E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild–type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.

中文翻译：

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无名指蛋白 138 抑制转录因子 C/EBPα 蛋白周转，导致急性髓系白血病分化停滞

E3 泛素连接酶、环指蛋白 138 (RNF138) 参与多种生物过程；然而，其在骨髓分化或肿瘤发生中的作用仍不清楚。 TNMplot 的 RNAseq 数据显示，与正常志愿者的骨髓相比，急性髓系白血病 (AML) 骨髓样本中的 RNF138 mRNA 水平高度升高。在这里，我们发现 RNF138 充当肿瘤抑制因子 CCAAT/增强子结合蛋白 (C/EBPα) 的 E3 连接酶，并促进其降解，导致 AML 中的骨髓分化停滞。野生型 RNF138 与 C/EBPα 发生物理相互作用，并促进其泛素依赖性蛋白酶体降解，而缺乏连接酶活性的突变 RNF-138 虽然与 C/EBPα 发生相互作用，但无法下调它。我们发现，RNF138 耗尽会增强从健康志愿者中分离的外周血单核细胞 (PBMC) 的内源性 C/EBPα 水平。我们的数据进一步表明，RNF138 介导的 C/EBPα 降解对其靶基因的反式激活潜力产生负面影响。此外，RNF138 过表达抑制全反式视黄酸诱导的 HL-60 细胞分化，而 RNF138 RNAi 则增强。与 RNF138 抑制 C/EBPα 蛋白周转一致，我们还观察到 RNF138 过表达抑制 β-雌二醇 (E2) 诱导的 C/EBPα 驱动的 C/EBPα 诱导型 K562-p42C/EBPα-雌激素受体细胞中的粒细胞分化。此外，我们还在从 AML 患者分离的 PBMC 中重述了这些发现，其中 RNF138 的消耗增加了骨髓分化标记物 CD11b 的表达。这些结果表明，RNF138 通过靶向 C/EBPα 进行蛋白酶体降解来抑制骨髓分化，并可能为髓系白血病中常见的 C/EBPα 表达缺失提供合理的机制。此外，靶向 RNF138 可以通过恢复 AML 中的 C/EBPα 表达来解决分化停滞问题。