Identification of a putative nuclear receptor involved in cytochrome P450 induction by cypermethrin in IPLB-LdFB cells

Cypermethrin, a pyrethroid insecticide, was administered to the fat body cell line of Lymantria dispar (IPLB-LdFB) to investigate cytochrome P450 (CYP450) induction at the cellular level. CYP450 activity increased according to the length of induction time in cypermethrin-treated IPLB-LdFB cells compared to non-treated cells. CYP450 activity was also increased by carbaryl and 3-methylcholanthrene, but not by monocrotophos. To search for a nuclear receptor that can mediate the cypermethrin-induced CYP450 expression, comparative 2-dimensional electrophoresis was conducted using soluble proteins from both the cytosolic and nuclear fractions of IPLB-LdFB, with or without cypermethrin pretreatment. Five protein spots were identified as more localized (over-expressed) in the cypermethrin-treated cells, particularly in the nuclear fraction, and were characterized by tandem mass spectrometry analysis. Among these five proteins, one was determined to nuclear receptor possessing both zinc finger and ligand-binding domain. The full-length cDNA fragment of a HNF4-like gene (LdFB-HNF4) was cloned and determined to possess both ligand- and DNA-binding domains. RNA interference (RNAi) using a 497-bp dsRNA was performed to silence the LdFB-HNF4 gene in IPLB-LdFB. RNAi resulted in ca. 43% reduction in the level of LdFB-HNF4 transcript and ca. 50% suppression of the CYP450 induction by cypermethrin in the dsRNA-treated cells. These findings suggest that LdFB-HNF4 in IPLB-LdFB is associated with CYP450 induction by cypermethrin.