Abstract
BRI3 (brain protein I3) is one of the Wnt/β-catenin pathway target genes as indicated by the results of serial analysis of gene expression (SAGE) and microarray analyses performed in our laboratory. The Wnt/β-catenin signaling pathway is an evolutionarily conserved pathway, which has important functions in early vertebrate development, axis formation, cellular proliferation, and morphogenesis. Previous studies showed that BRI3 expression is upregulated at both mRNA and protein levels upon β-catenin activation by various approaches, such as lithium treatment and overexpression of Wnt ligands in Huh7 (hepatocellular carcinoma) cell lines. Moreover, with regard to the previous literature, BRI3 was found to have a very important role in the TNFα-mediated cell death pathway. In this study, we screened a human liver cDNA library by yeast two-hybrid assay using BRI3 protein as bait, with the aim of finding novel interaction partners of BRI3. Library screening by yeast mating resulted in the identification of three candidate positive clones. Among these, IFITM3 and MGAT1 proteins were confirmed as interaction partners by using cotransformation in yeast cells and coimmunoprecipitation from mammalian cell lines. Considering the poor functional characterization of BRI3 to date, identification of novel BRI3-interacting proteins is an essential first step in determining the action mechanism of BRI3 with respect to the Wnt/β-catenin pathway.
Highlights
BRI3 was originally identified as a 125-amino-acid transmembrane protein that is overexpressed in TNFα-treated L929-murine fibrosarcoma cells (Wu et al, 2003)
BRI3 has been selected among the potential Wnt/βcatenin signaling pathway targets based on serial analysis of gene expression (SAGE) screening and an equivalent microarray screening (Kavak et al, 2010)
In order to identify novel transcriptional targets of the Wnt/β-catenin signaling pathway, these transcriptome profile analyses were performed in our laboratory using stable Huh7 cell lines overexpressing a mutant form of β-catenin, which is degradation-resistant
Summary
BRI3 (brain protein I3) was originally identified as a 125-amino-acid transmembrane protein that is overexpressed in TNFα-treated L929-murine fibrosarcoma cells (Wu et al, 2003). In order to identify novel transcriptional targets of the Wnt/β-catenin signaling pathway, these transcriptome profile analyses were performed in our laboratory using stable Huh (hepatocellular carcinoma) cell lines overexpressing a mutant form of β-catenin, which is degradation-resistant. BRI3 was among the several putative Wnt/β-catenin target genes that were detected with differential expression profiles upon β-catenin induction in the Huh cell line. Lithium treatment of Huh cell lines and overexpression of the Wnt ligands in the same cell lines resulted in the upregulation of BRI3 gene expression, as determined by quantitative RT-PCR (Kavak et al, 2010). The results obtained from luciferase reporter gene assay, in which BRI3 promoter activity was found to be increased due to overexpression of β-catenin, supported the previous data. Chromatin immunoprecipitation (ChIP) assays indicated that β-catenin interacts with the BRI3 promoter region in Huh cell lines and in mouse liver tissue
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