BSP and RANKL induce osteoclastogenesis and bone resorption synergistically.
RANKL and BSP are upregulated in several bone resorptive disorders. However, the mechanisms by which these two factors might induce osteoclastogenesis and bone resorption synergistically under pathological conditions remain largely unknown.


BACKGROUND
RANKL and bone sialoprotein II (BSP) have been shown to be upregulated in the serum of individuals with abnormally high osteoclastogenic and bone resorptive activities. Here we provide experimental evidence that RANKL and BSP induce osteoclastogenesis and bone resorption synergistically but mediate opposite effects in osteoclast survival and apoptosis.


METHODS
RAW264.7 cells and mouse bone marrow-derived monocytes/macrophages were treated with human recombinant BSP in the presence and absence of RANKL. TRACP stainings, bone resorption assays, Western blotting, immunoprecipitation analyses, and semiquantitative RT-PCR were used to evaluate the effects of BSP in osteoclast differentiation and bone resorption. Survival, DNA condensation, and caspase activity assays were used to determine the putative effects of BSP in osteoclast survival and apoptosis.


CONCLUSIONS
RANKL induced osteoclast differentiation and bone resorption at a higher extent in the presence than in the absence of BSP in RAW264.7 cells and bone marrow-derived monocytes/macrophages. c-Src-dependent c-Cbl phosphorylation was 8-fold higher in RAW264.7 cells treated with BSP and RANKL than in those treated with RANKL alone. Furthermore, BSP and RANKL activated the master regulator of osteoclastogenesis nuclear factor of activated T cells (NFAT)-2 and increased the mRNA expression of other differentiation markers such as cathepsin K or TRACP. Inhibition of c-Src activity or chelating intracellular calcium inhibited the synergistic effects in bone resorption and the phosphorylation of the c-Src substrate c-Cbl. Inhibition of calcineurin or intracellular calcium elevation inhibited the synergistic effects in osteoclastogenesis and decreased NFAT-2 nuclear levels. On the other hand, BSP and RANKL mediated opposite effects in osteoclast survival and apoptosis. Thus, BSP increased survival and decreased apoptosis markers in differentiated RANKL-treated RAW267.5 cells and RANKL/macrophage-colony stimulating factor (M-CSF)-treated bone marrow-derived monocytes/macrophages. In addition, RAW267.5 cells treated with BSP and RANKL exhibited decreased activation of the proapoptotic Jun N-terminal kinase pathway and increased activation of anti-apoptotic AKT pathway than cells treated with RANKL or BSP alone. Taken together, our findings suggest that BSP contributes to RANKL-mediated bone resorption by inducing osteoclastogenesis and osteoclast survival and decreasing osteoclast apoptosis.
Valverde
Paloma
P
Department of General Dentistry, Tufts University School of Dental Medicine, Boston, Massachusetts 02111, USA. paloma.valverde@tufts.edu
Tu
Qisheng
Q
Chen
Jake
J
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D001854:Bone Marrow Cells; D001862:Bone Resorption; D000081247:CSK Tyrosine-Protein Kinase; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D047468:Immunoprecipitation; D058574:Integrin-Binding Sialoprotein; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D019061:src-Family Kinases
D002352:Carrier Proteins; C546871:IBSP protein, human; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D008562:Membrane Glycoproteins; D050778:NFATC Transcription Factors; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D016173:Macrophage Colony-Stimulating Factor; D004247:DNA; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D002403:Cathepsins; D020169:Caspases; C534130:CTSK protein, human; D056657:Cathepsin K; C534132:Ctsk protein, mouse