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

描述（由申请人提供）：中枢神经系统的HIV-1感染导致超过30%的艾滋病患者发生痴呆，也称为HIV-1相关痴呆，HAD。单核细胞和巨噬细胞向脑的浸润与痴呆的发展和进展相关，是HAD的关键病理特征之一。单核细胞趋化蛋白1，MCP-1，一种β趋化因子，由于其在HAD患者的脑组织和脑脊液中的水平升高而受到特别的关注。据信，MCP-1在脑中的表达增加促进活化的单核细胞和巨噬细胞吸引到脑，在脑中它们通过释放几种炎性细胞因子和免疫调节剂引起神经元细胞死亡。包括我们在内的几个实验室的早期研究表明，HIV-1调节蛋白达特具有增强MCP-1基因转录和这种趋化蛋白在原代人星形胶质细胞中分泌的能力。然而，达特激活MCP-1的机制仍不清楚。我们最近的研究已经确定了C/EBP β 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等伴侣的结合来调节。此外，我们的研究结果表明，达特特异性地与C/EBP-β相互作用的能力，并与C/EBP-β合作，在人星形胶质细胞中增强MCP-1启动子的转录。另一方面，已显示达特刺激编码TNF α和TGF β-1的基因的转录，TNF α和TGF β-1的产物触发信号传导途径，分别引起NF κ B和Smads家族转录因子的p50/p65亚基的活化。在人星形胶质细胞中对MCP-1启动子上的NFrJ 3和Smads活性的检查揭示，通过与C/EBP-β结合，p50/p65提高MCP-1的转录，而Smad 3与C/EBP-β的相互作用抑制MCP-1基因转录的水平。这一观察结果表明，由达特诱导的两个信号事件之间的微妙平衡可以通过C/EBP-β决定MCP-1基因表达的水平。这些观察结果使我们假设达特通过与C/EBP-β及其配偶体直接相关和/或通过刺激与C/EBP-β通讯的细胞因子信号通路间接调节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的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诱导的星形胶质细胞和小胶质细胞中MCP-1活化的机制。这些机制研究的结果将提供重要的信息，可用于设计分子治疗工具，干扰MCP-1基因在中枢神经系统中的表达的激活。

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.