Jenny Matthew J, Karchner Sibel I, Franks Diana G, Woodin Bruce R, Stegeman John J, Hahn Mark E
Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543, USA.
Toxicol Sci. 2009 Aug;110(2):426-41. doi: 10.1093/toxsci/kfp116. Epub 2009 Jun 3.
The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to dimethyl sulfoxide (DMSO) displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.
芳烃受体（AHR）阻遏蛋白（AHRR）是一种与AHR相关的碱性螺旋-环-螺旋/Per-AHR核转运蛋白-Sim蛋白，受AHR依赖性机制调控，并作为AHR功能的转录阻遏物发挥作用。由于硬骨鱼特异性基因组复制，斑马鱼有两个AHRR基因（AHRRa和AHRRb），但其体内功能尚不清楚。我们在斑马鱼胚胎和斑马鱼肝细胞系（ZF-L）中使用反义吗啉代寡核苷酸（MO）来表征AHRR和AHR在正常胚胎发育、AHR信号传导以及2,3,7,8-四氯二苯并对二恶英（TCDD）毒性中的相互作用。在早期发育过程中暴露于TCDD（2和8 nM）的斑马鱼胚胎在受精后48和72小时（hpf）显示出CYP1A、AHRRa和AHRRb的强烈诱导。靶向AHR2的MO在48 hpf胚胎中抑制了TCDD诱导的CYP1A、AHRRa和AHRRb表达达84 - 95%，证明AHR2在介导AHRR诱导中起主要作用。ZF-L细胞中AHRR的双重MO敲低增强了TCDD对CYP1A的诱导，但未增强其他CYP1基因的诱导。在胚胎中，AHRR的双重敲低或单独敲低AHRRb增强了TCDD对CYP1A、CYP1B1和CYP1C1的诱导，并降低了Sox9b的组成型表达。相反，敲低AHRRa不影响Sox9b表达或CYP1诱导性。用两种靶向AHRRa的不同MO分别显微注射并暴露于二甲基亚砜（DMSO）的胚胎表现出类似于TCDD暴露胚胎的典型发育表型（心包水肿和下颌畸形）。相比之下，在暴露于DMSO的AHRRb吗啉代敲降胚胎中未观察到发育表型。这些数据证明了AHRRa和AHRRb在体内调节AHR信号传导中的不同作用，并表明自硬骨鱼特异性基因组复制以来它们经历了亚功能划分。
