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

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.