Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

Arthritis Rheum. 2005 Jul;52(7):2133-45. doi: 10.1002/art.21147.

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 alpha (TNFalpha) and the broad caspase inhibitor Z-VAD-FMK, and by immunoblotting of lysates from endothelial cells treated with HgCl2. 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 TNFalpha 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology
  • Animals
  • Cathepsins / metabolism*
  • Cattle
  • Cell Nucleus / drug effects
  • Cell Nucleus / enzymology
  • DNA Topoisomerases, Type I / metabolism*
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / enzymology*
  • Endothelium, Vascular / pathology
  • Fibroblasts / drug effects
  • Fibroblasts / enzymology*
  • Fibroblasts / pathology
  • Humans
  • Immunoblotting
  • L Cells
  • Lysosomes / enzymology*
  • Mercuric Chloride / pharmacology
  • Mice
  • Necrosis / enzymology*
  • Scleroderma, Systemic / immunology
  • Scleroderma, Systemic / physiopathology
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Amino Acid Chloromethyl Ketones
  • Tumor Necrosis Factor-alpha
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
  • Mercuric Chloride
  • Cathepsins
  • DNA Topoisomerases, Type I