Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi-shi 274-8510, Japan.
Zoolog Sci. 2024 Aug;41(4):329-341. doi: 10.2108/zs240004.
Enucleated erythrocytes are characteristic of adult mammals. In contrast, fish, amphibians, reptiles, birds, and fetal mammals possess nucleated erythrocytes in their circulation. Erythroid maturation is regulated by erythropoietin (EPO) and its receptor (EPOR), which are conserved among vertebrates. In mammals, EPOR on the erythroid progenitor membrane disappears after terminal differentiation. However, in western clawed frog, , mature erythrocytes maintain EPOR expression, suggesting that they have non-canonical functions of the EPO-EPOR axis rather than proliferation and differentiation. In this study, we investigated the non-canonical functions of EPOR in mature erythrocytes. EPO stimulation of peripheral erythrocytes did not induce proliferation but induced phosphorylation of intracellular proteins, including signal transducer and activator of transcription 5 (STAT5). RNA-Seq analysis of EPO-stimulated peripheral erythrocytes identified 45 differentially expressed genes (DEGs), including cytokine inducible SH2 containing protein gene () and suppressor of cytokine signaling 3 gene (), negative regulators of the EPOR-Janus kinase (JAK)-STAT pathway. These phosphorylation studies and pathway analysis demonstrated the activation of the JAK-STAT pathway through EPO-EPOR signaling in erythrocytes. Through comparison with EPO-responsive genes in mouse erythroid progenitors obtained from a public database, we identified 31 novel EPO-responsive genes indicating non-canonical functions. Among these, we focused on ornithine decarboxylase 1 gene (), which is the rate-limiting enzyme in polyamine synthesis and affects hematopoietic progenitor differentiation and the endothelial cell response to hypoxic stress. An EPO-supplemented culture of erythrocytes showed increased expression followed by a decrease in polyamine-rich erythrocytes, suggesting EPO-responsive polyamine excretion. These findings will advance our knowledge of the unknown regulatory systems under the EPO-EPOR axis and functional differences between vertebrates' nucleated and enucleated erythrocytes.
去核红细胞是成年哺乳动物的特征。相比之下，鱼类、两栖动物、爬行动物、鸟类和胎生哺乳动物的循环中存在有核红细胞。红细胞的成熟受红细胞生成素 (EPO) 和其受体 (EPOR) 的调节，这些在脊椎动物中是保守的。在哺乳动物中，EPO 受体在红细胞祖细胞膜上的表达在终末分化后消失。然而，在西方爪蟾中，成熟的红细胞仍然表达 EPOR，这表明它们具有非经典的 EPO-EPOR 轴功能，而不是增殖和分化。在这项研究中，我们研究了 EPOR 在成熟红细胞中的非经典功能。EPO 刺激外周红细胞不会诱导增殖，但会诱导细胞内蛋白的磷酸化，包括信号转导和转录激活因子 5 (STAT5)。EPO 刺激外周红细胞的 RNA-Seq 分析鉴定出 45 个差异表达基因 (DEGs)，包括细胞因子诱导的 SH2 结构域含有蛋白基因 () 和细胞因子信号转导抑制因子 3 基因 ()，它们是 EPOR-Janus 激酶 (JAK)-STAT 通路的负调节剂。这些磷酸化研究和通路分析表明，EPO-EPOR 信号通过在红细胞中激活 JAK-STAT 通路。通过与公共数据库中获得的来自小鼠红细胞祖细胞的 EPO 反应性基因进行比较，我们鉴定出 31 个新的 EPO 反应性基因，表明存在非经典功能。在这些基因中，我们重点关注鸟氨酸脱羧酶 1 基因 ()，它是多胺合成的限速酶，影响造血祖细胞的分化和内皮细胞对低氧应激的反应。在补充 EPO 的红细胞培养中，发现 表达增加，随后多胺丰富的红细胞减少，表明 EPO 反应性多胺排泄。这些发现将提高我们对 EPO-EPOR 轴下未知调控系统以及脊椎动物有核和去核红细胞之间功能差异的认识。
