Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH.
Department of Genetics, University of Pennsylvania, Philadelphia, PA.
Exp Hematol. 2020 Apr;84:29-44. doi: 10.1016/j.exphem.2020.03.003. Epub 2020 Apr 4.
Erythroid cell formation critically depends on signals transduced via erythropoietin (EPO)/EPO receptor (EPOR)/JAK2 complexes. This includes not only core response modules (e.g., JAK2/STAT5, RAS/MEK/ERK), but also specialized effectors (e.g., erythroferrone, ASCT2 glutamine transport, Spi2A). By using phospho-proteomics and a human erythroblastic cell model, we identify 121 new EPO target proteins, together with their EPO-modulated domains and phosphosites. Gene ontology (GO) enrichment for "Molecular Function" identified adaptor proteins as one top EPO target category. This includes a novel EPOR/JAK2-coupled network of actin assemblage modifiers, with adaptors DLG-1, DLG-3, WAS, WASL, and CD2AP as prime components. "Cellular Component" GO analysis further identified 19 new EPO-modulated cytoskeletal targets including the erythroid cytoskeletal targets spectrin A, spectrin B, adducin 2, and glycophorin C. In each, EPO-induced phosphorylation occurred at pY sites and subdomains, which suggests coordinated regulation by EPO of the erythroid cytoskeleton. GO analysis of "Biological Processes" further revealed metabolic regulators as a likewise unexpected EPO target set. Targets included aldolase A, pyruvate dehydrogenase α1, and thioredoxin-interacting protein (TXNIP), with EPO-modulated p-Y sites in each occurring within functional subdomains. In TXNIP, EPO-induced phosphorylation occurred at novel p-T349 and p-S358 sites, and was paralleled by rapid increases in TXNIP levels. In UT7epo-E and primary human stem cell (HSC)-derived erythroid progenitor cells, lentivirus-mediated short hairpin RNA knockdown studies revealed novel pro-erythropoietic roles for TXNIP. Specifically, TXNIP's knockdown sharply inhibited c-KIT expression; compromised EPO dose-dependent erythroblast proliferation and survival; and delayed late-stage erythroblast formation. Overall, new insight is provided into EPO's diverse action mechanisms and TXNIP's contributions to EPO-dependent human erythropoiesis.
红细胞的生成严重依赖于促红细胞生成素 (EPO)/EPO 受体 (EPOR)/JAK2 复合物转导的信号。这不仅包括核心反应模块（例如，JAK2/STAT5、RAS/MEK/ERK），还包括专门的效应物（例如，红细胞生成素、ASCT2 谷氨酰胺转运体、Spi2A）。通过使用磷酸化蛋白质组学和人类红系母细胞模型，我们鉴定了 121 种新的 EPO 靶蛋白，以及它们受 EPO 调节的结构域和磷酸化位点。GO 富集分析显示，“分子功能”中鉴定的衔接蛋白是 EPO 的一个主要靶标类别。这包括一个新的 EPOR/JAK2 偶联的肌动蛋白组装修饰物网络，其中衔接蛋白 DLG-1、DLG-3、WAS、WASL 和 CD2AP 是主要成分。GO 分析进一步鉴定了 19 种新的 EPO 调节的细胞骨架靶标，包括红细胞细胞骨架靶标血影蛋白 A、血影蛋白 B、踝蛋白 2 和血型糖蛋白 C。在每个靶标中，EPO 诱导的磷酸化发生在 pY 位点和亚结构域，这表明 EPO 对红细胞骨架进行协调调节。GO 分析进一步揭示，代谢调节剂也是 EPO 的一个意想不到的靶标组。这些靶标包括醛缩酶 A、丙酮酸脱氢酶α1 和硫氧还蛋白相互作用蛋白 (TXNIP)，EPO 调节的每个靶标在功能亚结构域中都有 p-Y 位点。在 TXNIP 中，EPO 诱导的磷酸化发生在 novel p-T349 和 p-S358 位点，同时 TXNIP 水平迅速增加。在 UT7epo-E 和原代人类干细胞 (HSC) 衍生的红细胞祖细胞中，慢病毒介导的短发夹 RNA 敲低研究揭示了 TXNIP 的新的促红细胞生成作用。具体来说，TXNIP 的敲低显著抑制 c-KIT 表达；损害 EPO 剂量依赖性红系母细胞增殖和存活；并延迟晚期红系母细胞的形成。总的来说，本研究为 EPO 的多种作用机制以及 TXNIP 对 EPO 依赖的人类红细胞生成的贡献提供了新的见解。
