Targeting cathepsin L (CL) by specific ribozymes decreases CL protein synthesis and cartilage destruction in rheumatoid arthritis

Abstract

The present study was undertaken to examine whether ribozymes cleaving specifically cathepsin L (CL) mRNA are able to decrease the synthesis of CL protease in rheumatoid arthritis synovial fibroblasts (RA-SF) and thereby reduce the invasiveness into cartilage both in vitro and in the SCID mouse coimplantation model of RA. Two different ribozymes that cleave CL mRNA specifically at positions 533 (RzCL533) and 790 (RzCL790) were generated. Using retroviral gene transfer, RA-SF were transduced with the ribozyme constructs or the empty vector. To examine the effect of the ribozymes on the mRNA level, quantitative analysis for CL mRNA was performed using real-time PCR. For evaluation on the protein level, ELISA using specific anti-CL antibodies was performed. In addition, transduced RA-SF were examined in vitro in a three-dimensional destruction assay evaluating their ability to degrade extracellular matrix produced by human chondrocytes. Matrix destruction was monitored by the release of soluble glycosaminoglycans (sGAG). Using the in vivo SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF and control cells were coimplanted with human cartilage for 60 days. After being killed, invasion of RA-SF into the cartilage was evaluated by using a semiquantitative score. Transduction of RA-SF with RzCL533 and RzCL790 ribozymes decreased significantly the expression of CL mRNA to 44% (range 25-62%) and 20% (range 1-43%), respectively, when compared to mocktransduced cells. The protein concentration of CL in the cell culture supernatants of transduced RA-SF was decreased from 16.0 ng/ml in the mock constructs to 4.1 and 8.2 ng/ml (mean), respectively. Using the in vitro cartilage destruction assay, the release of sGAG decreased to 46 and 60%, respectively, after 14 days when compared to mocktransduced cells. In the SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF revealed a significant lower cartilage invasion when compared to mock and untransduced cells. Using retroviral gene transfer, ribozymes cleaving CL mRNA inhibit specifically the synthesis of this matrix-degrading enzyme and reduce cartilage destruction in in vitro and in vivo models. Our study therefore suggests that ribozymes targeting CL could be a novel and efficient tool to inhibit joint destruction in RA.

Implication of clusterin in TNF-α response of rheumatoid synovitis: lesson from in vitro knock-down of clusterin in human synovial fibroblast cells

“…The upregulation of cathepsin L1 and cadherin 12 have been noted. Cathepsin L is known to be deleterious for cartilage in RA (15) and is clearly upregulated after inhibition of CLU, as already published (5). The family of cadherins is involved in cell-to-cell interaction and signaling, and cadherin 11 participates in the formation of layers in vitro; moreover, synovial cadherin-11 determines the behavior of synovial cells in their proinflammatory and destructive tissue response in inflammatory arthritis (9).…”

Cigarette smoke condensate increases cathepsin-mediated invasiveness of oral carcinoma cells

“…DNA primer sequences for qRT-PCR were obtained from the cited reference for the following primer pairs: cathepsin B forward (5′-GATCTGCATCCACACCAATG-3′) and reverse (5′-AACCAGGCCTTTTCTTGTCC-3′) (Wickramasinghe et al, 2005); cathepsin L forward (5′-GAGGCAACAGAAGAATCCTGTAAGT-3′) and reverse (5′-AGGGCCTTCTCCTGCTTAGG-3′) (Schedel et al, 2004). Cathepsin D forward (5′-CACCACAAGTACAACAGCGAC-3′) and reverse (5′-CTTGGCTGCGATGAAGGTGA-3′), and control β-actin forward (5′-AGAAAATCTGGCACCACACC-3′) and reverse (5′-GGGGTGTTGAAGGTCTCAAA-3′) primers were designed using PrimerBank software (http://pga.mgh.harvard.edu/primerbank/).…”

Gene targeting by ribozyme against TNF-α mRNA inhibits autoimmune arthritis

“…[22][23][24][25] Ribozyme-mediated therapy has also been beneficial in RA research, inhibiting different genes in synovial cells. [26][27][28][29][30] However, there is a need to explore the efficacy of in vivo ribozyme treatment in RA models. Here, for the first time, we have investigated the therapeutic activity of a ribozyme in a model of autoimmune arthritis.…”