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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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Related in: MedlinePlus

Markers of osteoclast are increased in cells derived from IKK2SSEEMYELO -tg mice.Bone marrow macrophages were isolated from WT and IKK2SSEE-tg mice and cultured in the presence of M-CSF for 4 days. RNA was then extracted and subjected to real-time Q-PCR to detect expression of the indicated markers. p<0.01 for NFATc1, p<0.01for TRAP, p<0.05 for Cathepsin K, p<0.01 for MMP9, and p<0.01 for calcitonin receptor (CTR).
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pone-0038694-g006: Markers of osteoclast are increased in cells derived from IKK2SSEEMYELO -tg mice.Bone marrow macrophages were isolated from WT and IKK2SSEE-tg mice and cultured in the presence of M-CSF for 4 days. RNA was then extracted and subjected to real-time Q-PCR to detect expression of the indicated markers. p<0.01 for NFATc1, p<0.01for TRAP, p<0.05 for Cathepsin K, p<0.01 for MMP9, and p<0.01 for calcitonin receptor (CTR).

Mentions: To determine the osteoclastogenic potential of IKK2SSEE transgenic bone marrow macrophages, these cells were cultured in vitro with M-CSF alone. Consistent with its intrinsic osteoclast effect in vitro, IKK2SSEE-expressing bone marrow macrophages isolated from these transgenic mice were capable of forming osteoclasts ex-vivo in the absence of RANKL (fig. 5). Furthermore, real-time PCR showed that IKK2SSEE upregulated NFATc1 expression in macrophage/osteoclast lineage (fig. 6). IKK2SSEE also significantly enhanced expression of major osteoclast markers including TRAP, Cathepsin K, MMP9 and calcitonin receptor expression compared to the wild type control (fig. 6).


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

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

Markers of osteoclast are increased in cells derived from IKK2SSEEMYELO -tg mice.Bone marrow macrophages were isolated from WT and IKK2SSEE-tg mice and cultured in the presence of M-CSF for 4 days. RNA was then extracted and subjected to real-time Q-PCR to detect expression of the indicated markers. p<0.01 for NFATc1, p<0.01for TRAP, p<0.05 for Cathepsin K, p<0.01 for MMP9, and p<0.01 for calcitonin receptor (CTR).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369901&req=5

pone-0038694-g006: Markers of osteoclast are increased in cells derived from IKK2SSEEMYELO -tg mice.Bone marrow macrophages were isolated from WT and IKK2SSEE-tg mice and cultured in the presence of M-CSF for 4 days. RNA was then extracted and subjected to real-time Q-PCR to detect expression of the indicated markers. p<0.01 for NFATc1, p<0.01for TRAP, p<0.05 for Cathepsin K, p<0.01 for MMP9, and p<0.01 for calcitonin receptor (CTR).
Mentions: To determine the osteoclastogenic potential of IKK2SSEE transgenic bone marrow macrophages, these cells were cultured in vitro with M-CSF alone. Consistent with its intrinsic osteoclast effect in vitro, IKK2SSEE-expressing bone marrow macrophages isolated from these transgenic mice were capable of forming osteoclasts ex-vivo in the absence of RANKL (fig. 5). Furthermore, real-time PCR showed that IKK2SSEE upregulated NFATc1 expression in macrophage/osteoclast lineage (fig. 6). IKK2SSEE also significantly enhanced expression of major osteoclast markers including TRAP, Cathepsin K, MMP9 and calcitonin receptor expression compared to the wild type control (fig. 6).

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