aryl hydrocarbon receptor nuclear translocator (ARNT，也稱為 HIF-1 β) 是 bHLH-PAS 的家族成員之一。ARNT 受環境因子的刺激扮演著關鍵性的角色並有很多的生物功能，像是血管的生成，癌症的形成與神經發育的形成等。從實驗室過去的研究中，我們發現 EGF 會增加 ARNT 在細胞核內的量並且 ARNT 有被磷酸化的現象。而過去的研究對於 ARNT 的後轉譯修飾作用與其在細胞核內分佈方面的探討目前是不清楚。首先我們利用蛋白質去磷酸化酶 2B (protein phosphatase 2B，PP2B 或者稱 calcineurin) 抑制劑，cyclosporin A (CsA) 來確認 ARNT 在細胞核內增加的量是否與後轉譯修飾作用有關係。我們的結果顯示 CsA 的作用會抑制 EGF 所誘發ARNT 的細胞核內累積並發現在蛋白質體抑制劑 (MG132) 的處理下回復了 CsA 所抑制的 ARNT 核內累積。EGF 與 CsA 的處理並不影響 ARNT 的轉錄及蛋白質的穩定性。我們發現 CsA 的作用會影響 ARNT 的核分佈，藉由降低 PP2B 蛋白表現之方式，發現 PP2B 會調控 ARNT 細胞核的分佈，進而利用免疫沉澱分析發現 ARNT 與 PP2B 之間沒有顯著的交互作用。此結果暗示著 PP2B 會調控 ARNT 的核分佈，並非透過直接的蛋白質交互作用而是可能透過其他未知的因子參與。另一方面，實驗室先前的研究結果發現在癌症的發展中 Skp2 與 ARNT 皆有高度的基因表現，並且已知 knockdown ARNT 會抑制細胞生長並且調控在細胞週期的 S 期，進而影響 DNA 的生合成。我們利用 luciferase assay 發現 ARNT 會活化 Skp2 的啟動子活性。除此之外，透過 CsA 的作用和剔除ARNT NLS的表現載體來影響 ARNT 的活性，則降低了 Skp2 啟動子的活性，也發現此調控機制發生在許多不同的細胞株中。這些結果證實 Skp2 啟動子的活化是受ARNT 所調控，並且暗示著 ARNT 在細胞週期的調控扮演了重要角色。

The aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF-1β) is a member of basic helix-loop-helix Per-AhR/ ARNT-Sim (bHLH-PAS) family of transcription factors. ARNT plays a critical role in responsible for environmental stimulation and involves in many cellular functions (such as angiogenesis, carcinogenesis and neuroigenesis). From our previous study, we found that epidermal growth factor (EGF) enhanced the nuclear accumulation and phosphorylation of ARNT. The relationship between post-translational modification and the nuclear accumulation of ARNT is unclear so far. In order to study whether post-translational modification of ARNT is essential for EGF-enhanced the increase of nuclear ARNT, protein phosphatase 2B (PP2B or calcineurin) inhibitor, cyclosporine A (CsA) was used. Our results showed that EGF-increased the nuclear accumulation of ARNT was inhibited by CsA and the inhibition effect of CsA was reversed by treating cells with the proteasome inhibitor MG132. However, the EGF and CsA treatment did not affect the transcription and protein stability of ARNT. CsA inhibits EGF-induced nuclear translocation and PP2B regulates the nuclear translocation of ARNT by knockdown of PP2B. No significant interaction between ARNT and PP2B was observed by immunopreciptation. These results indicate that PP2B-mediated the translocation of ARNT, instead of direct binding with it, may through the unknown factor. On the other hand, our previous results showed that both of the Skp2 and ARNT were highly expressed in cancer tissues and knockdown of ARNT disrupted S phase entry and DNA synthesis. To further identify the correlation between ARNT and Skp2 in tumorigenesis, we examined the probable effect of ARNT on Skp2 expression. We found that ARNT activated Skp2 promoter activity. In addition, we also found that impaired activity of ARNT by cells treated with CsA or transfected with ARNT NLS mutant led to reduce the Skp2 promoter activity in various cell lines. These results demonstrate that Skp2 promoter activity is regulated by ARNT and suggest that ARNT plays an important role in the regulation of cell cycle.

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