Egr-1 inhibits the expression of extracellular matrix genes in chondrocytes by TNFalpha-induced MEK/ERK signalling.
BACKGROUND
TNFalpha is increased in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. TNFalpha activates mitogen-activated kinase kinase (MEK)/extracellular regulated kinase (ERK) in chondrocytes; however, the overall functional relevance of MEK/ERK to TNFalpha-regulated gene expression in chondrocytes is unknown.


METHODS
Chondrocytes were treated with TNFalpha with or without the MEK1/2 inhibitor U0126 for 24 hours. Microarray analysis and real-time PCR analyses were used to identify genes regulated by TNFalpha in a MEK1/2-dependent fashion. Promoter/reporter, immunoblot, and electrophoretic mobility shift assays were used to identify transcription factors whose activity in response to TNFalpha was MEK1/2 dependent. Decoy oligodeoxynucleotides bearing consensus transcription factor binding sites were introduced into chondrocytes to determine the functionality of our results.


RESULTS
Approximately 20% of the genes regulated by TNFalpha in chondrocytes were sensitive to U0126. Transcript regulation of the cartilage-selective matrix genes Col2a1, Agc1 and Hapln1, and of the matrix metalloproteinase genes Mmp-12 and Mmp-9, were U0126 sensitive--whereas regulation of the inflammatory gene macrophage Csf-1 was U0126 insensitive. TNFalpha-induced regulation of Sox9 and NFkappaB activity was also U0126 insensitive. Conversely, TNFalpha-increased early growth response 1 (Egr-1) DNA binding was U0126 sensitive. Transfection of chondrocytes with cognate Egr-1 oligodeoxynucleotides attenuated the ability of TNFalpha to suppress Col2a1, Agc1 or Hapln1 mRNA expression.


CONCLUSIONS
Our results suggest that MEK/ERK and Egr1 are required for TNFalpha-regulated catabolic and anabolic genes of the cartilage extracellular matrix, and hence may represent potential targets for drug intervention in osteoarthritis or rheumatoid arthritis.
Rockel
Jason S
JS
Canadian Institutes of Health Research Group in Skeletal Development and Remodeling, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario N6A5C1, Canada. jsrockel@uwo.ca
Bernier
Suzanne M
SM
Leask
Andrew
A
D053674:Aggrecans / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002356:Cartilage / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D024043:Collagen Type II / Q000096:biosynthesis / Q000235:genetics; D051766:Early Growth Response Protein 1 / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000235:genetics* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D000099249:Proteoglycan Link Protein
D053674:Aggrecans; D024043:Collagen Type II; D051766:Early Growth Response Protein 1; D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D014409:Tumor Necrosis Factor-alpha; D000099249:Proteoglycan Link Protein; C508321:Acan protein, rat; C511513:COL2A1 protein, rat; C486110:Egr1 protein, rat