Shin Chan Soo, Her Sun-Ju, Kim Jeong-Ah, Kim Do Hee, Kim Sang Wan, Kim Seong Yeon, Kim Hyo-Soo, Park Ki Ho, Kim Jung Gu, Kitazawa Riko, Cheng Su-Li, Civitelli Roberto
Department of Internal Medicine, Seoul National University College of Medicine, Korea.
J Bone Miner Res. 2005 Dec;20(12):2200-12. doi: 10.1359/JBMR.050809. Epub 2005 Aug 8.
We studied the effects of dominant negative N-cadherin (NCadDeltaC) expression in ST2 cells on their ability to support osteoclastogenesis. Expression of NCadDeltaC in ST2 cells did not decrease cell-to-cell adhesion but significantly reduced osteoclast formation when co-cultured with BMMs. NCadDeltaC inhibited beta-catenin/TCF signaling, resulting in decreased RANKL expression, which could contribute to the reduced osteoclast formation.
Cadherin is a calcium-dependent cell adhesion molecule that plays major roles during embryonic development and morphogenesis. Classic cadherins interact with beta-catenin, which is also involved in the Wnt signaling pathway. We tested whether disruption of N-cadherin function in stromal cells by dominant negative N-cadherin affects their ability to support osteoclastogenesis by altering heterotypic interaction with osteoclast precursors.
ST2 cells were transduced with retrovirus encoding extracellular domain-truncated, dominant negative N-cadherin (NCadDeltaC) and co-cultured with bone marrow macrophages (BMMs) to study the ability to support osteoclastogenesis. As a downstream target of NCadDeltaC, beta-catenin/T-cell factor (TCF) transcriptional activity was analyzed using TOPflash reporter construct. Real-time RT-PCR analysis and RANKL-luciferase reporter assays were performed to study the effects of NCadDeltaC on the osteoprotegerin (OPG)/RANKL system.
Immunoblotting analysis showed that primary bone marrow stromal cells, ST2 cells, and BMMs expressed N-cadherin. Retroviral expression of NCadDeltaC in ST2 cells did not significantly inhibit cell adhesion but markedly impaired the formation of TRACP(+) osteoclasts (>40%) when co-cultured with BMMs. However, the inhibition of osteoclastogenesis was not reproduced by neutralizing antibody against N-cadherin. Expression of NCadDeltaC, however, strongly suppressed beta-catenin/TCF transcriptional activity in ST2 cells, which was rescued by constitutively active beta-catenin adenovirus (Ad DeltaN46 beta-catenin) or constitutively active TCF mutant (pCS2-VP16DeltabetaXTCF-3). As a potential downstream target of Wnt signaling, we found that the expression of RANKL was reduced in ST2 cells expressing NCadDeltaC. Moreover, Wnt-3A, Ad DeltaN46 beta-catenin, and VP16DeltabetaXTCF-3 increased the expression of RANKL and enhanced the transcriptional activity of mouse RANKL promoter in ST2 cells.
Our data suggest that expression of dominant negative N-cadherin in ST2 cells suppressed osteoclastogenesis by interfering with beta-catenin regulation of RANKL independent of cell-cell adhesion.
我们研究了在ST2细胞中表达显性负性N-钙黏蛋白（NCadDeltaC）对其支持破骨细胞生成能力的影响。在ST2细胞中表达NCadDeltaC并不会降低细胞间黏附，但与骨髓巨噬细胞（BMMs）共培养时，会显著减少破骨细胞的形成。NCadDeltaC抑制β-连环蛋白/TCF信号传导，导致RANKL表达降低，这可能是破骨细胞形成减少的原因。
钙黏蛋白是一种钙依赖性细胞黏附分子，在胚胎发育和形态发生过程中起主要作用。经典钙黏蛋白与β-连环蛋白相互作用，β-连环蛋白也参与Wnt信号通路。我们测试了通过显性负性N-钙黏蛋白破坏基质细胞中N-钙黏蛋白的功能是否会通过改变与破骨细胞前体的异型相互作用来影响其支持破骨细胞生成的能力。
用编码细胞外结构域截短的显性负性N-钙黏蛋白（NCadDeltaC）的逆转录病毒转导ST2细胞，并与骨髓巨噬细胞（BMMs）共培养，以研究其支持破骨细胞生成的能力。作为NCadDeltaC的下游靶点，使用TOPflash报告构建体分析β-连环蛋白/T细胞因子（TCF）转录活性。进行实时RT-PCR分析和RANKL-荧光素酶报告基因检测，以研究NCadDeltaC对骨保护素（OPG）/RANKL系统的影响。
免疫印迹分析表明，原代骨髓基质细胞、ST2细胞和BMMs表达N-钙黏蛋白。在ST2细胞中逆转录病毒表达NCadDeltaC不会显著抑制细胞黏附，但与BMMs共培养时，会明显损害TRACP(+)破骨细胞的形成（>40%）。然而，抗N-钙黏蛋白的中和抗体并未重现对破骨细胞生成的抑制作用。然而，NCadDeltaC的表达强烈抑制ST2细胞中的β-连环蛋白/TCF转录活性，这可通过组成型活性β-连环蛋白腺病毒（Ad DeltaN46β-连环蛋白）或组成型活性TCF突变体（pCS2-VP16DeltabetaXTCF-3）来挽救。作为Wnt信号的潜在下游靶点，我们发现表达NCadDeltaC的ST2细胞中RANKL的表达降低。此外，Wnt-3A、Ad DeltaN46β-连环蛋白和VP16DeltabetaXTCF-3增加了RANKL的表达，并增强了ST2细胞中小鼠RANKL启动子的转录活性。
我们的数据表明，在ST2细胞中表达显性负性N-钙黏蛋白通过干扰β-连环蛋白对RANKL的调节来抑制破骨细胞生成，而与细胞间黏附无关。
