Modulation of IFN action via novel regulatory factors

描述(申请人提供)：干扰素家族的细胞因子是关键的促进几个生理过程，如抗病毒，抗肿瘤和免疫反应。它们在临床上用于治疗一些癌症、病毒性疾病和神经退行性疾病。通过与其他细胞因子相互作用，IFN形成了一个巨大的细胞间信号分子网络，控制着肿瘤细胞的生长和宿主对病原体的防御。在此之前，我们已经发现了一个新的干扰素调控元件及其同源转录因子。其中一种蛋白质是CCAAT/增强子结合蛋白-β(C/EBP-2)，这是一种已知调节细胞分化、能量代谢、免疫反应、肿瘤生长和凋亡的转录因子。我们实验室的基因表达微阵列分析发现了几个受干扰素调控的基因，它们的表达需要C/EBP-2。其中之一是死亡相关蛋白Kase1(DAPK1)，它是细胞凋亡、细胞周期和转移的重要调节因子。DAPK1基因在多种人类肿瘤中经常缺失。DAPK1还调节自噬(一种新的死亡形式)，这对于去除受损的细胞器、对抗细胞内病原体、抗原呈递和抑制肿瘤至关重要。有趣的是，小鼠体内C/EBP-2基因的缺失导致了许多这些缺陷。在上一次资助期间，我们显示了C/EBP-2在调节DAPK1中的核心作用。在初步研究中，我们发现DAPK1的表达需要其他的调控因子。值得注意的是，激活转录因子6(ATF6)，内质网依赖的应激反应的关键调节因子，似乎调节DAPK1的表达。我们认为ATF6和C/EBP-2之间的直接相互作用通过自噬导致DAPK1的表达和生长抑制，其中MAP激酶，即凋亡刺激激酶1(ASK1)提供了关键的信号输入。在本提案的具体目标1中，我们将研究C/EBP-2和ATF6如何协同上调DAPK1的表达。在特定的目标2中，我们将研究ASK1如何通过ATF6调控DAPK1的表达。增强子结合转录因子(TF)利用转录共激活因子促进转录。其中，介体是一个由多个蛋白质组成的分子桥梁，将转录信号从转录因子传递到TATA盒上的通用转录因子复合体。上一次资助期间的研究还发现了MED1，它是干扰素诱导的C/EBP-2的结合伙伴Mediator的一个主要亚基。我们提供了DAPK家族成员ZIP-Kinase(ZIPK)参与调节DAPK1表达的初步证据。我们假设ZIPK通过调节MED1C/EBP结合域的磷酸化来调节DAPK1的表达。这方面将在特定目标3中进行研究。我们将使用RNAi、基因敲除小鼠、蛋白质相互作用、芯片分析、突变和转录分析来评估这些因素与DAPK1和自噬的关键相关性。通过了解这些途径所获得的知识，不仅将定义DAPK1的关键调控因子，还将为DAPK1的丢失如何发生提供指标。我们将调查这些通路与人类CLL的相关性，在这种疾病中，DAPK1似乎发挥着重要的肿瘤抑制作用。这些反过来将允许更好地设计治疗方法来对抗肿瘤的进展和转移。 公共卫生相关性：肿瘤转移是由于某些关键基因的丢失和相应的生物学过程的抑制造成的。在这项申请中提出的研究将调查抗转移基因DAPK1的调节，该基因对肿瘤抑制和自噬反应至关重要。

DESCRIPTION (provided by applicant): The interferon family of cytokines is critical for promoting several physiologic processes, such as antiviral, antitumor, and immune responses. They are in clinical use for the therapy of a number of cancers, viral diseases and neurodegenerative disorders. By interacting with other cytokines IFNs form a large network of intercellular signaling molecules that control neoplastic cell growth and host defenses against pathogens. Previously, we have identified a novel IFN-regulated element and its cognate transcription factors. One such protein is CCAAT/Enhancer Binding protein-beta (C/EBP-2), a transcription factor known to regulate cell differentiation, energy metabolism, immune response, tumor growth and apoptosis. A gene expression micro- array analysis in our lab identified several IFN regulated genes, whose expression required C/EBP-2. One of them is the death associated protein kinase1 (DAPK1), an important regulator of apoptosis, cell cycle, and metastasis. The expression of dapk1 gene is frequently lost in several human cancers. DAPK1 also regulates autophagy (a novel form of death), which is critical for the removal of damaged organelle, fighting intracellular pathogens, antigen presentation and tumor suppression. Interestingly, the loss of C/EBP-2 gene in mice causes many of these defects. During the last funding period, we have shown a central role for C/EBP-2 in regulating DAPK1. In preliminary studies, we show that the expression of DAPK1 requires other transacting factors. Notably, Activating transcription factor 6 (ATF6), a key regulator of endoplasmic reticulum-dependent stress responses, appears to regulate DAPK1 expression. We propose that a direct interaction between ATF6 and C/EBP-2 leads to DAPK1 expression and growth suppression via autophagy, wherein the MAP Kinase, apoptosis- stimulating kinase 1(ASK1), provides critical signal inputs. In specific aim 1 of this proposal we will investigate how C/EBP-2 and ATF6 collaborate to upregulate DAPK1 expression. In specific aim 2, we will investigate how ASK1 controls DAPK1 expression via ATF6. Enhancer bound transcription factors (TFs) promote transcription using transcriptional co-activators. One of them the Mediator, a molecular bridge comprised of multiple proteins, communicates the transcriptional signals from the TFs to general transcription factor complex at the TATA-box. Studies during the last funding period also identified Med1, a major subunit of Mediator, an IFN-induced binding partner of C/EBP- 2. We present preliminary evidence for the involvement of ZIP-kinase(ZIPK), a member of the DAPK family, in regulating DAPK1 expression in response to IFNs. We hypothesize that ZIPK regulates DAPK1 expression by modulating the phosphorylation of C/EBP-binding domain of Med1. This aspect will be investigated in specific aim 3. We will evaluate the critical relevance of these factors to DAPK1 and autophagy using RNAi, knockout mice, protein-interactions, ChIP assays, mutagenesis and transcriptional analyses. Knowledge gained from an understanding these pathways, will not only define the critical regulators of DAPK1, but also will provide indicators into how a loss of DAPK1 can occur. We will investigate the relevance of these pathways to human CLL, a disease in which dapk1 appears to play an important tumor suppressive role. These in turn will allow a better design of therapeutics to combat tumor progression and metastasis. PUBLIC HEALTH RELEVANCE: Tumor metastasis occurs due to a loss of certain critical genes and a suppression of corresponding biological processes. Studies proposed in this application will investigate the regulation of an anti-metastatic gene, DAPK1, which is critical for tumor suppression and autophagic responses.