Frizzled gene expression and negative regulation of canonical WNT–β-catenin signaling in mouse F9 teratocarcinoma cells

• Published in: Biochemistry and cell biology Vol. 95; no. 2; pp. 251 - 262
• Main Authors: Golenia, Gregory, Gatie, Mohamed I, Kelly, Gregory M
• Format: Journal Article
• Language: English
• Published: Canada NRC Research Press 01.04.2017; Canadian Science Publishing NRC Research Press
• Subjects: Adaptor Proteins, Vesicular Transport - genetics; Adaptor Proteins, Vesicular Transport - metabolism; Animals; beta Catenin - genetics; beta Catenin - metabolism; Biochemistry; Biology; Cancer; Cell Differentiation - drug effects; Cell Line, Tumor; Cells; Conditioning; Culture Media, Conditioned - pharmacology; Dickkopf; Differentiation; Dkk1 protein; embryon de souris; Endoderm; Endoderm - metabolism; Endoderm - pathology; endoderme extra-embryonnaire; extraembryonic endoderm; Feedback; Feedback loops; Feedback, Physiological; Frizzled gene; Frizzled protein; Gene expression; Gene Expression Regulation, Neoplastic; Genes, Reporter; Hepatocyte Nuclear Factor 1-alpha - genetics; Hepatocyte Nuclear Factor 1-alpha - metabolism; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; LEF protein; Lymphoid Enhancer-Binding Factor 1 - genetics; Lymphoid Enhancer-Binding Factor 1 - metabolism; Mice; mouse embryo; Negative feedback; Proteins; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Receptors, G-Protein-Coupled - antagonists & inhibitors; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Retinoic acid; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Rodents; Signal Transduction; siRNA; Studies; Teratocarcinoma; Teratocarcinoma - genetics; Teratocarcinoma - metabolism; Teratocarcinoma - pathology; Teratocarcinoma cells; Transcription activation; Tretinoin - pharmacology; WNT; Wnt protein; Wnt Proteins - genetics; Wnt Proteins - metabolism; β-Catenin; β-caténine; endoderme extra-embryonnaire; β-catenin; Dickkopf; β-caténine; embryon de souris; mouse embryo; extraembryonic endoderm; WNT
• ISSN: 0829-8211; 1208-6002
• DOI: 10.1139/bcb-2016-0150

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT–β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin–TCF–LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF–LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT–β-catenin signaling, thereby allowing PE cells to differentiate.