Inhibiting TRAF2-mediated Activation of NF-κB Facilitates Induction of AP-1

Sunil K. Manna,Banaganapalli Babajan,Pongali B. Raghavendra,Nune Raviprakash,Chitta Sureshkumar

doi:10.1074/jbc.m109.094961

Abstract

The compound 5-(4-methoxyarylimino)-2-N-(3,4-dichlorophenyl)-3-oxo-1,2,4-thiadiazolidine (P(3)-25) is known to possess anti-bacterial, anti-fungal, and anti-tubercular activities. In this report, we provide evidence that P(3)-25 inhibits NF-kappaB, known to induce inflammatory and tumorigenic responses. It activates AP-1, another transcription factor. It inhibits TRAF2-mediated NF-kappaB activation but not TRAF6-mediated NF-kappaB DNA binding by preventing its association with TANK (TRAF for NF-kappaB). It facilitates binding of MEKK1 with TRAF2 and thereby activates JNK and AP-1. We provide evidence, for the first time, that suggests that the interaction of P(3)-25 with TRAF2 leads to inhibition of the NF-kappaB pathway and activation of AP-1 pathway. These results suggest novel approaches to design of P(3)-25 as an anti-cancer/inflammatory drug for therapy through regulation of the TRAF2 pathway.

Highlights

Constitutively expressed nuclear transcription factor ␬B (NF-␬B) in tumor cells, the cell growth has been shown to be reduced dramatically both in vitro and in vivo [12,13,14]
P3-25 Inhibits NF-␬B but Increases AP-1 Activation Pathway— Jurkat and HuT-78 cells were treated with P3-25 (100 nM) for different times, and DNA binding to NF-␬B was measured by a gel shift assay
The amount of DNA binding to AP-1 was enhanced in Jurkat cells with increasing time of P3-25 treatment, whereas the high basal DNA binding to AP-1 (5.2-fold, p Ͻ 0.005) remained unaltered upon P3-25 treatment in HuT-78 cells (Fig. 1B1)

Summary

EXPERIMENTAL PROCEDURES

Sigma. 5-aryl TZD was obtained from Merck. Penicillin, streptomycin, neomycin, RPMI 1640 medium, and fetal bovine serum were obtained from Invitrogen. Jurkat and HuT-78 cells were treated with 100 nM P3-25 for different times, and nuclear extracts were prepared. The whole cell extracts (300 ␮g of protein) were immunoprecipitated with anti-IKK␣ and IKK␤ (1 ␮g each) antibodies, and kinase was assayed using GST-I␬B␣ as a substrate (C). The whole cell extracts (300 ␮g of protein) were immunoprecipitated with 1 ␮g of anti-JNK antibody, and JNK activity was assayed using GST-Jun as substrate (D, bottom). HuT-78 cells, transfected with the NF-␬B-SEAP construct were treated with 100 pM P3-25 or 5-aryl TZD for different times, and activity of SEAP was measured from culture supernatant (E). GFP-positive cells were counted and evaluated for transfection efficiency These cells were treated with P3-25 for different time periods. The cell pellets were extracted with lysis buffer (part of the luciferase assay kit from Promega), and the extracts were incubated with the firefly luciferin substrate (Promega)

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RESULTS

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DISCUSSION