BCL-2 Modifying Factor (BMF) Is a Central Regulator of Anoikis in Human Intestinal Epithelial Cells

Martin Hausmann,Katharina Leucht,Christian Ploner,Stephan Kiessling,Andreas Villunger,Helen Becker,Claudia Hofmann,Werner Falk,Michaela Krebs,Silvia Kellermeier,Michael Fried,Jürgen Schölmerich,Florian Obermeier,Gerhard Rogler

doi:10.1074/jbc.m111.265322

Abstract

BCL-2 modifying factor (BMF) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma 2. B-cell lymphoma 2 is neutralized by BMF and thereby facilitates cytochrome C release from mitochondria. We investigated the role of BMF for intestinal epithelial cell (IEC) homeostasis. Acute colitis was induced in Bmf-deficient mice (Bmf(-/-)) with dextran sulfate sodium. Colonic crypt length in Bmf(-/-) mice was significantly increased as compared with WT mice. Dextran sulfate sodium induced less signs of colitis in Bmf(-/-) mice, as weight loss was reduced compared with the WT. Primary human IEC exhibited increased BMF in the extrusion zone. Quantitative PCR showed a significant up-regulation of BMF expression after initiation of anoikis in primary human IEC. BMF was found on mitochondria during anoikis, as demonstrated by Western blot analysis. RNAi mediated knockdown of BMF reduced the number of apoptotic cells and led to reduced caspase 3 activity. A significant increase in phospho-AKT was determined after RNAi treatment. BMF knockdown supports survival of IEC. BMF is induced in human IEC by the loss of cell attachment and is likely to play an important role in the regulation of IEC survival.

Highlights

Intestinal epithelial cells (IEC)4 are generated from stem cells at the base of the crypt and migrate on the underlying basement membrane toward the intestinal lumen in 3–5 days
We investigated the role of BCL-2 modifying factor (BMF) for intestinal epithelial cell (IEC) homeostasis
Crypt Structures Are Protected in BmfϪ/Ϫ Mice upon DSS Colitis—We focused on the involvement of BMF in inflammatory responses and its property to induce apoptosis in IEC

Summary

Introduction

Intestinal epithelial cells (IEC)4 are generated from stem cells at the base of the crypt and migrate on the underlying basement membrane toward the intestinal lumen in 3–5 days. BMF mRNA in single cells from patients with inflammatory bowel disease was increased significantly (4.8 Ϯ 1.5-fold, Mann-Whitney rank sum test, p Ͻ 0.05) compared with crypts. Knockdown of BMF Maintains Phosphorylation of AKT and Blocks Activation of Caspases—Including the transport time of the resection from the department of surgery, isolation of crypts, and incubation on transwells, IEC were kept in viruscontaining media for 25.5 h.

Results

Conclusion