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Constitutively active canonical NF-κB pathway induces severe bone loss in mice.

Otero JE, Chen T, Zhang K, Abu-Amer Y - PLoS ONE (2012)

Bottom Line: Mechanistically, we observed that IKK2SSEE induced high expression of not only p65 but also p52 and RelB; the latter two molecules are considered exclusive members of the alternative NF-κB pathway.Furthermore, we found that NF-κB2/p100 is a potent inhibitor of IKK2SSEE-induced osteoclastogenesis.Deletion of p52 enabled more robust osteoclast formation by the active kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States of America.

ABSTRACT
Physiologic osteoclastogenesis entails activation of multiple signal transduction pathways distal to the cell membrane receptor RANK. However, atypical osteoclastogenesis driven by pro-inflammatory stimuli has been described. We have reported recently a novel mechanism whereby endogenous mutational activation of the classical NF-κB pathway is sufficient to induce RANKL/RANK-independent osteoclastogenesis. Here we investigate the physiologic relevance of this phenomenon in vivo. Using a knock-in approach, the active form of IKK2, namely IKK2SSEE, was introduced into the myeloid lineage with the aid of CD11b-cre mice. Phenotypic assessment revealed that expression of IKK2SSEE in the myeloid compartment induced significant bone loss in vivo. This observation was supported by a dramatic increase in the number and size of osteoclasts in trabecular regions, elevated levels of circulating TRACP-5b, and reduced bone volume. Mechanistically, we observed that IKK2SSEE induced high expression of not only p65 but also p52 and RelB; the latter two molecules are considered exclusive members of the alternative NF-κB pathway. Intriguingly, RelB and P52 were both required to mediate the osteoclastogenic effect of IKK2SSEE and co-expression of these two proteins was sufficient to recapitulate osteoclastogenesis in the absence of RANKL or IKK2SSEE. Furthermore, we found that NF-κB2/p100 is a potent inhibitor of IKK2SSEE-induced osteoclastogenesis. Deletion of p52 enabled more robust osteoclast formation by the active kinase. In summary, molecular activation of IKK2 may play a role in conditions of pathologic bone destruction, which may be refractory to therapeutic interventions targeting the proximal RANKL/RANK signal.

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NF-κB subunits RelB and p52 mediate the osteoclastogenic effect of IKK2SSEE.A) Marrow macrophages were isolated from WT and IKK2SSEE transgenic (TG) mice and plated in culture for 5 days in the presence of M-CSF. One set of WT cells was treated with RANKL (to serve as a positive control). Total cell lysates were subjected to Western blots as indicated. B) Marrow macrophages were isolated from mice with the indicated gene deletions (denoted by genotype at the lower part of the figure). Cells were infected with retrovirus expressing the indicated gene products (denoted by retrovirus) and were cultured with M-CSF in the absence of RANKL. Cell cultures were fixed and TRAP-stained on day 5. Multi-nucleated (≥3 nuclei/cells) TRAP-positive cells were counted from three different wells for each condition. The data represent the average of three independent experiments. *p<0.01, **p<0.05. C) Representative images of osteoclast cultures depicted in fig. 8B. D) IKK2SSEE transgenic mice alone or crossed with RelB-KO mice (hybrid denoted TG/RB-KO) were used. Osteoclasts were generated (in the absence of RANKL) from marrow macrophages isolated from IKK2SSEE-transgenic mice and from hybrid TG/RB-KO mice. In addition, osteoclasts were generated from marrow macrophages isolated from the latter mouse in which either P52-NF-κB or RelA were silenced using siRNA approach. Bottom panels of 8C are western blots for p52 and p65 (RelA) from control (scr = scramble) and silenced (si) conditions. Knockdown efficiency exceeded 80% of control.
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pone-0038694-g008: NF-κB subunits RelB and p52 mediate the osteoclastogenic effect of IKK2SSEE.A) Marrow macrophages were isolated from WT and IKK2SSEE transgenic (TG) mice and plated in culture for 5 days in the presence of M-CSF. One set of WT cells was treated with RANKL (to serve as a positive control). Total cell lysates were subjected to Western blots as indicated. B) Marrow macrophages were isolated from mice with the indicated gene deletions (denoted by genotype at the lower part of the figure). Cells were infected with retrovirus expressing the indicated gene products (denoted by retrovirus) and were cultured with M-CSF in the absence of RANKL. Cell cultures were fixed and TRAP-stained on day 5. Multi-nucleated (≥3 nuclei/cells) TRAP-positive cells were counted from three different wells for each condition. The data represent the average of three independent experiments. *p<0.01, **p<0.05. C) Representative images of osteoclast cultures depicted in fig. 8B. D) IKK2SSEE transgenic mice alone or crossed with RelB-KO mice (hybrid denoted TG/RB-KO) were used. Osteoclasts were generated (in the absence of RANKL) from marrow macrophages isolated from IKK2SSEE-transgenic mice and from hybrid TG/RB-KO mice. In addition, osteoclasts were generated from marrow macrophages isolated from the latter mouse in which either P52-NF-κB or RelA were silenced using siRNA approach. Bottom panels of 8C are western blots for p52 and p65 (RelA) from control (scr = scramble) and silenced (si) conditions. Knockdown efficiency exceeded 80% of control.

Mentions: We have shown previously that IKK2SSEE induces expression of RelA/p65 (canonical pathway) as well as RelB and p52 (non-canonical pathway) in IKK1- cells [18]. Consistent with these findings, we now show that expression of these NF-κB subunits is also elevated in cells derived from IKK2SSEE transgenic mice (Fig. 8A), suggesting that one or a combination of these subunits maybe responsible for the osteoclastogenic effect of IKK2SSEE. Thus, we surmised that expression of these NF-κB subunits at various combinations may recapitulate the IKK2SSEE osteoclastogenic effect. To this end, expression of RelA, RelB, or p52 individually did not alter the RANKL-independent osteoclastogenic potential of the cells, as was the case when RelA and RelB were co-expressed (not shown). Surprisingly, however, co-expression of p52 and RelB, both of which are components of the non-canonical NF-κB activation pathway (normally induced by IKK1), elicited RANKL-independent osteoclastogenesis, in vitro (fig. 8B, panel A, and corresponding images in fig. 8C). To determine if indeed p52 and RelB subunits are essential mediators of this osteoclastogenic process, we used P52-KO and p52/RelB double knockout cells. The results depicted in figure 8B (panels B–C) show that whereas IKK2SSEE induced robust osteoclastogenesis in p52 cells expressing RelB (fig. 8B, panel B), suggesting that NF-κB2/p100 is an endogenous inhibitor of IKK2SSEE, combined deletion of p52NF-κB and RelB abrogated the IKK2SSEE osteoclastogenic effect (gray bar). To further verify the specificity of this event, the osteoclastogenic effect of IKK2SSEE was examined using bone marrow macrophages isolated from IKK1- cells. Supporting a specific IKK2SSEE effect, combined expression of p52 and RelB NF-κB subunits in IKK1- cells mimicked IKK2SSEE-induced osteoclastogenesis (fig. 8B, panel D). Taken together, the data provides strong correlation between IKK2SSEE and P52/RelB-induced osteoclastogenesis. To further determine if this evidence holds true in the transgenic mouse model, IKK2SSEE-TG mouse was crossed with the RelB germ line knockout mouse. Marrow macrophages derived from the hybrid mouse, termed TG/RB-KO, were subjected to siRNA knockdown of either P52 or P65/RelA NF-κB subunits (siP52, siP65). Control conditions were treated with scrambled sequence (si-scr) or vehicle. Cells were then cultured in the presence of M-CSF, and osteoclasts were determined in 6 day old culture using TRAP staining assay. The results summarized in figure 8D indicate that whereas deletion of RelB reduced osteoclasts by approximately 31%, combined elimination of P52 (by siRNA knockdown) and RelB (by genetic deletion) significantly abrogated the IKK2SSEE-TG osteoclastogenic effect (∼87% reduction compared with TG control). On the other hand, knockdown of P65 subunit in the RelB-KO background had little effect on IKK2SSEE-TG-induced osteoclastogenesis. Altogether, our findings support the notion that p52 and RelB are the primary mediators of the IKK2SSEE osteoclastogenic effect in vitro and in vivo.


Constitutively active canonical NF-κB pathway induces severe bone loss in mice.

Otero JE, Chen T, Zhang K, Abu-Amer Y - PLoS ONE (2012)

NF-κB subunits RelB and p52 mediate the osteoclastogenic effect of IKK2SSEE.A) Marrow macrophages were isolated from WT and IKK2SSEE transgenic (TG) mice and plated in culture for 5 days in the presence of M-CSF. One set of WT cells was treated with RANKL (to serve as a positive control). Total cell lysates were subjected to Western blots as indicated. B) Marrow macrophages were isolated from mice with the indicated gene deletions (denoted by genotype at the lower part of the figure). Cells were infected with retrovirus expressing the indicated gene products (denoted by retrovirus) and were cultured with M-CSF in the absence of RANKL. Cell cultures were fixed and TRAP-stained on day 5. Multi-nucleated (≥3 nuclei/cells) TRAP-positive cells were counted from three different wells for each condition. The data represent the average of three independent experiments. *p<0.01, **p<0.05. C) Representative images of osteoclast cultures depicted in fig. 8B. D) IKK2SSEE transgenic mice alone or crossed with RelB-KO mice (hybrid denoted TG/RB-KO) were used. Osteoclasts were generated (in the absence of RANKL) from marrow macrophages isolated from IKK2SSEE-transgenic mice and from hybrid TG/RB-KO mice. In addition, osteoclasts were generated from marrow macrophages isolated from the latter mouse in which either P52-NF-κB or RelA were silenced using siRNA approach. Bottom panels of 8C are western blots for p52 and p65 (RelA) from control (scr = scramble) and silenced (si) conditions. Knockdown efficiency exceeded 80% of control.
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Related In: Results  -  Collection

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pone-0038694-g008: NF-κB subunits RelB and p52 mediate the osteoclastogenic effect of IKK2SSEE.A) Marrow macrophages were isolated from WT and IKK2SSEE transgenic (TG) mice and plated in culture for 5 days in the presence of M-CSF. One set of WT cells was treated with RANKL (to serve as a positive control). Total cell lysates were subjected to Western blots as indicated. B) Marrow macrophages were isolated from mice with the indicated gene deletions (denoted by genotype at the lower part of the figure). Cells were infected with retrovirus expressing the indicated gene products (denoted by retrovirus) and were cultured with M-CSF in the absence of RANKL. Cell cultures were fixed and TRAP-stained on day 5. Multi-nucleated (≥3 nuclei/cells) TRAP-positive cells were counted from three different wells for each condition. The data represent the average of three independent experiments. *p<0.01, **p<0.05. C) Representative images of osteoclast cultures depicted in fig. 8B. D) IKK2SSEE transgenic mice alone or crossed with RelB-KO mice (hybrid denoted TG/RB-KO) were used. Osteoclasts were generated (in the absence of RANKL) from marrow macrophages isolated from IKK2SSEE-transgenic mice and from hybrid TG/RB-KO mice. In addition, osteoclasts were generated from marrow macrophages isolated from the latter mouse in which either P52-NF-κB or RelA were silenced using siRNA approach. Bottom panels of 8C are western blots for p52 and p65 (RelA) from control (scr = scramble) and silenced (si) conditions. Knockdown efficiency exceeded 80% of control.
Mentions: We have shown previously that IKK2SSEE induces expression of RelA/p65 (canonical pathway) as well as RelB and p52 (non-canonical pathway) in IKK1- cells [18]. Consistent with these findings, we now show that expression of these NF-κB subunits is also elevated in cells derived from IKK2SSEE transgenic mice (Fig. 8A), suggesting that one or a combination of these subunits maybe responsible for the osteoclastogenic effect of IKK2SSEE. Thus, we surmised that expression of these NF-κB subunits at various combinations may recapitulate the IKK2SSEE osteoclastogenic effect. To this end, expression of RelA, RelB, or p52 individually did not alter the RANKL-independent osteoclastogenic potential of the cells, as was the case when RelA and RelB were co-expressed (not shown). Surprisingly, however, co-expression of p52 and RelB, both of which are components of the non-canonical NF-κB activation pathway (normally induced by IKK1), elicited RANKL-independent osteoclastogenesis, in vitro (fig. 8B, panel A, and corresponding images in fig. 8C). To determine if indeed p52 and RelB subunits are essential mediators of this osteoclastogenic process, we used P52-KO and p52/RelB double knockout cells. The results depicted in figure 8B (panels B–C) show that whereas IKK2SSEE induced robust osteoclastogenesis in p52 cells expressing RelB (fig. 8B, panel B), suggesting that NF-κB2/p100 is an endogenous inhibitor of IKK2SSEE, combined deletion of p52NF-κB and RelB abrogated the IKK2SSEE osteoclastogenic effect (gray bar). To further verify the specificity of this event, the osteoclastogenic effect of IKK2SSEE was examined using bone marrow macrophages isolated from IKK1- cells. Supporting a specific IKK2SSEE effect, combined expression of p52 and RelB NF-κB subunits in IKK1- cells mimicked IKK2SSEE-induced osteoclastogenesis (fig. 8B, panel D). Taken together, the data provides strong correlation between IKK2SSEE and P52/RelB-induced osteoclastogenesis. To further determine if this evidence holds true in the transgenic mouse model, IKK2SSEE-TG mouse was crossed with the RelB germ line knockout mouse. Marrow macrophages derived from the hybrid mouse, termed TG/RB-KO, were subjected to siRNA knockdown of either P52 or P65/RelA NF-κB subunits (siP52, siP65). Control conditions were treated with scrambled sequence (si-scr) or vehicle. Cells were then cultured in the presence of M-CSF, and osteoclasts were determined in 6 day old culture using TRAP staining assay. The results summarized in figure 8D indicate that whereas deletion of RelB reduced osteoclasts by approximately 31%, combined elimination of P52 (by siRNA knockdown) and RelB (by genetic deletion) significantly abrogated the IKK2SSEE-TG osteoclastogenic effect (∼87% reduction compared with TG control). On the other hand, knockdown of P65 subunit in the RelB-KO background had little effect on IKK2SSEE-TG-induced osteoclastogenesis. Altogether, our findings support the notion that p52 and RelB are the primary mediators of the IKK2SSEE osteoclastogenic effect in vitro and in vivo.

Bottom Line: Mechanistically, we observed that IKK2SSEE induced high expression of not only p65 but also p52 and RelB; the latter two molecules are considered exclusive members of the alternative NF-κB pathway.Furthermore, we found that NF-κB2/p100 is a potent inhibitor of IKK2SSEE-induced osteoclastogenesis.Deletion of p52 enabled more robust osteoclast formation by the active kinase.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States of America.

ABSTRACT
Physiologic osteoclastogenesis entails activation of multiple signal transduction pathways distal to the cell membrane receptor RANK. However, atypical osteoclastogenesis driven by pro-inflammatory stimuli has been described. We have reported recently a novel mechanism whereby endogenous mutational activation of the classical NF-κB pathway is sufficient to induce RANKL/RANK-independent osteoclastogenesis. Here we investigate the physiologic relevance of this phenomenon in vivo. Using a knock-in approach, the active form of IKK2, namely IKK2SSEE, was introduced into the myeloid lineage with the aid of CD11b-cre mice. Phenotypic assessment revealed that expression of IKK2SSEE in the myeloid compartment induced significant bone loss in vivo. This observation was supported by a dramatic increase in the number and size of osteoclasts in trabecular regions, elevated levels of circulating TRACP-5b, and reduced bone volume. Mechanistically, we observed that IKK2SSEE induced high expression of not only p65 but also p52 and RelB; the latter two molecules are considered exclusive members of the alternative NF-κB pathway. Intriguingly, RelB and P52 were both required to mediate the osteoclastogenic effect of IKK2SSEE and co-expression of these two proteins was sufficient to recapitulate osteoclastogenesis in the absence of RANKL or IKK2SSEE. Furthermore, we found that NF-κB2/p100 is a potent inhibitor of IKK2SSEE-induced osteoclastogenesis. Deletion of p52 enabled more robust osteoclast formation by the active kinase. In summary, molecular activation of IKK2 may play a role in conditions of pathologic bone destruction, which may be refractory to therapeutic interventions targeting the proximal RANKL/RANK signal.

Show MeSH
Related in: MedlinePlus