SummaryCells exposed to ER-stress undergo the Unfolded Protein Response (UPR) to avoid apoptosis, but may also activate autophagy. However, the signal for selection of one of these two protective responses is unknown. To clarify the key switch between autophagy and apoptosis, we examined the correlation of UPR-related signals with autophagy and/or apoptosis inductions in HepG2 cells exposed to three ER-stress inducers (NaF, tunicamycin, and thapsigargin) with time, including the effect of small interfering RNA on the cell responses. Thapsigargin-induced ER-stress caused only apoptosis after ∼2 hr with Ire1 phosphorylation, and Grp78, ATF4, and CHOP expressions. On the other hand, NaF- and tunicamycin-induced ER-stress caused only autophagy in the early stage by ∼8 hr with ATF4 expression and without CHOP expression. ATF4-siRNA completely inhibited the autophagy induced by NaF or tunicamycin with suppressed ATF4 protein and mRNA expressions, and also inhibited apoptosis by thapsigargin with suppression of both ATF4 and CHOP. CHOP-siRNA had no effect on autophagy activation by NaF and tunicamycin. On the other hand, CHOP-siRNA activated autophagy in thapsigargin-induced ER-stress with significant ATF4 expression, and suppressed apoptosis with CHOP suppression. These results showed that ATF4 is the key signal for autophagy induced by ER-stress, and that autophagy is switched to apoptosis by subsequent CHOP upregulation, suggesting that the changeover switch between autophagy and apoptosis is located between ATF4 to CHOP in the PERK pathway.
Enterotoxigenic Escherichia coli (ETEC) is a major cause of post-weaning diarrhea (PWD) in pigs and causes significant damage to the swine industry worldwide. In recent years, there has been increased regulation against the use of antibacterial agents in swine due to their health risks. Utilizing experimental models that consistently recapitulate PWD is important for the development of non-antibacterial agents against PWD in pigs. In this study, we established a highly reproducible PWD infection model by examining differences in adhesion of ETEC to the intestinal tissue as well as the association between MUC4 polymorphisms and sensitivity to PWD. Post-weaning diarrhea differences between pig breeds were also examined. The adhesion to enterocytes varied from 104.0 to 106.4 CFU/mL even among the F4 ETEC strains. Experimental infection revealed that PWD can be induced in all MUC4 genotypes after infection with 1010 CFU/pig of highly adherent ETEC, although there were variable sensitivities between the genotypes. Lowly adherent ETEC did not cause PWD as efficiently as did highly adherent ETEC. The incidence of PWD was confirmed for all pigs with the ETEC-susceptible MUC4 genotypes in all of the breeds. These results indicate that high-precision and reproducible experimental infection is possible regardless of pig breeds by controlling factors on the pig-end (MUC4 genotype) and the bacterial-end (adhesion ability).
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