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The IkappaB function of NF-kappaB2 p100 controls stimulated osteoclastogenesis.

Novack DV, Yin L, Hagen-Stapleton A, Schreiber RD, Goeddel DV, Ross FP, Teitelbaum SL - J. Exp. Med. (2003)

Bottom Line: We demonstrate that treatment of wild-type osteoclast precursors with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) increases both expression of p100 and its conversion to p52, resulting in unchanged net levels of p100.Conversely, p100-deficient osteoclast precursors show enhanced sensitivity to RANKL.These data demonstrate a novel, biologically relevant means of regulating NF-kappaB signaling, with upstream control and kinetics distinct from the classical IkappaBalpha pathway.

View Article: PubMed Central - PubMed

Affiliation: Washington University School of Medicine, 660 S. Euclid Ave., Box 8301, St. Louis, MO 63110, USA. novack@wustl.edu

ABSTRACT
The prototranscription factor p100 represents an intersection of the NF-kappaB and IkappaB families, potentially serving as both the precursor for the active NF-kappaB subunit p52 and as an IkappaB capable of retaining NF-kappaB in the cytoplasm. NF-kappaB-inducing kinase (NIK) controls processing of p100 to generate p52, and thus NIK-deficient mice can be used to examine the biological effects of a failure in such processing. We demonstrate that treatment of wild-type osteoclast precursors with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) increases both expression of p100 and its conversion to p52, resulting in unchanged net levels of p100. In the absence of NIK, p100 expression is increased by RANKL, but its conversion to p52 is blocked, leading to cytosolic accumulation of p100, which, acting as an IkappaB protein, binds NF-kappaB complexes and prevents their nuclear translocation. High levels of unprocessed p100 in osteoclast precursors from NIK-/- mice or a nonprocessable form of the protein in wild-type cells impair RANKL-mediated osteoclastogenesis. Conversely, p100-deficient osteoclast precursors show enhanced sensitivity to RANKL. These data demonstrate a novel, biologically relevant means of regulating NF-kappaB signaling, with upstream control and kinetics distinct from the classical IkappaBalpha pathway.

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NIK−/− mice have normal basal osteoclastogenesis and a blunted response to RANKL in vivo. (A) TRAP-stained sections of femurs from unmanipulated 3–4-mo-old WT and NIK−/− mice show no difference in trabecular architecture or OC number red-stained cells. Bar, 600 μm. (B) WT and NIK−/− mice were injected with 500 μg GST-RANKL or PBS daily for 7 d and killed on the 8th day. TRAP-stained coronal sections of calvaria adjacent to the sagittal suture show a dramatic increase in OCs along sutures and sinusoids only in WT RANKL-treated animals. Bar, 150 μm. (C) Quantitation of osteoclasts along inner (sinusoidal) calvarial surfaces for animals described in B confirms the significant difference in the response of WT and NIK−/− mice to RANKL injection. Error bars indicate SEM. *P < 0.0001 compared with WT sham. **P < 0.001 compared with NIK−/− sham and WT RANKL.
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fig2: NIK−/− mice have normal basal osteoclastogenesis and a blunted response to RANKL in vivo. (A) TRAP-stained sections of femurs from unmanipulated 3–4-mo-old WT and NIK−/− mice show no difference in trabecular architecture or OC number red-stained cells. Bar, 600 μm. (B) WT and NIK−/− mice were injected with 500 μg GST-RANKL or PBS daily for 7 d and killed on the 8th day. TRAP-stained coronal sections of calvaria adjacent to the sagittal suture show a dramatic increase in OCs along sutures and sinusoids only in WT RANKL-treated animals. Bar, 150 μm. (C) Quantitation of osteoclasts along inner (sinusoidal) calvarial surfaces for animals described in B confirms the significant difference in the response of WT and NIK−/− mice to RANKL injection. Error bars indicate SEM. *P < 0.0001 compared with WT sham. **P < 0.001 compared with NIK−/− sham and WT RANKL.

Mentions: Interestingly, in vivo basal osteoclastogenesis is unaltered in NIK−/− mice. Femurs of unmanipulated 3–4-mo-old WT and NIK−/− mice have similar cortical thickness, trabecular size and distribution, and number of osteoclasts (Fig. 2 A). On the other hand, and consistent with our in vitro observations, these mutants exhibit a blunted response to osteoclastogenic stimuli. Whereas WT mice generate a robust sevenfold increase in OC number along calvarial sinuses after 1 wk of RANKL treatment, NIK−/− mice show only a twofold rise (Fig. 2, B and C). Similarly, NIK−/− mice fail to show a significant osteoclastogenic response to a 3-d course of parathyroid hormone injection (unpublished data).


The IkappaB function of NF-kappaB2 p100 controls stimulated osteoclastogenesis.

Novack DV, Yin L, Hagen-Stapleton A, Schreiber RD, Goeddel DV, Ross FP, Teitelbaum SL - J. Exp. Med. (2003)

NIK−/− mice have normal basal osteoclastogenesis and a blunted response to RANKL in vivo. (A) TRAP-stained sections of femurs from unmanipulated 3–4-mo-old WT and NIK−/− mice show no difference in trabecular architecture or OC number red-stained cells. Bar, 600 μm. (B) WT and NIK−/− mice were injected with 500 μg GST-RANKL or PBS daily for 7 d and killed on the 8th day. TRAP-stained coronal sections of calvaria adjacent to the sagittal suture show a dramatic increase in OCs along sutures and sinusoids only in WT RANKL-treated animals. Bar, 150 μm. (C) Quantitation of osteoclasts along inner (sinusoidal) calvarial surfaces for animals described in B confirms the significant difference in the response of WT and NIK−/− mice to RANKL injection. Error bars indicate SEM. *P < 0.0001 compared with WT sham. **P < 0.001 compared with NIK−/− sham and WT RANKL.
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Related In: Results  -  Collection

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fig2: NIK−/− mice have normal basal osteoclastogenesis and a blunted response to RANKL in vivo. (A) TRAP-stained sections of femurs from unmanipulated 3–4-mo-old WT and NIK−/− mice show no difference in trabecular architecture or OC number red-stained cells. Bar, 600 μm. (B) WT and NIK−/− mice were injected with 500 μg GST-RANKL or PBS daily for 7 d and killed on the 8th day. TRAP-stained coronal sections of calvaria adjacent to the sagittal suture show a dramatic increase in OCs along sutures and sinusoids only in WT RANKL-treated animals. Bar, 150 μm. (C) Quantitation of osteoclasts along inner (sinusoidal) calvarial surfaces for animals described in B confirms the significant difference in the response of WT and NIK−/− mice to RANKL injection. Error bars indicate SEM. *P < 0.0001 compared with WT sham. **P < 0.001 compared with NIK−/− sham and WT RANKL.
Mentions: Interestingly, in vivo basal osteoclastogenesis is unaltered in NIK−/− mice. Femurs of unmanipulated 3–4-mo-old WT and NIK−/− mice have similar cortical thickness, trabecular size and distribution, and number of osteoclasts (Fig. 2 A). On the other hand, and consistent with our in vitro observations, these mutants exhibit a blunted response to osteoclastogenic stimuli. Whereas WT mice generate a robust sevenfold increase in OC number along calvarial sinuses after 1 wk of RANKL treatment, NIK−/− mice show only a twofold rise (Fig. 2, B and C). Similarly, NIK−/− mice fail to show a significant osteoclastogenic response to a 3-d course of parathyroid hormone injection (unpublished data).

Bottom Line: We demonstrate that treatment of wild-type osteoclast precursors with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) increases both expression of p100 and its conversion to p52, resulting in unchanged net levels of p100.Conversely, p100-deficient osteoclast precursors show enhanced sensitivity to RANKL.These data demonstrate a novel, biologically relevant means of regulating NF-kappaB signaling, with upstream control and kinetics distinct from the classical IkappaBalpha pathway.

View Article: PubMed Central - PubMed

Affiliation: Washington University School of Medicine, 660 S. Euclid Ave., Box 8301, St. Louis, MO 63110, USA. novack@wustl.edu

ABSTRACT
The prototranscription factor p100 represents an intersection of the NF-kappaB and IkappaB families, potentially serving as both the precursor for the active NF-kappaB subunit p52 and as an IkappaB capable of retaining NF-kappaB in the cytoplasm. NF-kappaB-inducing kinase (NIK) controls processing of p100 to generate p52, and thus NIK-deficient mice can be used to examine the biological effects of a failure in such processing. We demonstrate that treatment of wild-type osteoclast precursors with the osteoclastogenic cytokine receptor activator of NF-kappaB ligand (RANKL) increases both expression of p100 and its conversion to p52, resulting in unchanged net levels of p100. In the absence of NIK, p100 expression is increased by RANKL, but its conversion to p52 is blocked, leading to cytosolic accumulation of p100, which, acting as an IkappaB protein, binds NF-kappaB complexes and prevents their nuclear translocation. High levels of unprocessed p100 in osteoclast precursors from NIK-/- mice or a nonprocessable form of the protein in wild-type cells impair RANKL-mediated osteoclastogenesis. Conversely, p100-deficient osteoclast precursors show enhanced sensitivity to RANKL. These data demonstrate a novel, biologically relevant means of regulating NF-kappaB signaling, with upstream control and kinetics distinct from the classical IkappaBalpha pathway.

Show MeSH
Related in: MedlinePlus