P127 THE TRANS-GOLGI NETWORK PROTEIN AFTIPHILIN REGULATES COLONIC EPITHELIAL CELL MIGRATION AND IS CRUCIAL TO EMBRYONIC DEVELOPMENT

Abstract

Colonic epithelium forms a selective barrier between lumen and colonic mucosa. The integrity of colonic epithelium is maintained by cell proliferation, migration and expression of cellular junctions. A compromised colonic epithelium contributes to colonic inflammation in IBD patients. Previously, we showed that aftiphilin (AFTPH) is significantly downregulated in colonic biopsies from UC patients and involved in colonic epithelial cell permeability. In this study, we examined the role of AFTPH in cell proliferation and migration in vitro and in colonic inflammation by generation of total AFTPH knock-out mice. Human colonic epithelial NCM460 cells was transfected with si-RNA against AFTPH (si-AFTPH) and its control using lipofectamineRNAimax (Thermo Fisher Scientific). Two days post-transfection, cell migration and cell proliferation were assessed by scratch assay and MTT assay, respectively. Hemizygous AFTPH knock-out (AFTPH+/-) mice were generated by crossing heterozygous AFTPH-floxed mice (AFTPHflox/+, Australian Regenerative Medicine Institute, Monash University) and CMV-Cre mice (JAX no. 006054). Total AFTPH knock-out (AFTPH-/-) mice were generated by crossing AFTPH+/- mice. Neonates and embryos from Embryonic day (E) 13 and 17 were genotyped (Transnetyx). Gene-silencing of AFTPH in human colonic epithelial NCM460 cells reduced the rate of cell migration when compared to that of cells transfected with control si-RNA (area ratio of wound closure: 0.1 ± 0.04 vs. 0.2 ± 0.03). On contrary, AFTPH gene silencing did not affect cell proliferation when compared to control cells (OD570nm: 0.5 ± 0.01 vs. 0.5 ± 0.11). To study the role of AFTPH in colonic inflammation and intestinal permeability in vivo, we aimed to generate total AFTPH knock-out (AFTPH-/-) mice by crossbreeding AFTPH+/- mice. However, no AFTPH-/- mice were identified in neonates (AFTPH+/+: AFTPH+/-: AFTPH-/- = 5:16:0, average litter size = 3.0 ± 1.38). To study whether total AFTPH knock-out is embryonic lethal, embryos from E13 and E17 were collected and genotyped. Interestingly, our results showed that 7 AFTPH-/-mice were identified among the E13 embryos (AFTPH+/+: AFTPH+/-: AFTPH-/- = 5:14:7, average litter size of 8.7 ± 1.15); while 1 AFTPH-/- mouse were identified among the E17 embryos (AFTPH+/+: AFTPH+/-: AFTPH-/- = 10:11:1, average litter size of 7.3 ± 0.58). Our results indicated that AFTPH regulates colonic epithelial cell migration, but not cell proliferation, suggesting a potential role in wound healing during colitis. Importantly, total AFTPH knock-out is possibly embryonic lethal. An intestinal epithelial cell-specific (Villin-Cre) AFTPH knock-out mice is now being generated for studying the role of AFTPH in colonic cell function in vivo.