Ribozymes that inhibit the production of matrix metalloproteinase 1 reduce the invasiveness of rheumatoid arthritis synovial fibroblasts.
OBJECTIVE
To investigate whether retroviral gene transfer of ribozymes targeting matrix metalloproteinase 1 (MMP-1) inhibits the production of MMP-1 in rheumatoid arthritis synovial fibroblasts (RASFs) and reduces the invasiveness of these cells in vivo.


METHODS
MMP-1-specific ribozymes (RzMMP-1) were designed and cloned into the pLNSX retroviral vector. Cleavage of MMP-1 was determined in vitro, and the most effective ribozyme was selected for further investigation. RASFs were transduced with replication-deficient viruses carrying RzMMP-1 or with empty viruses (mock). Quantitative polymerase chain reaction with cleavage site-spanning fluorescent probes was used to measure the levels of MMP-1, MMP-9, and MMP-13 messenger RNA. In addition, protein levels of MMP-1 in cell culture supernatants were determined by enzyme linked immunosorbent assay. The effects of stimulation with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) on the production of MMP-1 were assessed accordingly. The invasiveness of RzMMP-1-transduced, mock-transduced, and untransduced RASFs was analyzed in the SCID mouse in vivo model of RA.


RESULTS
Transduction of RASFs with RzMMP-1 significantly decreased the production of MMP-1 in RASFs without affecting other MMPs, such as MMP-9 and MMP-13. RzMMP-1 not only reduced the spontaneous production of MMP-1, but also prevented the LPS- and TNFalpha-induced increase in MMP-1 production. Inhibition of MMP-1 was maintained for at least 2 months and was accompanied by a significant reduction of the invasiveness of RASFs in the SCID mouse model of RA.


CONCLUSIONS
Intracellular expression of ribozymes constitutes a feasible tool for inhibiting the production of matrix-degrading enzymes. Inhibition of MMP-1 alone results in a significant reduction of cartilage invasion by RASFs.
Rutkauskaite
Edita
E
Otto-von-Guericke-University, University Hospital Magdeburg, Leipziger-Strasse 44, D-39120 Magdeburg, Germany.
Zacharias
Wolfgang
W
Schedel
Jörg
J
Müller-Ladner
Ulf
U
Mawrin
Christian
C
Seemayer
Christian A
CA
Alexander
Dorothea
D
Gay
Renate E
RE
Aicher
Wilhelm K
WK
Michel
Beat A
BA
Gay
Steffen
S
Pap
Thomas
T
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology* / Q000628:therapy*; D002356:Cartilage / Q000473:pathology; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D015316:Genetic Therapy / Q000379:methods; D020781:Matrix Metalloproteinase 1 / Q000235:genetics* / Q000378:metabolism; D053509:Matrix Metalloproteinase 13; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013583:Synovial Membrane / Q000201:enzymology / Q000473:pathology
D016337:RNA, Catalytic; D017364:Collagenases; D053509:Matrix Metalloproteinase 13; C507081:Mmp13 protein, mouse; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1