Transforming growth factor β family signaling pathways in ovarian and uterine biology

项目摘要 转化生长因子β（TGFβ）超家族是哺乳动物中最大的分泌蛋白家族。 这些二聚体配体在几乎所有的发育、生理和病理生理中起作用， 过程，包括不育，通过2型和1型丝氨酸-苏氨酸的异二聚体复合物发出信号 激酶受体，其磷酸化下游调节SMAD蛋白并结合SMAD 4以调节 转录。在NICHD的支持下，从医生科学家奖（K11 HD 00960; 1991-93）开始， 这个R 01补助金（1994年至今），我们一直是富有成效的领导者，在确定和表征的 卵母细胞分泌的TGFβ家族成员、生长分化因子9（GDF 9）和骨形态发生蛋白 15（BMP 15），以及颗粒细胞分泌的激活素和促性腺激素。我们在这一领域发表了大量文章 包括在Nature、Nature Genetics、Nature Medicine、PLoS Biology、PLoS Genetics、PNAS和 科学尽管哺乳动物卵母细胞最初被假设为乘客而不是司机， 我们发现GDF 9对生育力至关重要，发现了X连锁的BMP 15基因， 并显示GDF 9：BMP异二聚体是小鼠和女性中最活跃的卵母细胞分泌配体。 这些见解定义了卵泡发生中的卵母细胞-体细胞对话。同时，我们展示了 α-受体结合蛋白基因敲除小鼠不育，发生卵巢癌，并死于激活素诱导的恶病质， 综合征BMP、GDF 9：BMP 15、激活素和肌生长抑制素共享共同的2型受体[激活素受体类型 2A型（ACVR 2A）或2B型（ACVR 2B）或BMPR 2]、1型受体（ALK 4和ALK 5）和受体调节的 SMAD（SMAD 1、2、3、5）。利用小鼠遗传学，我们已经证明这些蛋白质在垂体中起作用， 卵巢和子宫（例如，SMAD 2和SMAD 3的颗粒细胞特异性敲除导致卵丘缺陷， 由于GDF 9：BMP 15信号传导缺陷导致的不孕症，而子宫特异性敲除SMAD 2和SMAD 3 导致继发于子宫内膜增生的不孕症）。虽然SMAD 2和SMAD 3在 GDF 9：BMP 15，激活素和TGFβ信号，我们对转录复合物或DNA知之甚少 它们结合的序列。此外，没有ACVR 2A/2B和BMPR 2的小分子抑制剂。我们 总体假设是卵母细胞GDF 9：BMP 15、颗粒细胞激活素、子宫BMP和TGFβs信号传导 通过独特的SMAD介导的转录复合物调节小鼠的卵巢和子宫生理学 和妇女我们的提案将利用最先进的CRISPR/Cas9策略来操纵基因组。 小鼠基因组和DNA编码的化学文库，以产生新型ACVR 2A/2B和BMPR 2抑制剂， 执行后续的遗传学，蛋白质组学和生物化学方法，以达到我们的目标。在5年结束时， 我们希望能够解开女性中TGFβ家族配体所协调的关键分子事件， 生殖道，从而加速转化研究，以优化辅助生殖程序， 该研究旨在开发第一个ACVR 2A/2B和BMPR 2特异性小分子来调节女性生育能力。

PROJECT SUMMARY The transforming growth factor β (TGFβ) superfamily is the largest family of secreted proteins in mammals. These dimeric ligands, which function in nearly every developmental, physiologic, and pathophysiologic process, including infertility, signal through a heterodimeric complex of type 2 and type 1 serine-threonine kinase receptors that phosphorylate downstream regulatory SMAD proteins and bind SMAD4 to regulate transcription. With NICHD support that started with a physician scientists award (K11HD00960; 1991-93) and this R01 grant (1994-present), we have been productive leaders in the identification and characterization of the oocyte-secreted TGFβ family members, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), and granulosa cell-secreted activins and inhibins. We have published extensively in this field including >30 papers in Nature, Nature Genetics, Nature Medicine, PLoS Biology, PLoS Genetics, PNAS, and Science. Whereas mammalian oocytes were initially hypothesized to be passengers rather than drivers in ovarian folliculogenesis, we showed that GDF9 is essential for fertility, discovered the X-linked BMP15 gene, and showed that GDF9:BMP heterodimers are the most active oocyte-secreted ligand in mice and women. These insights have defined the oocyte-somatic cell dialogue in ovarian folliculogenesis. In parallel, we showed that inhibin α-knockout mice are infertile, develop ovarian cancers and die due to an activin-induced cachexia syndrome. BMPs, GDF9:BMP15, activins, and myostatin share common type 2 receptors [activin receptor type 2A (ACVR2A) or type 2B (ACVR2B) or BMPR2], type 1 receptors (ALK4 and ALK5), and receptor-regulated SMADs (SMAD1,2,3,5). Using mouse genetics, we have shown that these proteins function in the pituitary, ovaries, and uterus (e.g., granulosa-specific knockout of SMAD2 and SMAD3 leads to cumulus defects and infertility due to defective GDF9:BMP15 signaling, whereas uterine-specific knockout of SMAD2 and SMAD3 leads to infertility secondary to endometrial hyperplasia). Although SMAD2 and SMAD3 play redundant roles in GDF9:BMP15, activin, and TGFβ signaling, we know little about the transcriptional complexes or DNA sequences that they bind. In addition, there are no small molecule inhibitors of ACVR2A/2B and BMPR2. Our overall hypothesis is that oocyte GDF9:BMP15, granulosa cell activins, and uterine BMPs and TGFβs signal through unique SMAD-mediated transcriptional complexes to regulate ovarian and uterine physiology in mice and women. Our proposal will take advantage of state-of-the-art CRISPR/Cas9 strategies to manipulate the mouse genome and DNA-encoded chemical libraries to create novel inhibitors of ACVR2A/2B and BMPR2 and perform follow-up genetic, proteomic, and biochemical approaches to reach our goals. At the end of 5 years, we expect to have unlocked key molecular events that are orchestrated by TGFβ family ligands in the female reproductive tract, thereby accelerating translational research to optimize assisted reproductive procedures for women and to create the first ACVR2A/2B and BMPR2 specific small molecules to regulate female fertility.