RESUMEN
BACKGROUND: Osteoclasts are multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage lineage. During osteoclast differentiation, Rho GTPases are involved in various processes, including cell migration, adhesion, and polarity. However, the role of Rho-regulatory molecules in the regulation of osteoclast differentiation remains unclear. In this study, among these genes, we focused on active breakpoint cluster region-related (Abr) protein that is a multifunctional regulator of Rho GTPases. METHODS AND RESULTS: We examined using knockdown and overexpression experiments in RANKL-stimulated RAW-D macrophages whether Abr regulates osteoclast differentiation and cell morphology. We observed an increase in Abr expression during osteoclast differentiation and identified expression of a variant of the Abr gene in osteoclasts. Knockdown of Abr suppressed osteoclast differentiation and resorption. Abr knockdown markedly inhibited the expression of osteoclast markers, such as Nfatc1, c-fos, Src, and Ctsk in osteoclasts. Conversely, overexpression of Abr enhanced the formation of multinucleated osteoclasts, bone resorption activity, and osteoclast marker gene expression. Moreover, Abr overexpression accelerated lamellipodia formation and induced the formation of well-developed actin in osteoclasts. Importantly, the Abr protein interacted with poly(ADP-ribose) glycohydrolase (PARG) and Rho GTPases, including RhoA, Rac1/2/3, and Cdc42 in osteoclasts. CONCLUSIONS: Taken together, these results indicate that Abr modulates osteoclastogenesis by enhancing lamellipodia formation via its interaction with PARG.
Asunto(s)
Osteogénesis , Seudópodos , Diferenciación Celular/genética , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteogénesis/genética , Seudópodos/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
In inflammatory bone diseases such as periodontitis, the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome accelerates bone resorption by promoting proinflammatory cytokine IL-1ß production. However, the role of the NLRP3 inflammasome in physiological bone remodeling remains unclear. Here, we investigated its role in osteoclastogenesis in the presence and absence of lipopolysaccharide (LPS), a Gram-negative bacterial component. When bone marrow macrophages (BMMs) were treated with receptor activator of nuclear factor-κB ligand (RANKL) in the presence of NLRP3 inflammasome inhibitors, osteoclast formation was promoted in the absence of LPS but attenuated in its presence. BMMs treated with RANKL and LPS produced IL-1ß, and IL-1 receptor antagonist inhibited osteoclastogenesis, indicating IL-1ß involvement. BMMs treated with RANKL alone produced no IL-1ß but increased reactive oxygen species (ROS) production. A ROS inhibitor suppressed apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) speck formation and NLRP3 inflammasome inhibitors abrogated cytotoxicity in BMMs treated with RANKL, indicating that RANKL induces pyroptotic cell death in BMMs by activating the NLRP3 inflammasome via ROS. This suggests that the NLRP3 inflammasome promotes osteoclastogenesis via IL-1ß production under infectious conditions, but suppresses osteoclastogenesis by inducing pyroptosis in osteoclast precursors under physiological conditions.
Asunto(s)
Inflamasomas , Lipopolisacáridos , Animales , Médula Ósea/metabolismo , Caspasa 1/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Osteogénesis , Ligando RANK/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Periodontitis is an inflammatory disease initiated by dental deposits. Microorganisms in the dental biofilm induce cell death in epithelial cells, contributing to the breakdown of epithelial barrier function. Recently, dental calculus has also been implicated in pyroptotic cell death in oral epithelium. We analyzed the cytotoxic effects of dental calculus and freeze-dried periodontopathic bacteria on oral epithelial cells and macrophages. METHODS: HSC-2 (human oral squamous carcinoma cells) and phorbol 12-myristate 13-acetate-differentiated THP-1 macrophages were exposed to dental calculus or one of two species of freeze-dried bacterium, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. Following incubation for 24 hours, we measured cytotoxicity via lactate dehydrogenase release. Cells were then incubated with glyburide, an NLRP3 inflammasome inhibitor, to assess the potential role of pyroptosis. We also conducted a permeability assay to analyze the effects on epithelial barrier function. RESULTS: Dental calculus induced dose-dependent cell death in HSC-2 cells, whereas cell death induced by freeze-dried bacteria was insignificant. Conversely, freeze-dried bacteria induced more cell death than dental calculus in THP-1 macrophages. Cell death induced by dental calculus but not by freeze-dried bacteria was inhibited by glyburide, indicating that these are different types of cell death. In the permeability assays, dental calculus but not freeze-dried bacteria attenuated the barrier function of HSC-2 cell monolayers. CONCLUSION: Due to the low sensitivity of HSC-2 cells to microbial cytotoxicity, dental calculus had stronger cytotoxic effects on HSC-2 cell monolayers than freeze-dried A. actinomycetemcomitans and F. nucleatum, suggesting that it plays a critical role in the breakdown of crevicular/pocket epithelium integrity.
Asunto(s)
Aggregatibacter actinomycetemcomitans , Fusobacterium nucleatum , Cálculos Dentales , Células Epiteliales , Fusobacterium nucleatum/fisiología , Gliburida/farmacología , Humanos , Macrófagos , Porphyromonas gingivalisRESUMEN
Dental calculus (DC) is a common deposit in periodontitis patients. We have previously shown that DC contains both microbial components and calcium phosphate crystals that induce an osteoclastogenic cytokine IL-1ß via the NLRP3 inflammasome in macrophages. In this study, we examined the effects of cytokines produced by mouse macrophages stimulated with DC on osteoclastogenesis. The culture supernatants from wild-type (WT) mouse macrophages stimulated with DC accelerated osteoclastogenesis in RANKL-primed mouse bone marrow macrophages (BMMs), but inhibited osteoclastogenesis in RANKL-primed RAW-D cells. WT, but not NLRP3-deficient, mouse macrophages stimulated with DC produced IL-1ß and IL-18 in a dose-dependent manner, indicating the NLRP3 inflammasome-dependent production of IL-1ß and IL-18. Both WT and NLRP3-deficient mouse macrophages stimulated with DC produced IL-10, indicating the NLRP3 inflammasome-independent production of IL-10. Recombinant IL-1ß accelerated osteoclastogenesis in both RANKL-primed BMMs and RAW-D cells, whereas recombinant IL-18 and IL-10 inhibited osteoclastogenesis. These results indicate that DC induces osteoclastogenic IL-1ß in an NLRP3 inflammasome-dependent manner and anti-osteogenic IL-18 and IL-10 dependently and independently of the NLRP3 inflammasome, respectively. DC may promote alveolar bone resorption via IL-1ß induction in periodontitis patients, but suppress resorption via IL-18 and IL-10 induction in some circumstances.