Abstract
Objective
Autoantibodies to DNA topoisomerase I (topo I) are associated with diffuse systemic sclerosis (SSc), appear to be antigen driven, and may be triggered by cryptic epitopes exposed during in vivo topo I fragmentation. These autoantibodies recognize topo I and fragments of this autoantigen generated during apoptosis and necrosis. We undertook this study to determine whether lysosomal cathepsins are involved in topo I fragmentation during necrosis.
Methods
Topo I cleavage during necrosis was assessed by immunoblotting of lysates from L929 fibroblasts exposed to tumor necrosis factor α (TNFα) and the broad caspase inhibitor Z‐VAD‐FMK, and by immunoblotting of lysates from endothelial cells treated with HgCl~2~. Purified topo I and L929 nuclei were incubated with cathepsins B, D, G, H, and L, and topo I cleavage was detected by immunoblotting. The intracellular localization of cathepsin L activity and topo I in necrotic cells was examined using fluorescence microscopy.
Results
Treatment of L929 cells with TNFα and Z‐VAD‐FMK induced caspase‐independent cell death with necrotic morphology. This cell death involved topo I cleavage into fragments of approximately 70 kd and 45 kd. This cleavage profile was reproduced in vitro by cathepsins L and H and was inhibited by the cathepsin L inhibitor Z‐FY‐CHO. During necrosis, cathepsin L activity diffused from lysosomes into the cytoplasm and nucleus, whereas topo I partially relocalized to the cytoplasm. Z‐FY‐CHO delayed necrosis and partially blocked topo I cleavage. The topo I cleavage fragments were also detected in necrotic endothelial cells and recognized by SSc sera containing anti–topo I antibodies.
Conclusion
These results implicate cathepsins, particularly cathepsin L, in the cleavage of topo I during necrosis. This cleavage may generate potentially immunogenic fragments that could trigger anti–topo I immune responses in SSc.