Received for publication May 17, 2004.
Revised July 29, 2004.
Accepted for publication August 18, 2004.

Protein thiol modification by 15-deoxy-^12,14-prostaglandin J₂ addition in mesangial cells: role in the inhibition of pro-inflammatory genes

Abstract

The cyclopentenone prostaglandin (cyPG) and PPAR agonist 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂) displays anti-inflammatory effects in several experimental models. Direct modification of protein thiols is arising as an important mechanism of cyPG action. However, little is known about the extent or specificity of this process. Mesangial cells (MC) play a key role in glomerulonephritis. Here we have studied the selectivity of protein modification by 15d-PGJ₂ in MC, and the correlation with the modulation of several pro-inflammatory genes. MC incubation with biotinylated 15d-PGJ₂ results in the labeling of a distinct set of proteins as evidenced by 2D-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 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 nitric oxide 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 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 which plays an important role in the inhibition of MC responses to pro-inflammatory stimuli.

Key words: Prostanoid, PPARs, Structure/function/mechanism, Cyclooxygenases, Eicosanoids

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