Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone

Abstract

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of κB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.

Introduction

Giant cell tumors (GCT), also known as osteoclastomas, are rare primary neoplasms of the skeleton. Although mostly benign, GCT are locally destructive and cause extensive osteolysis. GCT contain, within the tumor mass, variable numbers of large, multinucleated cells that are phenotypically similar to osteoclasts.9 It is believed that the stromal cells of GCT are the tumor cells that induce osteoclastic bone resorption by recruiting osteoclast precursors and promoting their differentiation into functional osteoclasts.13 Although the mechanisms by which they do so are not fully understood, the stromal cells have been shown to release chemokines such as macrophage chemoattractant protein-1 and interleukin (IL)-8,27, 34 which may recruit osteoclast precursors into the tumor mass. In addition, we have reported recently3 that GCT express mRNA species encoding many known proosteoclastogenic cytokines, including macrophage colony stimulating factor (M-CSF), IL-1, IL-6, IL-11, and IL-17.19

As recently reviewed,11, 19 it is now known that many osteoclastogenic agents act by inducing the cell surface expression of the tumor necrosis factor (TNF)-ligand family member, receptor activator of nuclear factor κB ligand (RANKL),2 also known as TRANCE,31 ODF,33 and OPGL,18 which is thus central to physiological osteoclast development. RANKL promotes osteoclast development by binding to the TNF-receptor superfamily member, RANK,24 on osteoclast precursors, with the required presence of M-CSF.33 A soluble TNF-receptor family member, termed osteoprotegerin (OPG), is a natural RANKL antagonist,29 which can inhibit osteoclast formation and bone resorption. OPG-knockout mice have been shown to develop extensive osteoporosis, associated with an increased number of otherwise normal osteoclasts.4, 21 Likewise, deletion of the RANKL gene resulted in severe osteopetrosis and a complete lack of osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis.16

Mechanisms of tumor-induced osteolysis remain to be elucidated. In addition to osteoclast formation via the RANKL-RANK ligation pathway, recent evidence suggests that alternative pathways may exist in the mouse.15 Tumor-mediated osteolysis by human multiple myeloma and metastatic breast carcinoma appears to occur by an indirect mechanism, mediated in part by the expression of parathyroid hormone receptor protein (PTHrP) by the tumor cells, which in turn alters the expression of RANKL by otherwise normal osteoblasts.10, 26, 30 We have recently reported that the stromal cells within GCT samples express high levels of mRNA encoding RANKL relative to their expression of OPG, when compared to the levels of expression of these molecules in nonlytic tumors of bone.3 These results prompted us to investigate the function of RANKL and OPG within GCT. Our results indicate that both the formation of osteoclasts in GCT and the bone-resorptive activity of these tumor-derived osteoclasts can be prevented completely by exogenous OPG. Thus, GCT appear to represent a paradigm for the direct promotion of osteoclast formation and activity by tumor cells via the RANKL-RANK ligation pathway, in contrast to the osteolytic mechanisms previously reported for multiple myeloma and breast carcinoma.26, 30