Transforming growth factor β1 induction of Tissue Inhibitor of MetalloProteinases 3 in articular chondrocytes is mediated by reactive oxygen species

Abstract

Transforming growth factor β1 (TGF-β1) stimulates cartilage extracellular matrix synthesis but, in excess, evokes synovial inflammation, hyperplasia, and osteophyte formation in arthritic joints. TGF-β1 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-β1. In primary human and bovine chondrocytes, ROS scavenger and antioxidant N-acetylcysteine (NAC) inhibited TGF-β1-induced TIMP-3 mRNA and protein increases. Ebselen and ascorbate also reduced this induction. TGF-β1 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-β1 was partly Smad2-dependent. TGF-β1-stimulated Smad2 phosphorylation was inhibited by NAC. Reduced glutathione and L-cysteine also blocked Smad2 and TIMP-3 induction by TGF-β1, whereas a nonthiol, N-acetylalanine, did not. Smad2 was not activated by H 2O 2. Smad2 phosphorylation was independent, and TIMP-3 expression was dependent, on new protein synthesis. TGF-β-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-β1-induced TIMP-3 gene expression. Blocking TGF-β1-induced gene expression by modulating cellular redox status with thiols can be potentially beneficial for treating arthritic and other disorders caused by excessive TGF-β1.