Identifying the link between cholesterol homeostasis and type I interferon responses

• Main Authors: Ling-Chia Yen, 顏伶珈
• Other Authors: Hao-Sen Chiang
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/f35zm8

碩士 === 國立臺灣大學 === 生命科學系 === 106 === 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.