Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte.
The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-alpha) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-alpha, increased the number of cells exhibiting an elongated morphology. TNF-alpha potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-alpha and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-alpha and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-alpha or by EGF, alone or in combination. EGF alone did not activate NF-kappaB or alter NF-kappaB activation by TNF-alpha. Pharmacologic studies indicated that the effects of TNF-alpha and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-alpha attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-alpha or basal activity. TNF-alpha and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals.
Klooster
Aaron R
AR
CIHR Group in Skeletal Development and Remodeling, Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada. aaron.klooster@pharma.novartis.com
Bernier
Suzanne M
SM
D053674:Aggrecans; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002358:Cartilage, Articular / Q000166:cytology; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis / Q000235:genetics; D024043:Collagen Type II / Q000096:biosynthesis / Q000235:genetics; D003864:Depression, Chemical; D004357:Drug Synergism; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D006609:High Mobility Group Proteins / Q000502:physiology; D037181:Lectins, C-Type / Q000096:biosynthesis / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000187:drug effects; D016328:NF-kappa B / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D055758:SOX9 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
C508321:Acan protein, rat; D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D006609:High Mobility Group Proteins; D037181:Lectins, C-Type; D016328:NF-kappa B; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D055758:SOX9 Transcription Factor; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D004815:Epidermal Growth Factor; D011493:Protein Kinase C