CYB5R3 Controls Sex-Specific Stress Erythropoiesis via Heme-Biosynthesis

Cytochrome b5 reductase 3 (CYB5R3) or met-hemoglobin reductase is an oxidoreductase that maintains hemoprotein and cellular redox balance, yet its contribution to erythropoiesis under stress conditions remains unclear. Motivated by prior observations that the hypomorphic CYB5R3 T117S blunts hydroxyurea-induced fetal hemoglobin responses in patients with sickle cell disease, we tested whether CYB5R3 contributes to the regulation of erythropoiesis. Hematopoietic lineage-specific CYB5R3 knockout mice exhibited markedly impaired erythropoietic induction in response to chronic hypoxia compared to controls, with males showing a more pronounced deficit, and splenectomy further exacerbating this impairment. Genetic deletion of CYB5R3 in human CD34 progenitors reduced globin expression and disrupted terminal erythroid differentiation. Meanwhile, CYB5R3 knockdown in K562 cells produced a heme-deficient state whereby only exogenous heme but not hydroxyurea, iron, or upstream precursors restored globin synthesis. Transcriptomic profiling revealed coordinated downregulation of erythroid transcription factors and multiple enzymes in the heme biosynthetic pathway, which was reversed with heme treatment. Together, these results reveal an unexpected function for CYB5R3 beyond met-hemoglobin reduction, positioning it as a central metabolic regulator of sex-specific stress erythropoiesis and unveiling a heme-restricted vulnerability that may augment disease severity in anemia, hemoglobinopathies, and individuals carrying CYB5R3 loss-of-function variants. Key pointsO_LICYB5R3 is required for effective stress erythropoietic induction, with a more pronounced impact in males. C_LIO_LIErythroid-specific CYB5R3 deficiency creates a heme-limited state, impairing erythroblast differentiation and maturation. C_LI