Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis

Abstract

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

Figure 1.

TNF-α–induced osteoclastogenesis in WT or TRANCE-deficient cells. (A–C) Osteoclast formation from WT or TRANCE-deficient osteoclast precursors. (A and B) BMMs were derived from bone marrow cells of WT mice by culturing them for 3 d with M-CSF alone or with M-CSF + TGF-β as indicated. BMMs were further cultured with M-CSF alone or with M-CSF + TNF-α as indicated. (C) Splenocytes isolated from TRANCE KO mice were cultured for 3 d with M-CSF alone or with M-CSF + TGF-β in the presence of 5 μg/ml RANK-Fc to generate BMMs, which were subsequently treated with M-CSF alone, M-CSF/TNF-α, or M-CSF/TNF-α/RANK-Fc (5 μg/ml), as indicated. Cultured cells were fixed and stained for TRAP. (D and E) TRANCE-deficient osteoclast precursors were prepared by culturing HPCs with M-CSF and TGF-β. F-actin ring staining (D) and a pit formation assay (E) on TRANCE-deficient cells that were subsequently cultured for 3 d with the indicated conditions are shown.

Figure 2.

TNF-α–induced osteoclastogenesis from WT or RANK-deficient cells. (A) Osteoclast formation from WT or RANK-deficient osteoclast precursors. Spleen cells were incubated for 3 d with M-CSF alone or with M-CSF + TGF-β to generate osteoclast precursors as indicated. Precursors were further cultured with M-CSF alone, M-CSF/TRANCE, or M-CSF/TNF-α. Cultured cells were fixed and stained for TRAP. (B) Osteoclast precursors prepared with M-CSF and TGF-β were further cultured for 3 d with M-CSF/TNF-α and fixed and stained for F-actin rings and TRAP. (C) Osteoclast precursors were prepared by culturing HPCs with M-CSF and TGF-β for 3 d. BMMs were incubated for an additional 3 d with M-CSF alone or with M-CSF/TRANCE for WT cells. RANK-deficient osteoclast precursors prepared with M-CSF + TGF-β were cultured for 3 d with M-CSF/TNF-α and an additional day with M-CSF/TNF-α in the presence or absence of IL-1 as indicated. Cells were subjected to real-time PCR analysis for TRAP, cathepsin K, MMP-9, calcitonin receptor, carbonic anhydrase II, and NFATc1. Values were normalized to 18S RNA expression. (D) Osteoclast precursors were prepared and cultured as described in C with the indicated stimuli, and dentine slices were stained with toluidine blue.

Figure 3.

Induction of NFATc1. Osteoclast precursors were derived from bone marrow cells of WT mice by culture with M-CSF alone or with M-CSF/TGF-β and subsequently stimulated with TRANCE or TNF-α for the indicated times. M-CSF was present at all times. Samples were subjected to real-time PCR using NFATc1-specific primers. NFATc1 mRNA induction was normalized to HPRT expression.

Figure 4.

TRANCE-induced osteoclastogenesis in WT or TRAF6-deficient cells. (A and B) Osteoclast formation from WT or TRAF6-deficient cells. Spleen cells were incubated for 3 d with M-CSF alone or with M-CSF + TGF-β to generate osteoclast precursors, as indicated, and further cultured with M-CSF alone or M-CSF + TRANCE. Cultured cells were fixed and stained for TRAP. F-actin ring staining (C) and a pit formation assay (D) on WT or TRAF6-deficient TRAP⁺ MNCs derived from osteoclast precursors prepared with M-CSF and TGF-β are shown.