Hif1α and Rac1 Are Necessary For Enhanced HSPC Migration and Homing In Response To Prostaglandin E₂ Treatment

Hematopoietic stem cell (HSC) transplant is a lifesaving therapy for a number of different hematologic disorders, and takes advantage of the HSC’s ability to engraft and reconstitute the entire blood system. A critical step in the engraftment process is the ability of HSC’s to travel to or “home” to the bone marrow after transplant. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to delayed engraftment and immune reconstitution. Enhancing HSC homing is one strategy to improve HSC function and transplantation efficiency. We have previously shown that ex vivo treatment of mouse HSCs with 16-16 dimethyl Prostaglandin E₂ (dmPGE₂) increases their bone marrow homing efficiency and engraftment, resulting from upregulation of the homing receptor CXCR4. The exact mechanism behind this effect, however, has not been defined. The transcription factor Hypoxia Inducible Factor 1α (HIF1α) has been shown to transcriptionally regulate CXCR4 in hypoxia, and contains three hypoxia response elements within its promoter. Furthermore, PGE₂ has been shown to stabilize HIF1α under normoxic conditions in a number of transformed cell lines. We hypothesized that the effects of PGE₂ on enhancing HSC CXCR4 expression and homing might be mediated through stabilization of HIF1α. Here we show that pulse-treatment of mouse bone marrow cells with dmPGE₂ stabilizes HIF1α protein and increases HIF1α-dependent gene transcription in hematopoietic stem and progenitor cells (HSPCs). In addition, we show that similar treatment of HSPCs with the hypoxia mimetic dimethyloxalylglycine (DMOG) produces analogous effects to dmPGE₂ on enhanced CXCR4 expression, gene transcription and functional migration, and produces a 2-fold enhanced homing effect as a direct result of CXCR4 upregulation. Limiting-dilution competitive transplants demonstrated a significant increase in peripheral blood chimerism using DMOG-treated cells compared to vehicle, which resulted from a ∼2-fold increase in HSC frequency. Pharmacological inhibition of HIF1α stabilization in vitro with Sodium Nitroprusside (SNP) results in abrogation of dmPGE₂-induced CXCR4 upregulation and enhanced migration, confirming the requirement of HIF1α for these effects. In addition, the requirement for HIF1α in dmPGE₂-enhanced in vivo homing was confirmed using a mouse model of conditional HIF1α gene deletion. Finally, we validate that the hypoxia response element located 1.3kb upstream from the transcriptional start site within the CXCR4 promoter is required for enhanced CXCR4 expression after dmPGE₂ treatment. Unexpectedly, we also observed a significant increase in the small GTPase Rac1 after dmPGE₂ treatment. Rac1 is necessary for successful HSC engraftment, and has been shown to affect cell sensitivity to SDF-1 through colocalization with CXCR4. Rac1 is also required for HIF1 activity, suggesting that it may be involved in regulation of HIF1α and CXCR4 after dmPGE₂ treatment. Conditional knockout of Rac1, but not Rac2 in HSPCs results in lower basal levels of HIF1α protein, and loss of dmPGE₂-enhanced migration and CXCR4 expression, supporting the hypothesis that Rac1 is necessary for downstream effects on HIF1α and CXCR4. Using an ImageStream flow cytometer and Bright Detail Similarity analysis, we observed an increase in Rac1 and CXCR4 colocalization after dmPGE₂ treatment, which resulted in an approximate 10-fold enhanced HSPC sensitivity to SDF-1. Taken together, these data define a novel mechanism whereby ex vivo pulse treatment of HSPC with dmPGE₂ enhances HSPC function through alterations in cell motility and homing as a result of HIF1α and Rac1 modulation. We also define a new ex vivo pharmacologic strategy to improve HSC engraftment and repopulation, and for the first time, provide evidence that PGE₂ can enhance HSPC function through modulation of Rac1.