15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway

Abstract

Prostaglandin J(2) (PGJ(2)) and its metabolites Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are naturally occurring derivatives of prostaglandin D(2) that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ(2) is a high-affinity ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor alpha, in a PPARgamma-dependent manner. We report here that 15d-PGJ(2) potently inhibits NF-kappaB-dependent transcription by two additional PPARgamma-independent mechanisms. Several lines of evidence suggest that 15d-PGJ(2) directly inhibits NF-kappaB-dependent gene expression through covalent modifications of critical cysteine residues in IkappaB kinase and the DNA-binding domains of NF-kappaB subunits. These mechanisms act in combination to inhibit transactivation of the NF-kappaB target gene cyclooxygenase 2. Direct inhibition of NF-kappaB signaling by 15d-PGJ(2) may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs.

Figure 1

15d-PGJ₂ inhibits NF-κB by PPARγ-dependent and PPARγ-independent mechanisms. (A) 15d-PGJ₂ inhibits LPS induction of the iNOS promoter in PPARγ-negative RAW264.7 cells. Inhibition occurs at a lower concentration of 15d-PGJ₂ in the presence of coexpressed PPARγ. (B) HeLa cells lack endogenous PPARγ. Whole cell extracts were obtained from HeLa cells, from HeLa cells transfected with CMV-PPARγ expression plasmid, and from MCF-7 cells that contain endogenous PPARγ. PPARγ was detected by Western blotting with a PPARγ-specific antibody (Santa Cruz Biotechnology). (C) Inhibition of AP-1 activity by 15d-PGJ₂ is PPARγ-dependent. HeLa cells were transfected with the (AP-1)₃-TATA-Luc reporter and pCMV-β-gal (1 μg each), with or without 1 μg of pCMX-PPARγ. Transfected cells were treated with TPA (100 nM) with or without 15d-PGJ₂ (3 μM). (D) Effect of 15d-PGJ₂ on NF-κB activity in the presence or absence of PPARγ. HeLa cells were transfected with the (NF-κB)₃-TATA-Luc reporter and β-actin-β-gal (1 μg each), with or without pCMX-PPARγ (1 μg). Transfected cells were treated with TPA (100 nM) and BRL49653 or 15d-PGJ₂, as indicated. In D, results represent the mean of duplicate determinations, with the error bars representing the range for the duplicates.

Figure 2

Effects of 15d-PGJ₂ on activity of IKK and IκB degradation. (A) Structures of 15d-PGJ₂ (11-oxoprosta-5Z,9,12E,14Z-tetraen-1-oic acid) and cyclopentenone. The positions of chemically reactive, electrophilic carbons are indicated by asterisks. (B) Inhibition of NF-κB by cyclopentenone and the PPARγ-specific ligand BRL49653 is additive in the presence of PPARγ. HeLa cells were transfected with the (NF-κB)₃-TATA-Luc reporter and β-actin-β-gal (1 μg each), with pCMX-PPARγ (200 ng). Transfected cells were treated for 16 h with TPA (100 nM) and other additions as indicated. (C) 15d-PGJ₂ and cyclopentenone inhibit degradation of IκB in LPS-stimulated RAW264.7 cells. RAW cells were treated with 15d-PGJ₂ or cyclopentenone for 1 h. Cells were stimulated with LPS (1 μg/ml) for 30 min, and whole cell extracts were assayed for IκBα by Western blotting. (D) 15d-PGJ₂ inhibits IKK activity. PPARγ-negative resident murine peritoneal macrophages and RAW264.7 cells were incubated with 15d-PGJ₂ (6 μM) as indicated for 1 h and then stimulated with LPS (1 μg/ml). Whole cell extracts were prepared 10 min later, and IKK activity was assayed by using GST-IKBα (1–54) as a substrate. (E) 15d-PGJ₂ inhibits kinase activity of purified IKK. IκBα (1–54) was incubated with purified IKK and [γ-³²P]ATP in the presence of the indicated concentrations of 15d-PGJ₂ and 0.1 mM dithiothreitol. Incorporation of ³²P was determined by SDS/PAGE and phosphorimaging.

Figure 3

15d-PGJ₂ inhibits NF-κB DNA binding in HeLa cells without inhibiting nuclear entry of NF-κB. (A) (Upper) 15d-PGJ₂ (6 μM) and cyclopentenone (300 μM) prevent nuclear entry of p65 in LPS-treated (1 μg/ml) RAW264.7 cells. (Lower) Significant nuclear entry of NF-κB occurs in TNFα-stimulated (50 ng/ml) HeLa cells treated with 6 μM 15d-PGJ₂ or cyclopentenone (300 μM). (B) (Upper) 15d-PGJ₂, PGA₂ and cyclopentenone inhibit the DNA binding activity of NF-κB. HeLa cells were treated with TPA (100 nM) and the indicated additions, and nuclear extracts were assessed for DNA-binding activity by EMSA. (Lower) Western blot of p65 in nuclear extracts used in EMSA (Upper), lanes 1–6. Each lane was loaded with 10 μg of protein. (C) Specific NF-κB oligonucleotide (S) and anti-p65 antibody, but not mutant NF-κB oligonucleotide (M), abolish formation of the NF-κB/DNA complex. (D) Effect of 15d-PGJ₂ on IκB degradation in HeLa cells. Cells were treated with TNFα with or without 15d-PGJ₂, and IκB was quantified by Western blotting. (E) Effect of 15d-PGJ₂ on IKK activity in HeLa cells. Cells were treated with TNFα with or without 15d-PGJ₂, and IKK activity was assayed as described for Fig. 2D.

Figure 4

15d-PGJ₂ and cyclopentenone directly inhibit the DNA-binding activity of NF-κB. (A) Inhibition of NF-κB DNA binding by 15d-PGJ₂ involves the conserved Cys³⁸ in p65. (Upper) The indicated concentrations of 15d-PGJ₂ were incubated with 20 nM NF-κB p50/p65 and 200 pM labeled DNA, and protein–DNA complexes were assessed by EMSA. (Lower) Identical experimental setup as above using NF-κB p65 homodimers in which Cys³⁸ was mutated to Ser. (B) Inhibition of NF-κB DNA binding by cyclopentenone. The indicated concentrations of cyclopentenone were incubated with 20 nM of NF-κB p50/p65 and 200 pM labeled DNA.

Figure 5

15d-PGJ₂ inhibits production of PGE₂ and expression of COX-2 in macrophages. (A) 15d-PGJ₂ and cyclopentenone inhibit COX-2 promoter activity. RAW264.7 macrophages were transfected with the indicated COX-2 reporter construct (14) and stimulated with LPS (10 μg/ml) for 18 h. (B) RAW264.7 macrophages were treated with the indicated concentrations of 15d-PGJ₂ or cyclopentenone for 2 h and stimulated with LPS (30 ng/ml). The cells were harvested 8 h later, and RNA was analyzed by Northern blotting. (C) RAW264.7 macrophages were treated with the indicated concentrations of 15d-PGJ₂ or cyclopentenone, stimulated with LPS (1 μg/ml), and assayed for media content of PGE₂ 18 h later. (D) Model for mechanisms by which 15d-PGJ₂ exerts negative feedback on prostaglandin biosynthesis. COX-2-dependent synthesis of prostaglandin H provides substrate for production of PGD₂ in macrophages that express PGD₂ synthase. PGD₂ is further metabolized to PGJ₂ and 15d-PGJ₂. 15d-PGJ₂ inhibits NF-κB target genes, including COX-2, by inhibition of IKK, direct inhibition of DNA binding, and by PPARγ-dependent transrepression.