Anti-Apoptotic Effects of Caspase Inhibitors on Rat Intervertebral Disc Cells

Abstract

Apoptosis is thought to be a critical component of disc degeneration. Two main pathways of Fas-mediated apoptosis have been identified: Type I, which is the death-inducing signaling complex pathway, and Type II, which is the mitochondrial pathway. The apoptotic pathway for anulus fibrosus cells, which is phenotypically different from that of nucleus pulposus cells, has not been elucidated to our knowledge. The ultimate initiators or executioners of apoptosis are caspases. There are also inhibitors of caspases, which have the potential of being used as anti-apoptotic therapeutic agents. We therefore undertook this study to determine (1) the apoptotic pathway of anulus fibrosus cells and (2) the anti-apoptotic potential of caspase inhibitors. Rat anulus fibrosus cells were isolated, cultured, and placed in either 0% (apoptosis-promoting condition) or 10% (normal control) fetal bovine serum. We identified and quantified the presence of apoptotic cell death, caspase activities, and loss of mitochondrial membrane potential. In addition, we examined the cells for the expression of Fas, procaspases, and cytochrome-c. Finally, we analyzed the degree of anti-apoptotic effects of caspase inhibitors on the cells in 1% fetal bovine serum. The percentage of apoptosis and Fas expression in the cells incubated in 0% fetal bovine serum were increased compared with those in the cells incubated in 10% fetal bovine serum (both p < 0.001). Caspase-8, 9, and 3 activities were increased and expression of procaspases was decreased in the 0% fetal bovine serum compared with those in the 10% fetal bovine serum (all p < 0.001). In contrast, the loss of mitochondrial membrane potential and cytochrome-c release into the cytosol were unchanged in the 0% fetal bovine serum. Pancaspase and caspase-8 inhibitors reduced apoptotic cell death (p < 0.001 and p < 0.05, respectively), but caspase-9 inhibitor did not reduce apoptotic cell death. Our results suggest that, unlike nucleus pulposus cells, anulus fibrosus cells are Fas Type-I cells, which undergo apoptosis through the death-inducing signaling complex. We also found that apoptosis of intervertebral disc cells can be attenuated by caspase inhibitors.