Alpha actinin 4: its functions and regulation

描述（由申请人提供）：足细胞是肾小球中高度分化的上皮细胞，具有广泛的分支细胞突，交叉成独特的足突和狭缝膈，作为滤过屏障的关键组成部分。ACTN4基因突变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引起ACTN4突变的机制尚不清楚。根据我们的初步数据，我们假设ACTN4在细胞核和细胞质中都起作用，并且ACTN4能够调节足细胞细胞核中核受体的转录活性。具体目的是：1)表征ACTN4在条件永生化人足细胞（HPCs）中的作用。我们将敲低内源性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，与选定的靶基因建立直接结合，并通过ChIP（染色质免疫沉淀）-on-ChIP分析在全球范围内鉴定其相关基因。2)探讨ACTN4和核激素受体调控nephrin表达的机制。我们将通过瞬时转染报告基因测定法确定肾素启动子内对ACTN4、核受体及其配体有反应的序列决定因子。我们将通过ChIP实验验证这些数据，并描述组蛋白在激素作用下的修饰模式。我们将进一步测试已知的与人类疾病相关的PPAR突变是否对足细胞有影响。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。一旦我们在Aims 1和2中确定了ACTN4的正常功能，我们将以肾素启动子为模型，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以前未被重视的核作用，这对足细胞功能至关重要，并可能在未来的足细胞疾病治疗中具有重要意义。公共卫生相关性：肾脏的关键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。这个过程是由高度特化的足细胞介导的，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作为细胞核中的转录辅激活因子的新功能，该功能可调节几种激素敏感基因的转录。我们将研究这个核函数的细节。我们预计，我们的研究将揭示ACTN4先前未被重视的作用，这对足细胞功能至关重要，并可能在未来的肾脏疾病治疗中具有重要意义。

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.