Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Clinical Relevance: Caspase inhibitors may play a therapeutic role
in slowing disc degeneration that is due to inappropriate or excessive
apoptosis of intervertebral disc cells.