Dominant negative N-cadherin inhibits osteoclast differentiation by interfering with β-catenin regulation of RANKL, independent of cell-cell adhesion

Chan Soo Shin, Sun Ju Her, Jeong Ah Kim, Do Hee Kim, Sang Wan Kim, Seong Yeon Kim, Hyo Soo Kim, Ki Ho Park, Jung Gu Kim, Riko Kitazawa, Su Li Cheng, Roberto Civitelli

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

We studied the effects of dominant negative N-cadherin (NCadΔC) expression in ST2 cells on their ability to support osteoclastogenesis. Expression of NCadΔC in ST2 cells did not decrease cell-to-cell adhesion but significantly reduced osteoclast formation when co-cultured with BMMs. NCadΔC inhibited β-catenin/TCF signaling, resulting in decreased RANKL expression, which could contribute to the reduced osteoclast formation. Introduction: Cadherin is a calcium-dependent cell adhesion molecule that plays major roles during embryonic development and morphogenesis. Classic cadherins interact with β-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. Materials and Methods: ST2 cells were transduced with retrovirus encoding extracellular domain-truncated, dominant negative N-cadherin (NCadΔC) and co-cultured with bone marrow macrophages (BMMs) to study the ability to support osteoclastogenesis. As a downstream target of NCadΔC, β-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 NCadΔC on the osteoprotegerin (OPG)/RANKL system. Results: Immunoblotting analysis showed that primary bone marrow stromal cells, ST2 cells, and BMMs expressed N-cadherin. Retroviral expression of NCadΔC 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 NCadΔC, however, strongly suppressed β-catenin/TCF transcriptional activity in ST2 cells, which was rescued by constitutively active β-catenin adenovirus (Ad ΔN46 β-catenin) or constitutively active TCF mutant (pCS2-VP16ΔβXTCF-3). As a potential downstream target of Wnt signaling, we found that the expression of RANKL was reduced in ST2 cells expressing NCadΔC. Moreover, Wnt-3A, Ad ΔN46 β-catenin, and VP16ΔβXTCF-3 increased the expression of RANKL and enhanced the transcriptional activity of mouse RANKL promoter in ST2 cells. Conclusions: Our data suggest that expression of dominant negative N-cadherin in ST2 cells suppressed osteoclastogenesis by interfering with β-catenin regulation of RANKL independent of cell-cell adhesion.

Original languageEnglish
Pages (from-to)2200-2212
Number of pages13
JournalJournal of Bone and Mineral Research
Volume20
Issue number12
DOIs
StatePublished - Dec 2005

Keywords

  • Cadherin
  • Cell-cell adhesion
  • Osteoclastogenesis
  • Wnt signaling
  • β-catenin

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