The role of hepatocyte nuclear factors in the externalization of phosphatidylserine in sickle erythrocytes Free

In sickle cell disease (SCD), the asymmetric distribution of phospholipids on red blood cells (RBCs) leads to the exposure of phosphatidylserine (PS) on the outer membrane. Externalized PS contributes to SCD pathophysiology, including vasoocclusion, hemostatic activation and hemolysis. Activation of the calcium-dependent scramblase TMEM16F facilitates PS externalization. However, the mechanism by which sickle erythrocytes acquire altered scramblase expression during erythropoiesis in SCD remains unknown. Hepatocyte nuclear factor-1 alpha (HNF1a) and -4 alpha (HNF4a) are transcription factors that engage in reciprocal interactions, thereby regulating the activation and repression of genes associated with the development and differentiation of various cell types. Deficiency of HNF1a is associated with hemolytic anemia and high intracellular calcium accumulation in RBCs, while HNF4a regulates erythropoietin, γ-globin, and iron homeostasis—key modulators of erythropoiesis. We hypothesized that interaction of HNF1a and HNF4a regulates the expression of phospholipid transporters during erythropoiesis, enabling the externalization of PS in sickle RBCs. First, we tested the expression of PS in relation to that of HNF1a and HNF4a in RBCs isolated from SCD (SS) and normal control (AA) mice. We discovered reduced expression of HNF1a and HNF4a in the SS mice compared to the AA mice (n=4; p<0.01). This reduction was associated with a significant externalization of PS in sRBC. In vitro, we used differentiated K562 cells as representative erythroid progenitor cells. Upon exposure to heme to mimic in vivo hemolytic conditions, the K562 cells exhibited increased PS externalization and induction in TMEM16F expression while simultaneously displaying reduced HNF4a expression (n=3; p<0.01). Inhibition of HNF4a with BI6015 further elevated PS externalization in K562 cells. Conversely, pharmacological induction of HNF4a with oroxylin A (50 mg/kg bw; 3x/week for 2 weeks)–a flavonoid compound that activates HNF4a–significantly reduced PS exposure in RBCs of SS mice and increased total hemoglobin, RBC counts, and hematocrit compared to their baseline prior to OA treatment (n=4; p<0.05). These findings identify HNF4a as a key regulator of PS externalization in sickle erythroid cells. Ongoing research will elucidate the definitive role of HNF4a-HNF1a interaction in SCD erythropoiesis. Additionally, the induction of HNF4a can be developed as a therapeutic strategy to prevent the externalization of PS in sRBCs and its subsequent adhesion to the endothelium, thereby alleviating hemolysis and vasoocclusion in SCD.