AIM2 and IFI16 Innate Immune Sensors for Cytosolic DNA in Prostatic Diseases

描述(由申请人提供)： 研究表明，前列腺炎与前列腺增生症(BPH)和前列腺癌(PC)等与衰老相关的前列腺疾病的发生密切相关。然而，导致前列腺炎的分子机制在很大程度上仍不清楚。研究已经确定了胞质双链DNA(DsDNA)传感器蛋白在启动导致慢性炎症的先天性免疫反应中的作用。这些蛋白质包括NALP3和干扰素诱导的AIM2和IFI16。当AIM2和NALP3蛋白感应到胞浆dsDNA时，AIM2和NALP3蛋白招募接头蛋白ASC形成炎症小体，促进促炎细胞因子的分泌，如IL-1和IL-18。然而，当检测到胞浆dsDNA时，IFI16蛋白招募干扰素基因刺激物(STING)蛋白来刺激干扰素-β的表达，而当检测到核dsDNA时，IFI16蛋白招募ASC蛋白来形成炎症小体。根据我们的初步观察，我们假设前列腺上皮细胞(PrECs)中的AIM2和IFI16蛋白感应dsDNA会触发先天免疫反应，从而促进促炎细胞因子的产生。此外，我们推测，在PrECs中，AIM2和IFI16蛋白之间的物理和功能相互作用激活了核因子-βB的转录活性，从而促进了促炎细胞因子和趋化因子的表达，从而导致了慢性前列腺炎。目的1：我们试图确定I型和II型IFN调控AIM2蛋白表达水平以及AIM2和IFI16蛋白在人正常前EC和癌细胞中的亚细胞定位的分子机制。这些方法包括凝胶迁移率改变分析、启动子-报告分析、抑制STAT1表达、突变分析和染色质免疫沉淀分析(CHIP)。目的#2：研究AIM2和其他DNA感受器在人正常前皮样细胞、前列腺癌细胞系和前列腺病变细胞胞浆DNA触发的先天免疫反应中的作用。具体地说，我们建议：(I)研究胞浆dsDNA是否在PrECs和前列腺癌细胞系中触发AIM2和其他炎症体激活；以及(Ii)检测AIM2和其他DNA感受器蛋白在炎症相关前列腺疾病中的表达水平和亚细胞定位。将使用免疫组织化学、微阵列和实时定量聚合酶链式反应。目的#3：研究AIM2和IFI16蛋白之间的相互作用如何影响PrECs中的核因子-B活性。将使用包括芯片在内的生化和分子细胞生物学方法。意义：拟议的研究可能确定AIM2和IFI16双链DNA先天免疫传感器促进前列腺炎和炎症相关前列腺疾病发展的分子机制。

DESCRIPTION (provided by applicant): Studies indicate close links between prostatic inflammation and the development of aging-associated prostatic diseases such as benign prostate hyperplasia (BPH) and prostate cancer (PC). However, the molecular mechanisms that contribute to prostatic inflammation remain largely unknown. Studies have identified a role for cytosolic double-stranded DNA (dsDNA) sensor proteins in initiating innate immune responses that contribute to chronic inflammation. These proteins include NALP3 and the interferon (IFN) - inducible AIM2 and IFI16. Upon sensing cytosolic dsDNA, the AIM2 and NALP3 proteins recruit adaptor protein ASC to form an inflammasome, which promotes the secretion of pro-inflammatory cytokines such as IL-1¿ and IL-18. However, upon sensing cytosolic dsDNA, the IFI16 protein recruits the stimulator of interferon genes (STING) protein to stimulate the expression of IFN-¿, whereas upon sensing the nuclear dsDNA the protein recruits ASC protein to form an inflammasome. Based on our preliminary observations, we hypothesize that sensing of dsDNA by AIM2 and IFI16 proteins in prostate epithelial cells (PrECs) triggers innate immune responses that contribute to an increased production of proinflammatory cytokines. Additionally, we postulate that the physical and functional interactions between the AIM2 and IFI16 proteins in PrECs activate the transcriptional activity of NF-¿B that promotes the expression of the proinflammatory cytokines and chemokines; thus, leading to the chronic prostatic inflammation. Aim # 1: We seek to identify the molecular mechanisms by which type I and type II IFNs regulate the expression levels of AIM2 protein and subcellular localization of the AIM2 and IFI16 proteins in human normal PrECs and cancer cell lines. The approaches, including gel-mobility shift assays, promoter-reporter assays, knockdown of STAT1 expression, mutational analyses, and chromatin immunoprecipitation assays (ChIPs) will be used. Aim # 2: Characterize the role of AIM2 and other DNA sensors in cytosolic DNA-triggered innate immune responses in human normal PrECs, prostate cancer cell lines, and the prostatic lesions. Specifically, we propose to: (i) investigate whether cytosolic dsDNA triggers the AIM2 and other inflammasome activation in PrECs and prostate cancer cell lines; and (ii) examine expression levels and subcellular localization of the AIM2 and other DNA sensor proteins in inflammation-associated prostatic diseases. Immunohistochemistry, microarray, and quantitative real-time PCR will be used. Aim # 3: Investigate how interactions between the AIM2 and IFI16 proteins contribute to the modulation of NF-¿B activity in PrECs. Biochemical and molecular cell biology approaches including ChIPs will be used. Significance: Proposed studies are likely to identify the molecular mechanisms through which the AIM2 and IFI16 innate immune sensors for dsDNA contribute to prostatic inflammation and the development of inflammation-associated prostatic diseases.