Effects of sulfasalazine and its metabolites on steady state messenger RNA concentrations for inflammatory cytokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinase in rheumatoid synovial fibroblasts.
OBJECTIVE
To determine the effects of sulfasalazine (SASP) and its metabolites sulfapyridine (SP) and 5-amino salicylic acid (5ASA) on steady state mRNA levels of inflammatory cytokines [interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha)], matrix metalloproteinases [collagenase (MMP1), stromelysin (MMP3), gelatinase 72 kDa (MMP2)], tissue inhibitors of metalloproteinase (TIMP 1 and TIMP 2), and the TNF-alpha receptor in rheumatoid synovial fibroblasts.


METHODS
Cells were dosed with each compound for 24 h in the presence or absence of PMA inducer and messenger RNA (mRNA) extracted and subjected to Northern blot analysis. Messenger RNA levels were quantitated by densitometry and normalized to GAPDH or 18S rRNA.


RESULTS
We observed some modest effects of sulfasalazine and its metabolites on steady state mRNA levels including: (1) repressed mRNA levels for TNF-alpha [approximately 40% with 3x (drug median serum concentration) all 3 drugs], stromelysin (approximately 24% with 3x all 3 drugs and approximately 31% with 3x 5ASA), and collagenase (approximately 27% with 3x 5ASA); (2) elevated mRNA levels for TIMP 2 (3.5 kb transcript) (51% with 3x SP and 44% with 3x 5ASA), gelatinase (approximately 20% with 3x SP and 3x 5ASA), stromelysin (approximately 40% with 3x and 1x SASP), IL-1beta (approximately 31% with 0.1x 5ASA); and (3) no effect on mRNA levels for TNF-alpha receptor and TIMP 1.


CONCLUSIONS
(1) SASP and its metabolites showed varied effects on steady state mRNA concentrations for gene transcripts that fell into 3 categories: (a) repressed, (b) elevated, (c) no effect on mRNA levels. (2) No apparent linear dose response effect was observed for SASP or its metabolites, although a generalized suppression of mRNA levels at all doses was seen in some cases. (3) No predominant suppressive effect (> or = 50%) of mRNA levels by any of the drugs was observed for any of the genes studied; however, TIMP 2 mRNA levels increased 51% with 3x SP and 44% with 3x 5ASA.
Minghetti
P P
PP
Department of Rheumatology, Birmingham VA Medical Center, Alabama 35233, USA.
Blackburn
W D
WD
D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D018836:Inflammation Mediators / Q000502:physiology; D020782:Matrix Metalloproteinases / Q000235:genetics; D019804:Mesalamine / Q000494:pharmacology*; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism*; D013427:Sulfapyridine / Q000494:pharmacology*; D012460:Sulfasalazine / Q000494:pharmacology*; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics
D016207:Cytokines; D018836:Inflammation Mediators; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D012460:Sulfasalazine; D019804:Mesalamine; D020782:Matrix Metalloproteinases; D013427:Sulfapyridine