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Wnt/β-Catenin Signaling Regulates Cytokine-Induced Human Inducible Nitric Oxide Synthase Expression by Inhibiting Nuclear Factor-B Activation in Cancer Cells

Department of Surgery, T.E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, Pennsylvania

Requests for reprints: David A. Geller, University of Pittsburgh, UPMC Montefiore, 7 South, 3459 Fifth Avenue, Pittsburgh, PA 15213-2582. Phone: 412-692-2001; Fax: 412-692-2002; E-mail: gellerda{at}upmc.edu.

Key Words: iNOS • nitric oxide • NF-B • Wnt?β-catenin • hepatocellular carcinoma

The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor B (NF-B) and has recently been shown to be a target of the Wnt/β-catenin pathway. In this study, we tested the hypothesis that Wnt/β-catenin signaling might regulate cytokine- or tumor necrosis factor (TNF)–induced hiNOS expression through interaction with NF-B. A cytokine mixture of TNF + interleukin (IL)-1β + IFN induced a 2- to 3-fold increase in hiNOS promoter activity in HCT116 and DLD1 colon cells, but produced a 2-fold decrease in SW480 colon cancer cells. A similar differential activity was seen in liver cancer cells (HepG2, Huh7, and Hep3B). Overexpression of β-catenin produced a dose-dependent decrease in NF-B reporter activity and decreased cytokine mixture–induced hiNOS promoter activity. Gel shift for TNF-induced hiNOS NF-B activation showed decreased p50 binding and decreased NF-B reporter activity in the β-catenin–mutant HAβ18 cells. Conversely, enhanced p50 binding and increased NF-B reporter activity were seen in HAβ85 cells, which lack β-catenin signaling. Coimmunoprecipitation confirmed that β-catenin complexed with both p65 and p50 NF-B proteins. NF-B–dependent Traf1 protein expression also inversely correlated with the level of β-catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased β-catenin protein and increased TNF-induced p65 NF-B binding as well as iNOS and Traf1 expression. Finally, β-catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that β-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-B–dependent gene expression. These findings underscore the complex role of Wnt/β-catenin, NF-B, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis. [Cancer Res 2009;69(9):3764–71]