RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts

Xiaojun Wu, George Pan, Margaret A. McKenna, Majd Zayzafoon, Wencheng Xiong, Jay M. McDonald

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

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. Introduction: 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. Materials and 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-ê 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-κB 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-κB binding sites on the Fas promoter were mutated. Conclusion: RANKL upregulates Fas expression in osteoclast progenitors through NF-êÂ, 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.

Original languageEnglish (US)
Pages (from-to)107-116
Number of pages10
JournalJournal of Bone and Mineral Research
Volume20
Issue number1
DOIs
StatePublished - Dec 9 2005

Fingerprint

Osteoclasts
Apoptosis
Osteogenesis
Up-Regulation
Down-Regulation
Western Blotting
Polymerase Chain Reaction
Death Domain Receptors
Messenger RNA
Protein Stability
RNA Stability
Luciferases
Caspase 3
Monocytes
Flow Cytometry
Proteins
Bone Marrow
Macrophages

Keywords

  • Apoptosis
  • Cytokines
  • Fas
  • NF-κB
  • Osteoclasts
  • RANKL

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Orthopedics and Sports Medicine

Cite this

Wu, X., Pan, G., McKenna, M. A., Zayzafoon, M., Xiong, W., & McDonald, J. M. (2005). RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts. Journal of Bone and Mineral Research, 20(1), 107-116. https://doi.org/10.1359/jbmr.2005.20.1.107

RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts. / Wu, Xiaojun; Pan, George; McKenna, Margaret A.; Zayzafoon, Majd; Xiong, Wencheng; McDonald, Jay M.

In: Journal of Bone and Mineral Research, Vol. 20, No. 1, 09.12.2005, p. 107-116.

Research output: Contribution to journalArticle

Wu, X, Pan, G, McKenna, MA, Zayzafoon, M, Xiong, W & McDonald, JM 2005, 'RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts', Journal of Bone and Mineral Research, vol. 20, no. 1, pp. 107-116. https://doi.org/10.1359/jbmr.2005.20.1.107
Wu, Xiaojun ; Pan, George ; McKenna, Margaret A. ; Zayzafoon, Majd ; Xiong, Wencheng ; McDonald, Jay M. / RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts. In: Journal of Bone and Mineral Research. 2005 ; Vol. 20, No. 1. pp. 107-116.
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abstract = "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. Introduction: 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. Materials and 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-{\^e}{\^A} 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-κB 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-κB binding sites on the Fas promoter were mutated. Conclusion: RANKL upregulates Fas expression in osteoclast progenitors through NF-{\^e}{\^A}, 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.",
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AU - Pan, George

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AU - McDonald, Jay M.

PY - 2005/12/9

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N2 - 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. Introduction: 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. Materials and 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-ê 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-κB 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-κB binding sites on the Fas promoter were mutated. Conclusion: RANKL upregulates Fas expression in osteoclast progenitors through NF-êÂ, 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.

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KW - Osteoclasts

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