Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia
Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia
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DOI：
10.1016/j.bbrc.2014.10.050
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发表时间：
2014-11-07
影响因子：
3.1
通讯作者：
Harigae, Hideo
中科院分区：
文献类型：
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作者：
Fujiwara, Tohru;Okamoto, Koji;Harigae, Hideo
先天性铁粒幼细胞性贫血（CSA）是一种以小细胞性贫血和骨髓铁粒幼细胞为特征的遗传性疾病。CSA最常见的形式是由于X连锁基因5-氨基乙酰丙酸合酶2（ALAS 2）的突变。ALAS 2是一种线粒体酶，它利用甘氨酸和琥珀酰辅酶A形成5-氨基乙酰丙酸（ALA），这是血红素合成的关键前体。因此，ALA补充可能是一种有效的治疗策略，以恢复由ALAS 2缺陷引起的CSA的血红素合成。在一项临床前研究中，我们检测了ALA在人红系细胞中的作用，包括10562细胞和人诱导多能干细胞衍生的红系祖细胞（HiDEP）。ALA处理导致血红素在1 β 562细胞系中显著的剂量依赖性积累。同时，如使用联苯胺染色评估的，处理实质上诱导红系分化。定量逆转录聚合酶链反应（RT-PCR）分析证实，在1562个细胞中，血红素调节基因，如珠蛋白基因[血红蛋白α（HBA）和血红蛋白γ（HBG）]和血红素加氧酶I（HMOXI）基因显著上调。接下来，为了研究ALA转运到红系细胞中的机制，对先前鉴定的ALA转运蛋白（包括溶质载体家族15）进行定量RT-PCR分析（寡肽转运蛋白），成员（SLC 15 A）1，SLC 15 A2，溶质载体家族36（质子/氨基酸同向转运体），成员（SLC 36 A1）和溶质载体家族6（神经递质转运体），成员13（SLC 6A 13）。我们的分析表明SLC 36 A1在红系细胞中大量表达。因此，将γ-氨基丁酸（GABA）加入到1（562）细胞中以竞争性地抑制SLC 36 A1介导的转运。GABA处理显著阻碍ALA介导的血红蛋白化细胞数量的增加以及HBG、HBA和HMOXI的诱导。最后，在HiDEP细胞中小干扰RNA介导的ALAS 2敲低显著降低了HBA、HBG和HMOXI的表达，并且这些表达水平被ALA治疗挽救。总之，ALA似乎主要通过SLC 36 A1转运到红系细胞中并用于产生血红素。ALA可能代表CSA治疗的一种新的治疗选择，特别是对于携带ALAS 2突变的病例。(C)2014 Elsevier Inc. All rights reserved.
Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including 1(562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the 1(562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase I (HMOXI) gene, in 1(562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly expressed in erythroid cells. Thus, gamma-aminobutyric acid (GABA) was added to 1(562 cells to competitively inhibit SLC36A1-mediated transport. GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells as well as the induction of HBG, HBA, and HMOXI. Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA, HBG, and HMOXI, and these expression levels were rescued with ALA treatment. In summary, ALA appears to be transported into erythroid cells mainly by SLC36A1 and is utilized to generate heme. ALA may represent a novel therapeutic option for CSA treatment, particularly for cases harboring ALAS2 mutations. (C) 2014 Elsevier Inc. All rights reserved.