Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.
OBJECTIVE
Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs.


METHODS
Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA.


RESULTS
MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05).


CONCLUSIONS
The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.
Otto-von-Guericke-University, Magdeburg, Germany.
Edita
E
Rutkauskaite
Dagmar
D
Volkmer
Yukio
Y
Shigeyama
Jörg
J
Schedel
Geza
G
Pap
Ulf
U
Müller-Ladner
Ingmar
I
Meinecke
Dorothea
D
Alexander
Renate E
RE
Gay
Susanne
S
Drynda
Wolfram
W
Neumann
Beat A
BA
Michel
Wilhelm K
WK
Aicher
Steffen
S
Gay
Thomas
T
Pap
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000201:enzymology / Q000473:pathology* / Q000628:therapy; D002358:Cartilage, Articular / Q000201:enzymology / Q000473:pathology; D002465:Cell Movement; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D053511:Matrix Metalloproteinase 14; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D016372:RNA, Antisense / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013583:Synovial Membrane / Q000201:enzymology / Q000473:pathology*; D014162:Transfection
C507096:Mmp14 protein, mouse; D016372:RNA, Antisense; D012333:RNA, Messenger; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D053511:Matrix Metalloproteinase 14