Regulation of human nitric oxide synthase 2 expression by Wnt beta-catenin signaling.
Nitric oxide (NO.), an important mediator of inflammation, and beta-catenin, a component of the Wnt-adenomatous polyposis coli signaling pathway, contribute to the development of cancer. We have identified two T-cell factor 4 (Tcf-4)-binding elements (TBE1 and TBE2) in the promoter of human inducible NO synthase 2 (NOS2). We tested the hypothesis that beta-catenin regulates human NOS2 gene. Mutation in either of the two TBE sites decreased the basal and cytokine-induced NOS2 promoter activity in different cell lines. The promoter activity was significantly reduced when both TBE1 and TBE2 sites were mutated (P < 0.01). Nuclear extract from HCT116, HepG2, or DLD1 cells bound to NOS2 TBE1 or TBE2 oligonucleotides in electrophoretic mobility shift assays and the specific protein-DNA complexes were supershifted with anti-beta-catenin or anti-Tcf-4 antibody. Overexpression of beta-catenin and Tcf-4 significantly increased both basal and cytokine-induced NOS2 promoter activity (P < 0.01), and the induction was dependent on intact TBE sites. Overexpression of beta-catenin or Tcf-4 increased NOS2 mRNA and protein expression in HCT116 cells. Lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3beta, increased cytosolic and nuclear beta-catenin level, NOS2 expression, and NO. production in primary human and rat hepatocytes and cancer cell lines. Treatment with Wnt-3A-conditioned medium increased beta-catenin and NOS2 expression in fetal human hepatocytes. When administered in vivo, LiCl increased hepatic beta-catenin level in a dose-dependent manner with simultaneous increase in NOS2 expression. These data are consistent with the hypothesis that beta-catenin up-regulates NOS2 and suggest a novel mechanism by which the Wnt/beta-catenin signaling pathway may contribute to cancer by increasing NO. production.
Du
Qiang
Q
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Park
Kyung Soo
KS
Guo
Zhong
Z
He
Peijun
P
Nagashima
Makoto
M
Shao
Lifang
L
Sahai
Rohit
R
Geller
David A
DA
Hussain
S Perwez
SP
D000818:Animals; D045744:Cell Line, Tumor; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D045325:HCT116 Cells; D006801:Humans; D018021:Lithium Chloride / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008297:Male; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011917:Rats, Inbred Lew; D015398:Signal Transduction; D051820:TCF Transcription Factors / Q000096:biosynthesis / Q000378:metabolism; D058118:Transcription Factor 7-Like 2 Protein; D015533:Transcriptional Activation; D014162:Transfection; D051153:Wnt Proteins / Q000378:metabolism*; D051176:beta Catenin / Q000096:biosynthesis / Q000378:metabolism*
D012333:RNA, Messenger; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; C553059:Tcf7l2 protein, rat; D058118:Transcription Factor 7-Like 2 Protein; D051153:Wnt Proteins; D051176:beta Catenin; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride