IGF-1 also enhanced the stability of beta-catenin protein.
To investigate whether IGF-1 also enhanced transactivation potential, a reporter gene assay was performed in HEK293 cells. We used a reporter construct incorporating multimeric Lef/Tcf promoter sequences upstream of a luciferase reporter (TOPFLASH) and a control construct containing mutated promoter sequences (FOPFLASH, ref. 21). We cotransfected TOPFLASH or FOPFLASH, ?-catenin, and human wild-type Tcf-4 wild-type expression constructs into 293 cells. Optimized luciferase assays showed a basal level of TOP/FOP activity of between 4:1 and 20:1. Forty-eight hours after transfection, serum-starved 293 cells were treated with IGF-1 or insulin for 3 or 6 h. These factors alone failed to alter basal reporter activity (Fig. 6 b). A 16-h pretreatment of the transfectants with LiCl had a dramatic effect on reporter activity, with significant enhancement at 50 mM LiCl (P < 0.01 by Tukey's test for comparison with activity in the absence of LiCl). We saw a further increase in reporter activity when we added 50 ng/ml (6.5 nM) IGF-1 to transfectants pretreated with LiCl at 10 mM (P < 0.01 after 6 h of IGF-1 treatment) and at 50 mM (P < 0.01 after 3 h of IGF-1 treatment and P < 0.001 after 6 h).
Most colorectal cancers have loss of function mutations in the adenomatosis polyposis coli (APC) tumor suppressor gene. This leads to accumulation of beta-catenin, which together with the DNA binding protein TCF-4 functions as a transcriptional activator.
Here it is shown that one of the target genes of Tcf4 in epithelial cells is Tcf1 (NOTE: TCF7 is the current gene symbol for Tcf1) ....(from full text) ... Tcf1 expression is regulated by APC and beta -catenin-Tcf4. ...We sequenced 1/2 kb directly upstream of promoter I and found the region to be a CpG island containing two potential Tcf0binding motifs (Fig. 2B). The combination of beta -catenin and Tcf4 transactivated the enhancer three- to fourfold in a transient reporter assay (Fig. 2C). Furthermore, expression of a dominant-negative Tcf4 (Delta NTcf4, which lacks the beta -catenin interaction domain) inhibited enhancer activity in LS174T colorectal cancer cells (Fig. 2D).
IGFs can enhance cell migration, which is known to be influenced via regulation of the E-cadherin/beta-catenin complex. IGF-1 causes tyrosine phosphorylation and stabilization of beta-catenin. These effects may contribute to transformation, cell migration, and a propensity for metastasis in vivo.
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