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1.
World J Gastroenterol ; 21(15): 4499-508, 2015 Apr 21.
Article in English | MEDLINE | ID: mdl-25914458

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


Subject(s)
Caspase 8/biosynthesis , Cell Movement , Enterocytes/enzymology , Intestines/enzymology , Animals , Caco-2 Cells , Caspase 8/genetics , Enterocytes/pathology , Enzyme Repression , Gene Knockdown Techniques , Genotype , Humans , Inflammatory Bowel Diseases/enzymology , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/pathology , Intestines/pathology , Mice, Inbred C57BL , Mice, Knockout , Phenotype , RNA Interference , Signal Transduction , Time Factors , Transfection
2.
World J Gastroenterol ; 21(6): 1759-64, 2015 Feb 14.
Article in English | MEDLINE | ID: mdl-25684940

ABSTRACT

AIM: To hypothesize that beta-7 integrin affects cellular migration of both, lymphocytes and enterocytes. METHODS: The nucleoside analog BrdU was ip injected in beta-7-deficient mice (C57BL/6-Itgb(tmlcgn)/J) of male gender and age-matched male C57BL/J J mice (wild type) 4, 20, or 40 h before analysis. The total small intestine was isolated, dissected, and used for morphometrical studies. BrdU-positive epithelial cells were numbered in at least 15 hemi-crypts per duodenum, jejunum, and ileum of each animal. The outer most BrdU-positive cell (cell(max)) was determined per hemi-crypt, numerically documented, and statistically analysed. RESULTS: Integrins containing the beta-7-chain were exclusively expressed on leukocytes. In the small intestinal mucosa of beta-7 integrin-deficient mice the number of intraepithelial lymphocytes was drastically decreased. Moreover, the Peyer's patches of beta-7 integrin-deficient mice appeared hypoplastic. In beta-7 integrin-deficient mice the location of cell(max) was found in a higher position than it was the case for the controls. The difference was already detected at 4 h after BrdU application, but significantly increased with time (40 h after BrdU injection) in all small intestinal segments investigated, i.e., duodenum, jejunum, and ileum. Migration of small intestinal enterocytes was different between the experimental groups measured by cell(max) locations. CONCLUSION: The E-cadherin beta-7 integrin pathway probably controls migration of enterocytes within the small intestinal surface lining epithelial layer.


Subject(s)
Cell Movement , Enterocytes/metabolism , Integrin beta Chains/metabolism , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Animals , Integrin beta Chains/genetics , Intestinal Mucosa/cytology , Intestine, Small/cytology , Lymphocytes/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction , Time Factors
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