uggests a role for PKC in N-cadherin-mediated control of human osteoblast d ifferentiation. (C) 2001 Academic Press.