Recombinant equine interleukin-1beta induces putative mediators of articular cartilage degradation in equine chondrocytes.
Interleukin-1 is considered a central mediator of cartilage loss in osteoarthritis in several species, however an equine recombinant form of this cytokine is not readily available for in vitro use in equine osteoarthritis research. Equine recombinant interleukin-1beta was cloned and expressed and its effects on the expression and activity of selected chondrocytic proteins implicated in cartilage matrix degradation were characterized. Reverse transcriptase polymerase chain reaction methods were used to amplify the entire coding region of the equine IL-1beta mRNA, which was cloned into an expression vector, expressed in E. coli, and purified using a Ni2+ chromatographic method. The effects of the recombinant peptide on chondrocyte gene expression were determined by Northern blotting using RNA from equine chondrocyte cultures hybridized to probes for matrix metalloproteinases (MMP 1, MMP 3, MMP 13), tissue inhibitor of matrix metalloproteinases 1 (TIMP 1) and cyclooxygenase 2 (COX 2). Effects on selected mediators of cartilage degradation (nitrite concentrations and MMP activity) were determined using conditioned medium from reIL-1beta-treated equine cartilage explant cultures. A recombinant peptide of approximately 21 kd was obtained. Northern blotting analyses revealed a marked up-regulation of expression of all MMPs, TIMP 1, and COX 2 in mRNA from treated chondrocytes. Furthermore, cartilage explants exposed to reIL-1beta had augmented collagenase/gelatinase and stromelysin activities as well as increased concentration of nitrite in conditioned media. The development of a biologically active, species-specific IL-1beta provides a valuable tool in the study of osteoarthritis pathophysiology and its treatment in horses.
Tung
J T
JT
Department of Animal Science, Michigan State University, East Lansing 48824, USA.
Fenton
J I
JI
Arnold
C
C
Alexander
L
L
Yuzbasiyan-Gurkan
V
V
Venta
P J
PJ
Peters
T L
TL
Orth
M W
MW
Richardson
D W
DW
Caron
J P
JP
D000818:Animals; D015152:Blotting, Northern / Q000662:veterinary; D002358:Cartilage, Articular / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000201:enzymology; D051546:Cyclooxygenase 2; D015971:Gene Expression Regulation, Enzymologic; D006734:Horse Diseases / Q000503:physiopathology*; D006736:Horses; D007375:Interleukin-1 / Q000494:pharmacology / Q000502:physiology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D010003:Osteoarthritis / Q000503:physiopathology / Q000662:veterinary*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism
D007375:Interleukin-1; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D020782:Matrix Metalloproteinases