Annexin II stimulates RANKL expression through MAPK

Fanghong Li, Ho Yeon Chung, Sakamuri V. Reddy, Ganwei Lu, Noriyoshi Kurihara, Allan Z. Zhao, G. David Roodman

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. Introduction: Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte- macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. Materials and Methods: Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. Results and Conclusion: In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.

Original languageEnglish (US)
Pages (from-to)1161-1167
Number of pages7
JournalJournal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Volume20
Issue number7
DOIs
StatePublished - Jul 2005
Externally publishedYes

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Annexin A2
Granulocyte-Macrophage Colony-Stimulating Factor
Osteoclasts
Osteoprotegerin
Bone Marrow Cells
Stromal Cells
Bone Marrow
Conditioned Culture Medium
Mesenchymal Stromal Cells
Messenger RNA
Western Blotting
Membranes

Keywords

  • Annexin II
  • MAPK pathway
  • Osteoclast formation
  • Primary human bone marrow cells
  • RANKL

ASJC Scopus subject areas

  • Surgery

Cite this

Annexin II stimulates RANKL expression through MAPK. / Li, Fanghong; Chung, Ho Yeon; Reddy, Sakamuri V.; Lu, Ganwei; Kurihara, Noriyoshi; Zhao, Allan Z.; Roodman, G. David.

In: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 20, No. 7, 07.2005, p. 1161-1167.

Research output: Contribution to journalArticle

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abstract = "We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. Introduction: Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte- macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. Materials and Methods: Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. Results and Conclusion: In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.",
keywords = "Annexin II, MAPK pathway, Osteoclast formation, Primary human bone marrow cells, RANKL",
author = "Fanghong Li and Chung, {Ho Yeon} and Reddy, {Sakamuri V.} and Ganwei Lu and Noriyoshi Kurihara and Zhao, {Allan Z.} and Roodman, {G. David}",
year = "2005",
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T1 - Annexin II stimulates RANKL expression through MAPK

AU - Li, Fanghong

AU - Chung, Ho Yeon

AU - Reddy, Sakamuri V.

AU - Lu, Ganwei

AU - Kurihara, Noriyoshi

AU - Zhao, Allan Z.

AU - Roodman, G. David

PY - 2005/7

Y1 - 2005/7

N2 - We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. Introduction: Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte- macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. Materials and Methods: Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. Results and Conclusion: In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.

AB - We report that AX-II, in addition to inducing GM-CSF expression, also increases membrane-bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK-dependent pathway. Thus, both GM-CSF and RANKL are required for AX-II stimulation of OCL formation. Introduction: Annexin II (AX-II) is an autocrine/paracrine factor secreted by osteoclasts (OCLs) that stimulates human OCL formation and bone resorption in vitro by inducing bone marrow stromal cells and activated CD4+ T cells to produce granulocyte- macrophage colony-stimulating factor (GM-CSF). GM-CSF in turn increases OCL precursor proliferation and further enhances OCL formation. However, the induction of GM-CSF by AX-II cannot fully explain its effects on OCL formation. In this study, we tested the capacity of AX-II to induce the expression of RANKL and the corresponding signaling pathways AX-II employs in human marrow stromal cells to induce RANKL. We also showed that both GM-CSF and RANKL are required for OCL formation induced by AX-II. Materials and Methods: Real-time RT-PCR and Western blot analysis were used to detect RANKL and osteoprotegerin (OPG) mRNA and protein expression in unfractionated human bone marrow mononuclear cells stimulated with AX-II. Soluble RANKL in the conditioned medium was analyzed by ELISA. Activation of the MAPK pathway by AX-II was tested by Western blot. The effects of OPG and anti-GM-CSF on AX-II-induced OCL formation were also examined. Results and Conclusion: In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.

KW - Annexin II

KW - MAPK pathway

KW - Osteoclast formation

KW - Primary human bone marrow cells

KW - RANKL

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