Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 21, 2015; 21(15): 4499-4508
Published online Apr 21, 2015. doi: 10.3748/wjg.v21.i15.4499
Intestinal genetic inactivation of caspase-8 diminishes migration of enterocytes
Elke Kaemmerer, Paula Kuhn, Ursula Schneider, Min Kyung Jeon, Christina Klaus, Miriam Schiffer, Danika Weisner, Christian Liedtke, Jörg Jäkel, Lieven Nils Kennes, Ralf-Dieter Hilgers, Norbert Wagner, Nikolaus Gassler
Elke Kaemmerer, Paula Kuhn, Ursula Schneider, Min Kyung Jeon, Christina Klaus, Miriam Schiffer, Danika Weisner, Jörg Jäkel, Nikolaus Gassler, Institute of Pathology, RWTH Aachen University, 52074 Aachen, Germany
Elke Kaemmerer, Norbert Wagner, Department of Pediatrics, RWTH Aachen University, 52074 Aachen, Germany
Christian Liedtke, Department of Medicine III, RWTH Aachen University, 52074 Aachen, Germany
Lieven Nils Kennes, Ralf-Dieter Hilgers, Department of Medical Statistics, RWTH Aachen University, 52074 Aachen, Germany
Author contributions: Kaemmerer E and Kuhn P contributed equally; Kaemmerer E, Klaus C and Gassler N designed the research; Kaemmerer E, Kuhn P, Schneider U and Jeon MK performed the research; Schiffer M, Liedtke C, Jäkel J, Weisner D, Kennes LN and Hilgers RD contributed analytic tools; Kaemmerer E, Liedtke C, Hilgers RD, Wagner N and Gassler N wrote the paper.
Supported by Deutsche Forschungsgemeinschaft, No. DFG GA 785/5-1 (partially); and Deutsche Krebshilfe, No. GA 109313 (partially).
Ethics approval: The study was reviewed and approved by the Authority for Environment Conservation and Consumer Protection of the State North Rhine-Westfalia Institutional Review Board.
Institutional animal care and use committee: All procedures were approved by the Authority for Environment Conservation and Consumer Protection of the State North Rhine-Westfalia (IACUC protocol number: LANUV, Germany; 84-02.04.2013.A034).
Conflict-of-interest: The authors report no conflicts of interest.
Data sharing: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Nikolaus Gassler, MA, Professor, Institute of Pathology, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, Germany. ngassler@ukaachen.de
Telephone: +49-241-8088897 Fax: +49-241-8082439
Received: October 15, 2014
Peer-review started: October 18, 2014
First decision: November 14, 2014
Revised: December 2, 2014
Accepted: January 30, 2015
Article in press: January 30, 2015
Published online: April 21, 2015
Abstract

AIM: To verify the hypothesis that caspase-8 (Casp8), which regulates cellular apoptosis and necroptosis, is critically involved in enterocyte migration.

METHODS: Casp8-silenced Caco2 cells were used in migration assays. In addition, enterocyte-specific Casp8 heterozygous (Casp8+/∆int) or homozygous knockout mice (Casp8∆int) were generated by crossing genetically modified mice carrying loxP recombination sites in intron 2 and 4 of the murine Casp8 gene with transgenic animals expressing a cre-transgene under control of the villin promoter in a pure C57/BL6 genetic background. The nucleoside analog BrdU was injected i.p. in male Casp8+/∆int and Casp8∆int animals 4 h, 20 h, or 40 h before performing morphometric studies. Locations of anti-BrdU-immunostained cells (cellmax) in at least 50 hemi-crypts of 6 histoanatomically distinct intestinal mucosal regions were numbered and extracted for statistical procedures. For the mice cohort (n = 28), the walking distance of enterocytes was evaluated from cellmax within crypt (n = 57), plateau (n = 19), and villus (n = 172) positions, resulting in a total of 6838 observations. Data analysis was performed by fitting a three-level mixed effects model to the data.

RESULTS: In cell culture experiments with Caco2 cells, Casp8 knockdown efficiency mediated by RNA interference on Casp8 transcripts was 80% controlled as determined by Western blotting. In the scratch assay, migration of Casp8-deleted Caco2 cells was significantly diminished when compared with controls (Casp8∆scramble and Caco2). In BrdU-labeled Casp8∆int mice, cellmax locations were found along the hemi-crypts in a lower position than it was for Casp8+/∆int or control (cre-negative) animals. Statistical data analysis with a three-level mixed effects model revealed that in the six different intestinal locations (distinct segments of the small and large intestine), cell movement between the three mice groups differed widely. Especially in duodenal hemi-crypts, enterocyte movement was different between the groups. At 20 h, duodenal cellmax location was significantly lower in Casp8∆int (25.67 ± 2.49) than in Casp8+/∆int (35.67 ± 4.78; P < 0.05) or control littermates (44.33 ± 0.94; P < 0.01).

CONCLUSION: Casp8-dependent migration of enterocytes is likely involved in intestinal physiology and inflammation-related pathophysiology.

Keywords: Barrier function, Caspase 8, Cell migration, Inflammatory bowel disease, Intestinal morphogenesis

Core tip: Caspase 8 (Casp8) is involved in necroptosis and apoptosis of enterocytes. In addition, Casp8 is thought to be a mediator of cellular migration. Using cell culture experiments, migration of Casp8-deleted Caco2 cells is diminished. In vivo, enterocyte migration is significantly impaired in Casp8-deficient mice. The phenomenon is especially found along the duodenal crypt-villus axis. It is suggested that Casp8-dependent enterocyte migration could be involved in intestinal physiology and pathophysiology.