Prostaglandin J2 (PGJ2) and its metabolites ⌬ 12 -PGJ2 and 15-deoxy-⌬ 12,14 -PGJ2 (15d-PGJ2) are naturally occurring derivatives of prostaglandin D2 that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ 2 is a high-affinity ligand for the peroxisome proliferator-activated receptor ␥ (PPAR␥) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor ␣, in a PPAR␥-dependent manner. We report here that 15d-PGJ2 potently inhibits NF-B-dependent transcription by two additional PPAR␥-independent mechanisms. Several lines of evidence suggest that 15d-PGJ2 directly inhibits NF-B-dependent gene expression through covalent modifications of critical cysteine residues in IB kinase and the DNA-binding domains of NF-B subunits. These mechanisms act in combination to inhibit transactivation of the NF-B target gene cyclooxygenase 2. Direct inhibition of NF-B signaling by 15d-PGJ 2 may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs. P rostaglandin J 2 (PGJ 2 ) and its metabolites are naturally occurring derivatives of prostaglandin D 2 (PGD 2 ). The pathway for formation of these compounds involves sequential conversion of PGD 2 to PGJ 2 , ⌬ 12 -PGJ 2 , and 15-deoxy-⌬ 12,14 -PGJ 2 (15d-PGJ 2 ) (1). The last of these metabolites, 15d-PGJ 2 , is a high-affinity ligand for peroxisome proliferator-activated receptor ␥ (PPAR␥) (2, 3). 15d-PGJ 2 represses several genes in activated macrophages, including the inducible NO synthase (iNOS) and tumor necrosis factor ␣ (TNF␣) genes, and this repression is at least partly dependent on PPAR␥ expression (4-6). 15d-PGJ 2 is present in vivo during the resolution phase of inflammation, suggesting that it may function as a feedback regulator of the inflammatory response (7).Previous studies evaluating PPAR␥-dependent inhibition of iNOS expression indicated that 15d-PGJ 2 was significantly more effective than synthetic PPAR␥ ligands, despite binding to PPAR␥ with lower affinity (4). PGJ 2 and its metabolites are characterized by the presence of a cyclopentenone ring system that contains an electrophilic carbon that can react covalently by means of the Michael addition reaction with nucleophiles such as the free sulfhydryls of glutathione and cysteine residues in cellular proteins (1,8,9). This reactive center is not present in the synthetic PPAR␥ ligands and has been proposed to account for some of the receptor-independent biological actions of PGJ 2 , its metabolites, and the related cyclopentenone prostaglandins PGA 2 and PGA 1 (8, 9).The transcription factor NF-B plays a key role in the activation of inflammatory response genes (10). In resting cells, NF-B is sequestered in the cytoplasm by association with an inhibitory protein IB. In response to signaling by inflammatory cytokines, IB kinase (IKK) is activated and phosphorylates IB on two serine residues. IB is then ubiquitinated...

The DNA binding of three different NF-B dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-B p50/p65 heterodimer is shown here to bind the B DNA target site of the immunoglobulin enhancer (Ig-B) with an affinity of approximately 10 nM. The p50 and p65 homodimers bind to the same site with roughly 5-and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-B complex. The heterodimer binds to the Ig-B DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-B and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mM, with binding becoming virtually nonspecific at a salt concentration of 200 mM. Binding of the heterodimer to DNA was unchanged across a temperature range between 4°C and 42°C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-B p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.The Rel/NF-B transcription factors constitute one of the most important families of regulatory transcription factors. Members of the Rel/NF-B family are essential for diverse biological functions such as the regulation of innate and adaptive immunity, development, and apoptosis in a wide array of eukaryotes from Drosophila to man (1-4). Like most transcription factors, dimers of NF-B proteins modulate transcription by directly binding to enhancer sequences located in the regulatory regions of numerous genes. These DNA sequences are collectively known as B DNA sequences. In mammals, the Rel/NF-B dimers arise from five polypeptides, p50, p52, p65, c-Rel, and RelB. The most abundant of these dimers are the p50/p65 heterodimer and the p50 homodimer. The existences of some, but not all, of the other possible dimers have been shown to exist in cells.The NF-B family can be divided into two subgroups based on the presence or absence of an activation domain. p50 and p52 do not contain a distinct activation domain and belong to class I. The other three members constitute the class II subfamily. It is generally believed that the homodimers of p50 and p52 and the p50/p52 heterodimer function as transcriptional repressors. The remaining combinations of dimeric NF-B proteins, containing at least one monomer of p65, c-Rel, or RelB, act as activators. Rel/NF-B proteins share a region that shows over 45% sequence similarity across the entire family. This region, known as the Rel homology region (RHR), 1 is responsible for DNA binding and subunit dimerization. High resolution x-ray crystal structures of RHRs are known for four homodimers, p50, p52, p65, and c-Rel in their DNA-bound conformations (5-8). These struc...

X-ray crystal structures of the NF-B⅐IB␣ complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both IB␣ and NF-B. In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity. IB␣ preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker. Our results indicate that the nuclear localization sequence and the region C-terminal to it of the NF-B p65 subunit is a major contributor to NF-B⅐IB␣ complex formation. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and IB␣. A second set of interactions involving the acidic C-terminal/PEST-like region of IB␣ and the NF-B p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking. Phosphorylation of the C-terminal/ PEST-like region by casein kinase II further enhances binding.The Rel/NF-B family transcription factors perform a vital role in mediating the cellular response to stress, inflammation, the immune response, and apoptosis (1-4). Mammalian Rel/ NF-B family polypeptides include p65 (RelA), p50, p52, RelB, and the proto-oncoprotein c-Rel. These subunits associate in various combinations to form homodimers and heterodimers with distinct but overlapping functions. Among the most abundant and best understood of these dimers are the p50/p65 heterodimer and the homodimers of p50 and p65 (5). The functions of other NF-B dimers are generally restricted to specific cell types.In most cells the NF-B p50/p65 heterodimer exists in the cytoplasm as a complex with the inhibitor protein IB␣. IB␣ inactivates the NF-B p50/p65 heterodimer by masking the NF-B nuclear localization sequences (6). Activation of signaling pathways by extracellular signals, such as tumor necrosis factor or interleukin-1, leads to proteolysis of IB␣, allowing active NF-B p50/p65 heterodimer to translocate into the nucleus (7).IB␣ is composed of three distinct regions: an N-terminal signal receiving domain (SRD), 1 a central ankyrin repeat-containing domain (ARD), and an acidic C-terminal/PEST-like region rich in the amino acids proline, glutamic acid, serine, and threonine (8). IB␣ is phosphorylated my multiple proteins in vivo. The C-terminal/PEST-like region in constitutively phosphorylated by casein kinase II (CKII) (9, 10), and the SRD contains sites of inducible, signaling-dependent phosphorylation (11). X-ray crystal structures of IB␣ in complex with the NF-B p50/p65 heterodimer reveal an extensive and complex protein-protein interface (12, 13). The structures reveal that an apparently flexible segment of the NF-B p65 subunit, which contains the NLS, becomes ordered upon complex formation and makes several contacts with IB␣. Interestingly, no direc...

Taken together, these structures reveal that p65 exhibits the unique capability to specifically bind DNA targets of variable lengths from four to ten base pairs. Also, the small protein segment Arg41-Ser42-Ala43 is at least partially responsible for flexibility in DNA-binding modes.