RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts.
Osteoclast apoptosis is an influential determinant of osteoclast bone-resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown.


BACKGROUND
Fas, a death receptor, mediates apoptosis in multiple types of cells including osteoclasts. Here we report that RANKL acts as a survival factor in osteoclasts by downregulating Fas-mediated apoptosis and Fas expression in mature osteoclasts.


METHODS
RAW264.7 and mouse bone marrow macrophage/monocyte progenitors and progenitor-derived osteoclasts, in the presence of various concentrations of RANKL, were used in this study. Western blotting, semiquantitative RT-PCR, flow cytometry, nuclear staining, and a fluorescent caspase-3 activity assay were used to assess the effect of RANKL on Fas expression and Fas-mediated apoptosis. The involvement of NF-kappaB in the regulation of Fas by RANKL was analyzed by luciferase assay and EMSA.


RESULTS
Mature osteoclasts generated in the presence of a high concentration of RANKL (3.33 nM) failed to respond to Fas-induced apoptosis. The lack of responsiveness in mature osteoclasts is caused by the low level of Fas expression, as detected by both semiquantitative PCR and Western blotting. Fas protein and mRNA expression are inhibited by RANKL in concentration-dependent manners. The downregulation of Fas expression by RANKL is not because of modulation of the stability of Fas protein or mRNA. The regulation of Fas expression by RANKL is biphasic. During the early stage of osteoclastogenesis (1 day) when Fas is expressed at a very low level, RANKL upregulates Fas promoter activity by 2.4 +/- 0.1-fold in a concentration-dependent manner and increases Fas mRNA and protein. This event correlates with regulation of the binding activity of NF-kappaB to the Fas promoter by RANKL, as detected by EMSA. In osteoclast precursors, the induction of Fas promoter activity by RANKL was dramatically reduced when NF-kappaB binding sites on the Fas promoter were mutated.


CONCLUSIONS
RANKL upregulates Fas expression in osteoclast progenitors through NF-kappaB, making osteoclasts targets of Fas-stimulated apoptosis. In differentiated mature osteoclasts, RANKL reduces the levels of Fas expression and Fas-mediated apoptosis, acting as a survival factor.
Wu
Xiaojun
X
Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.
Pan
George
G
McKenna
Margaret A
MA
Zayzafoon
Majd
M
Xiong
Wen-Cheng
WC
McDonald
Jay M
JM
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001862:Bone Resorption / Q000378:metabolism*; D002352:Carrier Proteins / Q000494:pharmacology / Q000502:physiology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D008562:Membrane Glycoproteins / Q000494:pharmacology / Q000502:physiology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D010010:Osteoclasts / Q000187:drug effects / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D061945:Caspase Inhibitors; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D019014:fas Receptor; C505409:Casp3 protein, mouse; D053148:Caspase 3