Prostaglandins E2 and E1 inhibit cytokine-induced metalloprotease expression in human synovial fibroblasts. Mediation by cyclic-AMP signalling pathway.
BACKGROUND
Cytokine modulated matrix metalloprotease (MMP, i.e., collagenase and stromelysin) synthesis may be associated etiologically with osteoarthritic diseases. The aim of this study was to investigate the mode of action by which interleukin-1 beta (IL-1 beta) induced collagenase and stromelysin mRNA expression and synthesis in normal human synoviocytes and to explore mechanisms of suppression of these proteolytic enzymes by prostaglandins (PG).


METHODS
Collagenase and stromelysin expression and synthesis were induced in cultured human synoviocytes with rhIL-1 beta in the absence or presence of either chemical inhibitors of protein kinase (PK) A and C, PGE2 or PGE1, or cAMP mimetics. We used enzyme immunoassays to determine MMP antigen levels in spent culture medium and Northern hybridization to measure steady-state MMP mRNA expression.


RESULTS
Dose-response experiments revealed that 10 pg/ml of interleukin-1 beta was an effective sub-saturating concentration for the induction of collagenase and stromelysin expression in normal human synovial fibroblasts. The rate of collagenase synthesis and expression peaked at 18 to 24 hours after rhIL-1 beta stimulation, whereas stromelysin output increased steadily within the experimental time frame (72 hours). Protein kinase C inhibitors, H-7 and staurosporine, prevented the rhIL-1 beta induction of MMP mRNA expression and protein synthesis. Pretreatment of synoviocytes with phorbol myristate acetate for 18 hours abrogated the ability of rhIL-1 beta to induce MMP synthesis. Prostaglandins E2 and E1 potently inhibited in a dose-dependent fashion rhIL-1 beta induced MMP synthesis: PGE2, IC50, collagenase, 2.3 ng/ml; stromelysin, 21.2 ng/ml; PGE1, IC50, collagenase, 2.5 ng/ml; stromelysin, 13.4 ng/ml. MMP mRNA steady-state levels were suppressed in a fashion similar to that of the protein synthesis. Forskolin, dibutyryl cAMP, and 3-isobutyl-1-methyl xanthine mimicked the effects of the prostaglandins (PGs). [N-(2-methyl-amino)-5-isoquinoline-sulfonamide dihydrochloride] (H-8), an inhibitor of PKA activity, could reverse to a large extent, the suppressive effects of the PGs as did cycloheximide when preincubated with PGE2 before rhIL-1 beta activation of synoviocytes.


CONCLUSIONS
We conclude that IL-1 beta stimulates MMP synthesis by activating PKC but not PKA, and that the synthesis of collagenase and stromelysin is discoordinate on a temporal and quantitative basis. PGs inhibit rhIL-1 beta-induced MMP expression and synthesis by virtue of their ability to increase cAMP intracellular levels and subsequent activation of signal transduction mechanisms involving PKA. Homeostasis may be maintained in acute episodes of joint inflammation through feedback processes involving locally produced eicosanoids.
DiBattista
J A
JA
Rheumatic Disease Unit, Notre-Dame Hospital Research Center, Montreal, Canada.
Martel-Pelletier
J
J
Fujimoto
N
N
Obata
K
K
Zafarullah
M
M
Pelletier
J P
JP
D000527:Alprostadil / Q000494:pharmacology*; D017364:Collagenases / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology; D015232:Dinoprostone / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011994:Recombinant Proteins; D015398:Signal Transduction; D013583:Synovial Membrane / Q000166:cytology / Q000201:enzymology*
D007375:Interleukin-1; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017364:Collagenases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D000527:Alprostadil; D015232:Dinoprostone