Protein Thiol Modification by 15-deoxy- -Prostaglandin J2 Addition in Mesangial Cells: Role in the Inhibition of Pro-inflammatory Genes

The cyclopentenone prostaglandin and PPAR agonist 15-deoxy-prostaglandin J2 (15d-PGJ2) displays anti-inflammatory effects in several experimental models. Direct modification of protein thiols is arising as an important mechanism of cyclopentenone prostaglandin action. However, little is known about the extent or specificity of this process. Mesangial cells (MC) play a key role in glomerulonephritis. In this work, we have studied the selectivity of protein modification by 15d-PGJ2 in MC, and the correlation with the modulation of several proinflammatory genes. MC incubation with biotinylated 15d-PGJ2 results in the labeling of a distinct set of proteins as evidenced by two-dimensional electrophoresis. 15d-PGJ2 binds to nuclear and cytosolic targets as detected by fluorescence microscopy and subcellular fractionation. The pattern of biotinylated 15d-PGJ2-modified polypeptides is readily distinguishable from that of total protein staining or labeling with biotinylated iodoacetamide. 15d-PGJ2 addition requires the double bond in the cyclopentane ring. 9,10-Dihydro-15d-PGJ2, a 15d-PGJ2 analog that shows the same potency as peroxisome proliferator-activated receptor (PPAR) agonist in MC but lacks the cyclopentenone moiety, displays reduced ability to modify proteins and to block 15d-PGJ2 binding. Micromolar concentrations of 15d-PGJ2 inhibit cytokine-elicited levels of inducible nitricoxide synthase, cyclooxygenase-2, and intercellular adhesion molecule-1 in MC. In contrast, 9,10-dihydro-15d-PGJ2 does not reproduce this inhibition. 15d-PGJ2 effect is not blocked by the PPAR antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Moreover, compounds possessing an , -unsaturated carbonyl group, like 2-cyclopenten-1-one and 2-cyclohexen-1-one, reduce pro-inflammatory gene expression. These observations indicate that covalent modification of cellular thiols by 15d-PGJ2 is a selective process that plays an important role in the inhibition of MC responses to pro-inflammatory stimuli. Cyclopentenone prostaglandins (cyPG) are endogenous prostanoids that arise from the dehydration of their parent PG. Thus, dehydration of PGE2 gives rise to PGA2, whereas cyPG of the J series, like PGJ2 and 15-deoxy-PGJ2 (15d-PGJ2), arise from the dehydration of PGD2. The generation of 15d-PGJ2 has been reported to increase under situations associated with COX-2 induction, such as inflammatory processes (Gilroy et al., 1999). 15d-PGJ2 has been the subject of considerable study because of its identification as a ligand of the transcription factors known as peroxisome proliferator-activated receptors (PPAR), which are involved in the control of lipid metabolism and immune response (Forman et al., 1995). During the course of these studies it has been realized that 15d-PGJ2 may modulate multiple cellular functions by mechanisms dependent and independent of