Filamin A in the human ovary: roles in follicle maturation and in granulosa cell tumors

细丝蛋白A(Flana)是一种X染色体编码的细胞骨架蛋白。Flna使肌动蛋白细丝交叉连接，通过与多种结构和信号网络组件结合，作为细胞内信号支架。因此，它调节细胞的形状和命运，以及在发育和形态发生过程中的迁移。Flna在肿瘤生物学中的作用正在显现。Flna在哺乳动物性腺中的表达和作用在很大程度上是未知的。我们最近在人类睾丸、睾丸肿瘤和一种精原细胞瘤细胞系中描述了FLNA。在这些研究中，我们也使用了CRISPR/Cas9介导的基因敲除，证实了Flna在调节细胞表型和细胞迁移中的重要作用。由于缺乏关于雌性性腺中FLNA的信息，雌性性腺是成虫最活跃的器官，主持着多种形态发生事件，这促使我们搜索数据库并进行初步研究。很明显，Flna在人颗粒细胞(GCs)中表达，并在发育中的卵泡中显著增加，直到排卵。来源于排卵前卵泡的体外培养的人体外受精来源的GC在体外表达Flna，初步的免疫沉淀/质谱学研究表明，类固醇合成酶和缝隙连接蛋白是仅次于肌动蛋白和细胞骨架蛋白的潜在的新的Flna相互作用伙伴。在人类颗粒细胞肿瘤(GCTS)和被广泛研究的GCT细胞系KGN细胞中也发现了Flna。对缺失Flna的KGN细胞的初步研究(CRISPR/Cas9)表明，生物力学特性发生了巨大变化，细胞生长加快。基于这些结果，我们提出了假设：(1)FLNA是一种未知的、高度调控的因子，在人类卵泡发育和排卵过程中具有重要作用。此外，初步数据提示我们还假设(2)Flna可能在GCTS的生长和发展中发挥作用，并可能作为生物标志物。我们将研究Flna在卵巢中的表达，并使用原代人类GC和KGN细胞来破译卵巢细胞中Flna的调节、功能和相互作用伙伴。为此，我们将采用免疫沉淀/质谱学研究。我们将使用KGN细胞检测Flna在形态、细胞增殖和生物力学特性方面的作用，在KGN细胞中删除Flna。此外，对初级GCTS的研究，包括组织微阵列(TMA)，将解决这个问题，即Flna是否可以作为GCTS的生物标记物。通过国际合作对非人类灵长类卵巢进行研究是可能的，啮齿动物卵巢的可用性包括研究FLNA基因敲除小鼠的可能性。我们期待关于卵巢Flna在健康和疾病中的作用的新的翻译数据。

Filamin A (FLNA) is an X-chromosomal encoded, well-characterized cytoskeletal protein. FLNA cross-links actin filaments and acts as an intracellular signaling scaffold by binding to a multitude of structural and signaling network components. Thereby it regulates cell shape and fate, as well as migration during development and morphogenesis. FLNA has emerging roles in tumor biology. Expression and roles of FLNA in the mammalian gonads are largely unknown. We recently described FLNA in the human testis, testicular tumors and a seminoma cell line. The studies, in which we also employed CRISPR/Cas9-mediated knockouts, confirmed an essential involvement of FLNA in regulation of the cellular phenotype and cell migration. The absence of information on FLNA in the female gonad, the most dynamic organ of the adult hosting multiple morphogenic events, led us to search data bases and to perform pilot studies. It became apparent that FLNA is expressed by human granulosa cells (GCs), and increases strikingly in growing follicles up to ovulation. Cultured human IVF-derived GCs, stemming from pre-ovulatory follicles, express FLNA in vitro, and a pilot immunoprecipitation/mass spectrometry study indicated steroidogenic enzymes and gap junction proteins as potential, novel FLNA-interaction partners, next to actin and cytoskeletal proteins. FLNA was also found in granulosa cell tumors (GCTs), which are derived from human GCs, and in KGN cells, a widely studied GCT cell line. Preliminary studies in KGN cells, in which FLNA was deleted (CRISPR/Cas9) indicated massively altered biomechanical properties and increased cell growth. Based on these results we put forward the hypothesis (1) that FLNA is a previously unexplored, highly regulated factor with important roles in the growing human follicle and the ovulatory process. In addition, the preliminary data prompts us to also hypothesize (2) that FLNA may play a role in the growth and progression of GCTs and may serve as a biomarker. We will study expression in the ovary and use primary human GCs and KGN cells to decipher regulation, function and interaction partners of FLNA in ovarian cells. To this end we will employ immunoprecipitation/mass spectrometry studies. We will examine the role of FLNA for morphology, cell proliferation and biomechanical properties using KGN cells, in which FLNA is deleted. Further, investigations in primary GCTs, including a tissue microarray (TMA), will address the question, whether FLNA may serve as a biomarker for GCTs. Studies in non-human primate ovaries are possible via an international collaboration and the availability of rodent ovaries include the possibility to investigate FLNA knockout mice. We expect novel translational data on the role of ovarian FLNA in health and disease.