Effect of 5-aminolevulinic acid on erythropoiesis: a preclinical in vitro characterization for the treatment of congenital sideroblastic anemia.

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.

Regulation of the human HBA genes by KLF4 in erythroid cell lines.

KLF1/EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the HBB-like globin genes. Prompted by the observation that four KLF sites are distributed in the human alpha-globin gene (HBA) promoter, we investigated if KLFs could also act to modulate the expression of the HBA genes. Among the KLFs tested, only KLF4/GKLF bound specifically to three out of four alpha-globin KLF sites. The occupancy of the same sites by KLF4 in vivo was confirmed by chromatin immunoprecipitation assays with KLF4-specific antibodies. In luciferase reporter assays in MEL cells, high levels of the wild type HBA promoter, but not mutated promoters bearing point mutations that disrupted KLF4-DNA binding, were transactivated by over-expression of KLF4. In K562 cells, induced KLF4 expression with a Tet-off regulated cassette stimulated the expression of the endogenous HBA genes. In a complementary assay in the same cell line, knocking down KLF4 with lentiviral delivered sh-RNAs caused a parallel decrease in the transcription of the HBA genes. All experiments combined support a regulatory role of KLF4 in the control of HBA gene expression.

Molecular cloning and characterization of hemoglobin alpha and beta chains from plateau pika (Ochotona curzoniae) living at high altitude.

Hemoglobin (Hb) plays an important role in oxygen transfer from lung to tissues. Possession of a Hb with high oxygen affinity helps highland animals to adapt to high altitude, has been studied profoundly. Plateau pika (Ochotona curzoniae), a native species living at 3,000-5,000 m above sea level on Qinghai-Tibet Plateau, is a typical hypoxia and low temperature tolerant mammal. To investigate the possible mechanisms of plateau pika Hb in adaptation to high altitude, the complete cDNA and amino acid sequences of plateau pika hemoglobin alpha and beta chains have been described. Compared with human Hb, alterations in important regions can be noted: alpha111 Ala-->Asn, beta35 Tyr-->Phe, beta112 Cys-->Val, beta115 Ala-->Ser, and beta125 Pro-->Gln. Phylogenetic analysis of alpha and beta chains shows that plateau pika is closer to rabbit than to other species. This study provides essential information for elucidating the possible roles of hemoglobin in adaptation to extremely high altitude in plateau pika.

Arginine supplementation improves rotorod performance in sickle transgenic mice.

Patients with sickle cell disease (SCD) have been shown to have impaired visual-motor speed and coordination. Sensorimotor deficits in mice can be investigated by motor coordination tests that require whole body movements such as the rotorod. A sickle transgenic mouse model (S+S-Antilles) that expresses human alpha, human beta(S) and human beta(S-Antilles), is homozygous for the mouse beta(major) deletion, and has low plasma arginine was compared to control C57BL/6J mice and S+S-Antilles mice supplemented with 5% arginine on the rotorod. The rotorod consists of a 2.5 cm diameter, grooved rod turning at constant acceleration, requiring postural adjustments on the part of the mice to maintain equilibrium. C57BL mice on Purina mouse chow had an average latency to fall of S+S-Antilles mice on Purina mouse chow had an average of 127+/-56 s S+S-Antilles mice after 5% arginine supplementation had a mean latency of Arginine may improve rotorod performance in sickle transgenic mice by increasing NO synthesis thereby improving vasodilatation and blood flow with reversal of ischemia in brain and/or muscle. In conclusion, impaired rotorod performance in sickle transgenic mice presents an opportunity to apply this simple task to provide an efficient method to screen some types of therapeutic regimens for efficacy in SCD.

Phosphorus-31 nuclear magnetic resonance studies of human red blood cells.

Phosphorus-31 nuclear magnetic resonance spectroscopy is used to monitor the changes in the concentrations of 2,3-diphosphoglycerate and adenosine-5' triphosphate as a function of time and to measure the intracellular pH of normal and abnormal red blood cells in the presence of 25% oxygen, 5.6% carbon dioxide, and 69.4% nitrogen at 37 degrees C. Under these conditions, the intracellular pH values of normal AA, sickle SS, AS, SC, AC, and CC red cells are 7.24 +/- 0.07, 7.13 +/- 0.04, 7.15 +/- 0.03, 7.16 +/- 0.03, 7.24 +/- 0.05, and 7.14 +/- 0.03, respectively. The intracellular pH of SS red cells is about 0.1 pH unit more acidic than that of AA red cells. Time-dependent changes in the concentration of 2,3-diphosphoglycerate of normal human red cells show an initial lag phase, followed by a slow linear decrease. The duration of the initial lag phase decreases in the following order: AA approximately equal to AS approximately equal to AC greater than SC greater than SS approximately equal to CC red cells. The decay of 2,3-diphosphoglycerate is much faster in SS and CC red cells compared to other red cells studied. The time-dependent depletion of adenosine-5' triphosphate in these red cells is similar in nature to that of 2,3-diphosphoglycerate. The linewidths of 2-P and 3-P resonances of 2,3-diphosphoglycerate for fully oxygenated SS red cells are broader (approximately 20 Hz) than those for other red cells (approximately 10 Hz). However, the linewidths of 2-P and 3-P resonances of 2,3-diphosphoglycerate in the lysates of these red cells are narrower (approximately 4.5 Hz) than those in the intact red cells and are very similar in all types of red cells studied. The linewidths of the 31P resonances of adenosine-5' triphosphate are also similar (approximately 30-40 Hz) in all red cells studied. In addition, we have investigated the effect of carbamylation on the metabolism of 2,3-diphosphoglycerate and the intracellular pH in SS and AA red cells and have found that neither one is affected by this process. Our results provide further evidence that phosphorus-31 nuclear magnetic resonance spectroscopy offers a direct, non-invasive way to investigate the intracellular environment and the metabolism of phosphorylated metabolites in intact red blood cells.