Redox Regulation of PPAR in Polarized Macrophages

The peroxisome proliferator-activated receptor (PPAR gamma) is a central mediator of cellular lipid metabolism and immune cell responses during inflammation. This is facilitated by its role as a transcription factor as well as a DNA-independent protein interaction partner. We addressed how the cellular redox milieu in the cytosol and the nucleus of lipopolysaccharide (LPS)/interferon-gamma- (IFN gamma-) and interleukin-4- (IL4-) polarized macrophages (M phi) initiates posttranslational modifications of PPAR gamma, that in turn alter its protein function. Using the redox-sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative activation of M phi, while the redox status of PPAR gamma was determined via mass spectrometry. Cysteine residues located in the zinc finger regions (amino acid fragments AA 90-115, AA 116-130, and AA 160-167) of PPAR gamma were highly oxidized, accompanied by phosphorylation of serine 82 in response to LPS/IFN gamma, whereas IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, favoring a reductive milieu. Mutating these cysteines to alanine to mimic a redox modification decreased PPAR gamma-dependent reporter gene transactivation supporting a functional shift of PPAR gamma associated with the M phi phenotype. These data suggest distinct mechanisms for regulating PPAR gamma function based on the redox state of M phi.