Reactive Oxygen Species Control Hematopoietic Stem Cell-Niche Interaction through the Regulation of N-Cadherin.

Interaction of tissue stem cells with their particular microenvironments, known as stem cell niches, is critical for maintaining the stem cell properties, including self-renewal capacity and the ability of differentiation into single or multiple lineages. We previously reported that the regulation of reactive oxygen species (ROS) is critical for the self-renewal activity of HSCs (Ito et al., Nature 2004, Ito et al., Nat Med 2006). Accumulation of ROS in HSCs induced defects in repopulating ability and in maintenance of quiescence through the activation of p38MAPK and p16^Ink4a, indicating that the oxidative stress contributes to exhaustion of the stem cell population. From these findings, we hypothesized that loss of quiescence was associated with the impaired HSC-niche interaction. Oxidative stress may affect not only intrinsic function of HSC but the interaction between HSCs and their niche. In this study, we investigated the effects of ROS in the interaction of HSC with osteoblastic niche. We have found that the side-population (SP) in c-Kit⁺Sca-1⁺ Lin⁻ (KSL) fraction was the quiescent HSCs in the osteoblastic niche and KSL-SP cells expressed N-cadherin (NCAD) (Arai et al., Cell 2004), and NCAD mediated cell adhesion induced quiescence of HSCs. Then we analyzed the role of ROS in the maintenance of NCAD-mediated cell-cell adhesion and detachment of HSCs from the niche under the myelosuppressive condition. Administration of 5-FU to mice decreases the dividing cells (non-SP fraction), but not quiescent cells (SP fraction) in BM on day 2. On day 6, HSC population was shifted from SP to non-SP fraction. This event might be associated with the cell cycle activation and detachment of HSCs from the niche. We found that 5-FU treatment transiently increased a level of intracellular ROS in HSCs and downregulated the expression of NCAD in HSCs. Administration of anti-oxidant, N-Acetyl Cysteine (NAC), in 5-FU treated mice maintained NCAD expression in HSCs, suggesting that increased ROS suppressed the expression of NCAD in HSCs. In addition, NAC treatment inhibited the transition of HSCs from SP to non-SP fraction on day 6. Moreover, 5-FU induced upregulation of G-CSFR and Flt3 expression in HSCs on day 2, while NAC treatment inhibited the expressions of growth factor receptors. These data indicated that intracellular ROS was a trigger for the detachment of HSCs from the niche, and inhibition of oxidative stress in HSCs by ant-oxidant preserved NCAD-mediated cell adhesion and blocked cell cycle activation after myelosuppression.

Furthermore, these data led us the possibility that normal quiescent HSCs maintains low oxidative stress by existing in the low oxygen environment. To confirm this hypothesis, we evaluated the redox status of fractionated hematopoietic cells (Lin⁺, Lin⁻, KSL-non SP, and KSL-SP) by hypoxic marker pimonidazole. And we confirmed that >80 % of KSL-SP cells were pimonidazole positive, suggesting that quiescent HSCs resided in the hypoxic niche. Altogether, our data suggest that regulation of oxidative stress is critical for the interaction between HSCs and BM niche. And osteoblastic niche maintains HSCs in low oxidative stress.