15-Deoxy-Δ12,14-prostaglandin J2Inhibition of NF-κB-DNA Binding through Covalent Modification of the p50 Subunit

Eva Cernuda-Morollón,Estela Pineda-Molina,F Javier Cañada,Dolores Pérez-Sala

doi:10.1074/jbc.m104518200

Abstract

Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-kappaB. Here we explore the possibility that the NF-kappaB subunit p50 may be a target for the cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ(2) was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ(2) was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ(2)-treated p50, respectively. The interaction between p50 and 15d-PGJ(2) was relevant in intact cells. 15d-PGJ(2) effectively inhibited cytokine-elicited NF-kappaB activity in HeLa without reducing IkappaBalpha degradation or nuclear translocation of NF-kappaB subunits. 15d-PGJ(2) reduced NF-kappaB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-kappaB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ(2) resulted in the formation of a 15d-PGJ(2)-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ(2) that results in inhibition of DNA binding.

Highlights

Inflammatory conditions such as psoriasis, ulcerative colitis, and allergic eczema are associated with elevated levels of prostaglandins (PG)
Treatment of HeLa with biotinylated-15d-PGJ2 resulted in the formation of a 15d-PGJ2p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ2 that results in inhibition of DNA binding
Recent evidences indicate that COX-2 may play a dual role in inflammation, promoting at later stages the production of cyclopentenone PG, such as PGA2 and 15-deoxy-⌬12–14-PGJ2 (15d-PGJ2), that may favor resolution of inflammation [17]

Summary

EXPERIMENTAL PROCEDURES

Materials—15-Deoxy-⌬12,14-PGJ2 was from Calbiochem-Novabiochem. 15-Deoxy-⌬12,14-PGJ2 (mixture of isomers), carbaprostacyclin, PGE2, and PGA1 were from Cayman Chemical (Ann Arbor, MI). For EMSA of purified p50, 15 nM wild type or C62S mutant p50 treated with vehicle or cyclopentenone PG were incubated for 30 min at room temperature in the presence of labeled oligonucleotide, 0.2 mg/ml bovine serum albumin, and 1 ␮g of poly(dI-dC) carrier in binding buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5% glycerol, 0.01% Nonidet P-40). For EMSA of nuclear proteins, binding reactions containing 3.5 ␮g of nuclear extract were carried out for 20 min on ice. Unless otherwise stated, DTT final concentration was 0.1 mM. Cell lysates were immunoprecipitated with agarose-conjugated anti-p50 antibody according to the manufacturer’s instructions, and biotinylated proteins were subjected to Western blot and detection with HRP-conjugated streptavidin and ECL (Amersham Pharmacia Biotech)

RESULTS

Findings

DISCUSSION