Limits...
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.

Show MeSH

Related in: MedlinePlus

Bone mineral density is reduced in IKK2SSEEMYELO -tg mice.Long bones of 4-week old wild type (9 mice) and transgenic (8) mice were processed and analyzed by micro CT scanning as described under methods. Absolute values of bone mineral density (BMD), trabecular number (Tb.N), trabeculat thickness (Tb.Th), number of osteoclasts per bone surface (N.Oc/BS) and bone volume/total volume (BV/TV) between WT and TG mice are depicted. *P<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369901&req=5

pone-0038694-g003: Bone mineral density is reduced in IKK2SSEEMYELO -tg mice.Long bones of 4-week old wild type (9 mice) and transgenic (8) mice were processed and analyzed by micro CT scanning as described under methods. Absolute values of bone mineral density (BMD), trabecular number (Tb.N), trabeculat thickness (Tb.Th), number of osteoclasts per bone surface (N.Oc/BS) and bone volume/total volume (BV/TV) between WT and TG mice are depicted. *P<0.05.

Mentions: We have shown previously that the active form of IKK2, IKK2SSEE, induces RANKL-independent osteoclastogenesis, in vitro [18]. To determine the physiological significance of this finding, we examined its role in vivo through expression of constitutively active IKK2 in a tissue-specific manner. Specifically, we employed the R26StopIKK2SSEE mice in which a cDNA encoding IKK2 containing two serine to glutamate substitutions in the activation loop of the kinase domain, preceded by a loxP-flanked STOP cassette, was cloned into the ubiquitously expressed ROSA26 locus [19]. We crossed mice carrying this allele to the CD11b-cre mice in order to express IKK2SSEE exclusively in myeloid cells, inclusive of osteoclast progenitors. Mice were born and survived an average of 6 weeks of age. The IKK2SSEE transgenic mice (herein referred to as IKK2SSEEMYELO) exhibited growth retardation and severe skin inflammation (fig. 1A). Both macrophages and osteoclasts derived from IKK2SSEEMYELO express Flag-tagged IKK2SSEE detected by western blot (fig. 1B). Histological (fig. 2), micro-CT and histomorphometric (fig. 3) analyses showed that IKK2SSEEMYELO transgenic mice have significantly less trabecular bone compared with the wild type (WT) mice, while no significant reduction of cortical bone was observed (not shown). Specifically, bone mineral density (BMD) in transgenic mice was 58.9% of that in WT mice. BV/TV values were 0.177 mm3/mm3 in WT compared to 0.068 mm3/mm3 in transgenic mice. Closer examination revealed that trabecular bone sections of IKK2SSEE transgenic mice contain more and larger osteoclasts attached to trabeculi (fig. 2, insert). Indeed, histomorphometric quantification using Osteomeasure (Osteometrics, Inc) revealed that the trabecular region of transgenic mice contains nearly twice as much osteoclasts compared with WT mice (fig. 3). Supporting a functional significance of this observation, IKK2SSEEMYELO transgenic mice have two-fold increase of serum TRACP 5b activity (fig. 4) (10.59 U/L vs 4.93 U/L), reflecting an overall heightened bone resorptive activity of osteoclasts in these mice.


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

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

Bone mineral density is reduced in IKK2SSEEMYELO -tg mice.Long bones of 4-week old wild type (9 mice) and transgenic (8) mice were processed and analyzed by micro CT scanning as described under methods. Absolute values of bone mineral density (BMD), trabecular number (Tb.N), trabeculat thickness (Tb.Th), number of osteoclasts per bone surface (N.Oc/BS) and bone volume/total volume (BV/TV) between WT and TG mice are depicted. *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038694-g003: Bone mineral density is reduced in IKK2SSEEMYELO -tg mice.Long bones of 4-week old wild type (9 mice) and transgenic (8) mice were processed and analyzed by micro CT scanning as described under methods. Absolute values of bone mineral density (BMD), trabecular number (Tb.N), trabeculat thickness (Tb.Th), number of osteoclasts per bone surface (N.Oc/BS) and bone volume/total volume (BV/TV) between WT and TG mice are depicted. *P<0.05.
Mentions: We have shown previously that the active form of IKK2, IKK2SSEE, induces RANKL-independent osteoclastogenesis, in vitro [18]. To determine the physiological significance of this finding, we examined its role in vivo through expression of constitutively active IKK2 in a tissue-specific manner. Specifically, we employed the R26StopIKK2SSEE mice in which a cDNA encoding IKK2 containing two serine to glutamate substitutions in the activation loop of the kinase domain, preceded by a loxP-flanked STOP cassette, was cloned into the ubiquitously expressed ROSA26 locus [19]. We crossed mice carrying this allele to the CD11b-cre mice in order to express IKK2SSEE exclusively in myeloid cells, inclusive of osteoclast progenitors. Mice were born and survived an average of 6 weeks of age. The IKK2SSEE transgenic mice (herein referred to as IKK2SSEEMYELO) exhibited growth retardation and severe skin inflammation (fig. 1A). Both macrophages and osteoclasts derived from IKK2SSEEMYELO express Flag-tagged IKK2SSEE detected by western blot (fig. 1B). Histological (fig. 2), micro-CT and histomorphometric (fig. 3) analyses showed that IKK2SSEEMYELO transgenic mice have significantly less trabecular bone compared with the wild type (WT) mice, while no significant reduction of cortical bone was observed (not shown). Specifically, bone mineral density (BMD) in transgenic mice was 58.9% of that in WT mice. BV/TV values were 0.177 mm3/mm3 in WT compared to 0.068 mm3/mm3 in transgenic mice. Closer examination revealed that trabecular bone sections of IKK2SSEE transgenic mice contain more and larger osteoclasts attached to trabeculi (fig. 2, insert). Indeed, histomorphometric quantification using Osteomeasure (Osteometrics, Inc) revealed that the trabecular region of transgenic mice contains nearly twice as much osteoclasts compared with WT mice (fig. 3). Supporting a functional significance of this observation, IKK2SSEEMYELO transgenic mice have two-fold increase of serum TRACP 5b activity (fig. 4) (10.59 U/L vs 4.93 U/L), reflecting an overall heightened bone resorptive activity of osteoclasts in these mice.

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