Aggrecanases (a dystrophin and metalloproteinase with thrombospondin motif, ADAMTSs) are principal proteases involved in cartilage extracellular matrix aggrecan degradation. The role and relative contribution of MyD88, IRAK1, and TRAF6 adaptor proteins in IL-1β regulation of aggrecanase-1 (ADAMTS-4) is unknown. By small interfering RNAs-mediated knockdown, we show that IL-1β-induced up-regulation of ADAMTS-4 in chondrocytes requires MyD88, IRAK1, and TRAF6 adaptor proteins. However, partial inhibition of ADAMTS-4 induction by their knockdown suggested the involvement of additional signaling proteins. Because IL-1β is also known to induce reactive oxygen species (ROS) through Ras-mediated activation of NADPH oxidase, we investigated the implication of Ras in ADAMTS-4 regulation. Ras knockdown, or inhibition of ROS by antioxidants along with the ablation of MyD88, IRAK1, or TRAF6 more potently down-regulated IL-1β-induced ADAMTS-4. In addition, IL-1β-induced phosphorylation of downstream effectors, IκB kinase αβ, IκBβ, and activation of transcription factor NF-κB was significantly reduced in the MyD88-, IRAK1-, TRAF6-, or Ras-deficient cells. The combined knockdown of Ras and individual adaptor proteins strongly blocked the activation of IKKαβ, IκBβ, and NF-κB. These findings suggest that Ras, ROS along with MyD88, IRAK1, or TRAF6 synergistically mediate ADAMTS-4 regulation by IL1-β. Thus, complete ablation of ADAMTS-4 induction could be achieved by combined inhibition of Ras and individual adaptor proteins, which may be of therapeutic value in arthritis.