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. 2010 Nov 26;285(48):37427-35.
doi: 10.1074/jbc.M110.149484. Epub 2010 Sep 24.

Receptor activator of NF-{kappa}B (RANK) cytoplasmic IVVY535-538 motif plays an essential role in tumor necrosis factor-{alpha} (TNF)-mediated osteoclastogenesis

Joel Jules  1 , Zhenqi Shi, Jianzhong Liu, Duorong Xu, Shunqing Wang, Xu Feng
Affiliations

Receptor activator of NF-{kappa}B (RANK) cytoplasmic IVVY535-538 motif plays an essential role in tumor necrosis factor-{alpha} (TNF)-mediated osteoclastogenesis

Joel Jules et al. J Biol Chem. .

Abstract

Tumor necrosis factor-α (TNF) enhances osteoclast formation and activity leading to bone loss in various pathological conditions, but its precise role in osteoclastogenesis remains controversial. Although several groups showed that TNF can promote osteoclastogenesis independently of the receptor activator of NF-κB (RANK) ligand (RANKL), others demonstrated that TNF-mediated osteoclastogenesis needs permissive levels of RANKL. Here, we independently reveal that although TNF cannot stimulate osteoclastogenesis on bone slices, it can induce the formation of functional osteoclasts on bone slices in the presence of permissive levels of RANKL or from bone marrow macrophages (BMMs) pretreated by RANKL. TNF can still promote the formation of functional osteoclasts 2 days after transient RANKL pretreatment. These data have confirmed that TNF-mediated osteoclastogenesis requires priming of BMMs by RANKL. Moreover, we investigated the molecular mechanism underlying the dependence of TNF-mediated osteoclastogenesis on RANKL. RANK, the receptor for RANKL, contains an IVVY(535-538) motif that has been shown to play a vital role in osteoclastogenesis by committing BMMs to the osteoclast lineage. We show that TNF-induced osteoclastogenesis depends on RANKL to commit BMMs to the osteoclast lineage and RANKL regulates the lineage commitment through the IVVY motif. Mechanistically, the IVVY motif controls the lineage commitment by reprogramming osteoclast genes into an inducible state in which they can be activated by TNF. Our findings not only provide important mechanistic insights into the action of RANKL in TNF-mediated osteoclastogenesis but also establish that the IVVY motif may serve as an attractive therapeutic target for bone loss in various bone disorders.

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Figures

FIGURE 1.
FIGURE 1.
TNF can neither induce osteoclastogenesis nor activate osteoclast genes. A, BMMs were treated with M-CSF (M, 44 ng/ml), M-CSF (44 ng/ml) and RANKL (100 ng/ml) (M+R), or M-CSF (44 ng/ml) and TNF (10 ng/ml) (M+T) in a tissue culture dish for 4 days (d). The cultures were then stained for TRAP activity. B, BMMs on bone slices were cultured with M-CSF (44 ng/ml), M-CSF (44 ng/ml) and RANKL (100 ng/ml), or M-CSF (44 ng/ml) and TNF (10 ng/ml) for 4 days, and bone slices were then stained with Hoechst 33258 (Hoechst) or Alexa Fluor 488 phalloidin (Phalloidin). A separate set of cultures was continued for 4 additional days to perform bone resorption assays. C, BMMs were treated with M-CSF (44 ng/ml), M-CSF (44 ng/ml) and RANKL (100 ng/ml), or M-CSF (44 ng/ml) and TNF (10 ng/ml) in a tissue culture dish for 1, 2, or 4 days. Gene expression was determined by semiquantitative RT-PCR.
FIGURE 2.
FIGURE 2.
TNF can induce osteoclastogenesis and activate osteoclast genes in the presence of permissive levels of RANKL. A, BMMs were cultured with 44 ng/ml M-CSF (M) plus different doses of RANKL (R) with or without TNF (T, 5 ng/ml) for 4 days (d) in a tissue culture dish. The cultures were then stained for TRAP activity. B, BMMs were treated with M-CSF (44 ng/ml) and RANKL (10 ng/ml), M-CSF (44 ng/ml) and TNF (5 ng/ml), or M-CSF (44 ng/ml) + RANKL (10 ng/ml) + TNF (5 ng/ml) for 1, 2, or 4 days. Gene expression was determined by semiquantitative RT-PCR. C, BMMs on bone slices were treated with M-CSF (44 ng/ml) and RANKL (10 ng/ml), M-CSF (44 ng/ml) and TNF (5 ng/ml), M-CSF (44 ng/ml) + RANKL (10 ng/ml) + TNF (5 ng/ml), or M-CSF (44 ng/ml) and RANKL (100 ng/ml) for 4 days, and bone slices were stained with Hoechst 33258 (Hoechst) or Alexa Fluor 488 phalloidin (Phalloidin). A separate set of cultures was continued for 4 additional days to perform bone resorption assays.
FIGURE 3.
FIGURE 3.
TNF can promote osteoclastogenesis and activate osteoclast genes in BMMs pretreated by RANKL. A, BMMs were primed with M-CSF (M, 44 ng/ml) and RANKL (R, 100 ng/ml) for 6, 12, or 18 h and then continued with M-CSF (44 ng/ml) alone or M-CSF (44 ng/ml) plus TNF (T, 5 ng/ml) for 3 days (d). BMMs treated with M-CSF (44 ng/ml) plus RANKL (100 ng/ml) or M-CSF (44 ng/ml) plus TNF (5 ng/ml) served as positive and negative controls, respectively. All cultures were then stained for TRAP activity. B, BMMs on bone slices were primed with M-CSF (44 ng/ml) and RANKL (100 ng/ml) for 18 h and then treated with M-CSF (44 ng/ml) alone, M-CSF (44 ng/ml) and TNF (5 ng/ml), or M-CSF (44 ng/ml) and RANKL (100 ng/ml) for 4 days, and bone slices were then stained with Hoechst 33258 (Hoechst) or Alexa Fluor 488 phalloidin (Phalloidin). A separate set of cultures was continued for 4 additional days to perform bone resorption assays. C, quantification of the bone resorption assays is shown. Bars show averages ± S.D. D, BMMs were primed with M-CSF (44 ng/ml) and RANKL (100 ng/ml) for 18 h and then cultured with M-CSF (44 ng/ml) alone, M-CSF (44 ng/ml) and RANKL (100 ng/ml), or M-CSF (44 ng/ml) and TNF (5 ng/ml) for 1, 2, or 3 days. Gene expression was assessed by semiquantitative RT-PCR.
FIGURE 4.
FIGURE 4.
RANKL mediated-BMM priming is durable. A, BMMs were treated with M-CSF (M, 44 ng/ml) and RANKL (R, 100 ng/ml) for 18 h and then cultured with M-CSF alone for 0, 1, or 2 days (d) before treating with M-CSF (44 ng/ml) alone, M-CSF (44 ng/ml) and RANKL (100 ng/ml), or M-CSF (44 ng/ml) and TNF (T, 5 ng/ml) for 3 days. The cultures were stained for TRAP activity. B, BMMs were treated with M-CSF (44 ng/ml) and RANKL (100 ng/ml) for 18 h and then cultured with M-CSF alone for 0, 1 or 2 days before treating with M-CSF (44 ng/ml) alone, M-CSF (44 ng/ml) and RANKL (100 ng/ml), or M-CSF (44 ng/ml) and TNF (5 ng/ml) for 1 day. Gene expression was determined by semiquantitative RT-PCR. C, BMMs on bone slices were treated with M-CSF (44 ng/ml) and RANKL (100 ng/ml) for 18 h and then cultured with M-CSF alone for 2 days before treating with M-CSF (44 ng/ml) alone, M-CSF (44 ng/ml) and RANKL (100 ng/ml), or M-CSF (44 ng/ml) and TNF (5 ng/ml) for 3 days, and bone slices were then stained with Hoechst 33258 (Hoechst) or Alexa Fluor 488 phalloidin (Phalloidin). A separate set of cultures was continued for 4 additional days to perform bone resorption assays. D, quantification of the bone resorption assays is shown. Bars show averages ± S.D.
FIGURE 5.
FIGURE 5.
RANK IVVY535–538 motif plays a critical role in TNF-mediated osteoclastogenesis. A, schematic diagram of Ch1 and Ch2. B, flow cytometric analysis of BMMs infected with virus encoding GFP, Ch1, or Ch2. C, BMMs expressing GFP, Ch1, or Ch2 were treated with M-CSF (M, 44 ng/ml) and Fas-AB (F, 100 ng/ml) for 18 h. The cultures were then continued with M-CSF (44 ng/ml) alone or M-CSF (44 ng/ml) and TNF (T, 5 ng/ml) for 4 days (d). Infected BMMs treated with M-CSF (44 ng/ml) plus Fas-AB (100 ng/ml) or M-CSF (44 ng/ml) plus TNF (T, 5 ng/ml) for 5 days served as positive and negative controls, respectively. The cultures were then stained for TRAP activity.
FIGURE 6.
FIGURE 6.
RANK IVVY535–538 motif renders osteoclast genes responsive to TNF. A, BMMs expressing GFP, Ch1, or Ch2 were treated with M-CSF (M, 44 ng/ml) alone, M-CSF (44 ng/ml) and Fas-AB (F, 100 ng/ml), or M-CSF (44 ng/ml) and TNF (T, 5 ng/ml) for 18 h. B, BMMs expressing GFP, Ch1, or Ch2 were treated with M-CSF (44 ng/ml) and Fas-AB (100 ng/ml) for 18 h, and the cultures were then continued with M-CSF (44 ng/ml) alone or M-CSF (44 ng/ml) and TNF (5 ng/ml) for 4 days (d). Infected BMMs treated with M-CSF (44 ng/ml) plus Fas-AB (100 ng/ml) or M-CSF (44 ng/ml) plus TNF (5 ng/ml) for 5 days served as positive and negative controls, respectively. Gene expression in A and B was determined by semi-quantitative RT-PCR.
FIGURE 7.
FIGURE 7.
TNF-induced osteoclastogenesis attended by permissive Fas-AB dosage requires RANK IVVY535–538-mediated signaling. A, flow cytometric analysis of BMMs infected with virus encoding GFP, Ch1, or Ch2 is shown. B, BMMs expressing GFP, Ch1, or Ch2 were treated with M-CSF (M, 44 ng/ml) and Fas-AB (F, 10 ng/ml) with or without TNF (T, 5 ng/ml) for 5 days (d). The cultures were then stained for TRAP activity. C, BMMs expressing GFP, Ch1, or Ch2 were treated with M-CSF (44 ng/ml) and Fas-AB (10 ng/ml), M-CSF (44 ng/ml) and TNF (5 ng/ml), or M-CSF (44 ng/ml) + Fas-AB (10 ng/ml) + TNF (5 ng/ml) for 3 days. Gene expression was determined by semiquantitative RT-PCR.
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