Genetic Deletion of the Tumor Necrosis Factor Receptor p60 or p80 Sensitizes Macrophages to Lipopolysaccharide-induced Nuclear Factor-κB, Mitogen-activated Protein Kinases, and Apoptosis

Journal of Biological Chemistry

Kobayashi

Aggarwal

2005

Self Cite

171

102

Evodiamine, an alkaloidal component extracted from the fruit of Evodiae fructus (Evodia rutaecarpa Benth., Rutaceae), exhibits antiproliferative, antimetastatic, and apoptotic activities through a poorly defined mechanism. Because several genes that regulate cellular proliferation, carcinogenesis, metastasis, and survival are regulated by nuclear factor-B (NF-B), we postulated that evodiamine mediates its activity by modulating NF-B activation. In the present study, we investigated the effect of evodiamine on NF-B and NF-B-regulated gene expression activated by various carcinogens. We demonstrate that evodiamine was a highly potent inhibitor of NF-B activation, and it abrogated both inducible and constitutive NF-B activation. The inhibition corresponded with the sequential suppression of IB␣ kinase activity, IB␣ phosphorylation, IB␣ degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Evodiamine also inhibited tumor necrosis factor (TNF)-induced Akt activation and its association with IKK. Suppression of Akt activation was specific, because it had no effect on JNK or p38 MAPK activation.

Section: Methodsmentioning

confidence: 99%

Evodiamine Abolishes Constitutive and Inducible NF-κB Activation by Inhibiting IκBα Kinase Activation, Thereby Suppressing NF-κB-regulated Antiapoptotic and Metastatic Gene Expression, Up-regulating Apoptosis, and Inhibiting Invasion

Journal of Biological Chemistry

Kobayashi

Aggarwal

2005

Self Cite

171

102

“…Electrophoretic Mobility Shift Assays (EMSA)-To measure NF-B activation, we performed EMSA as described previously (44,45). Briefly, nuclear extracts prepared from TNF-treated cells (1 ϫ 10 6 /ml) were incubated with 32 P-end-labeled 45-mer double-stranded NF-B oligonucleotides (10 g of protein with 16 fmol of DNA) from the human immunodeficiency virus long terminal repeat, 5Ј-TTGTTACAAGGGA-CTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3Ј (boldface type indicates NF-B binding sites) for 30 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotides on 6.6% native polyacrylamide gels.…”

Section: Methodsmentioning

confidence: 99%

Identification of a p65 Peptide That Selectively Inhibits NF-κ B Activation Induced by Various Inflammatory Stimuli and Its Role in Down-regulation of NF-κB-mediated Gene Expression and Up-regulation of Apoptosis

Journal of Biological Chemistry

Singh²,

Aggarwal

2004

Self Cite

173

130

Because of the critical role of the nuclear transcription factor NF-B in inflammation, viral replication, carcinogenesis, antiapoptosis, invasion, and metastasis, specific inhibitors of this nuclear factor are being sought and tested as treatments. NF-B activation is known to require p65 phosphorylation at serine residues 276, 529, and 536 before it undergoes nuclear translocation. Small protein domains, termed protein transduction domains (PTDs), which are able to penetrate cell membranes can be used to transport other proteins across the cell membrane. We have identified two peptides from the p65 subunit of NF-B (P1 and P6 were from amino acid residues 271-282 and 525-537, respectively) that, when linked with a PTD derived from the third helix sequence of antennapedia, inhibited tumor necrosis factor (TNF)-induced NF-B activation in vivo. Linkage to the PTD was not, however, required to suppress the binding of the p50-p65-heterodimer to the DNA in vitro. PTD-p65-P1 had no effect on TNF-induced AP-1 activation. PTD-p65-P1 suppressed NF-B activation induced by lipopolysaccharide, interleukin-1, okadaic acid, phorbol 12-myristate 13-acetate, H 2 O 2 , and cigarette smoke condensate as well as that induced by TNF. PTD-p65-P1 had no effect on TNF-induced inhibitory subunit of NF-B (IB␣) phosphorylation, IB␣ degradation, or IB␣ kinase activation, but it blocked TNF-induced p65 phosphorylation and nuclear translocation. NF-B-regulated reporter gene expression induced by TNF, TNF receptor 1, TNF receptor-associated death domain, TNF receptor-associated factor-2, NF-B-inducing kinase, IB␣ kinase, and p65 was also suppressed by these peptides. Suppression of NF-B by PTD-p65-P1 enhanced the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results demonstrate the identification of a p65 peptide that can selectively inhibit NF-B activation induced by various inflammatory stimuli, down-regulate NF-B-mediated gene expression, and up-regulate apoptosis.NF-B represents a group of five proteins, namely c-Rel, RelA (p65), RelB, NF-B1 (p50 and p105), and NF-B2 (p52) (1). Ways to modulate NF-B expression therapeutically have focused on its active-inactive state transition mechanisms. NF-B is regulated by a family of inhibitors, called IB 1 (2). In an inactive state, NF-B is present in the cytoplasm as a heterotrimer consisting of p50, p65, and IB␣ subunits. In response to an activation signal, the IB␣ subunit is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteosomal pathway, thus exposing the nuclear localization signals on the p50-p65 heterodimer. The p65 is then phosphorylated, leading to the nuclear translocation and binding to a specific sequence in DNA, which in turn results in transcriptions of various genes including cyclin D1, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9.NF-B has been shown to regulate the expression of a number of genes whose products are involved in inflammation, viral replication, carcinogenesis, ant...

“…The concentration of stable nitrite was determined by Griess reaction, as previously described (14). The absorbance at 530 nm was determined on a plate reader (680XR; BioRad Laboratories, Hercules, CA).…”

Section: No Production Assaymentioning

confidence: 99%

“…NF-kB or AP-1 DNA-binding activity was assessed with EMSA, as described previously (14). Briefly, 3 mg nuclear extracts prepared from M-CSF-dependent macrophages (MDMs) and colon fragments was incubated for 30 min at 37˚C with [ 32 P]-end-labeled 27-mer double-stranded oligonucleotide containing the kB3 binding site of mouse TNF-a promoter (59-AGCTGAGGAGGGAAAGCCCCCTGTTTG-39) (15) or 28-mer double-stranded oligonucleotide containing the consensus AP-1 binding site of the human collagenase promoter (16).…”

Section: Emsamentioning

confidence: 99%

Fos Proteins Suppress Dextran Sulfate Sodium-Induced Colitis through Inhibition of NF-κB

The Journal of Immunology

Ray²,

Ikeda

et al. 2009

Self Cite

The Fos family proteins, c-Fos and Fra-1, are components of the dimeric transcription factor AP-1, which is typically composed of Fos and Jun family proteins. We have previously shown that mice lacking c-Fos (Fos−/− mice) respond more strongly to LPS injection than do wild-type (wt) controls. We then examined the sensitivity of Fos−/− mice to acute inflammatory stress in a dextran sulfate sodium (DSS)-induced colitis model. We found that Fos−/− mice exhibited more severe weight loss, bleeding, diarrhea, and colon shortening than did wt mice, in association with higher TNF-α production and NF-κB activity in colon segments of DSS-treated Fos−/− mice. Furthermore, NF-κB inhibition suppressed severe DSS-induced colitis in Fos−/− mice. In contrast, Fra-1 transgenic (Tg) mice responded poorly to LPS injection, and Fra-1–overexpressing macrophages and fibroblasts showed reduced production of proinflammatory cytokines, NO, and NF-κB activity. Remarkably, in the DSS-induced colitis model, Fra-1 Tg mice showed less severe clinical scores of colitis than did wt mice. Consistently, proinflammatory cytokine production and NF-κB activity in colon segments of DSS-treated Fra-1 Tg mice were lower than in wt controls. These findings reveal that the absence of c-Fos and overexpression of Fra-1 respectively enhance and suppress the activation of NF-κB in DSS-induced inflammatory stress. In this paper, we propose that AP-1 transcription factors containing c-Fos or Fra-1 are negative regulators of NF-κB–mediated stress responses.