Wnt/-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

^* To whom correspondence should be addressed. E-mail: gellerda{at}upmc.edu.

	Abstract

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/-catenin pathway. In this study, we tested the hypothesis that Wnt/-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)-1 + 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 -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 -catenin–mutant HA18 cells. Conversely, enhanced p50 binding and increased NF-B reporter activity were seen in HA85 cells, which lack -catenin signaling. Coimmunoprecipitation confirmed that -catenin complexed with both p65 and p50 NF-B proteins. NF-B–dependent Traf1 protein expression also inversely correlated with the level of -catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased -catenin protein and increased TNF-induced p65 NF-B binding as well as iNOS and Traf1 expression. Finally, -catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that -catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-B–dependent gene expression. These findings underscore the complex role of Wnt/-catenin, NF-B, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis. [Cancer Res 2009;69(9):3764–71]

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