Transcriptional regulation in chronic renal disease pathogenesis

描述(申请人提供)：核因子kappaB(核因子kB)是一种普遍存在的转录因子，介导免疫和炎症过程。我们已经证实，核因子-kB在HIVAN中持续激活，这是一种塌陷性肾小球疾病，在细胞水平上以上皮细胞增殖、凋亡和分化缺陷为特征。我们假设，在HIVAN中，一种病毒蛋白(可能是Nef)与核因子-kB激活信号体相关联，并减弱正常的失活机制，导致核因子-kB的持续激活，最终导致关键的HIVAN表型--上皮细胞增殖、免疫细胞募集和炎症。具体目标1将确定导致持续的核因子-kB激活的病毒蛋白，以及它“劫持”的宿主信号通路。我们的初步数据表明，HIV蛋白Nef的相互作用增强了IKK信号体的活性，允许持续的NF-kB激活，这可以通过抑制肿瘤坏死因子受体信号来抑制。特定的目标2和3将测试核因子-kB是否在一定程度上导致上皮细胞增殖(目标2)和免疫细胞招募和炎症(目标3)。上皮细胞异常增殖是HIVAN的主要病理表型。在特定的目标2中，我们提出了促进上皮细胞增殖的结果是依赖于核因子-kB的Cyclin D1转录调控。尽管我们在第一个资助阶段已经证明了HIVAN中细胞凋亡的增加，但我们预测并将检验HIVAN相关的细胞凋亡是否是促进上皮细胞增殖的代偿性反应。在特定的目标3中，我们将研究核因子-kB的激活在感染的上皮细胞的免疫细胞募集中的作用以及在炎症中的作用，炎症是HIVAN发病机制中一个尚未明确的方面。使用体外迁移试验作为免疫细胞募集模型的初步研究表明，使用小分子核因子-kB抑制剂，CYC202和BAY11-7082，可以有效地抑制感染的肾细胞产生的趋化物质。由于核因子-kB控制着许多免疫调节剂的表达，我们将利用靶向阵列筛选来研究核因子-kB在人肾细胞感染过程中诱导的转录反应，重点是那些促炎细胞因子。此外，初步研究表明，核因子-kB的小分子抑制剂以及肿瘤坏死因子受体拮抗剂(一种可能的上游刺激信号)抑制了感染肾细胞中的核因子-kB的激活。鉴于这些观察，我们将在HIVAN转基因小鼠模型上测试核因子-kB抑制剂或肿瘤坏死因子受体阻断是否抑制炎症并改善肾脏疾病的进展。明确核因子-kB失调的机制和病理后遗症将为HIVAN和其他以病理性增殖或炎症为特征的肾小球疾病确定新的治疗靶点。

DESCRIPTION (provided by applicant): Nuclear Factor kappa B (NF-kB) is a ubiquitous transcription factor that mediates immune and inflammatory processes. We have established that NF-kB is persistently activated in HIVAN, a collapsing glomerulopathy, which is characterized at the cellular level by defects in epithelial cell proliferation, apoptosis and differentiation. We hypothesize, in HIVAN, that a viral protein (likely Nef) associates with the NF-kB-activating signalsome and attenuates the normal, inactivating mechanism, resulting in persistence of NF-kB activation, and ultimately epithelial cell proliferation and immune cell recruitment and inflammation, key HIVAN phenotypes. Specific aim 1 will identify the viral protein that causes persistent NF-kB activation, as well as the host signaling pathway it "hijacks". Our preliminary data suggest interaction of the HIV protein Nef enhances IKK signalsome activity permitting persistent NF-kB activation, which can be suppressed by inhibition of TNF receptor signaling. Specific aims 2 and 3 will test if NF-kB causes, in part, epithelial cell proliferation (aim 2) and immune cell recruitment and inflammation (aim 3). Abnormal epithelial proliferation is the major pathologic phenotype in HIVAN. In specific aim 2, we propose increased epithelial proliferation results from NF-kB-dependent transcriptional regulation of Cyclin D1. Although we have shown in the first funding period that apoptosis is increased in HIVAN, we predict and will test if HIVAN-associated apoptosis is a compensatory response to increase epithelial cell proliferation. In specific aim 3, we will investigate the role of NF-kB activation in immune cell recruitment by infected epithelial cells and in inflammation, a poorly defined aspect of HIVAN pathogenesis. Preliminary studies using in vitro migration assays as a model for immune cell recruitment indicate that the use of small molecule NF-kB inhibitors, CYC202 and BAY11-7082, are effective at suppressing chemoattractants produced by infected renal cells. Since NF-kB controls the expression of many immune modulatory agents, we will use targeted array screening to characterize the transcriptional responses induced by NF-kB during infection of human renal cells, focusing on those pro-inflammatory cytokines. In addition, preliminary studies show that small molecule inhibitors of NF-kB, as well as TNF receptor blockade (a likely upstream stimulatory signaling) suppress NF-kB activation in infected renal cells. Given these observations, we will test in a transgenic mouse model of HIVAN if NF-kB inhibitors or TNF receptor blockade suppresses inflammation and ameliorates renal disease progression. Defining mechanisms and pathologic sequelae of NF-kB dysregulation will identify novel treatment targets for HIVAN and other glomerulopathies characterized by pathogenic proliferation or inflammation.