Regulation And Mechanism Of Srebf1c On The Fate Of Bone Marrow Hematopoietic Stem Cells Under Steady-state And Irradiation Conditions

While promoting social development,nuclear technology greatly increases the risk of people being exposed to ionizing radiation（IR）.Since the middle of the 20th century,there have been more than 400 radiological accidents,resulting in enormous human casualties.Bone marrow（BM）is one of the most sensitive tissues to IR and hematopoietic cells will be significantly damaged after exposure to irradiation more than 1.0 Gy.Hematopoietic stem cells（HSCs）are the foundation of hematopoietic recovery after irradiation,and hematopoietic stem cell transplantation is currently an effective measure for the treatment of severe and above radiation injury.Therefore,a growing number of researchers have focused on how to regulate the fate of HSCs.HSCs can continuously produce blood cells to meet the individual’s needs throughout life,which mainly depends on their self-renewal and differentiation properties.The majority of HSCs are quiescent and metabolically inactive in the steady state,which can preserve their self-renewal ability.At this time,HSCs mainly use glycolysis to generate energy due to the hypoxic BM microenvironment.Following stress conditions,such as irradiation,HSCs will switch to mitochondrial metabolism to meet the energy demands,driving HSCs into the cell cycle to repopulate hematopoiesis.However,hyperactivated mitochondrial metabolism can decrease the quiescence of HSCs,which in turn impairs the long-term hematopoietic reconstitution ability.In addition,some studies have reported that lipid metabolism and amino acid metabolism,such as glutamine and branched-chain amino acids（BCAA）,also play an important role in the modulation of HSC self-renewal and differentiation.Thus,the pattern of energy metabolism largely determines HSC fate.Sterol regulatory element binding factor-1c（Srebf1c）is a member of the highly conserved basic helix-loop-helix-leucine zipper family containing Srebf1a,Srebf1c and Srebf2 that are involved in important metabolic processes and possess incomplete overlapping functions.Previous studies have mainly reported the role of Srebf1c in regulating the energy metabolism of liver and adipocyte,but few have focused on its effects on the hematopoietic system.A recent study reported that Srebf1c can participate in glucose metabolism to regulate the response of NK cells to cytokines,suggesting that Srebf1c may be also a metabolic regulator in hematopoietic cells.Our previous sequencing showed that energy metabolism of HSCs was significantly changed upon irradiation,and the expression of Srebf1c was higher in normal HSCs but down-regulated after irradiation.Combined with the effect of Srebf1c on metabolism in other cell types,we speculate that Srebf1c may be participate in the regulation of HSC fate by affecting energy metabolism.In this study,we explored the regulation and underlying mechanism of Srebf1c on the fate of HSCs under steady-state and irradiation conditions using various techniques including real-time quantitative PCR,flow cytometry,BM transplantation（BMT）,whole transcriptome sequencing,transmission electron microscopy（TEM）,Seahorse analysis,immunofluorescence,Western blot,Ch IP-PCR and so on.The main results and conclusions of our study are as follows:1 Srebf1c is an important factor in maintaining HSC homeostasis.1.1 We found that Srebf1c was highly expressed in HSC relative to mature cells in the BM by real-time quantitative PCR and flow cytometry,suggesting that Srebf1c may play an important role in HSC biology.1.2 Flow cytometry analysis revealed that Srebf1c knockout significantly increased the proportion and number of HSCs,and changed the number of megakaryocyte-erythroid progenitor（MEP）and common myeloid progenitor（CMP）,but had no substantial effect on mature cells,in the BM.These results reveal that Srebf1c has an indispensable role in the maintenance of hematopoietic stem progenitor cell（HSPC）pool,but may be dispensable for mature cells.1.3 Further analysis showed that Srebf1c deficiency reduced the quiescence,increased the proliferation and accelerated apoptosis of HSCs,implying that Srebf1c may be involved in the maintenance of HSC quiescence and low apoptosis.2 Srebf1c can maintain HSC survival and function under stress conditions.2.1 We treated mice with stress stimuli and found that Srebf1c^-/-mice displayed a reduced survival and a delayed HSC and peripheral mature cell recovery under irradiation and5-fluorocrail（5-FU）conditions.Meanwhile,Srebf1c knockout aggravated DNA damage and apoptosis of HSCs upon irradiation.These results may be attributed to Srebf1c deletion-induced HSC proliferation.2.2 Competitive BM transplantation（BMT）and In vitro colonies formation analysis demonstrated that the long-term self-renewal capacity of HSCs was defective in the absence of Srebf1c.2.3 We largely ruled out the possibility that the functional defect of Srebf1c^-/-HSCs was caused by extrinsic factors,indicating that Srebf1c mainly maintains the long-term self-renewal of HSCs in an intrinsic manner.3 Srebf1c can regulate the fate of HSCs through the TSC1-m TORC1-mitochondrial signaling axis.3.1 RNA-sequencing showed that proliferation-related signatures and aerobic metabolic pathways were up-regulated,while quiescence-related signatures were down-regulated in Srebf1c^-/-HSCs.3.2 Flow cytometry,TEM and real-time quantitative PCR analysis revealed that the number,membrane potential and related genes expression of mitochondria were increased in HSCs after Srebf1c deletion.Meanwhile,we found that loss of Srebf1c enhanced mitochondrial metabolism and inhibited anaerobic glycolysis in HSCs,accompanied by elevating ATP and reactive oxygen species（ROS）levels.These results suggest that Srebf1c can maintain anaerobic glycolysis but limit mitochondrial metabolism in HSCs.3.3 Gene set enrichment analysis（GSEA）and flow cytometry analysis showed that Srebf1c ablation increased m TORC1 signaling pathway in HSCs,implying that Srebf1c may regulate HSC homeostasis via the limitation of m TORC1 activity.3.4 Srebf1c^-/-HSCs displayed a comparable expression of p-Akt but a decreased expression of TSC1 with WT HSCs.In addition,Ch IP-PCR analysis confirmed that Srebf1c can directly bind to the promoter region of TSC1.These results reveal that the inhibition of m TORC1 activity by Srebf1c in HSCs is the result of directly transcriptional regulation of TSC1 expression but not dependent on Akt.3.5 Treatment with rapamycin can significantly inhibit the activation of m TORC1 and mitochondria and it can reverse the increased number and abnormal proliferation of Srebf1c^-/-HSCs.More importantly,rapamycin treatment can improve the impaired function of HSCs after Srebf1c deletion.These data imply that Srebf1c can inhibit the hyperactivation of m TORC1 and its downstream mitochondrial metabolism,thereby maintain the quiescence and self-renewal capacity of HSCs.3.6 Antioxidant N-acetyl-L-cysteine（NAC）application can reduce the levels of ROS and largely rescue the phenotypic and functional defects of Srebf1c^-/-HSCs,indicating that abnormal mitochondrial activation-induced unbalanced homeostasis of Srebf1c^-/-HSCs is mainly dependent on ROS.In conclusion,our study firstly demonstrates the regulatory role of Srebf1c on HSC fate under steady-state and irradiation conditions,and elucidates its underlying molecular mechanisms.These findings expand the regulatory network of energy metabolism on HSCs and provide new ideas for the treatment of hematopoietic injury after irradiation.