Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.

Abstract 1492

Poster Board I-515

Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2^null mice and found increased total bone marrow cellularity, cKit⁺Sca1⁺Lin⁻ (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34⁻KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2^wt and Nrf2^null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2^null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2^null HSC. However, Nrf2^null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H₂O₂ resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2^null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2^wt or Nrf2^null HSC. Mice receiving Nrf2^null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2^wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2^wt and Nrf2^null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2^null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2^null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2^null HSC by treating mice with exogenous G-CSF. Nrf2^wt and Nrf2^null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2^null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2^wt or Nrf2^null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling.