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Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The FH gene encodes the fumarate hydratase of the Krebs cycle and functions as a homotetramer to catalyze the hydration of fumarate to malate. Mutations in FH result in uterine leiomyomas, a rare autosomal dominant inherited metabolic disease. However, how FH mutations result in this disease is poorly understood. Here, the FH mutation c.557G>A (p.S186N) was identified in a family with uterine leiomyomas phenotype. A series of studies were performed to confirm the pathogenicity of this mutation. Results showed that the FH mutant exhibited significantly lower fumarase enzyme activity and increased the fumarates level compared with the wildtype, which might be due to the impaired homotetramer formation in the native gel electrophoresis. Interestingly, the immunofluorescence study revealed that the overexpressed FH mutant exhibited puncta structures compared with the evenly expressed FH wildtype in cytoplasm suggesting that the altered amino acid might result in dysfunctional proteins which were accumulated to reduce its cytotoxicity. Importantly, the cells overexpressing the FH mutant exhibited higher proliferation and extracellular acidification rate value (ECAR) which might be caused by the upregulated HIF-1α indicating the tumor phenotype. Notably, phospho-mTOR was significantly increased and autophagy was inhibited in the FH mutant overexpression cells compared with the wildtype. Our work provides new insight into the FH mutation c.557G>A (p.S186N) underlies uterine leiomyomas and important information for accurate genetic counseling and clinical diagnosis of the disease. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) methylation is one of the most common internal modifications in eukaryotic messenger RNA occurring on N6 nitrogen of adenosine. However, the roles of m6A in temporomandibular joint osteoarthritis (TMJ OA) are still elusive. Here, we investigate the function and mechanism of methyltransferase-like 3 (Mettl3) in chondrocytes in inflammation. We found that the expression of Mettl3 decreased both in vivo TMJ OA mice and in vitro inflammatory stimulation. Functionally, loss and gain studies illustrated that Mettl3 inhibited the apoptosis and autophagy of chondrocytes induced by TNF-α stimulation in vitro. Mettl3 inhibitor, S-adenosylhomocysteine (SAH) promoted the apoptosis and autophagy of chondrocytes with inflammation in vitro and aggravated the degeneration of chondrocytes and subchondral bone in monosodium iodoacetate (MIA) induced TMJ OA mice in vivo. Mechanistically, the bioinformatics analysis, m6A-RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) were used to identify that Bcl2 mRNA was the downstream target of Mettl3 for m6A modification. Furthermore, the results revealed that Yth m6A RNA binding protein 1 (Ythdf1) mediated the stability of Bcl2 mRNA catalyzed by Mettl3. Co-immunoprecipitation (Co-IP) showed that Bcl2 protein interacted with Beclin1 protein in chondrocytes induced by TNF-α stimulation. In conclusion, our findings identify that Mettl3 inhibits the apoptosis and autophagy of chondrocytes in inflammation through m6A/Ythdf1/Bcl2 signal axis which provides promising therapeutic strategy for TMJ OA. • Mettl3 positively regulates Bcl2 to inhibit the apoptosis and autophagy of chondrocytes induced by TNF-α. • Ythdf1 mediates Bcl2 mRNA stability by an m6A-dependent manner in chondrocytes induced by TNF-α. • SAH restrains Mettl3 to play a role in chondrocytes with inflammation both in vivo and in vitro. [ABSTRACT FROM AUTHOR]