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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 osteoblastic cells. However, the mechanisms involved in this effect are not well characterized. In this study, we determined the role of the cell‐cell adhesion molecules N‐cadherin and E‐cadherin in the promotion of osteoblast differentiation by BMP‐2 in immortalized human neonatal calvaria (IHNC) cells. In cells cultured in aggregates, recombinant human BMP‐2 (rhBMP‐2) increased messenger RNA levels for alkaline phosphatase (ALP), the osteoblast specific transcription factor Osf2/Cbfa1 and osteocalcin, and enhanced in vitro osteogenesis in long‐term culture. RT‐PCR, immunocytochemical, and Western blot analyses showed that IHNC cells express E‐cadherin, N‐cadherin, and neural cell adhesion molecule (N‐CAM) mRNA and protein. Treatment with rhBMP‐2 induced a rapid and transient increase in N‐cadherin and E‐cadherin but not N‐CAM, mRNA, and protein levels. Incubation with the RNA polymerase II inhibitor 5,6‐dichloro‐1‐β‐D‐ribofuranosyl benzimidazole prevented the upregulation of N‐ and E‐cadherins induced by rhBMP‐2, suggesting that transcription 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–neutralizing antibodies. In addition, N‐ and E‐cadherin antibodies decreased the basal ALP activity and completely suppressed the rhBMP‐2‐induced increase in ALP activity and mRNA levels. Furthermore, anti‐N‐cadherin or anti‐E‐cadherin antibodies markedly decreased Osf2/Cbfa1 mRNA levels and abolished the rhBMP‐2‐induced increased Osf2/Cbfa1 expression, and reduced the increased osteocalcin mRNA levels induced by rhBMP‐2. We conclude that rhBMP‐2 rapidly and transiently increases N‐ and E‐cadherin expression, and this effect mediates the rhBMP‐2–induced early promotion of cell‐cell adhesion and osteoblast marker gene expression in human calvaria cells. J. Cell. Physiol. 183:117–128, 2000. © 2000 Wiley‐Liss, Inc.