CD44是高度保守的跨膜糖蛋白，通过其胞外区与透明质酸等配体结合，广泛参与调控细胞的增殖、分化和迁移等生物学过程。已有研究表明，CD44在造血系统中广泛表达，并参与调控造血干细胞的定植、淋巴细胞的归巢及白细胞的募集。在红系分化过程中，CD44的蛋白表达随着红细胞的成熟逐渐减少，被用作区分红系分化终末不同分化阶段的重要标志分子。然而CD44在红系终末分化中的功能和其作用机制尚不明确。

本研究中，我们利用CRISPR-Cas9介导的CD44敲除小鼠探讨CD44缺失对红系分化的影响。通过外周血常规检测、血涂片染色以及骨髓和脾脏细胞的流式分析等手段，我们发现CD44敲除小鼠呈轻度贫血，外周血不成熟网织红细胞增多。骨髓红系抑制且无核红细胞占比显著减少。骨髓红系分化在BFU-E/CFU-E祖细胞阶段增殖能力减弱并在终末分化的早幼红细胞阶段发生阻滞。脾脏大小未见显著变化，但其红系细胞比例显著增加，提示脾脏造血代偿。进一步分析应激造血状态下CD44敲除对红系分化的影响，使用苯肼诱导构建溶血性贫血模型，我们发现与野生型小鼠相比，CD44敲除小鼠的贫血更为严重，外周血红细胞大小和分布宽度增加，网织红细胞数目显著偏低。敲除鼠骨髓和脾脏红系代偿更为明显，但脱核率均显著低于野生型小鼠，提示红系造血生成障碍。

单细胞转录组测序和细胞周期分析显示CD44敲除小鼠骨髓有核红细胞增殖能力减弱并发生G2/M期阻滞。差异基因富集分析提示PERK介导的内质网应激通路及自噬和凋亡通路显著上调。我们首先通过Western Blotting证实PERK/eIF2α/ ATF4/CHOP通路在CD44敲除小鼠骨髓红系细胞中激活。内质网应激激活剂Tunicamycin和抑制剂TUDCA处理的小鼠实验表明，CD44敲除小鼠的红系细胞对内质网应激更为敏感，而抑制内质网应激明显改善敲除小鼠的骨髓红系造血，提示内质网应激是CD44缺失导致红系生成障碍的主要原因。流式细胞分析显示CD44敲除小鼠的骨髓红系细胞中Annexin V和7AAD双阳性的凋亡晚期细胞比例显著增高。电镜观察到敲除小鼠有核红细胞中受损线粒体及自噬小体明显增多。此外，自噬相关的FIP200、STX17和LC3蛋白表达水平均显著上调，与单细胞转录组分析结果一致。有趣的是，通过氯喹抑制自噬也可显著改善CD44敲除小鼠骨髓红系造血，提示自噬激活是敲除鼠红系造血异常的原因，而非内质网应激后的反馈性保护机制。

近年研究表明CD44在肿瘤细胞中参与氧化应激调控。为探索CD44敲除影响内质网应激的作用机制，我们对敲除小鼠骨髓有核红细胞的氧化应激进行了分析。DCFH-DA探针检测显示胞内活性氧（Reactive oxygen species ，ROS）水平显著升高。此外，CD44敲除细胞中抗氧化酶GPX1和GPX4在转录及蛋白水平上的表达均显著降低，而脂质过氧化物的含量则显著增加。相应的，氧化应激相关蛋白HO-1的蛋白表达显著上调，而NRF2的表达却明显降低，说明CD44敲除的红系细胞抗氧化能力降低，氧化应激水平升高。进一步研究表明，CD44缺失通过下调PI3K/AKT信号通路和激活自噬，抑制自噬接头蛋白p62表达，共同导致红系细胞NRF2表达的降低，从而引发细胞氧化损伤。最后，我们通过过氧化氢诱导小鼠胎肝原代红系细胞氧化应激，检测到药物浓度依赖性的PERK内质网应激通路激活和红系体外分化抑制。CD44敲除的胎肝原代红系细胞对氧化应激表现出更高的敏感性，在相同浓度的过氧化氢处理下，其ROS水平及PERK通路激活程度均显著高于野生型胎肝原代红系细胞，而NRF2蛋白表达则未见与野生型中类似的诱导上调。这些结果表明，在红系细胞中，CD44敲除抑制PI3K/AKT信号通路和p62蛋白水平，两者共同引起NRF2表达下调和细胞氧化应激增加，导致内质网应激和红系分化阻滞。此外，我们还发现敲除鼠中CD44配体透明质酸在骨髓中沉积增加，以及SWI2/SNF2家族成员BTAF1的红系表达下调，可能是红系细胞中CD44敲除的潜在代偿机制。本研究首次发现CD44缺失导致骨髓红系分化障碍，并探索了相关分子机制，为贫血性疾病和体外高效红系造血研究提供了新的线索。

CD44, a highly conserved transmembrane glycoprotein, regulates cellular proliferation, differentiation, and migration through interactions with ligands such as hyaluronic acid via its extracellular domain. CD44 is widely expressed in the hematopoietic system and critically regulates hematopoietic stem cell homing, lymphocyte trafficking, and leukocyte recruitment. During terminal erythroid differentiation, CD44 expression gradually decreases, and serves as a marker to distinguish different stages of erythroid precursors. However, the function and molecular mechanisms of CD44 in terminal erythropoiesis remains unclear.

In this study, CRISPR-Cas9-generated CD44 knockout (CD44KO) mice were employed to investigate the impact of CD44 deficiency on terminal erythropoiesis. Peripheral blood analysis revealed mild anemia and elevated immature reticulocytes in CD44KO mice. In bone marrow, erythropoiesis was impaired at both the BFU-E/CFU-E progenitors and basophilic erythroblast stages, accompanied by a significant reduction in enucleated erythroid cells. Compensatory splenic erythropoiesis was observed, evidenced by increased Ter119⁺ erythroid cells despite unaltered spleen size. Under phenylhydrazine-induced hemolytic stress, CD44KO mice exhibited exacerbated anemia, characterized byenlarged mean corpuscular volume and greater size variability of erythrocytes, with a significant decrease in enucleated erythroid cells in the bone marrow and spleen compared to wild-type mice.

Single-cell RNA sequencing combined with cell cycle analysis revealed reduced proliferative capacity and G2/M phase arrest of CD44KO bone marrow erythroblasts. Pathway enrichment analysis further indicateded activation of PERK-mediated endoplasmic reticulum (ER) stress, autophagy, and apoptosis. Western blotting confirmed upregulation of the PERK/eIF2α/ATF4/CHOP pathway in CD44KO erythroblasts. Experiments using the ER stress activator tunicamycin and the inhibitor TUDCA in mice demonstrated that erythroid cells from CD44KO mice were more sensitive to ER stress, while inhibition of ER stress significantly improved bone marrow erythropoiesis in CD44KO mice, suggesting that ER stress is the primary cause of erythropoietic impairment due to CD44 deficiency. Flow cytometry analysis showed a significant increase in the proportion of late apoptotic cells, marked by Annexin V and 7AAD double positivity, in bone marrow erythroid cells of CD44KO mice. Electron microscopy revealed a notable increase in damaged mitochondria and autophagosomes in erythroblats of CD44KO mice. Furthermore, the expression of autophagy-related proteins FIP200, STX17, and LC3 were significantly upregulated, consistent with the results of single-cell transcriptomic analysis. Interestingly, autophagy inhibition by chloroquine significantly improved bone marrow erythropoiesis in CD44KO mice, indicating that autophagy activation is a cause of the erythropoietic abnormalities in CD44KO mice, rather than a feedback protective mechanism following ER stress.

Recent studies have shown that CD44 is involved in limiting oxidative stress in tumor cells. To investigate the mechanisms by which CD44 deficiency affects ER stress, we analyzed oxidative stress in bone marrow erythroblasts from the CD44KO mice. DCFH-DA probe assays revealed significantly elevated intracellular reactive oxygen species (ROS) levels. Additionally, the expression of antioxidant enzymes GPX1 and GPX4 was markedly reduced at both the transcriptional and protein levels in CD44KO cells, while lipid peroxidation levels were significantly increased. Correspondingly, the expression of the oxidative stress-related protein HO-1 was significantly upregulated, whereas NRF2 expression was notably decreased, indicating that CD44 deficiency impairs the antioxidant capacity of erythroid cells and elevates oxidative stress levels. Further research revealed that CD44 deficiency downregulated the PI3K/AKT signaling pathway and activated autophagy, which suppressed the expression of the autophagy adaptor protein p62, collectively leading to the reduction of NRF2 in erythroid cells and causing oxidative damage. Finally, we induced oxidative stress in primary fetal liver-derived erythroid cells using hydrogen peroxide, and observed dose-dependent activation of the PERK-mediated ER stress pathway and inhibition of erythroid differentiation in vitro. CD44KO fetal liver-derived erythroid cells exhibited greater sensitivity to oxidative stress, as evidenced by significantly higher ROS levels and enhanced activation of the PERK pathway under the same concentration of hydrogen peroxide compared to wild-type cells. Unlike wild-type cells, CD44KO cells did not show the same induction of NRF2 protein expression under hydrogen peroxide treatment. These findings demonstrate that in erythroid cells, CD44 deficiency causes suppressed PI3K/AKT signaling pathway and p62 expression, which together lead to reduced NRF2 expression, increased oxidative stress, and subsequent ER stress, ultimately resulting in impaired erythroid differentiation. In addition, we observed increased deposition of the CD44 ligand hyaluronic acid in the bone marrow of CD44KO mice, as well as downregulated erythroid expression of BTAF1, a member of the SWI2/SNF2 family. Both of which may represent potential compensatory mechanisms in CD44-deficient erythroid cells. The present study is the first to reveal the CD44 deficiency-induced dyserythropoiesis and the underlying molecular mechanisms, providing new insights into the pathogenesis of anemia and strategies for optimizing erythropoiesis in vitro.