Attachment to laminin-111 facilitates transforming growth factor beta-induced expression of matrix metalloproteinase-3 in synovial fibroblasts.
BACKGROUND
In the synovial membrane of patients with rheumatoid arthritis (RA), a strong expression of laminins and matrix degrading proteases was reported.


OBJECTIVE
To investigate the regulation of matrix metalloproteinases (MMPs) in synovial fibroblasts (SFs) of patients with osteoarthritis (OA) and RA by attachment to laminin-1 (LM-111) and in the presence or absence of costimulatory signals provided by transforming growth factor beta (TGFbeta).


METHODS
SFs were seeded in laminin-coated flasks and activated by addition of TGFbeta. The expression of genes was investigated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunocytochemistry and ELISA, and intracellular signalling pathways by immunoblotting, and by poisoning p38MAPK by SB203580, MEK-ERK by PD98059 and SMAD2 by A-83-01.


RESULTS
Attachment of SF to LM-111 did not activate the expression of MMPs, but addition of TGFbeta induced a fivefold higher expression of MMP-3. Incubation of SF on LM-111 in the presence of TGFbeta induced a significant 12-fold higher expression of MMP-3 mRNA, and secretion of MMP-3 was elevated 20-fold above controls. Functional blocking of LM-111-integrin interaction reduced the laminin-activated MMP-3 expression significantly. Stimulation of SF by LM-111 and TGFbeta activated the p38MAPK, ERK and SMAD2 pathways, and inhibition of these pathways by using SB203580, PD98059 or A-83-01 confirmed the involvement of these pathways in the regulation of MMP-3.


CONCLUSIONS
Attachment of SF to LM-111 by itself has only minor effects on the expression of MMP-1 or MMP-3, but it facilitates the TGFbeta-induced expression of MMP-3 significantly. This mode of MMP-3 induction may therefore contribute to inflammatory joint destruction in RA independent of the proinflammatory cytokines interleukin (IL)1beta or tumour necrosis factor (TNF)alpha.
Hoberg
Maik
M
Department of Orthopedic Surgery, CRONA University Hospital, Tuebingen, Germany.
Rudert
Maximilian
M
Pap
Thomas
T
Klein
Gerd
G
Gay
Steffen
S
Aicher
Wilhelm K
WK
D000368:Aged; D001172:Arthritis, Rheumatoid / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007797:Laminin / Q000378:metabolism*; D008297:Male; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction; D013583:Synovial Membrane / Q000201:enzymology* / Q000473:pathology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D007797:Laminin; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C121346:laminin 1; D048049:Extracellular Signal-Regulated MAP Kinases; D019278:Matrix Metalloproteinase 3