Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Apr 25;97(9):4844-9.
doi: 10.1073/pnas.97.9.4844.

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

D S Straus  1 , G Pascual, M Li, J S Welch, M Ricote, C H Hsiang, L L Sengchanthalangsy, G Ghosh, C K Glass
Affiliations

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

D S Straus et al. Proc Natl Acad Sci U S A. .

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.

PubMed Disclaimer

Figures

Figure 1
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
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
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
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
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fukushima M. Prostaglandins Leukotrienes Essent Fatty Acids. 1992;47:1–12. - PubMed
    1. Forman B M, Tontonoz P, Chen J, Brun R P, Spiegelman B M, Evans R M. Cell. 1995;83:803–812. - PubMed
    1. Kliewer S A, Lenhard J M, Willson T M, Patel I, Morris D C, Lehmann J M. Cell. 1995;83:813–819. - PubMed
    1. Ricote M, Li A C, Willson T M, Kelly C J, Glass C K. Nature (London) 1998;391:79–82. - PubMed
    1. Jiang C, Ting A T, Seed B. Nature (London) 1998;391:82–86. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources