Plant Homeodomain Finger Protein 23 Inhibits Autophagy and Promotes Apoptosis of Chondrocytes in Osteoarthritis
Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by pain, stiffness, reduced mobility, and joint deformities, often leading to disability. It affects approximately 240 million people globally, making it a significant public health concern and economic burden. The pathogenesis of OA involves multiple factors, including mechanical stress, genetic predisposition, obesity, and aging, with aging being the most significant risk factor. Autophagy, a lysosome-dependent degradation process, plays a crucial role in maintaining cellular homeostasis and is implicated in the pathogenesis of many aging-related diseases, including OA. Chondrocytes, the only cell type present in cartilage, are critical for maintaining cartilage integrity. When external stimuli exceed a certain threshold, chondrocyte homeostasis is disrupted, leading to apoptosis and cartilage matrix degradation. Therefore, understanding the mechanisms that regulate chondrocyte survival and function is essential for preventing and delaying OA progression.
Plant Homeodomain Finger Protein 23 (PHF23) is a novel autophagy inhibitor gene that has not been extensively studied in the context of orthopedics. This study aimed to investigate the expression of PHF23 in articular cartilage and synovial tissue and analyze its relationship with chondrocyte autophagy in OA. The findings suggest that PHF23 expression is significantly increased in OA cartilage and synovium, and it is induced by interleukin-1b (IL-1b) through inflammatory stress. PHF23 suppresses autophagy in chondrocytes and accelerates apoptosis, highlighting its potential role in OA pathogenesis.
The study employed immunohistochemical staining and Western blot analysis to examine the expression of PHF23 in cartilage of different Outbridge grades and synovial tissue from OA patients and healthy controls. The results revealed that PHF23 expression was significantly increased in high-grade cartilage and synovial tissue of OA patients compared to normal controls. Specifically, the immunohistochemistry results showed that PHF23 expression was elevated in the synovial tissue of OA patients compared to normal synovial tissue from patients with acute meniscus tears. Similarly, PHF23 expression was greater in Outbridge grade III cartilage than in Outbridge grade I cartilage. Western blot analysis confirmed these findings, showing a significant increase in PHF23 expression in Outbridge grade III cartilage and OA synovial tissue compared to Outbridge grade I cartilage and normal synovial tissue.
To further understand the role of PHF23 in chondrocyte autophagy and apoptosis, the study utilized IL-1b to induce an OA-like condition in normal human chondrocytes. The results demonstrated that IL-1b-induced PHF23 expression increased over time, with the ratio of PHF23 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) being 0.31 ± 0.05, 0.56 ± 0.02, and 0.79 ± 0.05 for IL-1b stimulation at 12, 24, and 48 hours, respectively. The expression levels of autophagy-related proteins, including light chain 3B-I (LC3B-I), LC3B-II, and P62, were also examined. The ratios of LC3B-II/LC3B-I were 2.13 ± 0.04, 1.83 ± 0.03, and 1.76 ± 0.04 for IL-1b stimulation at 12, 24, and 48 hours, respectively. The ratios of P62/GAPDH were 0.07 ± 0.01, 0.14 ± 0.02, and 0.20 ± 0.03 for IL-1b stimulation at 12, 24, and 48 hours, respectively. These findings indicate that autophagy levels in chondrocytes increased significantly in the early stage of IL-1b stimulation (12 hours) but decreased gradually over time, while PHF23 expression increased significantly.
The study also constructed a PHF23 gene knockdown model using small interfering RNA (siRNA) to investigate the impact of PHF23 on autophagy and apoptosis in IL-1b-induced OA-like chondrocytes. The knockdown efficiency of PHF23 siRNA was confirmed by Western blot, showing a significant reduction in PHF23 expression. After PHF23 knockdown, the expression of autophagy-related proteins LC3B-II and Beclin-1 (BECN1) increased, indicating that PHF23 specifically inhibits autophagy in chondrocytes. Conversely, the expression of cleaved-caspase-3, an apoptosis-related protein, decreased in PHF23 knockdown chondrocytes compared to the control group, suggesting that PHF23 alleviates IL-1b-induced apoptosis in chondrocytes.
To further explore the relationship between autophagy and PHF23 expression, the study treated normal human chondrocytes with rapamycin, an autophagy enhancer, and 3-methyladenine (3-MA), an autophagy inhibitor, before IL-1b stimulation. The results showed that pre-treatment with rapamycin reduced IL-1b-induced chondrocyte apoptosis and mitochondrial damage, while 3-MA aggravated these effects. However, the expression of PHF23 was not affected by pre-treatment with rapamycin or 3-MA, indicating that PHF23 might influence OA chondrocyte apoptosis and mitochondrial damage through autophagy but is not affected by apoptosis and mitochondrial damage.
The study also examined the effect of rapamycin-induced high autophagy levels on PHF23 expression in normal chondrocytes. The results showed that rapamycin-induced high autophagy levels did not alter PHF23 expression, with the ratios of PHF23/GAPDH being 0.89 ± 0.02, 0.84 ± 0.06, and 0.80 ± 0.02 for rapamycin treatment at 12, 24, and 48 hours, respectively. This suggests that PHF23 can affect autophagy levels adversely without being influenced by autophagy levels.
In summary, the study provides evidence that PHF23 expression is significantly increased in human OA cartilage and synovium and is induced by IL-1b through inflammatory stress. PHF23 suppresses autophagy in chondrocytes and accelerates apoptosis, highlighting its potential role in OA pathogenesis. The findings suggest that targeting PHF23 could be a potential therapeutic strategy for preventing and treating OA. Further studies are needed to explore the molecular mechanisms underlying PHF23’s role in autophagy and apoptosis in chondrocytes and its potential as a therapeutic target in OA.
doi.org/10.1097/CM9.0000000000000402
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