Upregulated FOXM1 stimulates chondrocyte senescence in Acot12-/-Nudt7-/- double knockout mice.

Rationale: One of the hallmarks of osteoarthritis (OA), the most common degenerative joint disease, is increased numbers of senescent chondrocytes. Targeting senescent chondrocytes or signaling mechanisms leading to senescence could be a promising new therapeutic approach for OA treatment. However, understanding the key targets and links between chondrocyte senescence and OA remains unclear. Methods: Senescent chondrocytes were identified from Nudt7-/-, Acot12-/-, double-knockout mice lacking Acot12 and Nudt7 (dKO) and applied to microarray. The presence of forkhead transcription factor M1 (FOXM1) was detected in aged, dKO, and destabilization of the medial meniscus (DMM) cartilages and articular chondrocytes, and the effect of FoxM1 overexpression and acetyl-CoA treatment on cartilage homeostasis was examined using immunohistochemistry, quantitative real-time PCR (qRT-PCR), cell apoptosis and proliferation assay, and safranin O staining. Delivery of Rho@PAA-MnO2 (MnO2 nanosheet) or heparin-ACBP/COS-GA-siFoxM1 (ACBP-siFoxM1) nanoparticles into DMM cartilage was performed. Results: Here, we propose the specific capture of acetyl-CoA with the delivery of (FoxM1 siRNA (siFoxM1) to prevent cartilage degradation by inhibiting the axis of chondrocyte senescence. dKO stimulate chondrocyte senescence via the upregulation of FoxM1 and contribute to severe cartilage breakdown. We found that the accumulation of acetyl-CoA in the dKO mice may be responsible for the upregulation of FoxM1 during OA pathogenesis. Moreover, scavenging reactive oxygen species (ROS) induced by chondrocyte senescence via the implantation of MnO2 nanosheets or delivery of siFoxM1 functionalized with acetyl-CoA binding protein (ACBP) to capture acetyl-CoA using an injectable bioactive nanoparticle (siFoxM1-ACBP-NP) significantly suppressed DMM-induced cartilage destruction. Conclusion: We found that the loss of Acot12 and Nudt7 stimulates chondrocyte senescence via the upregulation of FoxM1 and accumulation of acetyl-CoA, and the application of siFoxM1-ACBP-NP is a potential therapeutic strategy for OA treatment.

Deficiency of peroxisomal NUDT7 stimulates de novo lipogenesis in hepatocytes.

Here, we found that heterozygous null of peroxisomal Nudt7 (Nudt7 +/- ) induced the typical NAFLD features, i.e. increased levels of hepatic triglyceride (TG) and fatty acid (FA), infiltration of inflammatory cells, impaired glucose tolerance and insulin sensitivity, and stimulation of lipolysis from adipose tissue. Particularly, in Nudt7 +/- hepatocytes, de novo lipogenesis (DNL) was significantly increased. Ingenuity pathway analysis (IPA) and KEGG pathway analysis of RNA sequencing data suggested the activation of PPAR signaling in the liver of Nudt7 +/- mice. Moreover, accumulation of palmitic acid in Nudt7 +/- hepatocyte increased the level of H3K4me3 on the promoters of PPARγ resulting in the activation of PPARγ and induced the DNL in the hepatocytes of Nudt7 +/- mice. Moreover, we found that liraglutide significantly reduced typical NAFLD features induced by NUDT7 deficiency. Our data suggest that dysregulation of peroxisomal NUDT7 is responsible for upregulation of hepatic DNL by accumulation of palmitic acid and PPARγ activation.

BNIP3-Dependent Mitophagy via PGC1α Promotes Cartilage Degradation.

Since mitochondria are suggested to be important regulators in maintaining cartilage homeostasis, turnover of mitochondria through mitochondrial biogenesis and mitochondrial degradation may play an important role in the pathogenesis of osteoarthritis (OA). Here, we found that mitochondrial dysfunction is closely associated with OA pathogenesis and identified the peroxisome proliferator-activated receptor-gamma co-activator 1-alpha (PGC1α) as a potent regulator. The expression level of PGC1α was significantly decreased under OA conditions, and knockdown of PGC1α dramatically elevated the cartilage degradation by upregulating cartilage degrading enzymes and apoptotic cell death. Interestingly, the knockdown of PGC1α activated the parkin RBR E3 ubiquitin protein ligase (PRKN)-independent selective mitochondria autophagy (mitophagy) pathway through the upregulation of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3). The overexpression of BNIP3 stimulated mitophagy and cartilage degradation by upregulating cartilage-degrading enzymes and chondrocyte death. We identified microRNA (miR)-126-5p as an upstream regulator for PGC1α and confirmed the direct binding between miR-126-5p and 3' untranslated region (UTR) of PGC1α. An in vivo OA mouse model induced by the destabilization of medial meniscus (DMM) surgery, and the delivery of antago-miR-126 via intra-articular injection significantly decreased cartilage degradation. In sum, the loss of PGC1α in chondrocytes due to upregulation of miR-126-5p during OA pathogenesis resulted in the activation of PRKN-independent mitophagy through the upregulation of BNIP3 and stimulated cartilage degradation and apoptotic death of chondrocytes. Therefore, the regulation of PGC1α:BNIP3 mitophagy axis could be of therapeutic benefit to cartilage-degrading diseases.

Loss of Acot12 contributes to NAFLD independent of lipolysis of adipose tissue.

In this study, we hypothesized that deregulation in the maintenance of the pool of coenzyme A (CoA) may play a crucial role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Specific deletion of Acot12 (Acot12-/-), the major acyl-CoA thioesterase, induced the accumulation of acetyl-CoA and resulted in the stimulation of de novo lipogenesis (DNL) and cholesterol biosynthesis in the liver. KEGG pathway analysis suggested PPARα signaling as the most significantly enriched pathway in Acot12-/- livers. Surprisingly, the exposure of Acot12-/- hepatocytes to fenofibrate significantly increased the accumulation of acetyl-CoA and resulted in the stimulation of cholesterol biosynthesis and DNL. Interaction analysis, including proximity-dependent biotin identification (BioID) analysis, suggested that ACOT12 may directly interact with vacuolar protein sorting-associated protein 33A (VPS33A) and play a role in vesicle-mediated cholesterol trafficking and the process of lysosomal degradation of cholesterol in hepatocytes. In summary, in this study, we found that ACOT12 deficiency is responsible for the pathogenesis of NAFLD through the accumulation of acetyl-CoA and the stimulation of DNL and cholesterol via activation of PPARα and inhibition of cholesterol trafficking.

NUDT7 Loss Promotes KrasG12D CRC Development.

Studies have suggested that dysregulation of peroxisomal lipid metabolism might play an important role in colorectal cancer (CRC) development. Here, we found that KrasG12D-driven CRC tumors demonstrate dysfunctional peroxisomal b-oxidation and identified Nudt7 (peroxisomal coenzyme A diphosphatase NUDT7) as one of responsible peroxisomal genes. In KrasG12D-driven CRC tumors, the expression level of Nudt7 was significantly decreased. Treatment of azoxymethane/dextran sulfate sodium (AOM/DSS) into Nudt7 knockout (Nudt7-/-) mice significantly induced lipid accumulation and the expression levels of CRC-related genes whereas xenografting of Nudt7-overexpressed LS-174T cells into mice significantly reduced lipid accumulation and the expression levels of CRC-related genes. Ingenuity pathway analysis of microarray using the colon of Nudt7-/- and Nudt7+/+ mice treated with AOM/DSS suggested Wnt signaling as one of activated signaling pathways in Nudt7-/- colons. Upregulated levels of ß-catenin were observed in the colons of KrasG12D and AOM/DSS-treated Nudt7-/- mice and downstream targets of ß-catenin such as Myc, Ccdn1, and Nos2, were also significantly increased in the colon of Nudt7-/- mice. We observed an increased level of palmitic acid in the colon of Nudt7-/- mice and attachment of palmitic acid-conjugated chitosan patch into the colon of mice induced the expression levels of b-catenin and CRC-related genes. Overall, our data reveal a novel role for peroxisomal NUDT7 in KrasG12D-driven CRC development.

Correction: TiO2 nanotube stimulate chondrogenic differentiation of limb mesenchymal cells by modulating focal activity.

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

Correction to: MicroRNA-9 regulates survival of chondroblasts and cartilage integrity by targeting protogenin.

Following publication of the original article [1], the authors reported that Figs. 3 and 6 are incorrect.

Correction to: Fis1 depletion in osteoarthritis impairs chondrocyte survival and peroxisomal and lysosomal function.

In Fig. 1C, the wrong si-control image was used by mistake.

Correction: HIF-1α:CRAT:miR-144-3p axis dysregulation promotes osteoarthritis chondrocyte apoptosis and VLCFA accumulation.

[This corrects the article DOI: 10.18632/oncotarget.20615.].

Dysregulation of the NUDT7-PGAM1 axis is responsible for chondrocyte death during osteoarthritis pathogenesis.

Osteoarthritis (OA) is the most common degenerative joint disease; however, its etiopathogenesis is not completely understood. Here we show a role for NUDT7 in OA pathogenesis. Knockdown of NUDT7 in normal human chondrocytes results in the disruption of lipid homeostasis. Moreover, Nudt7-/- mice display significant accumulation of lipids via peroxisomal dysfunction, upregulation of IL-1ß expression, and stimulation of apoptotic death of chondrocytes. Our genome-wide analysis reveals that NUDT7 knockout affects the glycolytic pathway, and we identify Pgam1 as a significantly altered gene. Consistent with the results obtained on the suppression of NUDT7, overexpression of PGAM1 in chondrocytes induces the accumulation of lipids, upregulation of IL-1ß expression, and apoptotic cell death. Furthermore, these negative actions of PGAM1 in maintaining cartilage homeostasis are reversed by the co-introduction of NUDT7. Our results suggest that NUDT7 could be a potential therapeutic target for controlling cartilage-degrading disorders.

HIF-1α:CRAT:miR-144-3p axis dysregulation promotes osteoarthritis chondrocyte apoptosis and VLCFA accumulation.

The functional role(s) of peroxisomes in osteoarthritis remains unclear. We demonstrated that peroxisomal dysfunction in osteoarthritis is responsible for very-long-chain fatty acid (VLCFA) accumulation. Through gene-profiling analyses, we identified CRAT as the gene responsible for this event. CRAT expression was suppressed in osteoarthritis chondrocytes, and its knockdown yielded pathological osteoarthritic characteristics, including VLCFA accumulation, apoptosis, autophagic inhibition, and mitochondrial dysfunction. Subsequent miRNA profiling revealed that peroxisomal dysfunction upregulates miR-144-3p, which overlapped with the osteoarthritis pathological characteristics observed upon CRAT knockdown. Moreover, knocking down HIF-1α in normal chondrocytes suppressed CRAT expression while stimulating miR-144-3p. Our data indicate that deregulation of a HIF-1a:CRAT:miR-144-3p axis impairs peroxisomal function during the pathogenesis of osteoarthritis.

Fis1 depletion in osteoarthritis impairs chondrocyte survival and peroxisomal and lysosomal function.

Cumulative evidence suggests the importance of organelle homeostasis in regulating metabolic functions in response to various cellular stresses. Particularly, the dynamism and health of the mitochondria-peroxisome network through fission and fusion are essential for cellular function; dysfunctional dynamism underlies the pathogenesis of several degenerative diseases including Parkinson's disease. Here, we investigated the role of Fis1 in cartilage homeostasis and its relevance to osteoarthritis (OA). We found that Fis1 is significantly suppressed in human OA chondrocytes compared to that in normal chondrocytes. Fis1 depletion through siRNA induced peroxisomal dysfunction. Moreover, Fis1 suppression altered miRNA profiles, especially those implicated in lysosomal regulation. Lysosomal destruction using LAMP-1-specific targeted nanorods or lysosomal dysfunction through chloroquine treatment resulted in enhanced chondrocyte apoptosis and/or suppression of autophagy. Accordingly, lysosomal activity and autophagy were severely decreased in OA chondrocytes despite abundant LAMP-1-positive organelles. Moreover, Fis1 morpholino-injected zebrafish embryos displayed lysosome accumulation, mitochondrial dysfunction, and peroxisome reduction. Collectively, these data suggest interconnected links among Fis1-modulated miRNA, lysosomes, and autophagy, which contributes to chondrocyte survival/apoptosis. This study represents the first functional study of Fis1 with its pathological relevance to OA. Our data suggest a new target for controlling cartilage-degenerative diseases, such as OA. KEY MESSAGE: Fis1 suppression in OA chondrocytes induces accumulation and inhibition of lysosomes. Fis1 suppression alters miRNAs, especially those implicated in lysosomal regulation. Lysosomal destruction results in chondrocyte apoptosis and suppression of autophagy. Fis1 depletion in zebrafish causes lysosome accumulation, mitochondrial dysfunction, and peroxisome reduction. This is the first functional study of Fis1 and its pathological relevance to OA.

PCGEM1 stimulates proliferation of osteoarthritic synoviocytes by acting as a sponge for miR-770.

Long non-coding RNAs (lncRNAs) have been reported to play important roles in cellular metabolism and development. Various diseases have been associated with aberrant expression of lncRNAs and the related dysregulation of mRNAs. An lncRNA profiling assay was carried out to identify the key lncRNA in osteoarthritic human synoviocytes; the results revealed that prostate cancer gene expression marker 1 (PCGEM1) was significantly overexpressed in osteoarthritic synoviocytes. Exogenous overexpression of PCGEM1 inhibited apoptosis, induced autophagy, and stimulated the proliferation of human synoviocytes. The increased expression of PCGEM1 in human synoviocytes also suppressed the expression of miR-770. Transfection of the miR-770 precursor resulted in reduced proliferation, and induced apoptosis of human synoviocytes. This effect of miR-770 expression was reversed by co-introduction of PCGEM1. Target validation showed a direct binding between PCGEM1 and miR-770. We demonstrate that PCGEM1 act as sponge lncRNA for miR-770 that regulates proliferation/apoptosis and autophagy, and suggest PCGEM1 as possible target for OA therapy.

MicroRNA-222 regulates MMP-13 via targeting HDAC-4 during osteoarthritis pathogenesis.

BACKGROUND: Even though increasing evidences on miRNA involvement in human pathological responses, the distinct roles and related mechanisms of miRNAs in the pathology of osteoarthritis (OA) are not yet fully understood. METHOD: RNA levels or protein levels of Apoptotic genes, HDACs, MMP-13, and miRNAs in human chondrocytes isolated from normal biopsy sample and OA cartilages were analyzed by real-time PCR or western blotting. Exogenous modulation of miR-222 level was performed using delivery of its specific precursor or specific inhibitor and target validation assay was applied to identify its potent target. In vivo study using DMM mice model was performed and assessed the degree of cartilage degradation. RESULTS: According to miRNA profiling, miR-222 was significantly down-regulated in OA chondrocytes. Over-expression of miR-222 significantly suppressed apoptotic death by down-regulating HDAC-4 and MMP-13 level. Moreover, 3'-UTR reporter assays showed that HDAC-4 is a direct target of miR-222. The treatment of chondrocytes with the HDAC inhibitor, trichostatin A (TSA), suppressed MMP-13 protein level and apoptosis, whereas the over-expression of HDAC-4 displayed opposite effects. The introduction of miR-222 into the cartilage of medial meniscus destabilized mice significantly reduced cartilage destruction and MMP-13 level. CONCLUSION: Taken together, our data suggest that miR-222 may be involved in cartilage destruction by targeting HDAC-4 and regulating MMP-13 level.