Annexin II Stimulates RANKL Expression Through MAPK

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.