Study On Regulation Of Hematopoietic Stem Cell Function By Cytochrome P450 Oxidoreductase (CPR) And Other Oxidoreductases

Background:Hematopoietic stem cells (HSCs) can self-renew, proliferate and differentiate. However the underlying regulatory mechanisms for HSCs remain to be further defined. Intracellular and extracellular redox metabolic microenvironment, an important component of the niche, plays essential roles in the regulation of HSCs. Cytochrome p450reductase (CPR), as the obligate electron donor to all known cytochrome P450(CYPs), may contribute to the maintenance of HSCs via both intrinsic and extrinsic mechanisms.Objective:This study is to evaluate the effects of CPR low expression on HSCs function using reverse CPR low (RCL) transgenic mouse model, which has global low CPR expression.Methods:Different hematopoietic cell populations including LT-HSC, ST-HSC, MPP, CMP, MEP, GMP, CLP, T lymphocyte, B lymphocyte, Myeliod cell and Granulocyte in BM and PB from WT and RCL mice were analyzed by flow cytometry. HSC repopulation capacity and differentiation abilities were assessed by bone marrow competitive transplantation and enriched HSC (LKS+) transplantation. The effects of CPR low microenvironment on hematopoiesis were examined by transplantation of normal bone marrow cells into RCL recipients. ROS levels, cell cycle status and apoptosis status was quantified by DCF-DA mean fluorescence intensity, Ki67protein and Annexin-V with flow cytometry respectively.Results:We found that the number of LT-HSC and ST-HSC was reduced while the number of CMP, GMP and MEP was increased in RCL mice. Competitive transplantation assay demonstrated that CPR low expression HSCs had enhanced repopulation capacity. Interestedly, the differention potential of the HSCs from RCL mice was not affected and the reactive oxygen species (ROS) levels in bone marrow (BM), HPCs and HSCs of RCL mice remained similar to that of WT. In addition, lymphoid differentiation ratio was reduced while myeloid differentiation ratio was increased without overt impact on overall hematopoietic repopulation in RCL mice. Conclusions:Our studies have demonstrated that low expression of CPR enhances repopulation efficiency of HSC, while expression microenvironment of low CPR favors the differentiation of myeloid over lymphoid lineages. Background:Hematopoietic stem cell (HSC) is the source of all blood cells. It can differentiate into various hematopoietic cells. While physiological reactive oxygen species (ROS) play an important role in regulating functions of HSC, excessive ROS is harmful to HSC. Oxidative reductases and antioxidants can eliminate cellular ROS to maintain ROS homeostasis and thus avoid excessive ROS damages. There are several types of oxidative reductases in cells, including Catalase、MnSOO (Manganese-superoxi-de dismutase)、GPX1(Glutathione peroxidase1)、Txrndl (thioredoxin reductase1)、Nqol [NAD(P)H dehydrogenenase quinone1]、 Hmox1(heme oxygenase1) and GSR (glutathione reductase). The Nrf2/Keap1/Cul3Complex involves in regulating of all above oxidative reductases. However, the functional roles of various oxidative reductases in hematopoietic cells and the roles of Nrf2in regulating to oxidative reductases in HSC remain unclear.Objectives:This study was to investigate the expression patterns of these oxidative reductases in murine hematopoietic cells, including LT-HSC, ST-HSC, MPP, CMP, MEP, GMP, CLP, T, B and M. Another goal of this study was to explore the role of Nrf2in modulating oxidative reductases in HSC.Methods:The expression of various oxidative reductases in hematopoietic cells that were sorted out via flow cytometry was investigated by qRT-PCR(semi-quantitative real-time PCR, qRT-PCR). c-Kit cells and LKS+(Lin-c-Kit+Sca-1+) cells were treated by small molecule sulforaphane (Nrf2activator) with various doses and at different time points. Genes regulated by Nrf2in these treated c-Kit-cells and LKS+cells were analyzed by qRT-PCR.Results:The expression levels of oxidative reductases (Catalase、MnSOD、GPX1、Nrf2、 Keapl、Cul3) in hematopoietic cells were relatively cell-type specific. However, there were no obvious correlation between the expression levels and differentiation stages. The expression of Nqol in LT-HSC was higher than in other cell populations. The expression of Txrndl increased in ST-HSC、MPP、CMP、GMP and myeloid cells, while reduced in T lymphocytes and B lymphocytes.The expression of Hmox1increased in CLP and myeloid cells, while reduced in ST-HSC、MPP、CMP、 GMP, MEP,T lymphocytes and B lymphocytes. The expression of GSR increased in ST-HSC、MPP、GMP and myeloid cells, while reduced in MEP and lymphocytes. The expression of Nrf2targeted genes, including Nqo1、Hmox1、Txrndl, can be induced in c-Kit-cells and LKS+cells, when treated with sulforaphane ex vivo. However, the doses and duration of sulforaphane treatment needed for higher expression of the Nrf2-targeted genes in c-Kit-cells and LKS+cells were different, in other words the LKS+cells needed higher dose and longer duration than c-Kit" cells. Interestingly, the expression of Keapl in all LKS+cell groups treated with sulforaphane, including the control was increased, when comparing with all c-Kit" cell groups.Conclusions:The expression levels of oxidative reductases (Catalase, MnSOD, GPX1, Nrf2, Keap1, Cu13, Nqo1, Hmox1, Txrnd1and GSR) in hematopoietic cells were relatively cell-type specific. This suggested that different reductases might play distinct roles in various hematopoietic cell populations. More importantly, the expression level of Nqo1in LT-HSC population was significantly increased than in other cell populations, thereby suggesting its unique role in HSCs. The expression of Nrf2-targeted genes can be induced in c-Kit" cells and LKS+cells when treated with sulforaphane ex vivo. This suggests that normal HSC function may be protected by sulforaphane under some conditions such as stress.