Pirogov Russian National Research Medical University, Moscow, Russia.
National Research Center "Kurchatov Institute", Moscow, Russia.
Toxicol Lett. 2023 Jun 1;382:58-65. doi: 10.1016/j.toxlet.2023.05.007. Epub 2023 May 20.
Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated by polycyclic aromatic hydrocarbons of synthetic and natural origin. While a number of novel AhR ligands have been recently identified, little is known about their possible influence on AhR levels and stability. We used western blot, qRT-PCR and immunocytochemistry to determine the effects of AhR ligands on AhR expression in N-TERT (N-TERT1) immortalized human keratinocytes, and immunohistochemistry to assess patterns of AhR expression in human and mouse skin and skin appendages. While AhR was highly expressed in cultured keratinocytes and in the skin, it was found primarily in the cytoplasm, but not in the nucleus, suggesting its inactivity. At the same time, treatment of N-TERT cells with proteasomal inhibitor MG132 and eventual inhibition of AhR degradation resulted in nuclear AhR accumulation. Treatment of keratinocytes with AhR ligands such as TCDD, FICZ, caused near-complete disappearance of AhR, and treatment with I3C resulted in substantially diminished level of AhR possibly due to ligand-induced AhR degradation. The AhR decay was blocked by proteasome inhibition, indicating degradation-based mechanism of regulation. Additionally, AhR decay was blocked by ligand-selective AhR antagonist CH223191, implying substrate-induced mechanism of degradation. Furthermore, degradation of AhR was blocked in N-TERT cells with knockdown of AhR dimerization partner ARNT (HIF1β), suggesting that ARNT is required for AhR proteolysis. However, addition of hypoxia mimetics (HIF1 pathway activators) CoCl and DMOG had only minor effects on degradation of AhR. Additionally, inhibition of HDACs with Trichostatin A resulted in enhanced expression of AhR in both untreated and ligand-treated cells. These results demonstrate that in immortalized epidermal keratinocytes AhR is primarily regulated post-translationally via proteasome-mediated degradation, and suggest potential means to manipulate AhR levels and signaling in the skin. Overall, the AhR is regulated via multiple mechanisms, including proteasomal ligand- and ARNT-dependent degradation, and transcriptional regulation by HDACs, implying complex system of balancing its expression and protein stability.
芳香烃受体 (AhR) 是一种基本螺旋-环-螺旋转录因子，可被合成和天然来源的多环芳烃激活。虽然最近已经鉴定出许多新型 AhR 配体，但它们对 AhR 水平和稳定性的可能影响知之甚少。我们使用 Western blot、qRT-PCR 和免疫细胞化学来确定 AhR 配体对 N-TERT（N-TERT1）永生化人角质形成细胞中 AhR 表达的影响，并使用免疫组织化学来评估 AhR 在人和小鼠皮肤和皮肤附属物中的表达模式。虽然 AhR 在培养的角质形成细胞和皮肤中高度表达，但主要存在于细胞质中，而不存在于细胞核中，表明其无活性。与此同时，用蛋白酶体抑制剂 MG132 处理 N-TERT 细胞并最终抑制 AhR 降解导致核 AhR 积累。用 AhR 配体如 TCDD、FICZ 处理角质形成细胞会导致 AhR 几乎完全消失，用 I3C 处理会导致 AhR 水平大幅降低，可能是由于配体诱导的 AhR 降解。AhR 的降解被蛋白酶体抑制所阻断，表明这是一种基于降解的调节机制。此外，AhR 降解被配体选择性 AhR 拮抗剂 CH223191 阻断，暗示降解的底物诱导机制。此外，在敲低 AhR 二聚化伙伴 ARNT（HIF1β）的 N-TERT 细胞中，AhR 降解被阻断，表明 ARNT 是 AhR 蛋白水解所必需的。然而，用缺氧模拟物（HIF1 途径激活剂）CoCl 和 DMOG 处理对 AhR 的降解只有很小的影响。此外，用 Trichostatin A 抑制组蛋白去乙酰化酶导致未处理和配体处理的细胞中 AhR 的表达增强。这些结果表明，在永生化表皮角质形成细胞中，AhR 主要通过蛋白酶体介导的降解进行翻译后调节，并提出了在皮肤中操纵 AhR 水平和信号的潜在方法。总的来说，AhR 通过多种机制进行调节，包括蛋白酶体配体和 ARNT 依赖性降解，以及 HDACs 的转录调节，这意味着其表达和蛋白质稳定性的平衡是一个复杂的系统。
