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

细丝蛋白A（FLNA）是一种X染色体编码的、特征明确的细胞骨架蛋白。FLNA交联肌动蛋白丝，并通过结合多种结构和信号网络组分作为细胞内信号传导支架。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在肿瘤生物学中具有新兴作用。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。我们最近描述了FLNA在人类睾丸，睾丸肿瘤和睾丸癌细胞系。这些研究中，我们还采用了CRISPR/Cas9介导的敲除，证实了FLNA在调节细胞表型和细胞迁移中的重要参与。在女性性腺，最有活力的器官的成人主办多个形态发生事件的信息FLNA的情况下，导致我们搜索数据库，并进行试点研究。很明显，FLNA由人颗粒细胞（GC）表达，并且在生长卵泡中显著增加直至排卵。培养的人IVF衍生的GC，源于排卵前卵泡，表达FLNA在体外，和试点免疫沉淀/质谱研究表明类固醇生成酶和间隙连接蛋白作为潜在的，新的FLNA相互作用的合作伙伴，旁边的肌动蛋白和细胞骨架蛋白。FLNA还在来源于人GCs的颗粒细胞瘤（GCT）和广泛研究的GCT细胞系KGN细胞中发现。在KGN细胞中的初步研究，其中FLNA被删除（CRISPR/Cas9）表明生物力学特性发生了巨大变化，细胞生长增加。基于这些结果，我们提出了以下假设：（1）FLNA是一种以前未被探索的高度调节的因子，在人类卵泡生长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GCT的生长和进展中发挥作用，并可能作为生物标志物。我们将研究卵巢中的表达，并使用原代人GCs和KGN细胞来破译卵巢细胞中FLNA的调控、功能和相互作用伴侣。为此，我们将采用免疫沉淀/质谱研究。我们将研究FLNA的形态学，细胞增殖和生物力学特性的作用，使用KGN细胞，其中FLNA被删除。此外，包括组织微阵列（TMA）在内的原发性GCT研究将解决FLNA是否可作为GCT生物标志物的问题。通过国际合作，可以对非人灵长类动物卵巢进行研究，啮齿类动物卵巢的可用性包括研究FLNA敲除小鼠的可能性。我们期待新的翻译数据卵巢FLNA在健康和疾病中的作用。

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.