HDAC4 represses ER stress induced chondrocyte apoptosis by inhibiting ATF4 and attenuates cartilage degeneration in an osteoarthritis rat model.

The present study evaluated whether the lack of histone deacetylase 4 (HDAC4) increases endoplasmic reticulum stress-induced chondrocyte apoptosis by releasing activating transcription factor 4 (ATF4) in human osteoarthritis (OA) cartilage degeneration. Articular cartilage from the tibial plateau was obtained from patients with OA during total knee replacement. Cartilage extracted from severely damaged regions was classified as degraded cartilage, and cartilage extracted from a relatively smooth region was classified as preserved cartilage. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to detect chondrocyte apoptosis. HDAC4, ATF4, and C/EBP homologous protein (CHOP) expression levels were measured using immunohistochemistry staining and real-time quantitative PCR. Chondrocytes were transfected with HDAC4 or HDAC4 siRNA for 24 h and stimulated with 300 µM H2O2 for 12 h. The chondrocyte apoptosis was measured using flow cytometry. ATF4, CHOP, and caspase 12 expression levels were measured using real-time quantitative PCR and western blotting. Male Sprague-Dawley rats (n = 15) were randomly divided into three groups and transduced with different vectors: ACLT + Ad-GFP, ACLT + Ad-HDAC4-GFP, and sham + Ad-GFP. All rats received intra-articular injections 48 h after the operation and every three weeks thereafter. Cartilage damage was assessed using Safranin O staining and quantified using the Osteoarthritis Research Society International score. ATF4, CHOP, and collagen II expression were detected using immunohistochemistry, and chondrocyte apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The chondrocyte apoptosis was higher in degraded cartilage than in preserved cartilage. HDAC4 expression was lower in degraded cartilage than in preserved cartilage. ATF4 and CHOP expression was increased in degraded cartilage. Upregulation of HDAC4 in chondrocytes decreased the expression of ATF4, while the expression of ATF4 was increased after downregulation of HDAC4. Upregulation of HDAC4 decreased the chondrocyte apoptosis under endoplasmic reticulum stress, and chondrocyte apoptosis was increased after downregulation of HDAC4. In a rat anterior cruciate ligament transection OA model, adenovirus-mediated transduction of HDAC4 was administered by intra-articular injection. We detected a stronger Safranin O staining with lower Osteoarthritis Research Society International scores, lower ATF4 and CHOP production, stronger collagen II expression, and lower chondrocyte apoptosis in rats treated with Ad-HDAC4. The lack of HDAC4 expression partially contributes to increased ATF4, CHOP, and endoplasmic reticulum stress-induced chondrocyte apoptosis in OA pathogenesis. HDAC4 attenuates cartilage damage by repressing ATF4-CHOP signaling-induced chondrocyte apoptosis in a rat model of OA.

Gu X ，Li F ，Che X ，Wei X ，Li P ... -  《BMC MUSCULOSKELETAL DISORDERS》

Adenovirus-mediated transduction with Histone Deacetylase 4 ameliorates disease progression in an osteoarthritis rat model.

Downregulation of histone deacetylase-4 (HDAC4) contributes to cartilage degeneration in osteoarthritis (OA) because it promotes upregulation of runt-related transcription factor-2 (Runx-2) and osteoarthritis-related genes. The effect of HDAC4 upregulation on cartilage damage in OA remains unknown. Rat chondrocytes were infected with Ad-GFP or Ad-HDAC4-GFP for 48 h, stimulated with interleukin-1β (IL-1β, 10 ng/mL) for 24 h, and then harvested for RT-qPCR. Male Sprague-Dawley rats in 3 groups were given anterior cruciate ligament transection (ACLT) or sham operation, and knee injections with different adenovirus (Ad) vectors at 48 h after surgery and every 3 weeks thereafter: ACLT+Ad-GFP (n = 17); ACLT+Ad-HDAC4-GFP (n = 20); and sham+Ad-GFP (n = 15). Three ACLT-Ad-HDAC4-GFP rats were sacrificed at different times to examine the expression of HDAC4. Two ACLT-Ad-GFP rats and two ACLT-Ad-HDAC4-GFP rats were euthanized at week-2; articular cartilage was harvested and expression of HDAC4 was determined by RT-qPCR. All other rats were euthanized at week-8. Cartilage damage and OA progression was assessed using radiography, fluorescence molecular tomography (FMT), histology, immunohistochemistry (IHC), ELISA, and RT-qPCR. Overexpression of HDAC4 in chondrocytes stimulated by IL-1β reduced the levels of Runx-2, MMP-13, and Collagen X, but increased the levels of Collagen II and Aggrecan. Upregulation of HDAC4 reduced osteophyte formation and cartilage damage, and increased articular cartilage anabolism. HDAC4 attenuated articular cartilage damage by repression of Runx-2, MMP-13, and collagen X and induction of collagen II and ACAN in this rat model of OA. Upregulation of HDAC4 may provide chondroprotection in OA patients.

Gu XD ，Wei L ，Li PC ，Che XD ，Zhao RP ，Han PF ，Lu JG ，Wei XC ... -  《-》

HDAC4 mutant represses chondrocyte hypertrophy by locating in the nucleus and attenuates disease progression of posttraumatic osteoarthritis.

The aim of this study was to evaluate whether histone deacetylase 4 S246/467/632A mutant (m-HDAC4) has enhanced function at histone deacetylase 4 (HDAC4) to attenuate cartilage degeneration in a rat model of osteoarthritis (OA). Chondrocytes were infected with Ad-m-HDAC4-GFP or Ad-HDAC4-GFP for 24 h, incubated with interleukin-1β (IL-1β 10 ng/mL) for 24 h, and then measured by RT-qPCR. Male Sprague-Dawley rats (n = 48) were randomly divided into four groups and transduced with different vectors: ACLT/Ad-GFP, ACLT/Ad-HDAC4-GFP, ACLT/Ad-m-HDAC4-GFP, and sham/Ad-GFP. All rats received intra-articular injections 48 h after the operation and every 3 weeks thereafter. Cartilage damage was assessed using radiography and Safranin O staining and quantified using the OARSI score. The hypertrophic and anabolic molecules were detected by immunohistochemistry and RT-qPCR. M-HDAC4 decreased the expression levels of Runx-2, Mmp-13, and Col 10a1, but increased the levels of Col 2a1 and ACAN more effectively than HDAC4 in the IL-1β-induced chondrocyte OA model; upregulation of HDAC4 and m-HDAC4 in the rat OA model suppressed Runx-2 and MMP-13 production, and enhanced Col 2a1 and ACAN synthesis. Stronger Safranin O staining was detected in rats treated with m-HDAC4 than in those treated with HDAC4. The resulting OARSI scores were lower in the Ad-m-HDAC4 group (5.80 ± 0.45) than in the Ad-HDAC4 group (9.67 ± 1.83, P = 0.045). The OARSI scores were highest in rat knees that underwent ACLT treated with Ad-GFP control adenovirus vector (14.93 ± 2.14, P = 0.019 compared with Ad-HDAC4 group; P = 0.003 compared with Ad-m-HDAC4 group). Lower Runx-2 and MMP-13 production, and stronger Col 2a1 and ACAN synthesis were detected in rats treated with m-HDAC4 than in those treated with HDAC4. M-HDAC4 repressed chondrocyte hypertrophy and induced chondrocyte anabolism in the nucleus. M-HDAC4 was more effective in attenuating articular cartilage damage than HDAC4.

Gu X ，Li F ，Gao Y ，Che X ，Li P ... -  《BMC MUSCULOSKELETAL DISORDERS》

Esculin suppresses the PERK-eIF2α-CHOP pathway by enhancing SIRT1 expression in oxidative stress-induced rat chondrocytes, mitigating osteoarthritis progression in a rat model.

Osteoarthritis (OA) is a degenerative disease characterized by the gradual degeneration of chondrocytes, involving endoplasmic reticulum (ER) stress. Esculin is a natural compound with antioxidant, anti-inflammatory and anti-tumor properties. However, its impact on ER stress in OA therapy has not been thoroughly investigated. We aim to determine the efficiency of Esculin in OA treatment and its underlying mechanism. We utilized the tert-butyl hydroperoxide (TBHP) to establish OA model in chondrocytes. The expression of SIRT1, PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins were detected by Western blot and Real-time PCR. The apoptosis was evaluated by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. X-ray imaging, Hematoxylin & Eosin staining, Safranin O staining and immunohistochemistry were used to assess the pharmacological effects of Esculin in the anterior cruciate ligament transection (ACLT) rat OA model. Esculin downregulated the expression of PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins, while upregulated the expression of SIRT1 and Bcl2 in the TBHP-induced OA model in vitro. It was coincident with the results of TUNEL staining and flow cytometry. We further confirmed the protective effect of Esculin in the rat ACLT-related model. Our results suggest the potential therapeutic value of Esculin on osteoarthritis. It probably inhibits the PERK-eIF2α-ATF4-CHOP pathway by upregulating SIRT1, thereby mitigating endoplasmic reticulum stress and protecting chondrocytes from apoptosis.

Cheng Z ，Liu Z ，Liu C ，Yang A ，Miao H ，Bai X ... -  《-》

The N-terminal fragment of histone deacetylase 4 (1-669aa) promotes chondrocyte apoptosis via the p53-dependent endoplasmic reticulum stress pathway.

Exogenous administration of the histone deacetylation 4 (HDAC4) protein can effectively delay osteoarthritis (OA) progression. However, HDAC4 is unstable and easily degrades into N-terminal (HDAC4-NT) and C-terminal fragments, and the HDAC4-NT can exert biological effects, but little is known about its role in chondrocytes and cartilage. Thus, the roles of HDAC4-NT fragments (1-289aa, 1-326aa and 1-669aa) in chondrocytes and cartilage were evaluated via real-time cell analysis (RTCA), safranin O staining, Sirius Red staining and nanoindentation. Molecular mechanisms were profiled via whole-transcriptome sequencing (RNA-seq) and verified in vitro and in vivo by a live cell real-time monitoring system, flow cytometry, western blotting and immunohistochemistry. The results showed that 1-669aa induced chondrocyte death and cartilage injury significantly, and the differentially expressed genes (DEGs) were enriched mainly in the apoptotic term and p53 signalling pathway. The validation experiments showed that 1-669aa induced chondrocyte apoptosis via the endoplasmic reticulum stress (ERS) pathway, and up-regulated p53 expression was essential for this process. Thus, we concluded that the HDAC4-NT fragment 1-669aa induces chondrocyte apoptosis via the p53-dependent ERS pathway, suggesting that in addition to overexpressing HDAC4, preventing it from degradation may be a new strategy for the treatment of OA.

Guo L ，Zhuo X ，Lu C ，Guo H ，Chen Z ，Wu G ，Liu F ，Wei X ，Rong X ，Li P ... -  《-》