title: Effect of 5-aminolevulinic acid on erythropoiesis: a preclinical in vitro characterization for the treatment of congenital sideroblastic anemia.
abstract: 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 K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 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 1 (HMOX1) gene, in K562 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 K562 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 HMOX1. Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA, HBG, and HMOX1, 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.
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan; Department of Molecular Hematology/Oncology, Tohoku University Graduate School, Sendai, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Fujiwara
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Okamoto
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan.
authors.forename: Ryoyu
authors.initials: R
authors.lastname: Niikuni
authors.affiliation: SBI Pharmaceuticals Co., Ltd., Tokyo, Japan.
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Okitsu
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Fukuhara
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Onishi
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan; Clinical Research, Innovation and Education Center, Tohoku University Hospital, Sendai, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ishizawa
authors.affiliation: Department of Hematopathology, Tohoku University Graduate School, Sendai, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Ichinohasama
authors.affiliation: Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: SBI Pharmaceuticals Co., Ltd., Tokyo, Japan.
authors.forename: Motowo
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: SBI Pharmaceuticals Co., Ltd., Tokyo, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai, Japan; Department of Molecular Hematology/Oncology, Tohoku University Graduate School, Sendai, Japan. Electronic address: harigae@med.tohoku.ac.jp.
authors.forename: Hideo
authors.initials: H
authors.lastname: Harigae
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism; D000622:Aminolevulinic Acid / Q000494:pharmacology*; D000756:Anemia, Sideroblastic / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D004353:Drug Evaluation, Preclinical; D004900:Erythroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004920:Erythropoiesis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D055785:Gene Knockdown Techniques; D040181:Genetic Diseases, X-Linked / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000096:biosynthesis; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D006441:Hemoglobin A / Q000235:genetics / Q000378:metabolism; D006455:Hemoglobins, Abnormal / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020014:K562 Cells; D051379:Mice; D027981:Symporters / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D026905:Amino Acid Transport Systems; D006455:Hemoglobins, Abnormal; C475905:SLC36A1 protein, human; D027981:Symporters; D006418:Heme; D005680:gamma-Aminobutyric Acid; D000622:Aminolevulinic Acid; D006441:Hemoglobin A; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D000624:5-Aminolevulinate Synthetase; C480079:ALAS2 protein, human
keywords: 5-Aminolevulinic acid; 5-Aminolevulinic acid synthase 2; Congenital sideroblastic anemia; Erythroid cells; Heme