Role of CYB5R3 in Erythropoiesis-Associated Heme Biosynthesis

Background: Cytochrome b5 reductase 3 (CYB5R3) is critical for sustaining vital physiological reactions connected to oxidative equilibrium, heme reduction, cholesterol biosynthesis, lipid desaturation and drug metabolism. Hemoglobin, Coenzyme Q, soluble guanylate cyclase (sGC), Vitamin E, Vitamin C, etc., are important substrates of CYB5R3 that get activated through reduction. With numerous genetic variants spotted till date, CYB5R3 T117S stands out with ~50% loss of function and a high allele frequency in persons of African origin, an ethnic group that mostly bears the burden of sickle cell disease (SCD). In an oxidative systemic phenomenon like SCD where stress erythropoiesis is induced to constantly replace hemolyzed erythrocytes, CYB5R3 may be critical. Hydroxyurea (HU) is the FDA-approved first line therapy for SCD. We have recently discovered that CYB5R3 T117S is a modifier of HU efficacy for treating anemia through fetal hemoglobin (HbF) synthesis and erythrocyte formation in SCD patients. Since NO-cGMP signaling is known to induce erythropoiesis and HbF under stress via CYB5R3-regulated sGC, this also makes the reductase a logical actor in this pathway. Hence, we hypothesized that CYB5R3 is crucial for erythropoiesis. Methodology and result: CRISPR was utilized to create a non-targeting (NT) control and CYB5R3 knockout (KO) CD34+ hematopoietic stem cells (HSCs). Cells were collected on day 11 to check the hemoglobin transcripts using qPCR and on day 15 to check protein level using western blot. In comparison to the NT group, the expression of beta globin (HBB) and gamma globin (HBG) were significantly lower in the KO cells (n=8-11, p<0.05). This was followed by a fewer percentages of CD44lowFSClow cells in the KO cells, representing fewer mature erythrocytes and enucleated reticulocytes on day 18 (n=4, p<0.05), determined using flow cytometry. Knocking down (KD) CYB5R3 in K562 cells using lentiviral transduction depleted the transcript (40-60%) and protein levels (60-80%) of HBB and HBG significantly (n=4, p<0.05). Treatment with HU did not induce globin accumulation as expected in the KD cells neither at transcript nor protein level, although there was a 1.5-fold induction in the NT group (n=4, p<0.05). Since hemoglobin is made up of heme and globin and heme induces globin formation, the level of heme and heme intermediate protoporphyrinogen were then measured in the cells using High Performance Liquid Chromatography-Mass-spectrometry (HPLC-MS). Both the protoporphyrinogen and heme levels were reduced by approximately 40% in the KD cells (n=8, p<0.05), demonstrating heme deficiency in absence of CYB5R3. Addition of hemin, an iron (III) chloride heme, although did not rescue the transcripts, significantly rescued the globin proteins in the KD cells in comparison to the baseline (n=4, p<0.05). However, treatment with iron alone did not have any effect on globins. Supplementing the cells with 5-amino-levulinic acid (5-ALA), the key substrate of the heme biosynthesis pathway, did boost the globin proteins by 2-3 fold in comparison to the untreated KD cells, but that was not enough to rescue the proteins back to the baseline as in NT (n=3). Both RNA-seq analysis and qPCR revealed suppressed levels of key erythroid transcription factors, including GATA-binding factor 1 (GATA1) and enzymes in heme biosynthesis pathway, including 5-amino-levulinate synthase 2 (ALAS2), Porphobilinogen Deaminase (PBGD), Coproporphyrinogen Oxidase (CPOX), along with other important erythroid specific genes (n=3, p<0.05) in the KD cells, thus leading to heme deficiency which was reversed to some extent by hemin supplementation (n=3, p<0.05). Conclusion: In conclusion, it can be said that the absence of CYB5R3 causes downregulation of erythroid specific transcription factors and heme biosynthesis enzymes, hindering heme formation. Lack of heme affects globin formation and stability, thereby affecting erythrocyte maturation in the long run. Thus, these new insights about the role of CYB5R3 in erythropoietic pathway may enable better understanding of the complex mechanisms regulating erythropoiesis and lead to novel drug development strategies for erythropoietic disorders.