A dominant negative cadherin inhibits osteoblast differentiation.
We have previously indicated that human osteoblasts express a repertoire of cadherins and that perturbation of cadherin-mediated cell-cell interaction reduces bone morphogenetic protein 2 (BMP-2) stimulation of alkaline phosphatase activity. To test whether inhibition of cadherin function interferes with osteoblast function, we expressed a truncated N-cadherin mutant (NCaddeltaC) with dominant negative action in MC3T3-E1 osteoblastic cells. In stably transfected clones, calcium-dependent cell-cell adhesion was decreased by 50%. Analysis of matrix protein expression during a 4-week culture period revealed that bone sialoprotein, osteocalcin, and type I collagen were substantially inhibited with time in culture, whereas osteopontin transiently increased. Basal alkaline phosphatase activity declined in cells expressing NCaddeltaC, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%). Finally, 45Ca uptake, an index of matrix mineralization, was decreased by 35% in NCaddeltaC-expressing cells compared with control cultures after 4 weeks in medium containing ascorbic acid and beta-glycerophosphate. Similarly, BMP-2 stimulation of alkaline phosphatase activity and bone sialoprotein and osteopontin expression also were curtailed in NCaddeltaC cells. Therefore, expression of dominant negative cadherin results in decreased cell-cell adhesion associated with altered bone matrix protein expression and decreased matrix mineralization. Cadherin-mediated cell-cell adhesion is involved in regulating the function of bone-forming cells.
Cheng
S L
SL
Department of Medicine, Washington University School of Medicine, Barnes-Jewish Hospital, St. Louis, Missouri 63110, USA.
Shin
C S
CS
Towler
D A
DA
Civitelli
R
R
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001857:Bone Matrix / Q000378:metabolism*; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002113:Calcification, Physiologic; D002448:Cell Adhesion; D002454:Cell Differentiation; D015536:Down-Regulation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D066298:In Vitro Techniques; D058574:Integrin-Binding Sialoprotein; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology / Q000378:metabolism*; D015675:Osteocalcin / Q000378:metabolism; D053495:Osteopontin; D012333:RNA, Messenger / Q000378:metabolism; D012795:Sialoglycoproteins / Q000378:metabolism; D015854:Up-Regulation
D015820:Cadherins; C546871:IBSP protein, human; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D012333:RNA, Messenger; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D015675:Osteocalcin; D053495:Osteopontin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000469:Alkaline Phosphatase