Strength of TRAF6 signalling determines osteoclastogenesis - PubMed (original) (raw)

Strength of TRAF6 signalling determines osteoclastogenesis

Yuho Kadono et al. EMBO Rep. 2005 Feb.

Abstract

TRANCE/TRAF6 signalling governs osteoclastogenesis in vivo. Only the TRANCE receptor (TRANCE-R) has been shown to induce osteoclastogenesis, even though other immune receptors, including CD40 and IL-1R/Toll-like receptor, use TRAF6 to activate overlapping signalling cascades. These observations led us to question whether qualitative or quantitative differences exist between the TRAF6-mediated signals induced by TRANCE and by other ligand-receptor pairs. Here we show that stimulation by overexpressed wild-type CD40 can induce osteoclastogenesis. Stimulation through modified CD40 containing increased numbers of TRAF6-binding sites in the cytoplasmic tails showed a dose-dependent increase in the activation of p38 kinase and more pronounced osteoclastogenesis. Moreover, precursors overexpressing TRAF6 differentiate into osteoclasts in the absence of additional signals from TRANCE. Our results suggest that differences in the osteoclastogenesis-inducing capacity of TRANCE-R versus other TRAF6-associated receptors may in part stem from a quantitative difference in the TRAF6-mediated signals.

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Figures

Figure 1

Figure 1

TRAF6-binding sites of the TRANCE receptor. (A) Schematic representation of modified TRANCE-R and its mutants. (B) Osteoclastogenesis mediated by TRANCE-R mutants. BMMs were transduced with pMX-puro empty vector (EV) or pMX-puro with wild-type TRANCE-R (TRWT) or mutants, and stimulated by anti-Flag antibody (10 μg/ml). Transduced BMMs were stained for TRAP after 4 days. Statistical analysis was performed using the Student's _t_-test versus TRWT. NS, not significant, *P<0.01, **P<0.001. Scale bars, 100 μm. (C) Transduced BMMs were serum starved for 1 h, stimulated by anti-Flag antibody for the period indicated and analysed by western blotting.

Figure 2

Figure 2

Stimulation of endogenous CD40 failed to induce osteoclastogenesis. (A) BMMs were stained for CD40 and analysed by FACS. Isotype (red line) and CD40-transduced (blue line) BMMs are shown. (B) BMMs were serum starved for 1 h, and stimulated with anti-CD40 (1 μg/ml) or TRANCE (500 ng/ml) for the period indicated. Total cell lysates were subjected to western blotting. (C) BMMs were cultured in the presence of anti-CD40 (0, 0.1, 0.3 or 1 μg/ml) and/or TRANCE (0, 15, 50, 150 or 500 ng/ml) for 4 days and stained for TRAP. Statistical analysis was performed using the Student's _t_-test versus anti-CD40 absent condition. NS, not significant.

Figure 3

Figure 3

Enhanced CD40 signalling has the potential to induce osteoclastogenesis. (A) BMMs were transduced with pMX-puro carrying WT CD40 and stained for CD40 after puromycin selection. Isotype (black line), empty vector-transduced (EV; red) and WT CD40-transduced (blue) BMMs are indicated. (B) Transduced BMMs were cultured in the absence or presence of TGF-β (1 ng/ml), stimulated with (closed bar) or without (open bar) anti-CD40 (1 μg/ml) for 4 days and stained for TRAP. (C) Osteoclasts from WT CD40-transduced BMMs with TGF-β pretreatment were stained for TRAP (top left) and F-actin (top right). Transduced BMMs were cultured on the bone slices with anti-CD40 for 6 days, and bone slices were stained with 0.5% toluidine blue (bottom). (D) NFATc1 mRNA induction. Transduced BMMs were stimulated with anti-CD40 or TRANCE (500 ng/ml) for 48 h. NFATc1 mRNA induction, normalized with HPRT expression, was determined by real-time PCR. (E) WT CD40-transduced BMMs, prepared in the absence or presence of TGF-β, were serum starved for 1 h, and stimulated with anti-CD40 for the period indicated. Total cell lysates were subjected to western blotting. (F) Transduced BMMs, prepared in the presence of TGF-β, were cultured with anti-CD40 or TRANCE in the absence or presence of TR-Fc (5 μg/ml) for 4 days, and stained for TRAP. Statistical analysis was performed using the Student's _t_-test versus the EV (B) or TR-Fc absent condition (F). NS, not significant; #P<0.05; **P<0.001. Scale bars, 100 μm.

Figure 4

Figure 4

CD40 TRAF6-binding site and osteoclastogenesis. (A) BMMs were transduced with pMX-puro empty vector (EV), WT CD40 or CD40 mutants with two (2T6) or three (3T6) TRAF6-binding sites. Total cell lysates were collected after puromycin selection and subjected to immunoprecipitation using anti-Flag antibody for transduced CD40 and western blotting for TRAF6. (B) Transduced BMMs were serum starved for 1 h and stimulated with anti-CD40 antibody (1 μg/ml) for the indicated period. Total cell lysates were subjected to western blotting. (C) Transduced BMMs were cultured with anti-CD40 for 4 days and stained for TRAP. Statistical analysis was performed using the Student's _t_-test versus EV. *P<0.01. Scale bars, 100 μm.

Figure 5

Figure 5

TRAF6-mediated signal is sufficient for osteoclastogenesis. (A) BMMs were transduced with pMX-puro empty vector (EV) or WT TRAF6 (T6). Total cell lysates were subjected to western blotting. (B) Transduced BMMs were cultured in the presence of only M-CSF for a further five days and stained for TRAP (top) or F-actin (bottom). (C) Transduced BMMs were cultured on the bone slices in the presence of only M-CSF for 7 days, and bone slices were stained with 0.5% toluidine blue. Statistical analysis was performed using the Student's _t_-test versus EV. **P<0.001. Scale bars, 100 μm.

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