HBP/O-GlcNAcylation Metabolic Axis Regulates Bone Resorption Outcome.

Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, αvintegrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt-deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Ogt-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt-deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome.

NLRP12 Attenuates Inflammatory Bone Loss in Experimental Apical Periodontitis.

Apical periodontitis is an inflammatory disorder that results from the host immune response to microbial infection through the dental pulp, leading to alveolar bone destruction. The nod-like receptor 12 (NLRP12) is an atypical intracellular sensor of the NLR family that is involved in the negative regulation of several inflammatory conditions and also osteoclastogenesis. However, the role of NLRP12 in the regulation of immune response and bone loss induced by bacterial infection remains unclear. Here we investigated the development of apical periodontitis in wild-type (WT) and NLRP12 knockout (NLRP12-/-) mice by using micro-computed tomography together with histological, immunohistochemical, and molecular analyses. We found that NLRP12-/- mice are highly susceptible to apical periodontitis induced by bacterial infection, which is associated with an elevated infiltration of neutrophils and macrophages, periapical lesion extension, and alveolar bone destruction. Furthermore, NLRP12-/- mice showed a high expression of inflammatory cytokines ( Il1b, Il6, and Tnfa) and the osteoclastogenic markers ( Rankl and Acp5) in the periapical tissues. Consistent with this observation, NLRP12-/- mice showed an increased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site, which was associated with augmented expression of the osteoclast effector genes, Ctsk and Mmp9. Mechanistically, NLRP12-deficient preosteoclasts showed elevated IκB-α degradation and p65 phosphorylation when stimulated with receptor activator of nuclear factor (NF)-κB ligand (RANKL). Similarly, increased IκB-α degradation was observed in the periapical tissue of NLRP12-/- mice. Furthermore, our in vitro study showed that preosteoclasts from NLRP12-/- mice exhibited higher RANKL-induced osteoclastogenesis, which was synergistically amplified by interleukin-1ß and tumor necrosis factor α (mimicking an inflammatory periapical milieu). In conclusion, our data show that NLRP12 exhibits a protective role in the periapical bone destruction by attenuating inflammation and osteoclastogenesis through negative regulation of the NF-κB pathway.

NOD2 contributes to Porphyromonas gingivalis-induced bone resorption.

The NOD-like receptors are cytoplasmic proteins that sense microbial by-products released by invasive bacteria. Although NOD1 and NOD2 are functionally expressed in cells from oral tissues and play a role triggering immune responses, the role of NOD2 receptor in the bone resorption and in the modulation of osteoclastogenesis is still unclear. We show that in an experimental model of periodontitis with Porphyromonas gingivalis W83, NOD2(-/-) mice showed lower bone resorption when compared to wild type. Quantitative polymerase chain reaction analysis revealed that wild-type infected mice showed an elevated RANKL/OPG ratio when compared to NOD2(-/-) infected mice. Moreover, the expression of 2 osteoclast activity markers-cathepsin K and matrix metalloproteinase 9-was significantly lower in gingival tissue from NOD2(-/-) infected mice compared to WT infected ones. The in vitro study reported an increase in the expression of the NOD2 receptor 24 hr after stimulation of hematopoietic bone marrow cells with M-CSF and RANKL. We also evaluated the effect of direct activation of NOD2 receptor on osteoclastogenesis, by the activation of this receptor in preosteoclasts culture, with different concentrations of muramyl dipeptide. The results show no difference in the number of TRAP-positive cells. Although it did not alter the osteoclasts differentiation, the activation of NOD2 receptor led to a significant increase of cathepsin K expression. We confirm that this enzyme was active, since the osteoclasts resorption capacity was enhanced by muramyl dipeptide stimulation, evaluated in osteoassay plate. These results show that the lack of NOD2 receptor impairs the bone resorption, suggesting that NOD2 receptor could contribute to the progression of bone resorption in experimental model of periodontitis. The stimulation of NOD2 by its agonist, muramyl dipeptide, did not affect osteoclastogenesis, but it does favor the bone resorption capacity identified by increased osteoclast activity.