Transcription factor CDX2 directly regulates the expression of Ctenopharyngodon idellus intestinal PepT1 to mediate the transportation of oligopeptide

Grass carp (Ctenopharyngodon idellus, C.idellus) is one of the most important globally cultured freshwater fish species, and its growth trait can be improved by dietary supplementation of oligopeptides which is transported through the intestinal oligopeptide transporter PepT1. However, many aspects of the molecular regulation mechanism of C.idellus PepT1 still remains elusive. In this study, the regulation mechanism of C.idellus PepT1 by CDX2 was extensively studied. Firstly, the interaction of the C.idellus CDX2 and SP1 was investigated by mammalian two hybrid assays and co-localization; The promoter sequence of PepT1 was cloned and its activity was directly up-regualted by CDX2. In the further study, the effect of CDX2 changes at both mRNA and protein levels on the expression of PepT1 was examined in vivo and in vitro. The mRNA expression of PepT1 was significantly decreased when CDX2 was repressed and its expression change profile was almost consistent with that of CDX2. To understand the regulation of CDX2 on the expression of PepT1 at protein level, the stability regulation mechanism of CDX2 was initially studied. The results showed that CDX2 protein was degraded via the ubiquitin-proteasome pathways, and increasing phosphorylation level of CDX2 protein declined its turnover and elevated its accumulation level in cells. The expression of PepT1 was significantly elevated with increasing CDX2 protein stability, and expressions of both CDX2 and PepT1 were stimulated by the oligopeptide. In conclusion, these observations may shed new light on the molecular regulation mechanism of PepT1 by CDX2 in C.idellus and provide new perceptions and strategies for the efficient transportation of intestinal oligopeptides in teleost fish.