Cbfß regulates Wnt/ß-catenin, Hippo/Yap, and Tgfß signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis.

As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that CBFB (subunit of a heterodimeric Cbfß/Runx1, Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfb in tamoxifen-induced Cbfbf/f;Col2a1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfß deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/Yap signaling and Tgfß signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfß and Yap expression and increased active ß-catenin expression in articular cartilage, while local AAV-mediated Cbfb overexpression promoted Yap expression and diminished active ß-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfb overexpression in knee joints of mice with OA showed the significant protective effect of Cbfß on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfß may be the cause of OA. Moreover, Local admission of Cbfb may rescue and protect OA through decreasing Wnt/ß-catenin signaling, and increasing Hippo/Yap signaling and Tgfß/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfb overexpression could be an effective strategy for treatment of OA.

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

[This corrects the article DOI: 10.1371/journal.pone.0058599.].

Cbfß regulates Wnt/ß-catenin, Hippo/Yap, and TGFß signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis.

As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfß, (subunit of a heterodimeric Cbfß/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfß in tamoxifen-induced Cbfßf/fCol2α1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfß deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-ß signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfß and Yap expression and increased active ß-catenin expression in articular cartilage, while local AAV-mediated Cbfß overexpression promoted Yap expression and diminished active ß-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfß overexpression in knee joints of mice with OA showed the significant protective effect of Cbfß on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfß may be the cause of OA. Moreover, Local admission of Cbfß may rescue and protect OA through decreasing Wnt/ß-catenin signaling, and increasing Hippo/Yap signaling and TGFß/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfß overexpression could be an effective strategy for treatment of OA.

Do Parents of Children with ADHD Know the Disease? Results from a Cross-Sectional Survey in Zhejiang, China.

Parents' knowledge, attitudes, and beliefs about attention-deficit/hyperactivity disorder (ADHD) are crucial in the selection of the treatment strategy and how to care for children with ADHD. However, little is known about parents' conception in mainland China. A semi-structured questionnaire was used to assess this information with 25 true/false questions regarding ADHD, and other questions investigating the methods of acquiring ADHD-related information, treatment preference, and concerns about ADHD treatment strategy. We found that the average score of all the participants was 17.42 ± 2.69 (total of 25 points) for the questionnaire on knowledge, attitudes, and beliefs about ADHD. This indicated that the parents had insufficient knowledge of this disease profile. They always accessed specialized information through mobile internet. For the treatment options, the investigated parents chose psychotherapy treatment rather than medications, in that they worried about the side effects of medication and expected to find alternative treatment strategies. The present investigation demonstrated that most parents lack knowledge about ADHD in treatment decision making in China. Medical professionals should provide parents with evidence-based ADHD-related information to help them understand this disease.

Correction: Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways.

[This corrects the article DOI: 10.1371/journal.pgen.1009233.].

Knockout and Double Knockout of Cathepsin K and Mmp9 reveals a novel function of Cathepsin K as a regulator of osteoclast gene expression and bone homeostasis.

Cathepsins play a role in regulation of cell function through their presence in the cell nucleus. However, the role of Cathepsin K (Ctsk) as an epigenetic regulator in osteoclasts remains unknown. Our data demonstrated that Ctsk-/-Mmp9-/- mice have a striking phenotype with a 5-fold increase in bone volume compared with WT. RNA-seq analysis of Ctsk-/- , Mmp9-/- and Ctsk-/-/Mmp9-/- osteoclasts revealed their distinct functions in gene expression regulation, including reduced Cebpa expression, increased Nfatc1 expression, and in signaling pathways activity regulation. Western blots and qPCR data validated these changes. ATAC-seq profiling of Ctsk-/- , Mmp9-/-, and Ctsk-/-/Mmp9-/- osteoclasts indicated the changes resulted from reduced chromatin openness in the promoter region of Cebpa and increased chromatin openness in Nfatc1 promoter in Ctsk-/-/Mmp9-/- osteoclasts compared to that in osteoclasts of WT, Ctsk/- and Mmp9-/- . We found co-localization of Ctsk with c-Fos and cleavage of H3K27me3 in wild-type osteoclasts. Remarkably, cleavage of H3K27me3 was blocked in osteoclasts of Ctsk-/- and Ctsk-/-/Mmp9-/- mice, suggesting that Ctsk may epigenetically regulate distinctive groups of genes' expression by regulating proteolysis of H3K27me3. Ctsk-/-/Mmp9-/- double knockout dramatically protects against ovariectomy induced bone loss. We found that Ctsk may function as an essential epigenetic regulator in modulating levels of H3K27me3 in osteoclast activation and maintaining bone homeostasis. Our study revealed complementary and unique functions of Ctsk as epigenetic regulators for maintaining osteoclast activation and bone homeostasis by orchestrating multiple signaling pathways and targeting both Ctsk and Mmp9 is a novel therapeutic approach for osteolytic diseases such as osteoporosis.

Mental health status of children with disorders of sexual development and their correlates.

Disorders of sexual development (DSD) refer to the congenital abnormalities of chromosomes, gonads, or gender anatomy. Children with DSD usually experience more stress. The present study aims to evaluate the mental health status of children with DSD, and to explore the potential relevant factors. We included 30 children with DSD and 30 age- and gender-matched children without DSD as the control group. All the children and their parents completed the scales of the Hamilton Anxiety Scale (HAMA). Children over 8 years old (n = 22) completed the Screen Scale for Child Anxiety Related Emotional Disorders (SCARED), the Depression Self-rating Scale for Children (DSRSC), and the Egna Minnen av Barndoms Uppfostran-own memories of parental rearing practices in childhood. DSD children had significantly higher somatic anxiety, mental anxiety, and total anxiety scores than the control group (p < 0.001). The scores of the SCARED, anxiety, and depression subscales of DSD children were higher than those of control children (p < 0.05 and p < 0.001, respectively). The correlation analysis showed that the score of generalized anxiety was positively related to age and entertainment. The regression analysis showed that age was a major factor that affected generalized anxiety in DSD children, and neuroticism was a major factor of anxiety disorder and separation anxiety in DSD children. Children with DSD have obvious anxiety problems, which are associated with family environmental factors (entertainment, success, and conflicts) and age. It is important to focus emphasis on emotional stability in children with DSD for detecting anxiety-related emotional disorders early.

GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways.

G-protein-coupled receptors (GPCRs) signaling is critical to cell differentiation and activation. However, the function of GPCRs in osteoclast differentiation and activation remains unclear. We found that the G-protein coupled receptor 125 (GPCR 125) gene (Gpr125) gene was highly expressed in osteoclasts through RNA-sequencing technology, qRT-PCR, and Western blot analysis. We characterized the role of GPCR125 in osteoclast differentiation and activation by loss-of-function and gain-of-function methods in osteoclasts. Osteoclasts with lentivirus-mediated GPR125 silencing demonstrated a dramatic reduction in differentiation and impaired bone resorption function. In contrast, overexpression of Gpr125 in osteoclasts increased NFATC1 expression and enhanced osteoclast differentiation and enhanced osteoclast-mediated bone resorption. These results indicated that GPCR125 positively regulates osteoclast formation and function. Following receptor activator of nuclear factor kappa-Β ligand (RANKL) stimulation, the expression levels of MAPK signaling pathway proteins phosphorylated-ERK (p-ERK) and phosphorylated-p38 (p-p38) were significantly decreased in the Gpr125 knockdown (sh-GPR125) group compared to its control group. We also found that phosphorylated AKT (p-AKT) expression was downregulated, as well as nuclear factor kappa-B (NF-κB) signaling pathway protein phosphorylated-IKB alpha (p-IKBα). Our results demonstrated that GPCR125 positively regulates osteoclasts via RANKL-stimulated MAPK and AKT-NF-κB signaling pathways, and GPCR125 could potentially be utilized as a novel therapeutic target in bone related diseases including osteoporosis.

Runx1 is a central regulator of osteogenesis for bone homeostasis by orchestrating BMP and WNT signaling pathways.

Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1α1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/ß-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast-adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/ß-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.

F2r negatively regulates osteoclastogenesis through inhibiting the Akt and NFκB signaling pathways.

G-protein-coupled receptors (GPCRs) are pivotal drug targets for many diseases. Coagulation Factor II Thrombin Receptor (F2R) is an important member of GPCR family that is highly expressed in osteoclasts. However, the role of F2r in osteoclasts is still unclear. Here, to examine the functions of F2r on osteoclast formation, differentiation, activation, survival, and acidification, we employed loss-of-function and gain-of-function approaches to study F2r using F2r-targeted short hairpin RNA (sh-F2r) lentivirus and overexpression plasmid pLX304-F2r lentivirus respectively, in mouse bone marrow cells (MBMs) induced osteoclasts. We used three shRNAs targeting F2r which had the ability to efficiently and consistently knock down the expression of F2r at different levels. Notably, F2r knockdown trigged a significant increase in osteoclast activity, number, and size, as well as promoted bone resorption and F-actin ring formation with increased osteoclast marker gene expression. Moreover, F2r overexpression blocked osteoclast formation, maturation, and acidification, indicating that F2r negatively regulates osteoclast formation and function. Furthermore, we investigated the mechanism(s) underlying the role of F2r in osteoclasts. We detected RANKL-induced signaling pathways related protein changes F2r knockdown cells and found significantly increased pAkt levels in sh-F2r infected cells, as well as significantly enhanced phosphorylation of p65 and IKBα in early stages of RANKL stimulation. These data demonstrated that F2r responds to RANKL stimulation to attenuate osteoclastogenesis through inhibiting the both F2r-Akt and F2r-NFκB signaling pathways, which lead a reduction in the expression of osteoclast genes. Our study suggests that targeting F2r may be a novel therapeutic approach for bone diseases, such as osteoporosis.

Author Correction: Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3ß-NFATc1 signalling pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bone resorption deficiency affects tooth root development in RANKL mutant mice due to attenuated IGF-1 signaling in radicular odontoblasts.

The tooth root is essential for normal tooth physiological function. Studies on mice with mutations or targeted gene deletions revealed that osteoclasts (OCs) play an important role in tooth root development. However, knowledge on the cellular and molecular mechanism underlying how OCs mediate root formation is limited. During bone formation, growth factors (e.g. Insulin-like growth factor-1, IGF-1) liberated from bone matrix by osteoclastic bone resorption stimulate osteoblast differentiation. Thus, we hypothesize that OC-osteoblast coupling may also apply to OC-odontoblast coupling; therefore OCs may have a direct impact on odontoblast differentiation through the release of growth factor(s) from bone matrix, and consequently regulate tooth root formation. To test this hypothesis, we used a receptor activator of NF-κB ligand (RANKL) knockout mouse model in which OC differentiation and function was entirely blocked. We found that molar root formation and tooth eruption were defective in RANKL-/- mice. Disrupted elongation and disorganization of Hertwig's epithelial root sheath (HERS) was observed in RANKL-/- mice. Reduced expression of nuclear factor I C (NFIC), osterix, and dentin sialoprotein, markers essential for radicular (root) odontogenic cell differentiation indicated that odontoblast differentiation was disrupted in RANKL deficient mice likely contributing to the defect in root formation. Moreover, down-regulation of IGF/AKT/mTOR activity in odontoblast indicated that IGF signaling transduction in odontoblasts of the mutant mice was impaired. Treating odontoblast cells in vitro with conditioned medium from RANKL-/- OCs cultured on bone slices resulted in inhibition of odontoblast differentiation. Moreover, depletion of IGF-1 in bone resorption-conditioned medium (BRCM) from wild-type (WT) OC significantly compromised the ability of WT osteoclastic BRCM to induce odontoblast differentiation while addition of IGF-1 into RANKL-/- osteoclastic BRCM rescued impaired odontoblast differentiation, confirming that root and eruption defect in RANKL deficiency mice may result from failure of releasing of IGF-1 from bone matrix through OC bone resorption. These results suggest that OCs are important for odontoblast differentiation and tooth root formation, possibly through IGF/AKT/mTOR signaling mediated by cell-bone matrix interaction. These findings provide significant insights into regulatory mechanism of tooth root development, and also lay the foundation for root regeneration studies.

C/ebpα controls osteoclast terminal differentiation, activation, function, and postnatal bone homeostasis through direct regulation of Nfatc1.

Osteoclast lineage commitment and differentiation have been studied extensively, although the mechanism by which transcription factor(s) control osteoclast terminal differentiation, activation, and function remains unclear. CCAAT/enhancer-binding protein α (C/ebpα) has been reported to be a key regulator of osteoclast cell lineage commitment, yet C/ebpα's roles in osteoclast terminal differentiation, activation and function, and bone homeostasis, under physiological or pathological conditions, have not been studied because newborn C/ebpα-null mice die within several hours after birth. Furthermore, the function of C/ebpα in osteoclast terminal differentiation, activation, and function is largely unknown. Herein, we generated and analyzed an osteoclast-specific C/ebpα conditional knockout (CKO) mouse model via Ctsk-Cre mice and found that C/ebpα-deficient mice exhibited a severe osteopetrosis phenotype due to impaired osteoclast terminal differentiation, activation, and function, including mildly reduced osteoclast number, impaired osteoclast polarization, actin formation, and bone resorption, which demonstrated the novel function of C/ebpα in cell function and terminal differentiation. Interestingly, C/ebpα deficiency did not affect bone formation or monocyte/macrophage development. Our results further demonstrated that C/ebpα deficiency suppressed the expression of osteoclast functional genes, e.g. encoding cathepsin K (Ctsk), Atp6i (Tcirg1), and osteoclast regulator genes, e.g. encoding c-fos (Fos), and nuclear factor of activated T-cells 1 (Nfatc1), while having no effect on Pu.1 (Spi1) expression. Promoter activity mapping and ChIP assay defined the critical cis-regulatory element (CCRE) in the promoter region of Nfatc1, and also showed that the CCREs were directly associated with C/ebpα, which enhanced the promoter's activity. The deficiency of C/ebpα in osteoclasts completely blocked ovariectomy-induced bone loss, indicating that C/ebpα is a promising new target for the treatment of osteolytic diseases. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Monocyte-Specific Knockout of C/ebpα Results in Osteopetrosis Phenotype, Blocks Bone Loss in Ovariectomized Mice, and Reveals an Important Function of C/ebpα in Osteoclast Differentiation and Function.

CCAAT/enhancer-binding protein α (C/ebpα) is critical for osteoclastogenesis by regulating osteoclast (OC) lineage commitment and is also important for OC differentiation and function in vitro. However, the role of C/ebpα in postnatal skeletal development has not been reported owing to lethality in C/ebpα-/- mice from hypoglycemia within 8 hours after birth. Herein, we generated conditional knockout mice by deleting the C/ebpα gene in monocyte via LysM-Cre to examine its role in OC differentiation and function. C/ebpαf/f LysM-Cre mice exhibited postnatal osteopetrosis due to impaired osteoclastogenesis, OC lineage priming defects, as well as defective OC differentiation and activity. Furthermore, our ex vivo analysis demonstrated that C/ebpα conditional deletion significantly reduced OC differentiation, maturation, and activity while mildly repressing macrophage development. At the molecular level, C/ebpα deficiency significantly suppresses the expressions of OC genes associated with early stages of osteoclastogenesis as well as genes associated with OC differentiation and activity. We also identified numerous C/ebpα critical cis-regulatory elements on the Cathepsin K promoter that allow C/ebpα to significantly upregulate Cathepsin K expression during OC differentiation and activity. In pathologically induced mouse model of osteoporosis, C/ebpα deficiency can protect mice against ovariectomy-induced bone loss, uncovering a central role for C/ebpα in osteolytic diseases. Collectively, our findings have further established C/ebpα as a promising therapeutic target for bone loss by concurrently targeting OC lineage priming, differentiation, and activity. © 2017 American Society for Bone and Mineral Research.

Osteoclast proton pump regulator Atp6v1c1 enhances breast cancer growth by activating the mTORC1 pathway and bone metastasis by increasing V-ATPase activity.

It is known that V-ATPases (vacuolar H+-ATPase) are involved in breast cancer growth and metastasis. Part of this action is similar to their role in osteoclasts, where they're involved in extracellular acidification and matrix destruction; however, the roles of their subunits in cancer cell proliferation, signaling, and other pro-tumor actions are not well established. Analysis of TCGA data shows that V-ATPase subunit Atp6v1c1 is overexpressed or amplified in 34% of human breast cancer cases, with a 2-fold decrease in survival at 12 years. Whereas other subunits, such as Atp6v1c2 and Atp6v0a3, are overexpressed or genomically amplified less often, 6% each respectively, and have less impact on survival. Experiments show that lentiviral-shRNA mediated ATP6v1c1 knockdown in 4T1 mouse mammary cancer cells significantly reduces orthotopic and intraosseous tumor growth. ATP6v1c1 knockdown also significantly reduces tumor stimulated bone resorption through osteoclastogenesis at the bone and metastasis in vivo, as well as V-ATPase activity, proliferation, and mTORC1 activation in vitro. To generalize the effects of ATP6v1c1 knockdown on proliferation and mTORC1 activation we used human cancer cell lines - MCF-7, MDA-MB-231, and MDA-MB-435s. ATP6V1C1 knockdown reduced cell proliferation and impaired mTORC1 pathway activation in cancer cells but not in the untransformed cell line C3H10T1/2. Our study reveals that V-ATPase activity may be mediated through mTORC1 and that ATP6v1c1 can be knocked down to block both V-ATPase and mTORC1 activity.

Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3ß-NFATc1 signalling pathway.

Many positive signalling pathways of osteoclastogenesis have been characterized, but negative signalling pathways are less well studied. Here we show by microarray and RNAi that guanine nucleotide-binding protein subunit α13 (Gα13) is a negative regulator of osteoclastogenesis. Osteoclast-lineage-specific Gna13 conditional knockout mice have a severe osteoporosis phenotype. Gna13-deficiency triggers a drastic increase in both osteoclast number and activity (hyper-activation), mechanistically through decreased RhoA activity and enhanced Akt/GSK3ß/NFATc1 signalling. Consistently, Akt inhibition or RhoA activation rescues hyper-activation of Gna13-deficient osteoclasts, and RhoA inhibition mimics the osteoclast hyperactivation resulting from Gna13-deficiency. Notably, Gα13 gain-of-function inhibits Akt activation and osteoclastogenesis, and protects mice from pathological bone loss in disease models. Collectively, we reveal that Gα13 is a master endogenous negative switch for osteoclastogenesis through regulation of the RhoA/Akt/GSK3ß/NFATc1 signalling pathway, and that manipulating Gα13 activity might be a therapeutic strategy for bone diseases.

The Triple Functions of D2 Silencing in Treatment of Periapical Disease.

INTRODUCTION: Dental caries is the most widespread chronic infectious disease. Inflammation in pulp tissues caused by dental caries will lead to periapical granulomas, bone erosion, loss of the tooth, and severe pain. Despite numerous efforts in recent studies to develop effective treatments for dental caries, the need for a potent therapy is still urgent. METHODS: In this study, we applied a gene-based therapy approach by administering recombinant adeno-associated virus (AAV)-mediated Atp6v0d2 (d2) RNA interference knockdown of d2 gene expression to prevent periapical bone loss and suppress periapical inflammation simultaneously. RESULTS: The results showed that d2 depletion is simultaneously capable of reducing bone resorption with 75% protection through reducing osteoclasts, enhancing bone formation by increasing osterix expression, and inhibiting inflammation by decreasing T-cell infiltration. Notably, AAV-mediated gene therapy of d2 knockdown significantly reduced proinflammatory cytokine expression, including tumor necrosis factor α, interferon-γ, interleukin-1α, and interleukin 6 levels in periapical diseases caused by bacterial infection. Quantitative real-time polymerase chain reaction revealed that d2 knockdown reduced osteoclast-specific functional genes (ie, Acp5 and Ctsk) and increased osteoblast marker genes (ie, Osx and Opg) in periapical tissues. CONCLUSIONS: Collectively, our results showed that AAV-mediated d2 depletion in the periapical lesion area can prevent the progression of endodontic disease and bone erosion while significantly reducing the inflammatory over-response. These findings show that the depletion of d2 simultaneously reduces bone resorption, enhances bone formation, and inhibits inflammation caused by periapical diseases and provide significant insights into the potential effectiveness of AAV-sh-d2-mediated d2 silencing gene therapy as a major endodontic treatment.

TET-mediated DNA demethylation controls gastrulation by regulating Lefty-Nodal signalling.

Mammalian genomes undergo epigenetic modifications, including cytosine methylation by DNA methyltransferases (DNMTs). Oxidation of 5-methylcytosine by the Ten-eleven translocation (TET) family of dioxygenases can lead to demethylation. Although cytosine methylation has key roles in several processes such as genomic imprinting and X-chromosome inactivation, the functional significance of cytosine methylation and demethylation in mouse embryogenesis remains to be fully determined. Here we show that inactivation of all three Tet genes in mice leads to gastrulation phenotypes, including primitive streak patterning defects in association with impaired maturation of axial mesoderm and failed specification of paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signalling. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signalling contributes to the gastrulation failure of Tet mutants. Increased Nodal signalling is probably due to diminished expression of the Lefty1 and Lefty2 genes, which encode inhibitors of Nodal signalling. Moreover, reduction in Lefty gene expression is linked to elevated DNA methylation, as both Lefty-Nodal signalling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, a point mutation in Tet that specifically abolishes the dioxygenase activity causes similar morphological and molecular abnormalities as the null mutation. Taken together, our results show that TET-mediated oxidation of 5-methylcytosine modulates Lefty-Nodal signalling by promoting demethylation in opposition to methylation by DNMT3A and DNMT3B. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation crucial to regulation of key signalling pathways in early body plan formation.

Ac45 silencing mediated by AAV-sh-Ac45-RNAi prevents both bone loss and inflammation caused by periodontitis.

AIM: Periodontitis induced by oral pathogens leads to severe periodontal tissue damage and osteoclast-mediated bone resorption caused by inflammation. On the basis of the importance of Ac45 in osteoclast formation and function, we performed this study to evaluate the therapeutic potential of periodontitis by local adeno-associated virus (AAV)-mediated Ac45 gene knockdown. MATERIAL AND METHODS: We used AAV-mediated short hairpin RNAi knockdown of Ac45 gene expression (AAV-sh-Ac45) to inhibit bone erosion and gingival inflammation simultaneously in a well-established periodontitis mouse model induced by Porphyromonas gingivalis W50. Histological studies were performed to evaluate the bone protection of AAV-sh-Ac45. Immunochemistry, ELISA and qRT-PCR were performed to reveal the role of Ac45 knockdown on inflammation, immune response and expression of cytokine. RESULTS: We found that Ac45 knockdown impaired osteoclast-mediated extracellular acidification and bone resorption in vitro and in vivo. Furthermore, local administration of AAV-sh-Ac45 protected mice from bone erosion by >85% and attenuated inflammation and decreased infiltration of T cells, dendritic cells and macrophages in the periodontal lesion. Notably, the expression of pro-inflammatory cytokines was also reduced. CONCLUSIONS: Local AAV-sh-Ac45 gene therapy efficiently protects against periodontal tissue damage and bone erosion through both inhibition of osteoclast function and attenuating inflammation, and may represent a powerful new treatment strategy for periodontitis.