Signaling in Cells Chronically Exposed to Interferons

描述(申请人提供)：在过去的十年里，关于干扰素和其他细胞因子通过JAK/STAT途径激活或下调即刻早期基因的机制，已经获得了丰富的知识。然而，与那些已经脱敏并随后对这些细胞因子的作用重新敏感的细胞相比，关于干扰素激活的基因在幼稚细胞中表达的信息很少，从而在临床环境中模拟重复给予干扰素。在幼稚细胞中，ISG54基因是通过IFNaB刺激的ISGF3的形成而激活的，ISGF3是由P48(IRF9)和酪氨酸磷酸化的STAT1和STAT2组成的异三聚体DNA结合复合体，它结合干扰素刺激反应元件(ISRE)。相反，在先前脱敏的细胞中，IFNB弱刺激ISGF3样复合体的组装，该复合体缺乏酪氨酸磷酸化的STAT1，尽管ISG54 mRNA的诱导与幼稚细胞相同。缺乏STAT1酪氨酸磷酸化和DNA结合是由于蛋白酪氨酸磷酸酶TC-PTP活性增加所致。尽管IFNA/B刺激的ISGF3的形成在先前脱敏的细胞中减少，但内毒素或双链RNA诱导ISG54表达的能力大大增强，可能是通过TLR3或TLR4-IRF3依赖的信号通路。我们假设，在先前对该细胞因子脱敏的细胞中，调节依赖ISRE的早期反应基因的1型干扰素(IFN(/())激活的信号通路本质上是不同的。IFNB反应的修饰会影响其他转录因子的能力，这些转录因子通过与Toll受体结合的配体来调节ISG54的激活。在接受这些细胞因子长期治疗的人类中，先前脱敏细胞的反应变化可能转化为IFNA/B和Toll受体介导的反应的变化。我们将通过执行以下特定目标来验证这一假设：1)确定在先前脱敏的细胞中对ISG54-诱导的差异要求。2)确定TC-PTP促进脱敏细胞中STAT1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.