Autophagy In Human Chondrocytes Research Articles

Knockdown of SGK1 alleviates the IL-1β-induced chondrocyte anabolic and catabolic imbalance by activating FoxO1-mediated autophagy in human chondrocytes.

Osteoarthritis (OA) is a common joint disease characterized by the progressive degeneration of articular cartilage with no effective treatment methods available. Cartilage degeneration is closely related to an anabolic and catabolic imbalance in chondrocytes, and accumulating evidence has revealed that autophagy is a crucial protective mechanism that maintains the balance of anabolic and catabolic activities. Therefore, studies aiming to identify additional genes that regulate autophagy as a promising therapeutic strategy for OA are needed. In this study, we analyzed the GSE113825 datasets from Gene Expression Omnibus and validated that serum- and glucocorticoid-regulated kinase 1 (SGK1) was upregulated in OA cartilage. Based on the results from loss-of-function studies, SGK1 silencing promoted the deposition of glycosaminoglycans in interleukin 1 beta (IL-1β)-treated chondrocytes, and significantly alleviated IL-1β-induced downregulation of Collagen II and Aggrecan, as well as the upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 5 and matrix metalloproteinase-13. Furthermore, SGK1 knockdown reversed the IL-1β-induced chondrocyte anabolic and catabolic imbalance by activating autophagy. Moreover, SGK1 directly bound to forkhead box protein O1 (FoxO1) and increased its phosphorylation, which in turn resulted in its translocation from the nucleus. The decreased FoxO1 levels led to a decrease in LC3-I/LC3-II conversion and Beclin-1 levels, subsequently inhibiting autophagosome formation and increasing P62 levels, thus indicating a downregulation of autophagy. Taken together, we identified a critical role of SGK1 in the IL-1β-induced chondrocyte anabolic and catabolic imbalance, which may represent a potential novel therapeutic target for OA.

Comparison of Apoptosis and Autophagy in Human Chondrocytes Induced by the T-2 and HT-2 Toxins.

In this report, we have investigated the apoptosis and autophagy of chondrocytes induced by the T-2 and HT-2 toxins. The viability of chondrocytes was measured by the MTT assay. Malondialdehyde (MDA) and superoxide dismutase (SOD) kits were used to measure the oxidative stress of chondrocytes. The apoptosis of chondrocytes was measured using flow cytometry. Hoechst 33258 and MDC staining agents were introduced to analyze apoptosis and autophagy induction in chondrocytes, respectively. Protein expression of Bax, caspase-9, caspase-3, and Beclin1 was examined by western blotting analysis. The T-2 and HT-2 toxins significantly decreased the viability of chondrocytes in a time-dependent manner. The level of oxidative stress in chondrocytes induced by the T-2 toxin was significantly higher when compared with that of the HT-2 toxin. The apoptosis rate of chondrocytes induced by the T-2 toxin increased from 3.26 ± 1.03%, 18.38 ± 1.28%, 34.5 ± 1.40% to 49.67 ± 5.31%, whereas apoptosis rate of chondrocytes induced by the HT-2 toxin increased from 3.82 ± 1.03%, 11.61 ± 1.27%, 25.72 ± 2.95% to 36.28 ± 2.81% in 48 h incubation time. Hoechst 33258 staining confirmed that apoptosis of chondrocytes induced by the T-2 toxin was significantly higher than that observed when the chondrocytes were incubated with the HT-2 toxin. MDC staining revealed that the autophagy rate of chondrocytes induced by the T-2 toxin increased from 6.38% to 63.02%, whereas this rate induced by the HT-2 toxin changed from 6.08% to 53.33%. The expression levels of apoptosis and autophagy related proteins, Bax, caspase-9, caspase-3, and Beclin1 in chondrocytes induced by the T-2 toxin were significantly higher when compared with those levels induced by the HT-2 toxin.

201 - Butein: A Plant Derived Small Molecule Activates Autophagy and Suppresses IL-1β Induced Oxidative Stress, Mitochondrial Dysfunction and Inflammatory Response in Human Chondrocytes

Osteoarthritis (OA) is the most common joint disease worldwide and is characterized by pain and stiffness due to the progressive loss of articular cartilage. There is increasing evidence that support a role of high levels of oxidative stress, increased mitochondrial dysfunction, inflammation and decline in autophagy in the pathogenesis of OA. Currently there is no disease modifying drug available for the treatment of OA. Autophagy is a cellular defense mechanism against stress-induced accumulation of protein aggregates and dysfunctional subcellular organelles. Here, we studied the effect of a plant derived small molecule, Butein, on human chondrocytes and cartilage explants (isolated from donors with and without known history of OA) against IL-1β induced oxidative stress, mitochondrial dysfunction and expression of inflammatory mediators (Cyclooxygenase 2 (COX-2), inducible Nitric Oxide Synthase (iNOS) and interleukin-6 (IL-6) in vitro . Butein blocked the IL-1β induced disruption of mitochondrial membrane potential and mitochondrial dysfunction and suppressed the generation of total and mitochondrial ROS and RNS levels in normal and OA chondrocytes. Additionally, IL-1β induced expression of iNOS, COX-2 and IL-6 was also inhibited by Butein in both normal and OA chondrocytes and cartilage explants. Butein activated AMPKα and inhibited mTOR activity to activate autophagy in human chondrocytes as evidenced by the increased levels of LC3-II, decreased levels of p62/SQSTM1 and increased numbers of autophagosomes. Activation of autophagy was critical to the observed chondroprotective effects as inhibition of autophagy abrogated the protective effects of Butein. These results suggest that Butein may be developed as a therapeutic agent for the prevention and or treatment of OA.

MicroRNA-155 suppresses autophagy in chondrocytes by modulating expression of autophagy proteins

Autophagy dysfunction has been reported in osteoarthritis (OA) cartilage. The objective of this study was to investigate the role of microRNA-155 (miR-155), which is overexpressed in OA, in the regulation of autophagy in human chondrocytes. Rapamycin (50nM) and 2-deoxyglucose (2-DG) (5mM) were used to stimulate autophagy in primary human articular chondrocytes and in the T/C28a2 human chondrocyte cell line. Cells were transfected with LNA GapmeR or mimic specific for miR-155 and autophagy flux was assessed by LC3 western blotting and by Cyto-ID(®) dye quantification in autophagic vacuoles. Expression of predicted miR-155 targets in the autophagy pathway were analyzed by real-time PCR and western blotting. Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy regulators including Ulk1, FoxO3, Atg14, Atg5, Atg3, Gabarapl1, and Map1lc3. MiR-155 is an inhibitor of autophagy in chondrocytes and contributes to the autophagy defects in OA.

Isosorbide dinitrate inhibits mechanical stress-induced cardiac hypertrophy and autophagy through downregulation of angiotensin II type 1 receptor.

Mechanical stress can induce cardiac hypertrophy and autophagy. Recently, it has been reported that nitric oxide donors inhibited autophagy in human chondrocytes. Therefore, the effect of isosorbide dinitrate (ISDN) on cardiac hypertrophy and autophagy induced by mechanical stress was investigated in this study. A 48-hour mechanical stretch and a 4-week transverse aortic constriction were performed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, before the assessment of myocardial autophagy using LC3b-II. ISDN was found to significantly reduce mechanical stretch-induced LC3b-II upregulation. Furthermore, mechanical stress was shown to upregulate angiotensin II (AngII) type 1 (AT1) receptor expression in both cultured cardiomyocytes and in mouse hearts, whereas ISDN was demonstrated to significantly suppress the upregulation of the AT1 receptor. It was concluded that ISDN could inhibit mechanical stress-induced cardiac hypertrophy and autophagy through the downregulation of AT1 receptor expression.

Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis.

IntroductionRecent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.MethodsDestabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).ResultsIntra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.ConclusionThese results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

Hyperglycemia-like culture conditions induce IL-1B and TNF-α expression and impair autophagy in human chondrocytes

Hyperglycemia-like culture conditions induce IL-1B and TNF-α expression and impair autophagy in human chondrocytes

A5.1 Culture OF human chondrocytes in high glucose induces inflammatory markers and impairs autophagy

Background and ObjectivesAccumulating evidence indicates that Diabetes Mellitus is an independent risk factor for severe osteoarthritis (OA). Understanding the mechanisms involved is essential for designing preventive strategies and targeted therapies...

Nitric oxide inhibits autophagy in human chondrocytes: Comment on the article by Sasaki et al

Nitric oxide inhibits autophagy in human chondrocytes: Comment on the article by Sasaki et al

Autophagy modulates osteoarthritis‐related gene expression in human chondrocytes

Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism. The purpose of this study was to elucidate the role of autophagy in human chondrocytes and pathophysiology of osteoarthritis (OA). Autophagy in articular cartilage and primary chondrocytes was assessed using antibodies for the autophagy markers light chain 3 and beclin 1. The states of autophagy under catabolic and nutritional stresses were examined. We also examined the effects of inhibition or induction of autophagy under stimulation with interleukin-1β. Autophagy was inhibited by small interfering RNA targeting ATG5, and autophagy was induced by rapamycin. The effects of inhibition or induction of autophagy were examined by real-time polymerase chain reaction for aggrecan, COL2A1, MMP13, and ADAMTS5 messenger RNA. To further examine the mechanism of autophagy regulation in OA human chondrocytes, we investigated whether autophagy modulates apoptosis and reactive oxygen species (ROS). Autophagy was increased in OA chondrocytes and cartilage. Catabolic and nutritional stresses increased autophagy. In addition, the inhibition of autophagy caused OA-like gene expression changes, while the induction of autophagy prevented them. Furthermore, the inhibition of autophagy increased the amount of cleaved poly(ADP-ribose) polymerase and cleaved caspase 9, while the induction of autophagy inhibited these increases. ROS activity was also decreased by induction of autophagy. These observations suggested that increased autophagy is an adaptive response to protect cells from stresses, and that autophagy regulates OA-like gene expression changes through the modulation of apoptosis and ROS. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA.

Paper # 93: Autophagy Modulates Osteoarthritis Related Gene Expressions in Human Chondrocytes

We investigated the role of autophagy in human chondrocytes. Autophagy was increased in human osteoarthritic cartilage. In addition, the inhibition of autophagy enhanced OA-like gene expression changes induced by IL-1β while the induction of autophagy prevented them. These observations suggested autophagy might play protective roles in human chondrocytes.