Mechanisms of Macrophage Activation

描述(申请人提供)：单核吞噬细胞在宿主对癌症的反应中的作用在很大程度上是通过对肿瘤微环境中遇到的刺激所诱导的基因表达模式来确定的。巨噬细胞对典型的促炎因子(IFNGamma和LPS)或抗炎药(IL-10和TGFbeta)反应的基因表达调控源于多种细胞内信号的整合，这些信号在转录和转录后水平上都有作用。目前对特定mRNA稳定性的转录后调控知识有限。许多细胞因子和趋化因子mRNAs的3‘非翻译区富含Au的序列元件(ARs)与ARE特异的RNA结合蛋白共同导致了mRNA的快速衰退。我们和其他人已经证明，促炎或抗炎刺激可以分别稳定或破坏选定的趋化因子mRNAs，特别是KC(小鼠CXCL1)。这些发现导致了一个普遍的假设，即对瞬时表达的炎症基因的转录后控制涉及到通过以序列特异的方式操作的过程诱导不稳定的mRNAs的稳定。抗炎药通过对抗这种反应发挥作用。目前，该模型的多个特征仍未定义。这些包括(A)刺激依赖反应的序列决定因素，(B)由稳定剂和/或去稳定剂启动的信号通路如何整合并与mRNA稳定性的控制相结合，(C)mRNA衰退中的哪些步骤被稳定或去稳定刺激所改变，以及(D)结合蛋白如何将信号事件与负责mRNA衰退变化的效应机制相结合。我们现在建议通过执行以下具体的实验目标来解决这些问题，作为对该假说的进一步检验。1.评价ARE依赖的、刺激介导的mRNA稳定性控制的序列基础。2.探讨内毒素、IL-10和转化生长因子β调控mRNA稳定性的信号通路。3.确定受刺激依赖调节的mRNA衰变机制。4.确定已知ARE/RNA结合蛋白在内毒素诱导的稳定化和IL-10或TGFβ介导的失稳中的作用

DESCRIPTION (provided by applicant): The contribution of mononuclear phagocytes in host response to cancer is determined in large part through the pattern of gene expression induced in response to stimuli encountered in the tumor microenvironment. Control of macrophage gene expression in response to prototypic pro-inflammatory (IFNgamma and LPS) or anti-inflammatory agents (IL-10 and TGFbeta) results from the integration of multiple intracellular signals that operate at both transcriptional and post-transcriptional levels. Current knowledge of post-transcriptional regulation of specific mRNA stability is limited. AU rich sequence elements (AREs) in the 3' untranslated regions (UTRs) of many cytokine and chemokine mRNAs in concert with ARE-specific RNA binding proteins lead to rapid mRNA decay. We and others have shown that pro-inflammatory or anti-inflammatory stimuli can stabilize or destabilize, respectively, selected chemokine mRNAs particularly KC (mouse CXCL1). These findings lead to the general hypothesis that post-transcriptional control of transiently expressed inflammatory genes involves the induced stabilization of unstable mRNAs through a process that operates in sequence specific fashion. Anti-inflammatory agents act by antagonizing this response. At present multiple features of this model remain undefined. These include (a) the sequence determinants for stimulus-dependent response, (b) how signaling pathways initiated by stabilizing and/or destabilizing agents are integrated and coupled with control of mRNA stability, (c) which steps in mRNA decay are altered by stabilizing or destabilizing stimuli and (d) how ARE-binding proteins couple signaling events to effector mechanisms responsible for the changes in mRNA decay. We now propose to address these issues as a further test of the hypothesis by performance of the following specific experimental aims. 1. Evaluate the sequence basis for ARE-dependent, stimulus-mediated control of mRNA stability. 2. Evaluate signaling pathways involved in the control of mRNA stability by LPS, IL-10 and TGFbeta. 3. Identify the mRNA decay mechanisms that are subject to stimulus-dependent modulation. 4. Determine the role of known ARE/RNA binding proteins in LPS-induced stabilization and IL-10 or TGFbeta-mediated destabilization