Department of Environmental & Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633, USA.
Mol Cell Endocrinol. 2010 Feb 5;315(1-2):208-18. doi: 10.1016/j.mce.2009.07.013. Epub 2009 Jul 23.
Ecdysteroids initiate signaling along multiple pathways that regulate various aspects of development, maturation, and reproduction in arthropods. Signaling often involves the induction of downstream transcription factors that either positively or negatively regulate aspects of the pathway. We tested the hypothesis that crustaceans express the nuclear receptors HR3 (ortholog to vertebrate ROR) and E75 (ortholog to vertebrate rev-erb) in response to ecdysteroid signaling. HR3 and E75 cDNAs were cloned from the crustacean Daphnia magna. The DNA-binding domain and ligand-binding domain of the daphnid HR3 were 95% and 61% identical to those of Drosophila melanogaster. The DNA-binding domain and ligand-binding domain of the daphnid E75 were 100% and 71% identical to those of D. melanogaster. Both receptors exhibited structural characteristics of binding to DNA as a monomer. The expression of these receptor mRNAs was evaluated through the adult molt cycle and during embryo development. E75 levels were relatively constant throughout the adult molt cycle and through embryo development. HR3 levels were comparable to those of E75 during the initial phases of the adult molt cycle but were elevated approximately 30-fold at a time in the cycle co-incident with the pre-molt surge in ecdysteroid levels. HR3 mRNA levels in embryos also varied co-incident with ecdysteroid levels. To substantiate a role of ecdysteroids in the expression of HR3, daphnids were continuously exposed to 20-hydroxyecdysone and changes in gene expression were measured. HR3 levels were significantly induced by 20-hydroxyecdysone; while E75 levels were minimally affected. These results are consistent with the premise that transcription of HR3 is regulated by ecdysteroids in the crustacean D. magna and that HR3 likely serves as a mediator of ecdysteroid regulatory action in crustaceans. The marginal induction of E75 by 20-hydroxyecdysone may represent limited, tissue or cell-type-specific induction of this transcription factor.
蜕皮甾类化合物通过多条信号通路发挥作用，调节节肢动物的发育、成熟和生殖的各个方面。信号通路通常涉及诱导下游转录因子，这些转录因子正向或负向调节该通路的各个方面。我们验证了这样一个假说，即甲壳类动物在蜕皮甾类信号转导中表达核受体 HR3（与脊椎动物 ROR 同源）和 E75（与脊椎动物 rev-erb 同源）。我们从甲壳类动物大型溞（Daphnia magna）中克隆了 HR3 和 E75 的 cDNA。大型溞 HR3 的 DNA 结合域和配体结合域与果蝇的分别有 95%和 61%的一致性。大型溞 E75 的 DNA 结合域和配体结合域与果蝇的分别有 100%和 71%的一致性。这两种受体都表现出作为单体与 DNA 结合的结构特征。通过成虫蜕皮周期和胚胎发育过程评估这些受体 mRNA 的表达。E75 的水平在整个成虫蜕皮周期和胚胎发育过程中相对稳定。HR3 的水平在成虫蜕皮周期的初始阶段与 E75 相当，但在该周期中与蜕皮甾类水平的预蜕皮激增同时升高约 30 倍。胚胎中 HR3 mRNA 的水平也与蜕皮甾类水平同时变化。为了证实蜕皮甾类化合物在 HR3 表达中的作用，连续暴露大型溞于 20-羟基蜕皮甾酮，并测量基因表达的变化。20-羟基蜕皮甾酮显著诱导 HR3 水平；而 E75 水平受影响最小。这些结果与这样一个前提一致，即 HR3 的转录受甲壳类动物大型溞中蜕皮甾类的调控，并且 HR3 可能作为蜕皮甾类调节作用在甲壳类动物中的介质。20-羟基蜕皮甾酮对 E75 的轻微诱导可能代表了该转录因子在组织或细胞类型上的有限诱导。
