| Abstract: | 胺基酸的角色被認為主要是參與代謝，例如，做為蛋白質生合成的原料或神經傳導物質的先質、修補體組織、或提供能量等，對生物體正常生理功能的維持有著重要角色。當必需胺基酸供應短缺，促使生物體改變許多基因表達，影響生理功能，有助於生物體適應胺基酸或蛋白質營養狀態的改變。但目前為止，對於調控胺基酸依賴型基因的表現，了解仍相當有限。先前研究發現：大鼠初代肝細胞培養於低濃度甲硫胺酸環境，其π屬麩胱甘肽硫轉移酶（π class of glutathione S-transferase, GSTP）蛋白質表現會受到誘發。本研究欲探討限制甲硫胺酸如何上調GSTP表現的分子機制。
初代肝細胞取自雄性Sprague-Dawley大鼠，培養於L-15培養液（500 μM L-methionine）或限制甲硫胺酸（20 μM L-methionine）的L-15培養液。限制甲硫胺酸隨著時間增加而誘發GSTP mRNA和蛋白質表現，並且伴隨ROS產生及胞內麩胱甘肽（GSH）減少。檢查上游訊號分子結果顯示：限制甲硫胺酸促進ERK磷酸化、增加細胞核內轉錄因子Nrf2蛋白質量、誘發antioxidant response element（ARE）報導基因活性。由於GSTP 啟動區含有一重要活化序列（GSTP enhancer I, GPEI）與ARE相似，因此進一步利用Electromobility gel shift assay (EMSA) 和 Chromatin immunoprecipitation (ChIP) 分析限制甲硫胺酸條件下，細胞核內Nrf2 與GPEI結合情況，結果發現：限制甲硫胺酸的確增加Nrf2 與 GPEI結合；若以small interfering RNA 靜默ERK2，不但減少Nrf2 轉移至細胞核內及ARE報導基因活性，同時也降低限制甲硫胺酸所誘發的GSTP表現。轉殖含有GPEI序列或是含有GSTP啟動區2713 bp序列的報導基因質體，更進一步證實限制甲硫胺酸透過活化ERK2，增加Nrf2結合至GPEI上調GSTP轉錄活性。
另外，以轉錄抑制劑actinomycin D預處理大鼠初代肝細胞，致使蛋白質誘發程度減低，但仍觀察到限制甲硫胺酸而上調GSTP蛋白質表現，這暗示著可能有轉錄後調控作用參與其中。限制甲硫胺酸會增加mTORC1活化及p70S6K、eIF4A1、eIF4E、eIF4G磷酸化；預處理mTOR抑制劑PP242抑制mTORC1也抑制限制甲硫胺酸所誘發的GSTP表現。限制甲硫胺酸也降低了PP2A活性並增加磷酸化eIF4E與GSTP mRNA 3’UTR 結合。檢視mTOR上游訊號分子發現：限制甲硫胺酸增加ERK及Akt (T308) 磷酸化。以siRNA 抑制ERK2表現，便無法觀察到限制甲硫胺酸所誘發的mTOR活化及GSTP表現；但以siRNA或抑制劑LY294002抑制PI3K 則不影響mTOR磷酸化，反而更加強了GSTP蛋白質表現。已知PI3K/Akt 透過磷酸化FOXO1使之出核而抑制其轉錄活性，我們觀察到限制甲硫胺酸會減少Akt (S473) 磷酸化並且增加細胞核內FOXO1蛋白質含量、上調錳型超氧化物歧化酶和過氧化氫酶蛋白質表現。FOXO1 siRNA會降低限制甲硫氨酸所誘發的GSTP蛋白質表現；PI3K siRNA則增強FOXO1留滯於細胞核，並增加DBE報導基因表現。染色質免疫沉澱進一步證實FOXO1結合於GSTP啟動區。
以上結果證明，限制甲硫胺酸上調GSTP基因表現與GSH耗竭引起氧化壓力有關，限制甲硫胺酸透過ERK-Nrf2及PI3K-Akt-FOXO訊號路徑增加GSTP轉錄作用；或是透過ERK-mTOR-eIF4E 增加GSTP蛋白質表現。由於GSTP參與藥物代謝反應，也在不正常增生細胞中大量表現影響藥物治療的效果，因此瞭解限制甲硫胺酸上調GSTP之調控機制可提供作為營養介入的參考。
Amino acids function as multiple roles including gluconeogenic substrates, protein turnover regulators, precursor of neurotransmitters or energy fuels. As amino acid supply is short, organisms have to change several physiological functions by regulating numerous gene expression, which help them adapting to amino acid limitation. But the molecular mechanisms involved in the amino acid-dependent gene expression remain need to be further investigated. L-Methionine, a sulfur-containing essential amino acid, acts as a methyl donor in the methylation of DNA and proteins and is a precursor of L-cysteine. Previous study showed the hepatic π class of glutathione S-transferase (GSTP) is up-regulated by L-methionine restriction. This study investigated the mechanism by which L-methionine restriction modulates the expression of the GSTP, an inducible phase II detoxification enzyme, in primary rat hepatocytes.
Hepatocytes isolated from male Sprague-Dawley rat were cultured in an L-15-based medium or in a L-methionine-restricted L-15 medium supplemented with 20 μM L-methionine up to 48 h. L-methionine restriction time-dependently induced GSTP mRNA and protein expression accompanied by ROS production and a decrease in the cellular glutathione (GSH) level. The phosphorylation of ERK, but not of JNK and p38, was stimulated by L-methionine restriction. Nrf2 translocation to the nucleus and antioxidant response element (ARE)-luciferase reporter activity were increased by L-methionine restriction. Electromobility gel shift assay and Chromatin immunoprecipitation assay confirmed that nuclear factor erythroid 2-related factor 2 (Nrf2) bound to the enhancer I of GSTP (GPEI) in cells exposed to L-methionine restriction. ERK2 siRNA abolished L-methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression.
In the second part, in the presence of actinomycin D, L-methionine restriction-increased GSTP protein was partially suppressed, although mRNA was almost abolished. L-methionine restriction increased mTORC1 activation and p70S6K, eIF4A1, eIF4E, and eIF4G phosphorylation. mTOR inhibitor PP242 abrogated L-methionine restriction-induced mTOR activation and GSTP induction. L-Methionine restriction suppressed protein phosphatase 2A activity and increased phosphorylated eIF4E binding to the 3’ untranslated region of GSTP mRNA. Knockdown of ERK2, but not of PI3K, attenuated L-methionine restriction-induced mTOR activation and GSTP expression. L-Methionine restriction increased FOXO1 nuclear accumulation and up-regulated expression of FOXO1 target genes, manganese superoxide dismutase and catalase. GSTP induction by L-methionine restriction was partially attenuated by silencing FOXO1. Moreover, PI3K siRNA enhanced FOXO1 nuclear retention and daf-16 family protein-binding element reporter activity. Chromatin immunoprecipitation revealed that L-methionine restriction increased the binding of FOXO1 to the GSTP promoter.
Taken together, results suggest that the regulation of GSTP gene by L-methionine restriction is related to oxidative stress initiated GSH depletion and via the ERK-mTOR-eIF4E and ERK-Nrf2/ PI3K-Akt-FOXO1 signaling pathways, which contributes to the stabilization of GSTP mRNA and up-regulation of GSTP transcription, respectively. |
