Activation of Wnt Signaling Protects Intestinal Epithelial Cells from Apoptosis By Increasing Cytoplasmic Levels of Rna-Binding Protein Hur
Emily C. Bellavance*1, Lan Liu1, Rao N. Jaladanki1, Tongtong Zou1, Douglas J. Turner1, Jian-Ying Wang1,2
1Department of Surgery, University of Maryland School of Medicine and Baltimore VA Medical Center, Baltimore, MD; 2Department of Pathology, University of Maryland School of Medicine and Baltimore VA Medical Center, Baltimore, MD
Apoptosis plays a critical role in maintenance of gut mucosal homeostasis, but the mechanism underlying this process remains unclear. Wnts are cysteine-rich glycoproteins that act as short-range ligands to locally activate receptor-mediated signaling pathways. Central to this signaling pathway is the stabilization of β-catenin and its interaction with DNA-binding factors of the T-cell factor family in the nucleus. Recently, genetic studies have shown an essential role for canonical Wnt signaling in gut development and intestinal epithelial cell (IEC) proliferation. Activation of Wnt signaling is implicated in controlling cell fate and carcinogenesis by modulating its target gene expression. HuR is the RNA-binding protein and plays a critical role in regulating apoptosis. Predominantly nuclear in unstimulated cells, HuR rapidly translocates to the cytoplasm where it induces expression of anti-apoptotic genes posttranscriptionally. We hypothesized that Wnt signaling activation protects IECs against apoptosis by altering HuR functions.
Methods: Studies were conducted in IEC-6 cells derived from rat small intestinal crypts. Wnt activation was induced by stable transfection with the expression vector containing Wnt-3 cDNA. Apoptosis was induced by exposure to tumor necrosis factor-α (TNF-α) in combination with cyclohexamide (CHX). Apoptosis was assessed by immunohistochemical staining for Annexin-V and by caspase-3 activity. Levels of cytoplasmic and nuclear HuR were determined by Western blot analysis.
Results: Stable Wnt3-transfected IEC-6 cells (Wnt-IECs) highly expressed Wnt3 protein. Levels of Wnt3 protein in Wnt-IECs were ~5-fold the value of parental IEC-6 cells (C-IECs) transfected with the vector containing no Wnt3 cDNA. After exposure to TNFα/CHX, Wnt-IECs had a significantly lower percentage of apoptotic cells and exhibited decreased levels of caspase-3 activity compared with C-IECs (p <0.001). C-IECs showed higher levels of caspase-3 activity and stronger Annexin-V staining, while Wnt-IECs displayed lower apoptotic indicators. Wnt-IECs also demonstrated 2-fold higher levels of cytoplasmic HuR, although there were no differences in total levels of HuR between C-IECs and Wnt-IECs.
Conclusion: These findings indicate that 1) activation of Wnt signaling pathway protects IECs against TNF-α/CHX-induced apoptosis and 2) increased resistant to apoptosis by Wnt3 overexpression is associated with an increase in levels of cytoplasmic HuR.
