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...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 lox P 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 Brd U 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-Brd U-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 modelto 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 Brd U-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: C a s p 8- d e p e n d e n t m i g ra t i o n o f enterocytes is likely involved in intestinal physiology and inflammation-related pathophysiology.展开更多
基金Supported by Deutsche Forschungsgemeinschaft,No.DFG GA 785/5-1(partially)Deutsche Krebshilfe,No.GA 109313(partially)
文摘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 lox P 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 Brd U 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-Brd U-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 modelto 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 Brd U-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: C a s p 8- d e p e n d e n t m i g ra t i o n o f enterocytes is likely involved in intestinal physiology and inflammation-related pathophysiology.
