Orai1 acts as a novel Ca²⁺ signal switch, balancing erythropoiesis through KLF1 regulation

Terminal erythropoiesis, the final stage of red blood cell maturation, is orchestrated by erythropoietin (EPO) and the master transcription factor, Kruppel-like factor 1 (KLF1). Recent studies highlight the importance of Ca²⁺ signaling in erythroid maturation; however, the underlying mechanisms remain elusive. Here we identify Orai1 as a novel EPO-responsive Ca²⁺ channel in erythroid cells, serving as a dynamic regulatory toggle that modulates KLF1 transcription and facilitates distinct phases of erythroid maturation. During the early stages, EPO-activated Orai1 suppresses KLF1 transcription through Ca²⁺-dependent NFAT2 activation and promoter binding, pausing erythroid maturation. As maturation progresses, Orai1 expression decreases, transitioning KLF1 regulation to an EPO-STAT5 pathway, thereby maintaining KLF1 expression and promoting terminal erythropoiesis. Using HUDEP-2 cells, umbilical cord blood and human pluripotent stem cell-derived CD71⁺ erythroblasts, we observed a progressive downregulation of Orai1 and reduction in intracellular Ca²⁺ levels during terminal maturation. The functional inactivation of Orai1 via R91W mutants and CRISPR-Cas9 knockout enhanced KLF1 expression, leading to increased erythroid-specific gene expression, accelerated erythroid maturation, higher levels of globin production and improved enucleation efficiency. This study unveils the EPO-Orai1-Ca²⁺-NFAT2-KLF1 axis as a critical regulatory checkpoint in erythropoiesis and highlights Orai1 downregulation as a potential strategy to enhance clinical red blood cell production by promoting erythrocyte maturation.