探討膽固醇體內平衡與第一型干擾素免疫反應的相互關係

Identifying the link between cholesterol homeostasis and type I interferon responses

顏伶珈(Ling-Chia Yen)

指導教授 ： 江皓森

國立臺灣大學/生命科學院/生命科學系/碩士(2017年)

https://doi.org/10.6342/NTU201704237

摘要

細胞內的膽固醇在免疫系統中扮演著重要的角色，研究已證實膽固醇可調控宿主抗病毒能力及多種發炎反應。第二型固醇調節元件結合蛋白 (sterol regulatory element-binding protein 2, SREBP2)是個已知調控膽固醇合成的轉錄因子，其活化時會經由固醇調節元件結合蛋白切割活化蛋白 (SREBP cleavage activating protein, SCAP)的運送離開內質網，並在高基氏體內被水解後進入細胞核。近期研究指出第一型干擾素 (type I interferon)會藉由調控SREBP2的活化去影響膽固醇代謝，但其中的機制尚未明瞭。透過大數據資料庫網站ImmuNet分析SREBP2後顯示，其和Rho型鳥嘌呤核苷酸交換因子 (Rho guanine nucleotide exchange factor 1, ARHGEF1)具有功能上的相關性。ARHGEF1主要表現在免疫細胞中，其參與調控的G蛋白偶聯受體 (G protein–coupled receptor, GPCR)被證明可作為細胞代謝物的受體，再加上同樣身為鳥嘌呤核苷酸交換因子蛋白的GEF-H1已被知道可影響第一型干擾素生成，因此我欲探討ARHGEF1是否可和藉由和SREBP2交互作用，進而調控膽固醇與第一型干擾素之間的反應。
在轉染SREBP2的猴子腎臟細胞COS-7中過表現ARHGEF1，可減少SREBP2前導蛋白 (precursor SREBP2)表現並使細胞內膽固醇含量維持在恆定值。在ARHGEF1基因減弱的人類單核球細胞THP-1中，第一型干擾素IFNβ1、其下游基因與促發炎因子表現皆上升；給予THP-1分化後的巨噬細胞干擾素基因(stimulator of interferon gene, STING)受體3’3’-cGAMP的刺激後，IFNβ1與膽固醇、脂肪酸合成相關基因的表現大幅上升，有趣的是，額外加入膽固醇培養可消除此上升表現。以CRISPR方式在人類肝癌細胞株Huh-7中減弱ARHGEF1的表現後亦可觀察到SREBP2前導蛋白與細胞內膽固醇含量增加的現象。在過表現ARHGEF1的Huh-7細胞中有胞內膽固醇含量上升、SREBP2前導蛋白表現下降的現象，過表現ARHGEF1亦可消除3’3’-cGAMP所引起IFNβ1與膽固醇、脂肪酸合成相關基因的高表現量。由以上結果推測ARHGEF1藉由改變SREBP2活化狀態影響細胞內膽固醇含量，進而調控第一型干擾素訊息途徑。可惜的是，實驗結果證明ARHGEF1與SREBP2之間並沒有直接或間接的蛋白交互作用，推測ARHGEF1並非藉由調控SREBP2而參與膽固醇與第一型干擾素之間的反應，或是此兩蛋白可能在特定環境條件下才會互相作用。總括來說，本研究揭露ARHGEF1可藉由改變細胞內膽固醇合成與含量影響第一型干擾素反應，藉此影響細胞抗病毒能力，至於其詳細作用機制仍有待探討。

關鍵字

Rho型鳥嘌呤核苷酸交換因子；第二型固醇調節元件結合蛋白 ； 膽固醇 ； 第一型干擾素 ； 干擾素基因途徑

並列摘要

Cellular cholesterol homeostasis plays a critical role in immunity that affects antiviral and inflammatory responses. The sterol regulatory element-binding protein 2 (SREBP2) is a transcriptional factor with a well-defined function in the regulation of cholesterol synthesis. SREBP cleavage activating protein (SCAP) is a sterol-regulated chaperone protein that escorts SREBP2 from the ER membrane to the Golgi apparatus where the active domains of SREBP2 are released proteolytically to enter the nucleus. Recent study has indicated that type I interferon (IFN) pathway reprograms cholesterol homeostasis by regulating SREBP2 activation. However, it remains unclear how cholesterol metabolism and type I IFN responses are co-regulated. In order to identify functionally important interactions with SREBP2, I first utilized an interactive big data resource (ImmuNet, http://immunet.princeton.edu) to generate a data-driven hypothesis that Rho Guanine Nucleotide Exchange Factor 1 (ARHGEF1), a GEF that is highly expressed in lymphocytes, is functionally related to SREBP2. ARHGEF1 is well known for its role in Gα12/13-ARHGEF1-RhoA signaling pathway. Previous studies also indicated that G protein–coupled receptors (GPCRs) act as receptors for metabolites including fatty acids and bile acids. Moreover, another GEF protein, GEF-H1, is demonstrated to regulate IFNβ expression. Therefore, I hypothesized that ARHGEF1 is involved in the interaction of cholesterol homeostasis and type I IFN responses via modulating SREBP2 maturation.
Here I showed that overexpression of ARHGEF1 in SREBP2-transfected COS-7 cells reduced the levels of SREBP2 precursor form and maintained total cellular cholesterol levels. Gene expression studies on unstimulated THP-1 cells revealed that knockdown of ARHGEF-1 resulted in the spontaneous induction of IFNβ1, interferon-stimulated genes (ISGs), and pro-inflammatory cytokines. The expression of IFNβ1 and cholesterol and fatty acid synthesis genes were further increased in ARHGEF1-reduced THP-1 macrophages in response to STING ligand 3’3’-cGAMP. Importantly, the addition of cholesterol significantly diminished this upregulation. It was also observed in Huh-7 cells that knockdown of ARHGEF1 reduced the level of precursor SREBP2 and cellular cholesterol. Overexpression of ARHGEF1 in Huh-7 cells increased cellular cholesterol level and decreased precursor SREBP2 expression. 3’3’-cGAMP-induced IFNβ and cholesterol and fatty acid synthesis genes expression were diminished in ARHGEF1-overexpressed Huh-7 cells. From the above results, it was inferred that ARHGEF1 affects the type I IFN pathway by regulating the activation of SREBP2. However, the results of co-immunoprecipitation assay and yeast-two hybrid assay indicated that there is no protein interaction between ARHGEF1 and SREBP2. ARHGEF1 may take part in the cholesterol homeostasis and type I IFN responses through other pathways, or ARHGEF1 interacts with SREBP2 only under specific conditions. Taken together, this study elucidate that ARHGEF1 is involved in the regulatory loop of cholesterol homeostasis and type I IFN responses, therefore regulates the antiviral responses.

並列關鍵字

ARHGEF1 ； SREBP2 ； cholesterol ； type I interferon ； STING pathway

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