Cartilage destruction mediated by synovial fibroblasts does not depend on proliferation in rheumatoid arthritis.
The aim of the study was to investigate the relationship between invasion and proliferation in rheumatoid arthritis synovial fibroblasts (RASFs). In vitro, RASFs, normal synovial fibroblasts (NSFs), and RASFs transformed with SV40 T-antigen (RASF(SV40)) were analyzed for the expression of cell surface markers (Thy1, VCAM-1, ICAM-1, CD40, CD44) and their proliferation by flow cytometry. Furthermore, colony-forming unit assays were performed and the expression of matrix metalloproteinases (MMP)-14 and cathepsin K mRNA were determined by real-time polymerase chain reaction. In vivo, in the severe combined immunodeficiency (SCID) mouse co-implantation model, RASFs, NSFs, and RASF(SV40) were tested for cartilage invasion, cellular density, and for their expression of the cell cycle-associated protein Ki67. In the SCID mouse co-implantation model, RASFs invaded significantly stronger into the cartilage than NSFs and RASF(SV40). Of note, RASF(SV40) cells formed tumor-like tissues, and the cellular density adjacent to the cartilage was significantly higher than in RASFs or NSFs. In turn, the proliferation marker Ki67 was strongly expressed in the SV40-transformed synoviocytes in SCID mice, but not in RASFs, and specifically not at sites of cartilage invasion. Using the colony-forming unit assay, RASFs and NSFs did not form colonies, whereas RASF(SV40) lost contact inhibition. In vitro, the proliferative rate of RASFs was low (4.3% S phase) in contrast to RASF(SV40) (24.4%). Expression of VCAM-1 was significantly higher, whereas of ICAM-1 was significantly lower, in RASFs than in RASF(SV40). CD40 was significantly stronger expressed in RASF(SV40), whereas CD44 and AS02 were present at the same degree in almost all synoviocytes. Expression of cathepsin K and matrix metalloproteinase-14 mRNA was significantly higher in RASFs than in the RASF(SV40). Our data demonstrate clearly that invasion of cartilage is mediated by activated RASFs characterized by increased expression of adhesion molecules, matrix-degrading enzymes, but does not depend on cellular proliferation, suggesting the dissociation of invasion and proliferation in RASFs.
Seemayer
Christian A
CA
Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zürich, Switzerland. christian.seemayer@ruz.usz.ch
Kuchen
Stefan
S
Kuenzler
Peter
P
Rihosková
Veronika
V
Rethage
Janine
J
Aicher
Wilhelm K
WK
Michel
Beat A
BA
Gay
Renate E
RE
Kyburz
Diego
D
Neidhart
Michel
M
Gay
Steffen
S
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000473:pathology*; D001483:Base Sequence; D015415:Biomarkers / Q000032:analysis; D002358:Cartilage, Articular / Q000473:pathology*; D056657:Cathepsin K; D002403:Cathepsins / Q000235:genetics; D002455:Cell Division; D002478:Cells, Cultured; D017931:DNA Primers; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D010003:Osteoarthritis / Q000235:genetics / Q000473:pathology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D013583:Synovial Membrane / Q000473:pathology*
D015415:Biomarkers; D017931:DNA Primers; D012333:RNA, Messenger; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases