Protein thiol modification by 15-deoxy-Delta12,14-prostaglandin J2 addition in mesangial cells: role in the inhibition of pro-inflammatory genes.
The cyclopentenone prostaglandin and PPARgamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) 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-PGJ(2) in MC, and the correlation with the modulation of several proinflammatory genes. MC incubation with biotinylated 15d-PGJ(2) results in the labeling of a distinct set of proteins as evidenced by two-dimensional electrophoresis. 15d-PGJ(2) binds to nuclear and cytosolic targets as detected by fluorescence microscopy and subcellular fractionation. The pattern of biotinylated 15d-PGJ(2)-modified polypeptides is readily distinguishable from that of total protein staining or labeling with biotinylated iodoacetamide. 15d-PGJ(2) addition requires the double bond in the cyclopentane ring. 9,10-Dihydro-15d-PGJ(2), a 15d-PGJ(2) 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-PGJ(2) binding. Micromolar concentrations of 15d-PGJ(2) inhibit cytokine-elicited levels of inducible nitricoxide synthase, cyclooxygenase-2, and intercellular adhesion molecule-1 in MC. In contrast, 9,10-dihydro-15d-PGJ(2) does not reproduce this inhibition. 15d-PGJ(2) effect is not blocked by the PPARgamma antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Moreover, compounds possessing an alpha,beta-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-PGJ(2) is a selective process that plays an important role in the inhibition of MC responses to pro-inflammatory stimuli.
Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.
Francisco J
FJ
Sánchez-Gómez
Eva
E
Cernuda-Morollón
Konstantinos
K
Stamatakis
Dolores
D
Pérez-Sala
D000818:Animals; D019921:Biotinylation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D003517:Cyclopentanes / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects*; D005920:Glomerular Mesangium / Q000166:cytology* / Q000378:metabolism; D007249:Inflammation / Q000235:genetics; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D047492:Peroxisome Proliferator-Activated Receptors / Q000378:metabolism; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014162:Transfection
C097240:15-deoxy-delta(12,14)-prostaglandin J2; D003517:Cyclopentanes; D007527:Isoenzymes; D047492:Peroxisome Proliferator-Activated Receptors; D011506:Proteins; D013438:Sulfhydryl Compounds; D018799:Intercellular Adhesion Molecule-1; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C013905:cyclopentenone; D015230:Prostaglandin D2