RNAi-Mediated Silencing of Atp6i and Atp6i Haploinsufficiency Prevents Both Bone Loss and Inflammation in a Mouse Model of Periodontal Disease

Periodontal disease affects about 80% of adults in America, and is characterized by oral bacterial infection-induced gingival inflammation, oral bone resorption, and tooth loss. Periodontitis is also associated with other diseases such as rheumatoid arthritis, diabetes, and heart disease. Although many efforts have been made to develop effective therapies for this disease, none have been very effective and there is still an urgent need for better treatments and preventative strategies. Herein we explored for the first time the possibility that adeno-associated virus (AAV)-mediated RNAi knockdown could be used to treat periodontal disease with improved efficacy. For this purpose, we used AAV-mediated RNAi knockdown of Atp6i/TIRC7 gene expression to target bone resorption and gingival inflammation simultaneously. Mice were infected with the oral pathogen Porphyromonas gingivalis W50 (P. gingivalis) in the maxillary periodontium to induce periodontitis. We found that Atp6i depletion impaired extracellular acidification and osteoclast-mediated bone resorption. Furthermore, local injection of AAV-shRNA-Atp6i/TIRC7 into the periodontal tissues in vivo protected mice from P. gingivalis infection-stimulated bone resorption by >85% and decreased the T-cell number in periodontal tissues. Notably, AAV-mediated Atp6i/TIRC7 knockdown also reduced the expression of osteoclast marker genes and inflammation-induced cytokine genes. Atp6i^+/− mice with haploinsufficiency were similarly protected from P. gingivalis infection-stimulated bone loss and gingival inflammation. This suggests that AAV-shRNA-Atp6i/TIRC7 therapeutic treatment may significantly improve the health of millions who suffer from P. gingivalis-mediated periodontal disease.     

Silencing of Atp6v1c1 Prevents Breast Cancer Growth and Bone Metastasis

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1''s expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.     

Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts

Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-kappaB (nuclear factor kappaB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3(-/-) osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.     

Adrm1 interacts with Atp6v0d2 and regulates osteoclast differentiation

Bone homeostasis is tightly regulated by matrix-producing osteoblasts and bone-resorbing osteoclasts. During osteoclast development, mononuclear preosteoclasts derived from myeloid cells fuse together to form multinucleated, giant cells. Previously, we reported that the d2 isoform of the vacuolar (H⁺) ATPase V0 domain (Atp6v0d2) plays an important role in osteoclast maturation and bone formation. To understand how Atp6v0d2 controls osteoclast maturation, we have performed a yeast two-hybrid screen using full-length Atp6v0d2 as the bait, and identified adhesion-regulating molecule 1 protein (Adrm1) as a potential functional partner of Atp6v0d2. The interaction between Atp6v0d2 and Adrm1 was confirmed in yeast and invivo using immunoprecipitation assays. We also show that Adrm1 is required for cell migration and osteoclast maturation.     

Atp6v1c1 May Regulate Filament Actin Arrangement in Breast Cancer Cells

Previous studies have shown that the rate of breast cancer metastasis correlates with the expression of vacuolar H⁺-ATPases (V-ATPases). However, how V-ATPase is involved in breast cancer metastasis remains unknown. Our previous study showed that Atp6v1c1-depleted osteoclasts did not form organized actin rings and that Atp6v1c1 co-localizes with F-actin. In this study, we found that the normal arrangement of filamentous actin is disrupted in Atp6v1c1-depleted 4T1 mouse breast cancer cells and in the ATP6V1C1-depleted human breast cancer cell lines MDA-MB-231 and MDA-MB-435s. We further found that Atp6v1c1 co-localizes with F-actin in 4T1 cells. The results of our study suggest that high expression of Atp6v1c1 affects the actin structure of cancer cells such that it facilitates breast cancer metastasis. The findings also indicate that Atp6v1c1 could be a novel target for breast cancer metastasis therapy.     

The Pros1/Tyro3 axis protects against periodontitis by modulating STAT/SOCS signalling

Periodontitis, an oral inflammatory disease caused by periodontal pathogen infection, is the most prevalent chronic inflammatory disease and a major burden on healthcare. The TAM receptor tyrosine kinases (Tyro3, Axl and Mertk) and their ligands (Gas6 and Pros1) play a pivotal role in the resolution of inflammation and have been associated with chronic inflammatory and autoimmune diseases. In this study, we evaluated the effects of exogenous Pros1 in in vitro and in vivo models of periodontitis. We detected higher Pros1 but lower Tyro3 levels in inflamed gingival specimens of periodontitis patients compared with healthy controls. Moreover, Pros1 was mostly localized in the gingival epithelium of all specimens. In cultured human gingival epithelial cells (hGECs), Porphyromonas gingivalis LPS (p.g‐LPS) stimulation down‐regulated Pros1 and Tyro3. Exogenous Pros1 inhibited p.g‐LPS–induced production of TNF‐α, IL‐6, IL‐1β, MMP9/2 and RANKL in a Tyro3‐dependent manner as revealed by PCR, Western blot analysis, ELISA and gelatin zymography. Pros1 also restored Tyro3 expression down‐regulated by p.g‐LPS in hGECs. In rats treated with ligature and p.g‐LPS, administration of Pros1 attenuated periodontitis‐associated gingival inflammation and alveolar bone loss. Our mechanistic studies implicated SOCS1/3 and STAT1/3 as mediators of the in vitro and in vivo anti‐inflammatory effects of Pros1. Collectively, the findings from this work supported Pros1 as a novel anti‐inflammatory therapy for periodontitis.     

NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP)

NFATc1 has been characterized as a master regulator of nuclear factor kappaB ligand-induced osteoclast differentiation. Herein, we demonstrate a novel role for NFATc1 as a positive regulator of nuclear factor kappaB ligand-mediated osteoclast fusion as well as other fusion-inducing factors such as TNF-alpha. Exogenous overexpression of a constitutively active form of NFATc1 in bone marrow-derived monocyte/macrophage cells (BMMs) induces formation of multinucleated osteoclasts as well as the expression of fusion-mediating molecules such as the d2 isoform of vacuolar ATPase V(o) domain (Atp6v0d2) and the dendritic cell-specific transmembrane protein (DC-STAMP). Moreover, inactivation of NFATc1 by cyclosporin A treatment attenuates expression of Atp6v0d2 and DC-STAMP and subsequent fusion process of osteoclasts. We show that NFATc1 binds to the promoter regions of Atp6v0d2 and DC-STAMP in osteoclasts and directly induces their expression. Furthermore, overexpression of Atp6v0d2 and DC-STAMP rescues cell-cell fusion of preosteoclasts despite reduced NFATc1 activity. Our data indicate for the first time that the NFATc1/Atp6v0d2 and DC-STAMP signaling axis plays a key role in the osteoclast multinucleation process, which is essential for efficient bone resorption.     

v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation

Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H(+)) ATPase (v-ATPase) V(0) domain (Atp6v0d2). Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.     

AZD8835 inhibits osteoclastogenesis and periodontitis-induced alveolar bone loss in rats

Chronic periodontitis (CP) is one of the most common oral diseases, which is characterized by the loss of connective tissue and alveolar bone in adults. AZD8835, a novel dual phosphoinositide-3-kinase (PI3K) inhibitor, is currently in phase 1 clinical evaluation to treat breast cancer. However, whether AZD8835 has any effect on teeth and alveolar bone health remains unclear. In the current study, we aimed to investigate the potential effect of AZD8835 in treating CP in vitro and in vivo. We found that AZD8835 could inhibit osteoclast differentiation, bone resorption, and downregulate the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (Trap), cathepsin K (Ctsk), V-ATPase d2 (Atp6v0d2), and calcitonin receptor (Ctr). In addition, AZD8835 suppressed osteoclastogenesis by inhibiting receptor activator of nuclear factor kappa B ligand (RANKL)-induced PI3K/protein kinase B (AKT), extracellular signal-regulated kinase, and nuclear factor-κB signaling in BMMs. In vivo, AZD8835 greatly ameliorated alveolar bone (ABL) loss in rats with CP. Meanwhile, histological examination showed fewer osteoclasts in the treatment group. In conclusion, these results indicated that AZD8835 is a promising agent to reduce ABL in CP.     

Natural uranium impairs the differentiation and the resorbing function of osteoclasts

BackgroundUranium is a naturally occurring radionuclide ubiquitously present in the environment. The skeleton is the main site of uranium long-term accumulation. While it has been shown that natural uranium is able to perturb bone metabolism through its chemical toxicity, its impact on bone resorption by osteoclasts has been poorly explored. Here, we examined for the first time in vitro effects of natural uranium on osteoclasts.MethodsThe effects of uranium on the RAW 264.7 monocyte/macrophage mouse cell line and primary murine osteoclastic cells were characterized by biochemical, molecular and functional analyses.ResultsWe observed a cytotoxicity effect of uranium on osteoclast precursors. Uranium concentrations in the μM range are able to inhibit osteoclast formation, mature osteoclast survival and mineral resorption but don't affect the expression of the osteoclast gene markers Nfatc1, Dc-stamp, Ctsk, Acp5, Atp6v0a3 or Atp6v0d2 in RAW 274.7 cells. Instead, we observed that uranium induces a dose-dependent accumulation of SQSTM1/p62 during osteoclastogenesis.ConclusionsWe show here that uranium impairs osteoclast formation and function in vitro. The decrease in available precursor cells, as well as the reduced viability of mature osteoclasts appears to account for these effects of uranium. The SQSTM1/p62 level increase observed in response to uranium exposure is of particular interest since this protein is a known regulator of osteoclast formation. A tempting hypothesis discussed herein is that SQSTM1/p62 dysregulation contributes to uranium effects on osteoclastogenesis.General significanceWe describe cellular and molecular effects of uranium that potentially affect bone homeostasis.     

YTHDF2 mediates LPS-induced osteoclastogenesis and inflammatory response via the NF-κB and MAPK signaling pathways

Aberrant elevation of osteoclast differentiation and function is responsible for disrupting bone homeostasis in various inflammatory bone diseases. YTH domain family 2 (YTHDF2) is a well-known m⁶A-binding protein that plays an essential role in regulating cell differentiation and inflammatory processes by mediating mRNA degradation. However, the regulatory role of YTHDF2 in inflammatory osteoclast differentiation remains unelucidated. Here, we detected the expression of m⁶A-related genes and found that YTHDF2 was upregulated in RANKL-primed osteoclast precursors stimulated with lipopolysaccharide (LPS). Ythdf2 knockdown in RAW264.7 cells and primary bone marrow-derived macrophages (BMMs) enhanced osteoclast formation and bone resorption, which was assessed by TRAP staining assay and pit formation assay. Ythdf2 depletion upregulated osteoclast-related gene expression and proinflammatory cytokine secretion. In contrast, overexpression of Ythdf2 produced the reverse effect. Furthermore, Ythdf2 knockdown enhanced the phosphorylation of IKKα/β, IκBα, ERK, P38 and JNK. NF-κB and MAPK signaling pathway inhibitors effectively abrogated the enhanced expression of Nfact1, c-Fos, IL-1β and TNF-α caused by Ythdf2 knockdown. Mechanistically, the mRNA stability assay revealed that Ythdf2 depletion led to stabilization of Tnfrsf11a, Traf6, Map4k4, Map2k3, Map2k4 and Nfatc1 mRNA. In summary, our findings demonstrated that YTHDF2 has a negative regulatory role in LPS-induced osteoclast differentiation and the inflammatory response via the NF-κB and MAPK signaling pathways.     

Neutrophil Mobilization by Surface-Glycan Altered Th17-Skewing Bacteria Mitigates Periodontal Pathogen Persistence and Associated Alveolar Bone Loss

Alveolar bone (tooth-supporting bone) erosion is a hallmark of periodontitis, an inflammatory disease that often leads to tooth loss. Periodontitis is caused by a select group of pathogens that form biofilms in subgingival crevices between the gums and teeth. It is well-recognized that the periodontal pathogen Porphyromonas gingivalis in these biofilms is responsible for modeling a microbial dysbiotic state, which then initiates an inflammatory response destructive to the periodontal tissues and bone. Eradication of this pathogen is thus critical for the treatment of periodontitis. Previous studies have shown that oral inoculation in mice with an attenuated strain of the periodontal pathogen Tannerella forsythia altered in O-glycan surface composition induces a Th17-linked mobilization of neutrophils to the gingival tissues. In this study, we sought to determine if immune priming with such a Th17-biasing strain would elicit a productive neutrophil response against P. gingivalis. Our data show that inoculation with a Th17-biasing T. forsythia strain is effective in blocking P. gingivalis-persistence and associated alveolar bone loss in mice. This work demonstrates the potential of O-glycan modified Tannerella strains or their O-glycan components for harnessing Th17-mediated immunity against periodontal and other mucosal pathogens.     

Vasohibin-1 promotes osteoclast differentiation in periodontal disease by stimulating the expression of RANKL in gingival fibroblasts

Vasohibin-1 (VASH1) is a key inhibitor of vascular endothelial growth factor-induced angiogenesis. Although the involvement of VASH1 in various pathological processes has been extensively studied, its role in periodontal disease (PD) remains unclear. We aimed to investigate the role of VASH1 in PD by focusing on osteoclastogenesis regulation. We investigated VASH1 expression in PD by analyzing data from the online Gene Expression Omnibus (GEO) database and using a mouse ligature-induced periodontitis model. The effects of VASH1 on osteoclast differentiation and osteoclastogenesis-supporting cells were assessed in mouse bone marrow-derived macrophages (BMMs) and human gingival fibroblasts (GFs). To identify the stimulant of VASH1, we used culture broth from Porphyromonas gingivalis (Pg), a periopathogen. The GEO database and mouse periodontitis model revealed that VASH1 expression was upregulated in periodontitis-affected gingival tissues, which was further supported by immunohistochemistry and qRT-PCR analyses. VASH1 expression was significantly stimulated in GFs after treatment with the Pg broth. Direct treatment with recombinant VASH1 protein did not stimulate osteoclast differentiation in BMMs but did contribute to osteoclast differentiation by inducing RANKL expression in GFs through a paracrine mechanism. Small interfering RNA-mediated silencing of VASH1 in GFs abrogated RANKL-mediated osteoclast differentiation in BMMs. Additionally, VASH1-activated RANKL expression in GFs was significantly suppressed by MK-2206, a selective inhibitor of AKT. These results suggest that Pg-induced VASH1 may be associated with RANKL expression in GFs in a paracrine manner, contributing to osteoclastogenesis via an AKT-dependent mechanism during PD progression.     

Induction of prostaglandin E(2) and interleukin-6 in gingival fibroblasts by oral biofilms

Polymicrobial oral biofilms attaching on tooth surfaces can trigger inflammatory responses by the neighbouring tooth-supporting periodontal tissues. An excessive inflammatory response can cause destruction of the periodontal tissues, including the alveolar bone, thus resulting in periodontitis. Mediators of inflammation, such as prostaglandin E(2) (PGE(2) ) and interleukin-6, are primary regulators of alveolar bone destruction in periodontitis. The present study aimed to comparatively investigate the effects of in vitro supragingival and subgingival biofilms, on the regulation of PGE(2) and interleukin-6 in human gingival fibroblasts. The cells were challenged with culture supernatants of the two biofilms for 6?h. Cyclo-oxygenase (COX)-2, an enzyme responsible for the conversion of PGE(2) , and interleukin-6 gene expression were analysed by quantitative real-time PCR. The production of PGE(2) and interleukin-6 by the cells was analysed by ELISA. While the supragingival biofilm did not induce significant changes, the subgingival biofilm caused an 8.6- and 2.9-fold enhancement of COX-2 and interleukin-6 gene expression, respectively, and a 72.5- and 1.5-fold enhancement of PGE(2) and interleukin-6 production, respectively. In conclusion, subgingival biofilms are potent inducers of PGE(2) in gingival fibroblasts, providing further mechanistic insights into the association of subgingival biofilms with bone resorption periodontitis.