Tat-ATOX1 inhibits inflammatory responses via regulation of MAPK and NF-κB pathways

Abstract

Antioxidant 1 (ATOX1) protein has been reported to exhibit various protective functions, including antioxidant and chaperone. However, the effects of ATOX1 on the inflammatory response has not been fully elucidated. Thus, we prepared cell permeable Tat-ATOX1 and studied the effects on lipopolysaccharide (LPS)- and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced inflammation. Experimental results showed that transduced Tat-ATOX1 protein significantly suppressed LPS-induced intracellular reactive oxygen species (ROS). Also, Tat-ATOX1 protein markedly inhibited LPS- and TPA-induced inflammatory responses by decreasing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and further inhibited phosphorylation of mitogen activated protein kinases (MAPKs; JNK, ERK and p38) and the nuclear factor-kappaB (NF-κB) signaling pathway. These results indicate that the Tat-ATOX1 protein has a pivotal role in inflammation via inhibition of inflammatory responses, suggesting Tat-ATOX1 protein may offer a therapeutic strategy for inflammation.

PEP-1-GLRX1 Protein Exhibits Anti-Inflammatory Effects by Inhibiting the Activation of MAPK and NF-κB Pathways in Raw 264.7 Cells

“…These results indicate that PEP-1-GLRX1 plays an anti-inflammatory role in TPA-induced skin inflammation. We have demonstrated that cell-permeable antioxidant PTD-fusion protein markedly inhibits TPA-induced inflammation in a mice model (27,48). In addition, other studies have reported that antioxidant proteins play protective roles in inflammation (51,52).…”

“…Since protein transduction is important for the development of therapeutic proteins, PTD-fused protein transduction can be used for the intracellular application of therapeutic proteins (20)(21)(22)(23)(24). In previous studies, we have demonstrated that PTD-fusion proteins were transduced into various cells (25)(26)(27)(28)(29)(30). In this study, we first identified the distribution of transduced PEP-1-GLRX1 using fluorescence analysis.…”

“…PEP-1-GLRX1 and control GLRX1 proteins were purified as described previously (19). To remove endotoxins from purified proteins, we treated the proteins with Detoxi-Gel TM (Pierce, Rockford, IL, USA) according to the manufacturer's instruction and confirmed the proteins (＜ 0.03 EU/ml) using a Limulus amoebocyte lysate assay (BioWhitaker, Walkersville, MD, USA) (27,48). Then, the protein concentration was measured using the Bradford assay (53).…”

“…We did Western blot analysis as described previously (27,48). Equal amounts of sample proteins were separated with 15% SDS-PAGE and transferred to a nitrocellulose membrane, which was blocked with 5% nonfat dry milk in TBST buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h. The membranes were immunoblotted with the indicated primary and HRP-conjugated secondary antibodies as recommended by the manufacturer.…”

“…Fluorescence microscopy analysis was done as described previously (27,48). Raw 264.7 cells were grown on coverslips and treated with 0.4 M of PEP-1-GLRX1 for 1 h at 37 o C. Then, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 5 min at room temperature.…”