Pitha-Rowe Ian, Hassel Bret A, Dmitrovsky Ethan
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 2004 Apr 30;279(18):18178-87. doi: 10.1074/jbc.M309259200. Epub 2004 Feb 18.
Acute promyelocytic leukemia (APL) cases expressing the t(15,17) product, promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARalpha), have clinical remissions through leukemic cell differentiation after all-trans-retinoic acid (RA) treatment. This differentiation therapy propelled interest in uncovering molecular mechanisms for RA-dependent APL differentiation. We previously identified the ubiquitin-activating enzyme-E1-like protein (UBE1L) as an RA-regulated target gene in APL that triggers PML/RARalpha degradation and apoptosis. This study reports that conjugation of the ubiquitin-like species, interferon-stimulated gene, 15-kDa protein (ISG15), also occurs during RA-induced APL differentiation. Knock-down of UBE1L expression inhibited this conjugation. RA treatment of APL and other RA-responsive leukemic cells induced expression of UBE1L and ISG15 as well as intracellular ISG15 conjugates. Notably, ISG15 conjugation did not occur in RA-resistant NB4-R1 APL cells. Induction of UBE1L and ISG15 along with ISG15 conjugation in RA-sensitive NB4-S1 APL cells were detected following treatment with specific retinoids and type I interferon (IFN). UBE1L and ISG15 mRNAs were co-expressed in normal human tissues that were examined. In contrast, UBE1L mRNA expression was markedly repressed in several cancer cell lines. A physical association was found between UBE1L and ISG15 in vivo. This required the conserved diglycine motif in the carboxyl terminus of ISG15. Targeting UBE1L expression with small inhibitory RNA or small hairpin RNA inhibited IFN and RA-induced ISG15 conjugation. Formation of ISG15 conjugates through induction of an activating enzyme represents a novel pharmacologic mechanism for regulation of this ubiquitin-related species. Taken together, the observed rela tionship between expression of UBE1L and ISG15, their physical association and coordinate regulation, and induced ISG15 conjugation during leukemic cell differentiation implicate an important role for these proteins in retinoid response.
表达t(15;17)产物早幼粒细胞白血病(PML)/维甲酸受体α(RARα)的急性早幼粒细胞白血病(APL)病例，在全反式维甲酸(RA)治疗后通过白血病细胞分化实现临床缓解。这种分化疗法激发了人们对揭示RA依赖性APL分化分子机制的兴趣。我们之前鉴定出泛素激活酶E1样蛋白(UBE1L)是APL中一种受RA调控的靶基因，它能触发PML/RARα降解和凋亡。本研究报道，在RA诱导的APL分化过程中，也会发生类泛素物质干扰素刺激基因15 kDa蛋白(ISG15)的缀合。敲低UBE1L表达会抑制这种缀合。RA处理APL和其他对RA有反应的白血病细胞会诱导UBE1L和ISG15的表达以及细胞内ISG15缀合物的形成。值得注意的是，在对RA耐药的NB4-R1 APL细胞中未发生ISG15缀合。在用特定类维生素A和I型干扰素(IFN)处理后，在对RA敏感的NB4-S1 APL细胞中检测到UBE1L和ISG15的诱导以及ISG15缀合。在被检测的正常人体组织中，UBE1L和ISG15 mRNA共同表达。相比之下，UBE1L mRNA表达在几种癌细胞系中明显受到抑制。在体内发现UBE1L和ISG15之间存在物理关联。这需要ISG15羧基末端保守的双甘氨酸基序。用小干扰RNA或小发夹RNA靶向UBE1L表达可抑制IFN和RA诱导的ISG15缀合。通过诱导激活酶形成ISG15缀合物代表了一种调控这种泛素相关物质的新药理学机制。综上所述，观察到的UBE1L和ISG15表达之间的关系、它们的物理关联和协同调控，以及白血病细胞分化过程中诱导的ISG15缀合，暗示了这些蛋白在类维生素A反应中起重要作用。
