A dominant negative cadherin inhibits osteoblast differentiation
A dominant negative cadherin inhibits osteoblast differentiation
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DOI：
10.1359/jbmr.2000.15.12.2362
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发表时间：
2000-12-01
影响因子：
6.2
通讯作者：
Civitelli, R
中科院分区：
文献类型：
--
作者：
Cheng, SL;Shin, CS;Civitelli, R
我们以前已经表明，人成骨细胞表达的钙粘蛋白和钙粘蛋白介导的细胞-细胞相互作用的扰动减少骨形态发生蛋白2（BMP-2）刺激碱性磷酸酶活性的剧目。为了测试钙粘蛋白功能的抑制是否会干扰成骨细胞的功能，我们表达了截短的N-钙粘蛋白突变体，在稳定转染的克隆中，钙依赖性细胞-细胞粘附降低了50%，在4周培养期间基质蛋白表达的分析显示，骨唾液蛋白，骨钙素，和I型胶原蛋白在培养中随时间的推移而受到实质性抑制，而骨桥蛋白则短暂增加。在培养3周后，相对于对照细胞，表达NCad DeltaC的细胞中的基础碱性磷酸酶活性下降，并且它们的细胞增殖速率适度降低（17%）。最后，在含有抗坏血酸和β-甘油磷酸的培养基中4周后，与对照培养物相比，表达NCad DeltaC的细胞中的Ca-45摄取（基质矿化的指标）降低了35%。同样，NCad DeltaC细胞中BMP-2对碱性磷酸酶活性以及骨唾液酸蛋白和骨桥蛋白表达的刺激也被抑制。因此，显性负性钙粘蛋白的表达导致与改变的骨基质蛋白表达和降低的基质矿化相关的细胞-细胞粘附降低。钙粘蛋白介导的细胞-细胞粘附参与调节骨形成细胞的功能。
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 (NCad DeltaC) 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 NCad DeltaC, relative to control cells, after 3 weeks in culture, and their cell proliferation rate was reduced moderately (17%), Finally, Ca-45 uptake, an index of matrix mineralization, was decreased by 35% in NCad DeltaC-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 NCad DeltaC 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.