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Research article

Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes

Roman ZWICKY, Kathrin MÜNTENER, Mary B. GOLDRING, Antonio BAICI
Biochemical Journal Oct 01, 2002, 367 (1) 209-217; DOI: 10.1042/bj20020210
Roman ZWICKY
Institute of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland,
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Kathrin MÜNTENER
Institute of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland,
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Mary B. GOLDRING
Beth Israel Deaconess Medical Center, New England Baptist Bone & Joint Institute, Harvard Institutes of Medicine, Rm 246, 4 Blackfan Circle, Boston, MA 02115-5713, U.S.A.
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Antonio BAICI
Institute of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland,
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Abstract

Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies.

  • cartilage
  • cysteine peptidase
  • osteoarthritis
  • proteolysis
  • RNA interference

Footnotes

  • Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; dsRNA, double-stranded RNA; eIF-2, eukaryotic initiation factor-2; FCS, fetal calf serum; RNAi, RNA interference; RT, reverse transcriptase; siRNA, small interfering RNA; ssDNA, single-stranded DNA; 5′-UTR, 5′-untranslated region.

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October 2002

Volume: 367 Issue: 1

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Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes
Roman ZWICKY, Kathrin MÜNTENER, Mary B. GOLDRING, Antonio BAICI
Biochemical Journal Oct 2002, 367 (1) 209-217; DOI: 10.1042/bj20020210
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Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes
Roman ZWICKY, Kathrin MÜNTENER, Mary B. GOLDRING, Antonio BAICI
Biochemical Journal Oct 2002, 367 (1) 209-217; DOI: 10.1042/bj20020210

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Keywords

cartilage
cysteine peptidase
osteoarthritis
proteolysis
RNA interference

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