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


BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Shin
Chan Soo
CS
Department of Internal Medicine, Seoul National University College of Medicine, Korea. csshin@snu.ac.kr
Her
Sun-Ju
SJ
Kim
Jeong-Ah
JA
Kim
Do Hee
DH
Kim
Sang Wan
SW
Kim
Seong Yeon
SY
Kim
Hyo-Soo
HS
Park
Ki Ho
KH
Kim
Jung Gu
JG
Kitazawa
Riko
R
Cheng
Su-Li
SL
Civitelli
Roberto
R
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000378:metabolism; D015820:Cadherins; D002117:Calcitriol / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D018920:Coculture Techniques; D003907:Dexamethasone / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000235:genetics; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D017154:Stromal Cells / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D015820:Cadherins; D002352:Carrier Proteins; C489083:Cdh2 protein, mouse; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D051176:beta Catenin; D003907:Dexamethasone; D002117:Calcitriol