Elsevier

Bone

Volume 34, Issue 1, January 2004, Pages 57-64
Bone

Transforming growth factor-β induces osteoclast formation in the absence of RANKL

https://doi.org/10.1016/j.bone.2003.08.008Get rights and content

Abstract

Transforming growth factor beta (TGFβ) is a multifunctional growth factor that is produced by many cells in bone and is abundant in the bone matrix. TGFβ is known to regulate RANKL-induced osteoclast formation and bone resorbing activity. In this study we sought to determine whether TGFβ could directly induce osteoclast formation by a RANKL-independent mechanism. We found that the addition of TGFβ to cultures of human monocytes and RAW 264.7 cells (in the presence of M-CSF and the absence of RANKL, TNFα or IL-6/IL-11) was sufficient to induce the formation of TRAP+ and VNR+ cells, which formed actin rings and were capable of extensive lacunar resorption. The addition of osteoprotegerin or antibodies to TNFα and its receptors, as well as antibodies to gp130, did not inhibit lacunar resorption, indicating that TGFβ did not act by stimulating RANKL, TNF or IL-6 production by monocytes. TGFβ-induced osteoclast formation was qualitatively different from that induced by RANKL with numerous TRAP+/VNR+ mononuclear and small multinucleated cells being formed; these cells produced many small resorption lacunae. Our results indicate that TGFβ, which is abundant in the bone matrix, can, in the presence of M-CSF, directly induce mononuclear phagocyte osteoclast precursors to differentiate into osteoclastic cells capable of lacunar resorption.

Introduction

Osteoclasts are highly specialized multinucleated cells that are uniquely capable of lacunar bone resorption. Mononuclear osteoclast precursors are bone-marrow-derived cells that circulate in the monocyte fraction and differentiate into functional osteoclasts at the bone surface [1]. Osteoclast formation requires the presence of macrophage-colony stimulating factor (M-CSF) and involves an interaction between the receptor activator of nuclear factor κB (RANK), which is expressed on osteoclast precursors, and RANK ligand (RANKL), which is expressed by several cell types including osteoblasts [2], [3]. Several cytokine factors are known to influence RANKL-induced osteoclast formation and function [2], [4]. It has also recently been shown that cytokines, such as tumor necrosis factor-α (TNFα), interleukin-6 (IL-6) and IL-11 are capable of inducing osteoclast formation from mouse marrow and human circulating precursors by a RANKL-independent mechanism [5], [6], [7], [8].

Transforming growth factor β (TGFβ) is a multifunctional growth factor that is abundant in bone; it is produced by many cells in the bone microenvironment, including osteoblasts, fibroblasts and osteoclasts [9]. TGFβ induces bone formation and has been reported both to enhance and to inhibit osteoclast formation and resorption, depending on the assay system used to measure these parameters [10], [11], [12], [13], [14]. It is thought that these mononuclear phagocytes are essentially uncommitted precursors and that exposure to different cytokines or growth factors markedly influences the extent of their commitment to the osteoclast lineage [15].

Whether TGFβ, like TNFα and IL-6/IL-11, is capable of directly inducing osteoclast formation by a RANKL-independent mechanism is not known. In this study we have sought to determine whether, in the absence of RANKL, TNFα and IL-6/IL-11, TGFβ is capable of directly inducing the formation of osteoclasts from circulating human precursors and murine RAW 264.7 cells; the latter is a macrophage cell line that expresses high levels of RANK and has been shown to form osteoclasts when incubated with sRANKL [16]. We have also characterized the nature and extent of osteoclast formation and lacunar resorption carried out by cells formed in this way and compared it with that of RANKL-induced osteoclasts.

Section snippets

Reagents

Cells were cultured in α-minimal essential medium (MEM) (Invitrogen, UK) containing penicillin (100 U/ml), streptomycin (100 μg/ml) and 10% heat-inactivated fetal bovine serum (Invitrogen) (MEM–FBS). Recombinant human M-CSF, TGFβ1, IL-1α and antibodies to TNFα, TNF receptor I and II and anti-gp130 were purchased from R&D Systems, Inc. (Abingdon, UK). Human soluble RANKL (sRANKL) and osteoprotegerin (OPG) were kindly provided by Dr. Lacey, Amgen (Thousand Oaks, CA, USA). RAW 264.7 cells were a

TGFβ induces osteoclast formation from human PBMCs in the absence of sRANKL

Twenty-four-hour cultures of either sorted (CD14+) or unsorted (CD14+/CD14) PBMCs contained numerous CD14+ cells, which were negative for TRAP and VNR. There was no evidence of lacunar resorption on dentine slices in these 24-h cultures. These cells thus expressed the phenotype of monocytes and did not express the cytochemical and functional features of osteoclasts.

After 14 days of incubation, TRAP+ and VNR+ mononuclear and multinucleated cells (MNC) were seen on coverslips of sorted (CD14+)

Discussion

TGFβ is a multifunctional growth factor produced by many cells in the bone microenvironment, including osteoblasts, fibroblasts, macrophages and osteoclasts [9]. TGFβ is abundant in the bone matrix; it is released during bone resorption and is thought to act as a coupling factor between bone resorption and bone formation [21]. The effects of TGFβ on osteoclast formation and bone resorption are controversial with both stimulation and inhibition being reported in various biological assay systems.

Acknowledgements

This study was supported in part by the Wellcome Trust, Action Research and the Frances and Augustus Newman Foundation and the TR Golden Charitable Trust. We are also grateful to Amgen for supplying human soluble RANKL and OPG.

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