| PurposeHematopoietic stem cells(HSCs)exst predominantly in quiescence under physiological condition,a state enabling them to maintain lifelong hematopoietic and regenerative capacities..However,under stress conditions HSCs are activated and adapted to specific hematopoiesis for body’s requirement.So far,the molecular mechanisms of hematopoietic stem sell quiescence are not clear.The slfn family members are regulators of cell cycle.It has been reported that slfn2 mutation could lead to loss of quiescent state of various immune cell populations including T cell and myeloid cells.The aim of the present study is to determine whether slfn2 plays a role in the maintenance of hematopoietic stem cell quiescence.Methods(1)Establishment of mouse DNA damage,inflammation and tumor models: In this study,C57BL/6J mice(6-8 weeks old)were used as experimental animals.Mouse DNA damage was induced by intraperitoneal injection of 5-FU(150 mg/kg).Acute inflammation in mice was induced by intraperitoneal injection of LPS(0.5 mg/kg).The tumor models,including mouse lung cancer(LLC),breast cancer(4T1)and AOM/DSS-induced colon cancer were created according to literatures or established methods in our laboratory.(2)Isolation and preparation of mouse bone marrow cells: Mice were sacrificed at scheduled time points,and bone marrows were isolated from the tibia and fibula and single cell suspension prepared.s.(3)Mouse hematopoietic stem cell sorting: bone marrow single cell suspension was stained with a panel of fluorescent-labelled antibodies and sorted for HSCs by flow cytometry.(4)Slfn2 knockdown and overexpression: FACS-purified HSCs from normal mice(for slfn2 knockdown)or tumor-bearing mice(for slfn2 overexpression)were infected respectively with lentivirus expressing slfn2 sh RNA-Pruo-GFP or slfn2 c DNA-Pruo-GFP.(5)Hematopoietic stem cell cycle analysis: Hematopoietic stem cells were stained with 7-AAD and KI67 and analyzed by FACS.(6)Analysis of hematopoietic stem cell proliferation: HSCs were incubated in the presence of Ed U,and incorporation of Ed U was detected by FACS.(7)Statistical methods: Data were analyzed using Prism statistical software for independent sample T-test analysis,and the results were expressed as mean±standard deviation(SD).Results(1)Activation of mouse hematopoietic stem cells under different stress conditions.We examined the effect of different stress conditions on hematopoietic stem cell activation.Cell cycle analysis showed that in lung cancer,breast cancer,and colon cancer models,the proportion of hematopoietic stem cells in G0 phase isolated from tumor-bearing mice was significantly reduced,and the percentages were 79.83 ± 2.28% vs91.57 ± 0.28%,(P < 0.01,tumor-bearing mice versus normal mice,the same below),89.63± 1.68% vs 97.30 ± 0.76%(P <0.05),81.37 ± 4.59% VS 94.60 ± 1.15%(P <0.05),respectively(three independent experiments,).We observed similar changes under DNA damage,acute inflammation,and AOM/DSS-induced chronic inflammation,and the percentages of HSCs in the G0 phase of treated mice were 83.00 ± 2.02%(VS 90.43 ±1.10%,normal HSCs,the same below;P <0.05),83.00 ± 2.02%(VS 90.43 ± 1.10%,P<0.05),85.75 ± 1.75%(VS 97.43 ± 1.68%,P <0.05),respectively.These results indicate that HSCs are activated under different stress or pathological conditions such as DNA damage,inflammation,and tumors.(2)Changes of slfn2 expression in mouse hematopoietic stem cells under different stress conditions.To understand the relationship between slfn2 expression and the quiescence of hematopoietic stem cells,we examined the expression of slfn2 gene in mouse bone marrow hematopoietic stem cells using the above-mentioned experimental models.The results showed that DNA damage and acute inflammation caused more than 3 times(0.26±0.07,P<0.01))and 2 times(0.40±0.14,P<0.05)downregulation of slfn2 in HSCs,respectively.During development of lung cancer,breast cancer,and colon cancer,the expression of slfn2 in HSCs was also significantly down-regulated,and the values were 0.59±0.11(P<0.05),0.71±0.08(P<0.05),and 0.51±0.13(P<0.01)folds of normal mouse HSCs,respectively.(3)Effect of slfn2 knockdown on hematopoietic stem cell quiescence.To demonstrate a role of slfn2 in quiescent regulation of hematopoietic stem cells,we analyzed the effect of slfn2 knockdown using normal mouse HSCs.Compared with control cells,the percentage of cells in G0 phase of the slfn2 knockdowned HSCs was significantly decreased(7.66±3.5% vs 21.67±6.67%,P<0.05);meanwhile,the proliferation of HSCs was significantly increased after slfn2 knockdown(42.00±1.41% VS13.75±0.78%,P<0.01).These results indicate that down-regulation of slfn2 results in exit from quiescence and rapid proliferation in hematopoietic stem cells.(4)Effects of slfn2 overexpression on hematopoietic stem cells quiescence.Given the effect of slfn2 knockdown on the quiescence of HSCs,we further tested whether overexpression of slfn2 could reverse the activation of HSCs and stimulate them to return to normal quiescent state.For this purpose,we performed slfn2 overexpression experiments using hematopoietic stem cells isolated from tumor-bearing mice.The results showed that upon increased expression of slfn2 gene,the proportion of HSCs in G0 phase waswas significantly higher than that of blank transfected cells(39.9±4.33% vs23.43±5.16%,P<0.05).Consistent with their activation state,the proliferation of HSCs was inhibited after slfn2 overexpression(33.23±1.80% VS 44.20±1.57%,P<0.01).ConclusionHematopoietic stem cells undergo a quiescence-activated transition in response to environmental stimuli.This study revealed that DNA damage,inflammation,and tumor could lead to activation of hematopoietic stem cells,a cellular process depending on the down-regulation of slfn2 expression.Gene expression modification experiments further showed that artificially down-regulating or up-regulating the expression leveles of slfn2 gene caused,respectively,the hematopoietic stem cells exit or re-enter the quiescent state as well as changes of cell proliferation activity.Altogher,these results point to a critical role for slfn2 in the maintenance of hematopoietic stem cell quiescence.Our findings not only revealed an unrecognized molecular event involving the slfn2 control of HSCs resting state,but also provided novel clues for addressing important issues of abnormal activation and functional exhaution of HSCs under pathological conditions. |
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