Effect And Mechanism Of SRC-3 On The Regulation Of Hematopoietic Stem Cell Homeostasis And Radiation Injury Of Bone Marrow

With the extensive application of nuclear energy and nuclear-related technologies in industrial and agricultural production,medical treatment,military industry and other fields,ionizing radiation-caused damage frequently occurs.Bone marrow(BM)is extremely sensitive to irradiation,and the exposure of more than 1.0 Gy in a short time is enough to cause evident hematopoietic dysfunction.Moderate-to-high doses of ionizing radiation can even lead to a dramatic decrease in the number of hematopoietic stem cell(HSC)due to the serious imbalance of HSC homeostasis,eventually resulting in hematopoietic failure.However,the intrinsic mechanism has not been clarified,and the effective measures in treatment of this disease is still lacking.Therefore,it is of great significance to deeply explore the critical factors involved in the regulation of HSC homeostasis as well as the underlying mechanisms,which may provide important guidance on developing new strategies and drugs to treat irradiation-induced hematopoietic injury.Mature blood cells have a limited life span,which need to be continuously supplemented by HSC proliferation and differentiation.HSCs have the ability to self-renew and differentiate into all kinds of hematopoietic cells in lifetime.Based on the different survival time and self-renewal ability,HSCs can be further divided into three subsets including long-term HSC(LT-HSC),short-term HSC(ST-HSC)and multipotent progenitor(MPP).Adult HSCs are mainly located in a hypoxic BM niche and have a unique metabolic characteristic,mainly relying on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy supplement.Under various stress conditions,such as ionizing radiation,infection and chemotherapy,the metabolic pattern of HSCs changes and gradually transitions toward mitochondrial metabolism in order to meet the increased energy requirements.Under normal conditions,most of the HSCs,especially the LT-HSCs,are retained in a quiescent state,which is required to maintain their homeostasis and prevent them from ionizing radiation and cytotoxic damage.Although HSCs have been studied for more than 50 years and many critical hematopoietic regulators have found,how to precisely regulate HSC homeostasis has not been fully clarified.Thus,it is very urgent to have an in-depth understanding of the underlying mechanism in the regulation of HSC homeostasis.Steroid receptor coactivator 3(SRC-3)is a member of the SRC family consisting of three cognate proteins(SRC-1,SRC-2 and SRC-3).As a coactivator of nuclear receptor and other transcription factors,SRC-3 can recruit histone acetylase and other cofactors to regulate the transcription of target genes,therefore playing an important role in multiple biological processes,including somatic growth,sexual maturity and energy metabolism.Due to the fact that SRC-3 is highly expressed in a variety of tumor cells(such as breast cancer,prostate cancer,colon cancer and leukemia),previous studies focused more on exploring its function on tumor oncogenesis and development.However,little is known about its role in the hematopoietic system.Of note,it has been reported that SRC-3 deficiency leads to increased white blood cells and decreased platelets in the peripheral blood of mice.Moreover,our previous studies found that the number of polyploid megakaryocytes was significantly reduced in mice BM when SRC-3 was deleted and SRC-3 knockout(SRC-3^-/-)mice were highly sensitive to irradiation.These findings prompt us to speculate that the abnormity of hematopoietic system caused by SRC-3 deficiency may result from the defect in hematopoietic stem progenitor cells.At the same time,we observed that the expression of SRC-3 was significantly decreased in HSCs purified from the BM of normal C57 mice after irradiation exposure,which probably contributes to the irradiation-induced imbalance of HSC homeostasis.In addition,recent studies have shown that SRC-3 is a critical regulator of the pluripotent network of embryonic stem cells and tumor stem cells.Hence,we reasonably speculate that SRC-3 may be involved in regulating the homeostasis and function of HSCs and it may be a potential target for the treatment of the imbalance of HSC homeostasis following irradiation exposure.Here,we first detected the expression of SRC-3 in HSCs from mouse BM by quantitative polymerase chain reaction(q PCR),flow cytometry,immunofluorescence and Western blot,respectively.Then,flow cytometry was used to comprehensively analyze the proportion,number,cell cycle,proliferation,apoptosis,differentiation and mobilization of HSCs in the BM of wild-type and SRC-3^-/-mice.Further,a mouse model of acute radiation injury was established to evaluate the radioprotection effect of SRC-3 on HSCs,and the role of SRC-3 in the modulation of HSC long-term hematopoietic reconstruction ability was assessed mainly through non-competitive and competitive BM transplantation assays.Finally,the intrinsic mechanism by which SRC-3 regulates HSC homeostasis and function was deeply analyzed and discussed by using a series of experimental techniques,including microarry,flow cytometry,immunoprecipitation,Seahorse analysis,lentivirus transfection and so on.The main results and conclusions obtained from this study are as follows:1.qPCR,flow cytometry,immunofluorescence and Western blot analyses showed that SRC-3 was highly enriched in HSCs compared with lineage-restricted progenitors and differentiated cells in the BM of normal wild-type mice.This specific expression pattern suggests that SRC-3 may play a potential role in HSC biology.2.Flow cytometry analysis revealed that the percentage and number of total HSCs were significantly increased in the BM of mice after SRC-3 knockout.Among HSC subpopulations,the percentage of LT-HSC was increased and the percentage of MPP was decreased,while the percentage of ST-HSC was not significantly changed.Among hematopoietic progenitor cells,the percentage of common myeloid progenitor(CMP)was significantly reduced,but the percentages of granulocyte monocyte progenitors(GMP),megakaryocyte erythroid progenitors(MEP)and common lymphoid progenitors(CLP)were unchanged.These data indicate that SRC-3 is required to maintain a normal hematopoietic stem and progenitor cell(HSPC)pool.3.Using Ki67 and Brd U staining,we found that SRC-3 deficiency significantly reduced the quiescence and increased the proliferation of HSCs.Meantime,q PCR analysis showed that cell cycle-related genes(Cyclin E1 and Cyclin E2)were up-regulated and cell cycle inhibitors(P21 and P27)were down-regulated in HSCs after SRC-3 knockout.These illustrate that SRC-3 is involved in maintain the quiescence of HSCs in mice.4.Compared with wild-type controls,SRC-3^-/-mice displayed significantly increased proportion of HSCs in the spleen and peripheral blood,indicating that SRC-3 deficiency promotes the mobilization of HSCs from the BM to the periphery.5.By establishing the mouse model of acute radiation injury,we found that deletion of SRC-3 significantly slowed down the recovery of HSC number in the BM of mice after irradiation exposure and SRC-3 deficiency resulted in more significant increases in DNA damage and apoptosis in HSCs,which demonstrate that SRC-3 can protect HSCs from irradiation injury.In addition,loss of SRC-3 increased the sensitivity of HSCs to5-fluorouracil(5-FU)treatment.These observations may be caused by the impaired quiescence and increased proliferation of HSCs in the absence of SRC-3.6.By non-competitive,competitive,reverse BM transplantation assays and other experiments,we noticed that SRC-3 deficiency significantly impaired the long-term reconstruction ability of HSCs and SRC-3 regulated HSC homeostasis in a cell-intrinsic manner.7.Microarray analysis revealed that mitochondrial-related components were increased and oxidative phosphorylation was activated in HSCs after SRC-3 knockout.Further investigations confirmed that SRC-3 deficiency led to significantly increased mitochondria number,membrane potential and metabolic activity in HSCs.8.We observed that knockout of SRC-3 significantly elevated the levels of reactive oxygen species(ROS)in HSCs from mice BM.Of note,Mito Sox staining exhibited that the increased ROS was mainly derived from mitochondria.Importantly,treatment of antioxidant N-acetyl-l-cysteine(NAC)could partially reverse the phenotypic and functional defects in HSCs induced by SRC-3 deficiency.9.SRC-3 deficiency significantly decreased the expression of acetylation enzyme GCN5,which led to the activation of peroxisome proliferator-activated receptor-?coactivators 1?(PGC-1?)by reducing its acetylation level,therefby promoting mitochondrial metabolism.Finally,overexpression of GCN5 by lentivirus transfection significantly improved the phenotypic and functional defects in SRC-3-null HSCs.Conclusion:We show for the first time that SRC-3 is required to maintain HSC homeostasis.Meahwhile,SRC-3 is involved in protecting HSCs from ionizing radiation damage as well as maintaining HSC function.Furthermore,our data uncovers the molecular mechanism by which SRC-3 regulates HSC homeostasis and function.Thus,our findings may provide a new insight or target for the prevention and treatment of hematopoietic injury induced by ionizing radiation.