N- and E-cadherin mediate early human calvaria osteoblast differentiation promoted by bone morphogenetic protein-2

Bone morphogenetic protein-2 (BMP-2) stimulates the differentiation of oste oblastic cells. However, the mechanisms involved in this effect are not: we ll characterized. In this study, we determined the role of the cell-cell ad hesion molecules N-cadherin and E-cadherin in the promotion of osteoblast d ifferentiation by BMP-2 in immortalized human neonatal calvaria (IHNC) cell s. In cells cultured in aggregates, recombinant human BMP-2 (rhBMP-2) incre ased messenger RNA levels for alkaline phosphatase (ALP), the osteoblast sp ecific transcription factor Osf2/Cbfa1 and osteocalcin, and enhanced in vit ro osteogenesis in long-term culture. RT-PCR, immunocytochemical, and Weste rn blot analyses showed that IHNC cells express E-cadherin, N-cadherin, and neural cell adhesion molecule (N-CAM) mRNA and protein. Treatment with rhB MP-2 induced a rapid and transient increase in N-cadherin and E-cadherin bu t not N-CAM, mRNA, and protein levels. Incubation with the RNA polymerase I I inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole prevented the upregulation of N- and E-cadherins induced by rhBMP-2, suggesting that tra nscription is necessary for this effect. N- and E-cadherins were functional because rhBMP-2 increased cell-cell adhesion in a cell aggregation assay, and this effect was largely blocked by N-cadherin- and E-cadherin-neutraliz ing antibodies. In addition, N- and E-cadherin antibodies decreased the bas al ALP activity and completely suppressed the rhBMP-2-induced increase in A LP activity and mRNA levels. Furthermore, anti-N-cadherin or anti-E-cadheri n antibodies markedly decreased Osf2/Cbfa1 mRNA levels and abolished the rh BMP-2-induced increased Osf2/Cbfa1 expression, and reduced the increased os teocalcin mRNA levels induced by rhBMP-2. We conclude that rhBMP-2 rapidly and transiently increases N- and E-cadherin expression, and this effect med iates the rhBMP-2-induced early promotion of cell-cell adhesion and osteobl ast marker gene expression in human calvaria cells. (C) 2000 Wiley-Liss, In c.