Identification of Novel Protein Targets for Modification by 15-Deoxy-Δ^12,14-Prostaglandin J₂ in Mesangial Cells Reveals Multiple Interactions with the Cytoskeleton

Abstract

The cyclopentenone prostaglandin 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) has been shown to display protective effects against renal injury or inflammation. In cultured mesangial cells (MC), 15d-PGJ₂ inhibits the expression of proinflammatory genes and modulates cell proliferation. Therefore, cyclopentenone prostaglandins (cyPG) have been envisaged as a promise in the treatment of renal disease. The effects of 15d-PGJ₂ may be dependent on or independent from its role as a peroxisome proliferator–activated receptor agonist. It was shown recently that an important determinant for the peroxisome proliferator–activated receptor–independent effects of 15d-PGJ₂ is the capacity to modify proteins covalently and alter their function. However, a limited number of protein targets have been identified to date. Herein is shown that a biotinylated derivative of 15d-PGJ₂ recapitulates the effects of 15d-PGJ₂ on the stress response and inhibition of inducible nitric oxide synthase levels and forms stable adducts with proteins in intact MC. Biotinylated 15d-PGJ₂ was then used to identify proteins that potentially are involved in cyPG biologic effects. Extracts from biotinylated 15d-PGJ₂–treated MC were separated by two-dimensional electrophoresis, and the spots of interest were analyzed by mass spectrometry. Identified targets include proteins that are regulated by oxidative stress, such as heat-shock protein 90 and nucleoside diphosphate kinase, as well as proteins that are involved in cytoskeletal organization, such as actin, tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ₂ binding to several targets was confirmed by avidin pull-down. Consistent with these findings, 15d-PGJ₂ induced early reorganization of vimentin and tubulin in MC. The cyclopentenone moiety and the presence of cysteine were important for vimentin rearrangement. These studies may contribute to the understanding of the mechanism of action and therapeutic potential of cyPG.