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Although alpha alpha B-crystallin is expressed in articular chondrocytes, little is known about its role in these cells. Protein kinase casein kinase 2 (CK2) inhibition induces articular chondrocyte death. The present study examines whether alpha B-crystallin exerts anti-apoptotic activity in articular chondrocytes. Primary rat articular chondrocytes were isolated from knee joint slices. Cells were treated with CK2 inhibitors with or without alpha B-crystallin si RNA. To examine whether the silencing of aB-crystallin sensitizes rat articular chondrocytes to CK2 inhibition induced apoptosis, we assessed apoptosis by performing viability assays, mitochondrial membrane potential measurements, flow cytometry, nuclear morphology observations, and western blot analysis. To investigate the mechanism by which aB-crystallin modulates the extent of CK2 inhibition-mediated chondrocyte death, we utilized confocal microscopy to observe the subcellular location of aB-crystallin and its phosphorylated forms and performed a co-immunoprecipitation assay to observe the interaction between aB-crystallin and CK2. Immunochemistry was employed to examine aB-crystallin expression in cartilage obtained from rats with experimentally induced osteoarthritis (OA). Our results demonstrated that silencing of aB-crystallin sensitized rat articular chondrocytes to CK2 inhibitor -induced apoptosis. Furthermore, CK2 inhibition modulated the expression and subcellular localization of aB-crystallin and its phosphorylated forms and dissociated aB-crystallin from CK2. The population of rat articular chondrocytes expressing aB-crystallin and its phosphorylated forms was reduced in an experimentally induced rat model of OA. In summary, aB-crystallin protects rat articular chondrocytes against CK2 inhibition -induced apoptosis. aB-crystallin may represent a suitable target for pharmacological interventions to prevent OA.

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.