Alpha actinin 4: its functions and regulation

描述（由申请方提供）：足细胞是肾小球中高度分化的上皮细胞，具有广泛分支的细胞突起，交错成独特的足突起和狭缝隔膜，其作为过滤屏障的关键组分。ACTN 4的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，ACTN 4在足细胞中的正常功能以及致病ACTN 4突变的潜在机制尚不清楚。基于我们的初步数据，我们假设ACTN 4在细胞核和细胞质中均起作用，并且ACTN 4能够调节足细胞核中核受体的转录活性。本研究的具体目的是：1）研究ACTN 4在条件永生化人足细胞（HPC）中的作用。我们将敲低内源性ACTN 4，并确定对足细胞标记基因表达的影响。我们还将建立表达ACTN 4的细胞系，以建立与选定靶基因的直接结合，并通过ChIP（染色质免疫沉淀）-on-ChIP分析全面鉴定其相关基因。2)探讨ACTN 4和核激素受体对nephrin表达的调控机制。我们将确定nephrin启动子内的序列决定簇，这些决定簇通过瞬时转染报告基因分析对ACTN 4、核受体及其配体有反应。我们将通过ChIP检测来验证这些数据，并描述组蛋白对激素的反应模式。我们将进一步测试已知的人类疾病相关的PPAR突变是否对足细胞有影响。3)探讨FSGS引起的ACTN 4突变对正常足细胞行为和基因表达的作用。一旦我们在目标1和2中建立了ACTN 4的正常功能，我们将使用nephrin启动子作为模型来检查与疾病相关的ACTN 4突变体及其在转录调节、组蛋白修饰和激素应答中的作用。我们预计，我们对ACTN 4及其相互作用蛋白的研究将揭示ACTN 4以前被低估的核作用，这对足细胞功能至关重要，并可能在足细胞疾病中具有未来的治疗意义。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，同时防止比白蛋白大的蛋白质进入尿液。这一过程是由称为足细胞的高度特化细胞介导的，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ACTN 4的基因突变与几种肾小球疾病有关。我们已经确定了ACTN 4作为细胞核中转录共激活因子的一种新功能，可以调节几种激素敏感基因的转录。我们将研究这种核功能的细节。我们预计，我们的研究将揭示ACTN 4以前被低估的作用，这对足细胞功能至关重要，并可能在肾脏疾病中具有未来的治疗意义。

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.