Stromelysin-1 and mesothelin are differentially regulated by Wnt-5a and Wnt-1 in C57mg mouse mammary epithelial cells

Abstract

Background: The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca2+ pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca2+ pathway and whether it regulates gene expression.

Results: To test the hypothesis that a specific cell line can respond to distinct Wnts with different patterns of gene expression, we over-expressed Wnt-5a and Rfz-2 in C57mg mammary epithelial cells and compared this cell line to C57mg cells over-expressing Wnt-1. These Wnts were chosen since previous studies suggest that C57mg cells respond differently to these Wnts, and since these Wnts can activate different signaling pathways in other systems. Using DNA microarray analysis, we identified several genes that are regulated by Wnt-5a and Rfz-2 as well as by Wnt-1. We then focused on two genes previously linked to various cancers, mesothelin and stromelysin-1, which are respectively up-regulated by Wnt-1 and Wnt-5a in C57mg cells.

Conclusion: Different Wnts have distinct effects on gene expression in a single cell line.

Figure 1

Induction of mesothelin and SL-1 expression by Wnt-1 or Wnt-5a signaling. A. RT-PCR was performed on RNA obtained from 3 separate experiments in which C57mg cells were co-cultured with 293T cells expressing Wnt-1, Wnt-5a, or CS2+ as control. The amounts of mesothelin and SL-1 mRNAs were normalized to HPRT and plotted as fold increase relative to C57mg cells co-culture with 293T cells expressing CS2+. B. C57mg cells (wt) or C57mg cells stably expressing the Rfz-2/β₂AR chimeric receptor were treated with the β₂AR agonist isoproterenol or the antagonist propranolol as control for 8 hours prior to harvesting for RNA extraction and RT-PCR analysis. The amounts of mesothelin and SL-1 mRNAs were normalized to HPRT and plotted as fold increase relative to propranolol treated cells. Mean levels were determined from 3 separate experiments.

Figure 2

Effect of Li+ or PMA on mesothelin and SL-1 expression in C57mg cells. For Li+ treatment, C57mg cells were treated with 10 mM LiCl or 10 mM KCl as control for 6 hours. For PMA treatment, C57mg cells were treated with 1 μM PMA or ethanol as control for 6 hours. RNA was extracted and RT-PCR analysis was performed using primers specific to mesothelin or SL-1 mRNAs. mRNA levels were normalized to HPRT and fold increase shown is relative to KCl treatment in the case of Li+ and to ethanol treatment in the case of PMA. Mean levels were determined from 3 independent experiments.