Liu Zhilin, Rudd Michael D, Hernandez-Gonzalez Inmaculata, Gonzalez-Robayna Ignacio, Fan Heng-Yu, Zeleznik Anthony J, Richards JoAnne S
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Mol Endocrinol. 2009 May;23(5):649-61. doi: 10.1210/me.2008-0412. Epub 2009 Feb 5.
The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear and FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3-mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development.
叉头框转录因子FOXO1在生长卵泡的颗粒细胞中高表达，但在体外培养时会被促卵泡激素（FSH）下调，在体内会被促黄体生成素（LH）诱导的黄体化下调。为了分析FOXO1的功能，我们用表达两种FOXO1突变体的腺病毒载体感染大鼠和小鼠颗粒细胞：一种功能获得性突变体FOXOA3，它有两个丝氨酸残基和一个苏氨酸残基突变为丙氨酸，使该蛋白组成性激活并定位于细胞核；另一种是FOXOA3突变体DNA结合结构域（mDBD），其中DNA结合结构域发生了突变。然后用溶剂或FSH对感染的细胞进行特定时间的处理。颗粒细胞的感染效率很高，仅引起极少的细胞凋亡，并将FOXO1蛋白维持在未接触FSH的细胞中观察到的内源性蛋白水平。从用溶剂或FSH处理12小时和24小时的对照细胞和腺病毒感染细胞中提取RNA。Affymetrix微阵列和数据库分析确定，并通过实时逆转录聚合酶链反应（RT-PCR）验证，脂质、固醇和类固醇生物合成途径（Hmgcs1、Hmgcr、Mvk、Sqle、Lss、Cyp51、Tm7sf2、Dhcr24和Star、Cyp11a1和Cyp19）中的基因，包括胆固醇生物合成和类固醇生成的两个关键转录调节因子Srebf1和Srebf2（Nr5a1、Nr5a2），是FSH诱导并在培养的颗粒细胞中被FOXOA3和FOXOA3-mDBD抑制的主要靶标。相比之下，FOXOA3和FOXOA3-mDBD诱导了编码参与胆固醇分解为氧固醇的一种酶的Cyp27a1 mRNA的表达。在培养的颗粒细胞中被FSH上调的基因，在分别用FSH（马绒毛膜促性腺激素）和LH（人绒毛膜促性腺激素）预处理的未成熟小鼠的排卵前卵泡和黄体的颗粒细胞中也被诱导。相反，相同处理使Foxo1和Cyp27a1 mRNA减少。总体而言，这些数据提供了新的证据，表明FOXO1可能在颗粒细胞中发挥关键作用，调节脂质和固醇生物合成，从而在卵泡发育早期防止类固醇生成升高。
