苯芘對於缺氧狀態下多環芳香烴受體與缺氧誘導因子-1α間關係之影響:核受體輔活化子角色之探討

Abstract

苯芘為多環芳香烴 (PAHs) 這類環境汙染物的其中之一員，可透過與多環芳香烴受體 (Aryl Hydrocarbon Receptor, AhR) 結合，啟動下游基因的表現。過去文獻指出許多心血管疾病患者暴露苯芘之後，在缺氧環境下病情會有惡化的趨勢，可能機轉為苯芘會抑制缺氧狀態下的訊息傳遞。缺氧誘導因子-1α(Hypoxia- inducible Factor-1α, HIF-1α)為一種對氧分壓敏感的轉錄因子，於正常氧分壓下會被快速降解，但缺氧環境 (hypoxia) 會促使 HIF-1α 穩定存在並與芳香烴受體核轉位蛋白 (aryl hydrocarbon receptor nuclear translocator, ARNT) 形成複合體啟動下游基因轉錄。先前本實驗室觀察到 HUVECs 細胞前處理苯芘 (B[a]P) 後，接著進行缺氧狀態時會抑制 HIF-1α 蛋白穩定性；並證實在 HepG2 細胞株內 AhR 蛋白的表現量確實會對缺氧反應元件 (Hypoxia- Responsive Element, HRE) 活性及其下游基因表現造成影響，顯示同時暴露 PAHs 與缺氧環境下，可能導致 HIF-1α與 AhR 共同競爭和 ARNT 的結合，進而對彼此啟動下游轉錄活化形成干擾，但詳細機轉目前仍未明瞭。HIF-1α、ARNT 及 AhR 三者在結構上皆屬於 PAS (PER-ARNT-SIM) 家族中的 bHLH (basic helix -loop-helix) 轉錄因子。在過去的研究中發現，核受體輔活化子(Nuclear Receptor Coactivator 2, NcoA2)，有可能透過與 bHLH-PAS 類的蛋白-蛋白間交互作用 (protein-protein interaction) 來調控轉錄因子間的活化與抑制，進而在轉錄因子的活化、影響胚胎的成形和改變腫瘤的發育等過程中扮演重要的角色；因此本研究興趣在於探討 NcoA2 在 HIF-1α 與 AhR 兩條訊息傳遞路徑中所扮演之角色。本研究以HEK293T 和 HepG2 兩株細胞為模型，主要因為兩株細胞 AhR 表現量不同；利用干擾性核糖核酸 (RNA interference, RNAi) 技術使 NcoA2 蛋白靜默，並以冷光酶分析法 (Luciferase assay) 觀察結果，發現 HRE 活性在缺氧狀態下NcoA2 的靜默會減少 1.5 倍的轉錄活性，顯示 NcoA2 在細胞缺氧狀態下調控的重要性。此外，氯化亞鈷或 0.5% 氧分壓的缺氧環境下會使 NcoA2 蛋白表現量有減少趨勢，但透過大量表現 HIF-1α並不影響 NcoA2 蛋白表現量，顯示NcoA2 並非透過 HIF-1α，而是受到缺氧狀態下誘導的訊息傳遞所調控；而利用 HepG2 處理苯芘後也可以發現 NcoA2 有減少的趨勢，但在 HEK293T 卻看不到相同結果，顯示 AhR 的表現量會對 NcoA2 造成影響。除此之外，我們也觀察到前處理苯芘後在缺氧狀態下會使 NcoA2 在核內的表現量更加減少的現象，顯示 AhR 活化後所導致的 NcoA2 蛋白減少可能會降低缺氧狀態下 HRE的轉錄活性；免疫沉澱法分析的結果也得知， NcoA2 可能可以透過 AhR 的表現量或是自身不正常的大量表現，來增加與 ARNT 的作用進而抑制 HIF-1α- ARNT 複合體的形成，但 NcoA2 本身對於 HRE 轉錄活化有一定的促進作用，因此 NcoA2 的靜默也會導致 HIF-1α 與 ARNT 的作用不完全，進而影響HRE的轉錄活性。總結以上結果，NcoA2 確實在HRE 的轉錄活化扮演重要角色，但是 AhR 的表現量可能是影響 NcoA2 功能的主要因素，並且 NcoA2 的表現量差異也會雙向調節HRE的轉錄活性。

Benzo[a]pyrene (B[a]P), one of the polycyclic aromatic hydrocarbons family (PAHs), and is a well-known aryl hydrocarbon receptor (AhR) agonist. Such of studies indicate that when exposure to B[a]P, many patients with CVD (cardiovascular disease) have worsening trend in oxygen- deficient environment condition, the reason is B[a]P may inhibite the signal transduction induced by hypoxia. Hypoxia-inducible Factor-1α (HIF-1α) a transcriptional factor composed of α- and β-subunits (ARNT), is a key regulator of metabolic adaptation to hypoxia. In our previous studies, hypoxia –induced HIF-1α was suppressed in human umbilical vein endothelial cells (HUVECs) after treated with Benzo[a]pyrene (B[a] P), the decreased in AhR protein level enhanced the activity of HRE and HRE-related genes induction under hypoxia treatment in HepG2. The data suggest when exposure to PAHs under hypoxia, AhR might compete to ARNT with HIF-1α forming the complex for transactivation. However, the detailed mechanism was still unknown. Nuclear Receptor Coactivator 2 (NcoA2), a transcription coactivator which could regulate the bHLH-PAS family proteins (HIF-1α, ARNT, AhR) transcriptional activity by protein-protien interactions to regulate transcription factor activation and inhibition, and thus play an important role in the activation of the transcription factor, affecting the embryo forming and change the course of tumor development. The aim of this study is to understand the role of NcoA2 between AhR and HIF-1α pathway.In this study, based on the basal level of AhR, we use the HEK293T and HepG2 as the cell model. NcoA2 was silenced by using NcoA2 shRNA, following analysis with Luciferase assay observed the HRE activity was decreased obviously; moreover,the HIF-1α protein accumulation (6hr after hypoxia treatment) and NcoA2 degradation was found in hypoxia –treated HEK293T cells. The decreased NcoA2 protein was mainly found in nuclear fraction rather than cytosol, these results indicated NcoA2 may regulate the gene transcription under hypoxia in a HIF-1α - independent manner. In contrast to hypoxia, the treatment of B[a]P could also repress NcoA2 expression in HepG2, but not in HEK293T. interestingly, HepG2 was pre-treated B[a]P follow by hypoxia incubating for 6 hours, the result showed that NcoA2 significantly decreased in nuclear fraction, indicating that the activation of AhR caused NcoA2 protein reduction may reduce the activity of the transcription of HRE under hypoxic. Immunoprecipitation analysis showed AhR expression or NcoA2 abnormal expression may influence NcoA2-ARNT interaction thereby inhibit HIF-1α-ARNT complex formation. In conclusion, NcoA2 indeed plays an important role on HRE activation; but the expression of the AhR may be a major factor affecting NcoA2 function. These results lead NcoA2 colud both up-regulate and down –regulate the transcriptional activity of the HRE.