Oxidative stress promotes ligand-independent and enhanced ligand-dependent tumor necrosis factor receptor signaling.
Tumor necrosis factor (TNF) receptor 1 (TNFR1, p55) and 2 (TNFR2, p75) are characterized by several cysteine-rich modules in the extracellular domain, raising the possibility that redox-induced modifications of these cysteine residues might alter TNFR function. To test this possibility, we examined fluorescence resonance energy transfer (FRET) in 293T cells transfected with CFP- and YFP-tagged TNFRs exposed to the thiol oxidant diamide. Treatment with high concentrations of diamide (1 mm) resulted in an increase in the FRET signal that was sensitive to inhibition with the reducing agent dithiothreitol, suggesting that oxidative stress resulted in TNFR self-association. Treatment of cells with low concentrations of diamide (1 mum) that was not sufficient to provoke TNFR self-association resulted in increased TNF-induced FRET signals relative to the untreated cells, suggesting that oxidative stress enhanced ligand-dependent TNFR signaling. Similar findings were obtained when the TNFR1- and TNFR2-transfected cells were pretreated with a cell-impermeable oxidase, DsbA, that catalyzes disulfide bond formation between thiol groups on cysteine residues. The changes in TNFR self-association were functionally significant, because pretreating the HeLa cells and 293T cells resulted in increased TNF-induced NF-kappaB activation and TNF-induced expression of IkappaB and syndecan-4 mRNA levels. Although pretreatment with DsbA did not result in an increase in TNF binding to TNFRs, it resulted in increased TNF-induced activation of NF-kappaB, consistent with an allosteric modification of the TNFRs. Taken together, these results suggest that oxidative stress promotes TNFR receptor self-interaction and ligand-independent and enhanced ligand-dependent TNF signaling.
Department of Medicine and Molecular Physiology and Biophysics, Winters Center for Heart Failure Research, Baylor College of Medicine, Houston, Texas 77030, USA.
Hatice Z
HZ
Ozsoy
Natarajan
N
Sivasubramanian
Eric D
ED
Wieder
Steen
S
Pedersen
Douglas L
DL
Mann
D002460:Cell Line; D045744:Cell Line, Tumor; D002469:Cell Separation; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D018384:Oxidative Stress; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D015398:Signal Transduction
D008024:Ligands; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II