The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression.
OBJECTIVE
To elucidate the influence and mode of action of HMR1726 (the active metabolite of leflunomide) on TNF-alpha and IL-17 activated metalloproteinases expression in synoviocytes.


METHODS
Synovial fibroblasts from RA and OA patients were stimulated with both cytokines and altered gene expression in the presence or absence of leflunomide was detected by microarray analyses and quantitative RT-PCR. Protein expression was detected by western blotting and commercial ELISAs.


RESULTS
Microarray analyses revealed that the addition of HMR1726 (50 microM) to TNF-alpha and IL-17- stimulated synoviocytes induced gene expression of metallo-proteinases, especially MMP-1 and -3 in comparison to activated synoviocytes in the absence of leflunomide. To confirm these data, we examined the influence of different concentrations of HMR1726 in synoviocytes from further 5 OA and 7 RA patients by quantitative PCR. HMR1726 gradually induced MMP-1 and MMP-3 gene expression in a dose-dosedependent manner. Similar results were observed on protein levels. Examination of signal transduction pathways participating in the regulation of leflunomideinduced MMPs expression showed that the mechanism underlying activation of MMP-1 is in part p38- and activation of MMP-3 was MEK1/2- dependent.


CONCLUSIONS
Leflunomide was not able to abolish expression of metallo-proteinases in synoviocytes activated with TNF-a and IL-17.
Alexander
Dorothea
D
Center for Orthopaedic Surgery, University Hospital Tuebingen, Research Laboratory, Germany.
Friedrich
Bjorn
B
Abruzzese
Tanja
T
Gondolph-Zink
Bernd
B
Wülker
Nikolaus
N
Aicher
Wilhelm K
WK
D000328:Adult; D000368:Aged; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics*; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D020381:Interleukin-17 / Q000494:pharmacology*; D007555:Isoxazoles / Q000494:pharmacology*; D000077339:Leflunomide; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000235:genetics* / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010003:Osteoarthritis / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013583:Synovial Membrane / Q000187:drug effects* / Q000201:enzymology* / Q000473:pathology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D020381:Interleukin-17; D007555:Isoxazoles; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D014409:Tumor Necrosis Factor-alpha; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1; D000077339:Leflunomide