Alpha actinin 4: its functions and regulation

描述(申请人提供)：足细胞是肾小球中高度分化的上皮细胞，具有广泛的分枝细胞突起，交错形成独特的足突和裂隙横隔膜，是过滤屏障的关键组件。ACTN4的基因突变会破坏足细胞的细胞骨架结构，并与几种肾小球疾病有关。然而，ACTN4在足细胞中的正常功能以及ACTN4突变致病的机制尚不清楚。根据我们的初步数据，我们假设ACTN4在细胞核和细胞质中都发挥作用，并且ACTN4能够调节足细胞核中核受体的转录活性。其具体目的是：1)研究ACTN4在条件永生化人足细胞中的作用。我们将敲除内源性ACTN4，并确定其对足细胞标记基因表达的影响。我们还将建立表达ACTN4的细胞系，以建立与选定的目标基因的直接结合，并通过芯片(染色质免疫沉淀)芯片分析全球鉴定其相关基因。2)剖析ACTN4和核激素受体调节neparin表达的机制。我们将通过瞬时转染报告分析来确定neparin启动子中对ACTN4、核受体及其配体作出反应的序列决定因素。我们将通过芯片分析来验证这些数据，并描绘出组蛋白对激素的修饰模式。我们将进一步测试已知的与人类疾病相关的PPAR突变是否会对足细胞产生影响。3)探讨FSGS引起的ACTN4突变对正常足细胞行为和基因表达的影响。一旦我们确定了ACTN4在AIMS 1和AIMS 2中的正常功能，我们将以newitin启动子为模型，研究与疾病相关的ACTN4突变体及其在转录调节、组蛋白修饰和激素反应中的作用。我们预计，我们对ACTN4及其相互作用蛋白的研究将揭示ACTN4以前被低估的核作用，它对足细胞功能至关重要，并可能在未来对足细胞疾病具有治疗意义。与公共健康相关：肾脏的主要功能之一是清除毒素和代谢废物，同时防止大于白蛋白的蛋白质进入尿液。这一过程是由高度专门化的足细胞介导的，足细胞产生肾小球滤过屏障的关键成分。已知的细胞骨架结构蛋白ACTN4的基因突变与几种肾小球疾病有关。我们已经确定了ACTN4的一个新功能，它是细胞核中的转录辅助激活因子，调节几个激素敏感基因的转录。我们将调查这一核功能的细节。我们预计，我们的研究将揭示ACTN4以前被低估的作用，它对足细胞功能至关重要，并可能在未来对肾脏疾病产生治疗意义。

DESCRIPTION (provided by applicant): Podocytes are highly differentiated epithelial cells in the kidney glomerulus that possess extensively branched cell processes, interdigitating into the unique foot processes and slit diaphragms which function as key components of the filtration barrier. Genetic mutations in ACTN4 disrupt cytoskeletal structure of the podocytes and have been linked to several glomerular diseases. However, the normal function of ACTN4 in podocytes as well as the mechanism underlying disease-causing ACTN4 mutations is not clearly understood. Based on our preliminary data, we hypothesize that ACTN4 plays a role in both the nucleus and the cytoplasm and that ACTN4 is capable of modulating transcriptional activity of nuclear receptors in the nucleus of podocytes. The Specific Aims are: 1) To characterize the role of ACTN4 in conditionally immortalized human podocytes (HPCs). We will knockdown endogenous ACTN4 and determine the effect on the expression of podocyte marker genes. We will also establish cell lines expressing ACTN4 to establish direct binding to selected target genes and globally identify its associated genes by ChIP (chromatin immunoprecipitation)-on-ChIP analyses. 2) To dissect the mechanisms by which ACTN4 and nuclear hormone receptors regulate the expression of nephrin. We will determine the sequence determinants within the nephrin promoter that are responsive to ACTN4, nuclear receptors, and their ligands by transient transfection reporter assays. We will verify these data by ChIP assays and delineate histone modification patterns in response to hormones. We will further test whether known human disease-linked PPAR mutations have an effect on podocytes. 3) To explore the role of FSGS- causing ACTN4 mutations on normal podocyte behavior and gene expression. Once we have established the normal functions of ACTN4 in Aims 1 & 2, we will examine the disease-linked ACTN4 mutants and their role in transcriptional regulation, histone modifications, and hormone response using the nephrin promoter as a model. We anticipate that our studies on ACTN4 and its interacting proteins will uncover a previously underappreciated nuclear role for ACTN4 that is critical for podocyte functions and may have future therapeutic implications in podocyte diseases. PUBLIC HEALTH RELEVANCE: One of the key functions of the kidney is to remove toxins and metabolic waste while preventing proteins larger than albumin from entering the urine. This process is mediated by highly specialized cells known as podocytes that produce critical components of the filtration barrier in glomeruli. Genetic mutations in a known cytoskeletal structural protein, ACTN4, have been linked to several glomerular diseases. We have identified a novel function of ACTN4 as a transcriptional coactivator in the nucleus that modulates the transcription of several hormone-sensitive genes. We will investigate the details of this nuclear function. We anticipate that our studies will uncover a previously underappreciated role for ACTN4 that is critical for podocyte functions and may have future therapeutic implications in kidney diseases.