Molecular and functional characterization of aryl hydrocarbon receptor nuclear translocator 1 (ARNT1) and ARNT2 in chicken (Gallus gallus)

Our previous studies have provided evidence that birds have two isoforms of aryl hydrocarbon receptors (AHR1 and AHR2) and AHR nuclear translocators (ARNT1 and ARNT2) that potentially mediate toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. We have also shown that while both in vitro-expressed chicken AHR1 (ckAHR1) and AHR2 (ckAHR2) exhibit binding affinities to TCDD, only ckAHR1 but not ckAHR2 showed a TCDD-dose-dependent transactivation potency of chicken cytochrome P450 1A5 (ckCYP1A5) in in vitro reporter gene assays. To explore the molecular mechanism of functional difference in the two ckAHRs, the present study investigated the molecular characteristics and function of chicken ARNT (ckARNT) that is a potential dimerization partner for the activation of ckAHR. The full-length ckARNT1 and ckARNT2 cDNAs were isolated and their alternative splice variants were also identified. The ckARNT1 transcript was ubiquitously expressed in various tissues, but ckARNT2 showed restricted expressions in brain, kidney and eye, indicating a similar expression pattern to mammalian ARNTs. The expressions of tagged-ckARNT1 and -ckARNT2 were confirmed in a chicken hepatoma LMH cells by western blot analyses, and their interactions with each ckAHR and a specific recognition DNA element, xenobiotic response element (XRE), were examined by gel shift assays. The result showed that ckARNT1 and ckARNT2 dimerize with each ckAHR isoform and bind with the XRE in a TCDD-dependent manner. Hence, we conclude that functional loss on the dimerization with ckARNTs or the XRE binding is not the major cause of the deficient TCDD-dependency of ckAHR2 for the transactivation. Furthermore, in vitro reporter gene assays showed that transfected ckARNT1 failed to modulate the transcriptional induction of ckAHR-mediated ckCYP1A5 gene by TCDD in COS-7 and LMH cells, whereas ckARNT2 could potentiate the TCDD-dependent response in COS-7 but not in LMH cells. This suggests that ckARNT2 has a distinct role from ckARNT1 in AHR signaling pathway and in a cell-specific mode of action.