Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, South Carolina 29208, USA.
Biol Reprod. 2010 Dec;83(6):1015-26. doi: 10.1095/biolreprod.110.085969. Epub 2010 Aug 25.
GATA4 and GATA6 are zinc-finger transcription factors that regulate specific genes involved in steroidogenesis. Using RNA interference (RNAi)-mediated reduction of GATA4 and/or GATA6 with microarray analysis, we aimed to identify novel GATA target genes in luteinizing porcine granulosa cells under vehicle- and cAMP-treated conditions. Microarray analysis identified IGF1 mRNA to be cAMP- and GATA-responsive, and real-time PCR demonstrated that the cAMP-induced increase in IGF1 mRNA was reduced under conditions of GATA6 depletion and GATA4 plus GATA6 depletion, but not GATA4 depletion. Insulin-like growth factor 1 protein levels in media were also decreased by GATA6 or GATA4 plus GATA6 reduction. IGFBP2 and IGFBP4 mRNAs were increased and IGFBP5 mRNA decreased with vehicle and cAMP treatment under GATA4 plus GATA6 RNAi conditions. GATA6 reduction alone increased basal IGFBP4 and decreased IGFBP5 with both vehicle and cAMP, and GATA4 reduction alone lowered cAMP IGFBP5 levels with cAMP. No changes in IGFBP3 mRNA were observed with GATA reduction relative to the control RNAi condition. Levels of insulin-like growth factor binding proteins 2-5 in media as assessed by Western ligand blotting were not altered by GATA reduction. Electromobility gel shift assays with two GATA-containing oligonucleotides of the IGF1 5'-regulatory region showed GATA4 and GATA6 could bind the more proximal GATA-B site. These studies indicate that although GATA4 and GATA6 can bind the porcine IGF1 5'-region, GATA6 is functionally most important for cAMP-stimulated mRNA levels. Using microarray analysis, we identified other mRNAs that were altered by GATA-reduced conditions, including ALDH1, DIO2, and EDNRB. Our findings further support GATA as a coordinator of endocrine/paracrine/autocrine signals in the ovary.
GATA4 和 GATA6 是锌指转录因子，可调节类固醇生成过程中涉及的特定基因。我们使用 RNA 干扰 (RNAi) 介导的 GATA4 和/或 GATA6 减少，并结合微阵列分析，旨在确定在载体和 cAMP 处理条件下，猪黄体化颗粒细胞中新型 GATA 靶基因。微阵列分析表明 IGF1 mRNA 对 cAMP 和 GATA 有反应，实时 PCR 表明，在 GATA6 耗竭和 GATA4 加 GATA6 耗竭的情况下，cAMP 诱导的 IGF1 mRNA 增加减少，但 GATA4 耗竭则不然。在 GATA6 或 GATA4 加 GATA6 减少的情况下，培养基中的胰岛素样生长因子 1 蛋白水平也降低。IGFBP2 和 IGFBP4 mRNA 在载体和 cAMP 处理下增加，IGFBP5 mRNA 在 GATA4 加 GATA6 RNAi 条件下减少。GATA6 单独减少可增加基础 IGFBP4 并降低载体和 cAMP 下的 IGFBP5，而 GATA4 单独减少可降低 cAMP IGFBP5 与 cAMP。与对照 RNAi 条件相比，GATA 减少时 IGFBP3 mRNA 没有变化。通过 Western 配体印迹法评估的 IGFBP2-5 水平在培养基中没有因 GATA 减少而改变。使用包含 IGF1 5'-调控区的两个 GATA 结合寡核苷酸的电泳凝胶迁移率变动分析表明，GATA4 和 GATA6 可以结合更接近的 GATA-B 位点。这些研究表明，尽管 GATA4 和 GATA6 可以结合猪 IGF1 5'-区，但 GATA6 对 cAMP 刺激的 mRNA 水平最为重要。使用微阵列分析，我们确定了其他受 GATA 减少条件改变的 mRNA，包括 ALDH1、DIO2 和 EDNRB。我们的发现进一步支持 GATA 作为卵巢内分泌/旁分泌/自分泌信号的协调者。
