And it has been proved to be modulated by pro-inflammatory cytokines, IL-1 and TNF-, in chondrocytes [38], [44]. of PKR was observed in damaged OA cartilages as well as in TNF–stimulated chondrocytes. Phosphorylation of PKC (protein kinase C) was found after TNF- administration or PKR activation using poly(I:C), indicating PKC was regulated by PKR. The subsequent increased activity of NADPH oxidase led to oxidative stress accumulation and antioxidant capacity downregulation followed by an exaggerated inflammatory response with elevated levels of COX-2 and IL-8 via ERK/NF-B pathway. Activated ERK pathway also impeded the inhibition of MMP-13 by PPAR-. These findings exhibited that TNF–induced PKR activation brought on oxidative stress-mediated inflammation and MMP-13 in human chondrocytes. Unraveling these deregulated signaling cascades will deepen our knowledge of OA pathophysiology and provide aid in the development of novel therapies. < .05 compared to non-damaged cartilage or control group). 3.2. Increased PKC expression after inflammation is usually mediated by PKR Previously, elevated Sulfabromomethazine expression of protein kinase C (PKC) was found in human OA articular cartilages and was required for TNF- or IL-1-induced NF-B activation in chondrocytes [14]. Therefore, we sought to examine the relationship between PKR and PKC. As shown in Fig. 2A and B, protein expression of phospho-PKC was up-regulated in the mid-damaged and damaged cartilages. And the increased expression levels of phospho-PKC and phospho-PKR were observed after TNF- treatment Sulfabromomethazine in human chondrocytes which were isolated from non-damaged cartilage (Fig. 2C and D). Next, we assessed the effect of a synthetic analog of dsRNA polyinosinic-polycytidylic acid, poly(I:C), around the expression of PKC and PKR in chondrocytes. As expected, poly(I:C) enhanced the expression of phospho-PKR (Fig. 2C and D). It was noteworthy that this expression of phospho-PKC was up-regulated as well, indicating that activation of PKR possibly led to phosphorylation of PKC. As such, we utilized si-PKR to hinder Sulfabromomethazine the expression of PKR and found that the TNF–induced activation of PKC was abrogated by si-PKR (Fig. 2E and F). These results exhibited that increased expression of PKC after inflammation was via up-regulation of phospho-PKR. Open in a separate windows Fig. 2 Increased expression of PKC after cartilage inflammation is due to PKR upregulation Protein expression (A) and the ratio (B) of p-PKC to total PKC from three different regions; Protein expression Rabbit Polyclonal to ATG4C (C) and quantification (D) of PKR as well as PKC activation by addition of Sulfabromomethazine TNF- and poly(I:C), which is known to activate PKR. Protein expression (E) and the ratio (F) of p-PKC to total PKC after treatment of TNF- with or without the addition of si-PKR. (G)Western blotting confirming PKR knockdown efficiency. (n = 3; * p < .05 compared to non-damaged cartilage or no treatment control group; & p < .05 compared to TNF--treated group). 3.3. Upregulation of NADPH oxidase (NOX) activity under the inflammatory condition is usually regulated by PKR Reactive oxygen species (ROS) could be generated by chondrocytes following activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [15] and oxidative stress has been shown to induce the expression of OA markers [16]. Moreover, IL-1-mediated MMP secretion in chondrocytes has been proven by up-regulation of NADPH oxidase (NOX) [17]. In the current study, we examined whether the effect of inflammatory stimulation on NOX activity was via PKR signaling pathway. First, we exhibited that this subunits (p47 and Rac-1) as well as the isoform (NOX-1) of NADPH oxidase were Sulfabromomethazine elevated in the mid-damaged and damaged cartilages (Fig. 3A and B). Likewise, the activity of NOX was also increased in these damaged cartilages (Fig. 3C). Next, we showed the TNF--induced up-regulation of subunits and isoform (Fig. 3D and E) as well as NOX activity (Fig. 3F) in chondrocytes using si-PKR or si-PKC. Together, these findings suggested that the.