Signaling in Cells Chronically Exposed to Interferons

描述（由申请人提供）：在过去的十年中，已经获得了关于干扰素和其他细胞因子通过Jak/Stat途径激活或下调立即早期基因的机制的大量知识。然而，与已经脱敏并随后对这些细胞因子的作用重新致敏的细胞相比，关于初始细胞中干扰素激活的基因表达的信息很少，从而在临床环境中模拟重复的干扰素给药。在幼稚细胞中，ISG 54基因通过IFNaB刺激的ISGF 3的形成而被激活，ISGF 3是一种异源三聚体DNA结合复合物，由p48（IRF 9）和酪氨酸磷酸化的Stat 1和Stat 2组成，其结合干扰素刺激的反应元件（ISRE）。相比之下，在先前去致敏的细胞中，IFNB微弱地刺激缺乏酪氨酸磷酸化的Stat 1的ISGF 3样复合物的组装，即使ISG 54 mRNA诱导与幼稚细胞中相同。Stat 1酪氨酸磷酸化和DNA结合的缺乏是由于蛋白酪氨酸磷酸酶Tc-PTP的活性增加。尽管在先前脱敏的细胞中IFN α/B刺激的ISGF 3形成减少，但LPS或双链RNA诱导ISG 54表达的能力（推测是通过TLR 3或TLR 4-IRF 3依赖性信号传导途径）大大增强。我们假设，调节1型干扰素（IFN α）激活ISRE依赖性早期反应基因的信号通路在先前已对这种细胞因子脱敏的细胞中是实质性不同的。IFNB反应的修饰影响通过与Toll受体结合的配体调节的其他转录因子调节ISG 54活化的能力。先前脱敏的细胞中改变的应答可能转化为长期用这些细胞因子治疗的人中IFN α/B和Toll受体介导的应答的变化。我们将通过执行以下具体目标来测试该假设：1）确定先前去致敏细胞中ISG 54诱导的差异要求。2)确定Tc-PTP增强先前脱敏细胞中Stat 1去磷酸化的机制。

DESCRIPTION (provided by applicant): Over the past decade, a wealth of knowledge has been obtained concerning the mechanisms by which interferons and other cytokines activate or downregulate immediate early genes via the Jak/Stat pathway. However, little information is available on interferon-activated gene expression in naive cells compared to cells that have been desensitized and subsequently re-sensitized to the actions of these cytokines, thereby mimicking repeated interferon administration in a clinical setting. In naive cells, the ISG54 gene is activated via IFNaB-stimulated formation of ISGF3, a heterotrimeric DNA binding complex consisting of p48 (IRF9) and tyrosine-phosphorylated Stat1 and Stat2, which binds the Interferon Stimulated Response Element (ISRE). In contrast, in previously de-sensitized cells, IFNB weakly stimulates the assembly of an ISGF3-like complex that lacks tyrosine phosphorylated Stat1, even though ISG54 mRNA induction is the same as in naive cells. The lack of Stat1 tyrosine phosphorylation and DNA binding is due to increased activity of the protein tyrosine phosphatase Tc-PTP. Although IFNa/B stimulated formation of ISGF3 is decreased in previously desensitized cells, the ability of LPS or double stranded RNA to induce ISG54 expression, presumably through a TLR3- or TLR4-IRF3-dependent signaling pathway, is greatly enhanced. We hypothesize that the signaling pathways that regulate type 1 interferon (IFN(/() activation of ISRE-dependent early response genes are substantially different in cells that have been previously desensitized to this cytokine. Modification of the IFNB response impinges on the ability of other transcription factors regulated through ligands that bind to Toll receptors to regulate activation of ISG54. The altered responses in previously desensitized cells likely translates into changes in IFNa/B and Toll receptor-mediated responses in humans who are chronically treated with these cytokines. We will test this hypothesis by performing the following specific aims: 1) Determine the differential requirements for ISG54-induction in previously de-sensitized cells. 2) Determine the mechanisms by which Tc-PTP enhances dephosphorylation of Stat1 in previously desensitized cells.