Abstract
Transforming growth factor beta1 (TGF-beta1) stimulates cartilage extracellular matrix synthesis but, in excess, evokes synovial inflammation, hyperplasia, and osteophyte formation in arthritic joints. TGF-beta1 induces tissue inhibitor of metalloproteinases 3 (TIMP-3), an inhibitor of cartilage-damaging matrix metalloproteianases and aggrecanases. We investigated the role of reactive oxygen species (ROS) in TIMP-3 induction by TGF-beta1. In primary human and bovine chondrocytes, ROS scavenger and antioxidant N-acetylcysteine (NAC) inhibited TGF-beta1-induced TIMP-3 mRNA and protein increases. Ebselen and ascorbate also reduced this induction. TGF-beta1 time-dependently induced ROS production that was suppressed by NAC. Hydrogen peroxide, a ROS, induced TIMP-3 RNA. The TIMP-3 increase induced by TGF-beta1 was partly Smad2-dependent. TGF-beta1-stimulated Smad2 phosphorylation was inhibited by NAC. Reduced glutathione and L-cysteine also blocked Smad2 and TIMP-3 induction by TGF-beta1, whereas a nonthiol, N-acetylalanine, did not. Smad2 was not activated by H2O2. Smad2 phosphorylation was independent, and TIMP-3 expression was dependent, on new protein synthesis. TGF-beta-stimulated ERK and JNK phosphorylation was also inhibited by NAC. However, inhibitory actions of NAC were not mediated by ERK activation. Thus, ROS mediate TGF-beta1-induced TIMP-3 gene expression. Blocking TGF-beta1-induced gene expression by modulating cellular redox status with thiols can be potentially beneficial for treating arthritic and other disorders caused by excessive TGF-beta1.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acetylcysteine / pharmacology
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Animals
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Ascorbic Acid / pharmacology
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Azoles / pharmacology
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Blotting, Northern
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Blotting, Western
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Cartilage, Articular / metabolism*
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Cattle
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Cells, Cultured
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Chondrocytes / metabolism*
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Culture Media, Serum-Free / pharmacology
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Cycloheximide / pharmacology
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DNA-Binding Proteins / metabolism
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Down-Regulation
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Free Radicals
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Growth Substances / metabolism
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Humans
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Hydrogen Peroxide / pharmacology
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Isoindoles
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JNK Mitogen-Activated Protein Kinases / metabolism
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MAP Kinase Kinase 4
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Mitogen-Activated Protein Kinase 3 / metabolism
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Mitogen-Activated Protein Kinase Kinases / metabolism
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Oligonucleotides, Antisense / pharmacology
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Organoselenium Compounds / pharmacology
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Oxidation-Reduction
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Phosphorylation
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RNA / chemistry
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RNA / metabolism
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RNA, Messenger / metabolism
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Reactive Oxygen Species*
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Signal Transduction
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Smad2 Protein
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Species Specificity
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Time Factors
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Tissue Inhibitor of Metalloproteinase-3 / metabolism*
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Trans-Activators / metabolism
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Transfection
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Transforming Growth Factor beta / metabolism*
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Transforming Growth Factor beta1
Substances
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Azoles
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Culture Media, Serum-Free
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DNA-Binding Proteins
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Free Radicals
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Growth Substances
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Isoindoles
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Oligonucleotides, Antisense
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Organoselenium Compounds
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RNA, Messenger
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Reactive Oxygen Species
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SMAD2 protein, human
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Smad2 Protein
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TGFB1 protein, human
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Tissue Inhibitor of Metalloproteinase-3
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Trans-Activators
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Transforming Growth Factor beta
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Transforming Growth Factor beta1
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ebselen
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RNA
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Cycloheximide
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Hydrogen Peroxide
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JNK Mitogen-Activated Protein Kinases
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Mitogen-Activated Protein Kinase 3
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MAP Kinase Kinase 4
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Mitogen-Activated Protein Kinase Kinases
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Ascorbic Acid
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Acetylcysteine