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. 2008 Sep 5;283(36):24546-53.
doi: 10.1074/jbc.M800434200. Epub 2008 Jun 19.

Defective osteoclastogenesis by IKKbeta-null precursors is a result of receptor activator of NF-kappaB ligand (RANKL)-induced JNK-dependent apoptosis and impaired differentiation

Jesse E Otero  1 , Simon Dai, Domenica Foglia, Muhammad Alhawagri, Jean Vacher, Manolis Pasparakis, Yousef Abu-Amer
Affiliations

Defective osteoclastogenesis by IKKbeta-null precursors is a result of receptor activator of NF-kappaB ligand (RANKL)-induced JNK-dependent apoptosis and impaired differentiation

Jesse E Otero et al. J Biol Chem. .

Abstract

It has been reported previously that inhibitory kappaB kinase (IKK) supports osteoclastogenesis through NF-kappaB-mediated prevention of apoptosis. This finding suggests that the ligand for receptor activator of NF-kappaB (RANKL), the master osteoclastogenic cytokine, induces apoptosis of osteoclast precursors (OCPs) in the absence of IKKbeta/NF-kappaB competency. To validate this hypothesis, we sought to determine the pro-apoptotic signaling factors induced by RANKL in IKKbeta-null osteoclast OCPs and to rescue osteoclast differentiation in the absence of IKKbeta through their inhibition. To accomplish this, we generated mice that lack IKKbeta in multiple hematopoietic lineages, including OCPs. We found that these mice possess both in vitro and in vivo defects in osteoclast generation, in concurrence with previous reports, and that this defect is a result of susceptibility to RANKL-mediated apoptosis as a result of gain-of-function of JNK activation. We demonstrate that differentiation of OCPs depends on IKKbeta because reduced IKKbeta mRNA expression correlates with impaired induction of osteoclast differentiation markers in response to RANKL stimulation. We further show that fine-tuned inhibition of JNK activation in these cells inhibits RANKL-induced apoptosis and restores the ability of IKKbeta-null OCPs to become mature osteoclasts. Our data highlight the pro-osteoclastogenic and anti-apoptotic roles of IKKbeta in OCPs and identify a pro-apoptotic mechanism activated within the RANK signalosome.

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Figures

FIGURE 1.
FIGURE 1.
Mice with IKKβ-deleted osteoclast precursors possess a defect in in vivo and in vitro osteoclastogenesis. A, histochemical TRAP stain at growth plate of femur of CD11b Cre-positive WT/WT IKKβ and CD11b Cre-positive floxed/floxed IKKβ (OCPΔIKKβ) mice to visualize osteoclasts. Arrows indicate osteoclasts. B, Western blot for indicated proteins in total cell lysates of osteoclast precursors from CD11b Cre-negative, CD11b Cre-positive WT/floxed IKKβ, and CD11b Cre-positive floxed/floxed (f/f) IKKβ mice. C and D, osteoclast precursors from CD11b Cre-positive wt/wtIKKβ, CD11b Cre-positive WT/floxed IKKβ, and CD11b Cre-positive floxed/floxed IKKβ mice were cultured in osteoclastogenic conditions. C, cells were either not stimulated (panels a, d, and g) or were further stimulated with 10 ng/ml TNF-α (panels b, e, and h) or 100 ng/ml LPS (panels c, f, and i) on day 4 of culture. Cells were fixed and histochemically stained for TRAP to visualize osteoclasts on day 6 of culture. D, quantification of C. TRAP-positive multinucleated cells (MNCs) with three or more nuclei were scored as osteoclasts. Asterisk indicates p < 0.005 for difference between number of TRAP-positive MNCs in wells represented by panels d and g.
FIGURE 2.
FIGURE 2.
OCPΔIKKβ are defective in osteoclast differentiation and demonstrate increased susceptibility to apoptosis. OCPΔIKKβ and control OCPs were plated in whole media supplemented with 10 ng/ml m-CSF. Cells were either not stimulated (Unstim) or were stimulated with 10 ng/ml RANKL for 5 days to induce osteoclast differentiation. mRNA was collected and analyzed by real time PCR for markers of osteoclast differentiation: A, β3 integrin; B, cathepsin K (Cath K); C, calcitonin receptor (CtR); D, matrix metalloproteinase 9 (MMP9); E, tartrate-resistant acid phosphatase (TRAP); F, IKKβ (IKK2). GAPDH served as the internal standard for cDNA normalization. Data are presented as relative quantification with WT nonstimulated levels serving as the reference point (relative expression value of 1). Values represent mean quantification plus the S.E. G, OCPΔIKKβ and control OCPs were plated in whole media supplemented with 10 ng/ml m-CSF. mRNA was collected and analyzed by real time PCR for the indicated NF-κB-regulated anti-apoptotic proteins as follows: A20, cIAP2 (cellular inhibitor of apoptosis 2); cIAP1 (cellular inhibitor of apoptosis 1); XIAP (X-linked inhibitor of apoptosis); and Bcl-xL. β-Actin served as the internal standard for cDNA normalization. Data are presented as relative quantification with control levels serving as the reference point (relative expression value of 1). Values represent mean quantification plus the S.E. Data are representative of three independent experiments. H, OCPΔIKKβ and control OCPs were serum-starved and were either not stimulated or were stimulated with 10 ng/ml RANKL for 15, 30, 45, 60, or 240 min. Total cell lysates were analyzed by Western blot for integrity of full-length PARP (f-PARP). β-Actin served as the loading control.
FIGURE 3.
FIGURE 3.
Apoptosis contributes to the in vivo deficiency of osteoclasts in OCPΔIKKβ mice. A, immunoperoxidase TUNEL stain and hematoxylin counterstain of histological sections of growth plate of humerus from OCPΔIKKβ and control mice to visualize apoptosis of peritrabecular osteoclasts and OCPs. Upper images were taken at ×10 magnification, and lower images are panels from upper images taken at ×40 magnification. B, graph depicting quantification of TUNEL-positive peritrabecular nuclei per ×40 field visualized by light microscopy. Arrows indicate apoptotic nuclei. Asterisk denotes p < 0.001. C, TRAP stain of sections taken from same paraffin-embedded bones used for TUNEL stain in A to demonstrate correlation between apoptosis and defective in vivo osteoclastogenesis.
FIGURE 4.
FIGURE 4.
Loss of IKKβ in OCPs results in a gain-of-function in JNK phosphorylation. OCPΔIKKβ and control OCPs were serum-starved for 4–6 h. A, cells were either not stimulated or were stimulated with 10 ng/ml RANKL or TNF-α for 5, 10, or 30 min. B, cells were either not stimulated or were stimulated with 10 ng/ml RANKL for 7.5, 15, 30, or 60 min. Total cell lysates were then analyzed by Western blot for the indicated phosphorylated proteins and whole proteins. Equal loading for phosphorylated proteins was determined by stripping the membrane and re-probing for the respective whole protein (A and B). C, cells were stimulated with 10 ng/ml RANKL for 10, 15, 30, or 120 min. Total cell lysates were then analyzed by Western blot for MKP5 and MKP1. β-Actin served as the loading control.
FIGURE 5.
FIGURE 5.
Inhibition of RANKL-mediated JNK-induced apoptosis rescues osteoclastogenesis defect in OCPs deficient in IKKβ. A, OCPs from Cre+ wild-type/floxed IKKβ mice were plated in whole media supplemented with 10 ng/ml m-CSF. Four groups of cells were treated at the time of plating with either no TAT-TI-JIP or with 0.4, 1.0, or 2.0 μm TAT-TI-JIP. Also at the time of plating, one sample in each group was stimulated with 20 ng/ml RANKL. Cells were lysed after 24 h of stimulation, and total cell lysates were analyzed by Western blot for cleaved PARP (c-PARP), phosphorylated c-Jun (p-c jun), and total c-Jun. Cleaved PARP and phospho-c-Jun quantification in the different conditions is shown in numerical and graph form under the corresponding blot image. B, OCPΔIKKβ and control OCPs were plated in osteoclastogenic conditions. At the time of plating, one group of cells from each population was either left untreated (panels a and c) or treated with 0.4 μm TAT-TI-JIP (panels b and d). Cells were fixed and histochemically stained for TRAP to visualize osteoclasts on day 6 of culture. Quantification is shown in graph below. TRAP-positive multinucleated cells (MNCs) with three or more nuclei were scored as osteoclasts. Data are representative of three independent experiments, and error bars represent S.E. Asterisk indicates p < 0.0001 for difference between number of TRAP-positive MNCs in wells represented by panels a and c. No significant difference exists between panels a and d.
FIGURE 6.
FIGURE 6.
Inhibition of JNK in OCPΔIKKβ cells rescues bone resorption. Control (a and c) and OCPΔIKKβ (b and d) osteoclast precursors were plated onto BD Biocoat Osteologic tissue culture slides in osteoclastogenic conditions in the absence (a and b) or presence (c and d) of 0.4 μm TAT-TI-JIP. Cells were removed from the slides with deionized water, and resorption pits were noted as clear areas. Images were taken at ×10 magnification.
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