Abstract
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.
Highlights
Osteoarthritis (OA) is characterized by the destruction of extracellular matrix and the loss of chondrocyte function [1]
To examine whether the inhibition of CK2 activity modulates the extent of tumor necrosis factor (TNF)-amediated chondrocyte death, cells were co-treated with one of three CK2 inhibitors and TNF-a
Cells that were co-treated with dichlorobenzimidazol riboside (DRB) and TNF-a failed to show ladder-like DNA fragments from their genomic DNA on a standard agarose gel, Pulsed-field gel electrophoresis (PFGE) revealed the disintegration of nuclear DNA into giant fragments of 1-2 Mbp and high molecular-weight fragments of 100–1000 Kbp (Fig. 2D)
Summary
Osteoarthritis (OA) is characterized by the destruction of extracellular matrix and the loss of chondrocyte function [1]. Chondrocyte depletion was found to be a persistent and important event in OA. Mechanical injury, loss of extracellular matrix, loss of growth factors or excessive reactive oxygen species can induce chondrocyte depletion [2]. Because articular chondrocytes are solely responsible for the production and maintenance of the extracellular matrix, chondrocyte depletion is implicated in cartilage degeneration, which pertains to OA pathogenesis [3,4]. An important question regarding the extent of the contribution of apoptotic cell death to chondrocyte depletion during OA progression remains unresolved. Several studies support the idea that another type of cell death, necrosis, can be involved in chondrocyte death during OA progression [2]
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