Regulation of MCP-1 expression by C/EBPbeta and HIV Tat

描述（由申请人提供）：在超过30%的艾滋病患者中，HIV-1中枢神经系统感染导致痴呆的发展，也称为HIV-1相关痴呆，HAD。单核细胞和巨噬细胞向大脑的浸润与痴呆的发生和进展相关，是HAD的关键病理特征之一。单核细胞趋化蛋白1 （MCP-1）是一种β趋化因子，由于其在HAD患者脑组织和脑脊液中的水平升高而受到特别关注。人们认为，MCP-1在大脑中的表达增加，促进了活化的单核细胞和巨噬细胞向大脑的吸引，在那里它们通过释放几种炎症细胞因子和免疫调节剂导致神经元细胞死亡。包括我们在内的几个实验室的早期研究表明，HIV-1调节蛋白Tat能够增强MCP-1基因的转录和人类星形胶质细胞中这种化学引诱蛋白的分泌。然而，Tat激活MCP-1的机制尚不清楚。我们最近的研究已经证实了C/EBPbeta DNA结合转录因子在星形胶质细胞中刺激MCP-1启动子表达的能力。C/ ebp - β是一种普遍存在的调节蛋白，其活性可以通过与CHOP、cmyb和Spl等伙伴蛋白的关联来调节。此外，我们的研究结果表明Tat能够特异性地与C/ ebp - β相互作用，并与C/ ebp - β合作，增强人类星形胶质细胞中MCP-1启动子的转录。另一方面，Tat已被证明可以刺激编码TNFalpha和TGFbeta-1的基因的转录，其产物分别触发信号通路，导致NFKappaB和Smads转录因子家族的p50/p65亚基的激活。对人类星形胶质细胞中MCP-1启动子上NFrJ3和Smads活性的检测显示，p50/p65通过与C/ ebp - β结合，可提高MCP-1的转录水平，而Smad3与C/ ebp - β相互作用可抑制MCP-1基因的转录水平。这一观察结果表明，Tat诱导的两种信号事件之间的微妙平衡可以通过C/ ebp - β决定MCP-1基因的表达水平。这些观察结果使我们假设Tat直接通过与C/ ebp - β及其伙伴相关和/或间接通过刺激与C/ ebp - β通信的细胞因子信号通路来调节CNS中MCP-1的转录。为了验证这一假设，我们将使用各种分子生物学和细胞生物学体外和体内技术进行一系列全面的结构和功能实验，以破译tat诱导星形胶质细胞和小胶质细胞中MCP-1激活的机制。这些机制研究的结果将为设计干扰CNS中MCP-1基因表达激活的分子治疗工具提供重要信息。

DESCRIPTION (provided by applicant): HIV-1 infection of the central nervous system leads to the development of dementia, also known as HIV-1 associated dementia, HAD, in greater than 30% of AIDS patients. Infiltration of monocytes and macrophages to the brain correlating with the development and progression of dementia is among the key pathologic features of the HAD. Monocyte chemoattractant protein 1, MCP-1, a beta chemokine, has received special attention due to its elevated levels in brain tissue and in cerebrospinal fluid of patients with HAD. It is believed that the increased expression of MCP-1 in the brain promotes the attraction of activated monocytes and macrophages to the brain where they cause neuronal cell death by releasing several inflammatory cytokines and immunomodulators. Earlier studies by several laboratories including ours have indicated that the HIV-1 regulatory protein, Tat, has the ability to augment transcription of the MCP-1 gene and the secretion of this chemoattractant protein in primary human astrocytes. However, the mechanism involved in the activation of MCP-1 by Tat remains unknown. Our recent studies have established the capacity of C/EBPbeta DNA binding transcription factor in stimulating expression of the MCP-1 promoter in astrocytes. C/EBP-beta is a ubiquitous regulatory protein whose activity can be modulated through its association with partners such as CHOP, cmyb, and Spl. Further, our results demonstrate the ability of Tat to specifically interact with C/EBP-beta, and cooperate with C/EBP-beta in enhancing transcription of the MCP-1 promoter in human astrocytes. On the other hand, Tat has been shown to stimulate transcription of genes encoding TNFalpha and TGFbeta-1 whose products trigger the signaling pathways causing the activation of p50/p65 subunits of NFKappaB and Smads family of transcription factors, respectively. Examination of NFrJ3 and Smads activities upon the MCP-1 promoter in human astrocytes revealed that by associating with C/EBP-beta, p50/p65 elevates transcription of MCP-1, whereas the interaction of Smad3 with C/EBPbeta suppresses the level of MCP-1 gene transcription. This observation implies that the delicate balance between the two signaling event induced by Tat can dictate, via C/EBP-beta, the level of MCP-1 gene expression. These observations led us to hypothesize that Tat modulates transcription of MCP-1 in the CNS directly by associating with C/EBP-beta and its partners and/or indirectly by stimulating cytokine signaling pathways that communicate with C/EBP-beta. To test this hypothesis, we will perform a comprehensive series of structural and functional experiments using a variety of molecular biology and cell biology in vitro and in vivo techniques to decipher the mechanism involved in Tat-induced activation of MCP-1 in astrocytes and microglia. The outcome of these mechanistic studies will provide important information that can be utilized for devising molecular therapeutic tools for interfering with the activation of MCP-1 gene expression in the CNS.