Exploring the Potential Usefulness of 5-Aminolevulinic Acid for X-Linked Sideroblastic Anemia

Abstract

(Background) Congenital sideroblastic anemia (CSA) is an inherited disease; it is a microcytic type of anemia characterized by bone marrow sideroblasts with excess iron deposition in the mitochondria. The most common form of CSA is XLSA (X-linked sideroblastic anemia), which is attributed to mutations in the X-linked gene ALAS2 (erythroid-specific 5-aminolevulinate synthase). ALAS2 encodes the first and rate-limiting enzyme involved in heme biosynthesis in erythroid cells, which utilizes glycine and acetyl-coenzyme A to form 5-aminolevulinic acid (ALA) and also requires pyridoxal 5′-phosphate (PLP, vitamin B6) as a cofactor. Based on the evidence that half of the XLSA cases were unresponsive to PLP (Ohba et al. Ann Hematol 2013), ALA supplementation could emerge as an alternative therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. As a preclinical study, we focused our study on the effect of ALA on human erythroid cells. Furthermore, we investigated the molecular mechanism by which ALA is transported into erythroid cells.(Method) Human K562 erythroid cells as well as human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2013) were used for the analysis. We investigated the effects of ALA (0.01, 0.1, and 0.5 mM for 72 h) on heme content, hemoglobinization, and erythroid-related gene expression. Heme content was determined fluorometrically at 400 nm (excitation) and 662 nm (emission). Small interfering RNA (siRNA)-mediated knockdown of ALAS2 was conducted using Amaxa Nucleofector™ (Amaxa Biosystems, Koln, Germany). For transcription profiling, Human Oligo chip 25K (Toray, Tokyo, Japan) was used for control and ALAS2 siRNA-treated HiDEP cells. Gamma-aminobutyric acid (GABA) (Sigma, St. Louis, MO, USA) was used at concentrations of 10 and 20 mM.(Results) First, we demonstrated that ALA treatment resulted in significant dose-dependent accumulation of heme in K562 cells. Concomitantly, the treatment substantially induces erythroid differentiation as assessed using hemoglobin (benzidine) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes such as the globin genes (HBA and HBG) and the heme oxygenase 1 (HMOX1) gene in K562 cells. To investigate the mechanism by which ALA was transported into erythroid cells, we conducted quantitative RT-PCR analysis for previously reported ALA transporters, including SLC15A1, SLC15A2, SLC36A1, and SLC6A13 (Frølund et al. Br J Pharmacol 2010; Ahlin et al. Drug Metab Dispos 2009; Moretti et al. Br J Cancer 2002). The analysis revealed that SLC36A1 was abundantly expressed in K562 and HiDEP cells. Thus, GABA was added to K562 cells to competitively inhibit SLC36A1-mediated transport (Frølund et al. Br J Pharmacol 2010). GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells.Next, siRNA-mediated knockdown of ALAS2 in HiDEP cells resulted in a significant decrease in the expression of globin genes as well as HMOX1; however, ringed sideroblasts were not observed. Microarray analysis revealed >2-fold up- and down-regulation of 38 and 68 genes caused by ALAS2 knockdown, respectively. The downregulated gene ensemble included globins (HBZ, HBG, HBE, HBD, and HBM) as well as genes involved in iron metabolism (ferritin heavy chain 1: FTH1, transferrin receptor: TFRC and glutaredoxin-1: GLRX5). Gene ontology analysis revealed significant enrichment of cellular iron ion homeostasis (p = 0.000076), cell division (p = 0.00062), DNA repair (p = 0.0006) and translation (p = 0.018), implying that heme was involved in various biological processes in erythroid cells. Interestingly, ALA treatment significantly improved the consequences of ALAS2 knockdown-mediated downregulation of HBA, HBG, and HMOX1.(Conclusion) ALA appears to enter into erythroid cells mainly by SLC36A1 and utilized to generate heme precursor. Thus,ALA may represent a novel therapeutic option for CSA, particularly for cases harboring ALAS2 mutations. DisclosuresFujiwara:Chugai Pharmaceutical, CO., LTD.: Research Funding.