Intestinal genetic inactivation of caspase-8 diminishes migration of enterocytes.
OBJECTIVE
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 (cell(max)) 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 cell(max) 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, cell(max) 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 cell(max) 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).


CONCLUSIONS
Casp8-dependent migration of enterocytes is likely involved in intestinal physiology and inflammation-related pathophysiology.
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
E
Kaemmerer
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.
Paula
P
Kuhn
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.
Ursula
U
Schneider
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.
Min Kyung
MK
Jeon
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.
Christina
C
Klaus
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.
Miriam
M
Schiffer
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.
Danika
D
Weisner
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.
Christian
C
Liedtke
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.
Jörg
J
Jäkel
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.
Lieven Nils
LN
Kennes
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.
Ralf-Dieter
RD
Hilgers
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.
Norbert
N
Wagner
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.
Nikolaus
N
Gassler
D000818:Animals; D018938:Caco-2 Cells; D053181:Caspase 8 / Q000096:biosynthesis* / Q000235:genetics; D002465:Cell Movement; D020895:Enterocytes / Q000201:enzymology* / Q000473:pathology; D004794:Enzyme Repression; D055785:Gene Knockdown Techniques; D005838:Genotype; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D007422:Intestines / Q000201:enzymology* / Q000473:pathology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8
Barrier function; Caspase 8; Cell migration; Inflammatory bowel disease; Intestinal morphogenesis