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-beta-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.
Haÿ
E
E
INSERM U 349, Affiliated CNRS, Cell and Molecular Biology of Bone and Cartilage, Paris, France.
Lemonnier
J
J
Modrowski
D
D
Lomri
A
A
Lasmoles
F
F
Marie
P J
PJ
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D015820:Cadherins / Q000235:genetics / Q000502:physiology*; D002448:Cell Adhesion; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012886:Skull; D013997:Time Factors; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D015820:Cadherins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2