Experimental Study Of SRC-3on Regulative And Protective Effect And Its Mechanism In Radiation-induced Hematopoietic Injury Of Mice

Background and Objectives：It is well known that hematopoietic system is a critical organ of radiation exposure.After a lethal dose of total body irradiation （TBI）, hematopoietic cells in bone marrow （BM）underwent apoptosis or radiation-induced mitotic death. Meanwhile, cell cycle andproliferation in hematopoietic cells were delayed. Furthermore, the bone marrow stromalcells （BMSCs） were decreased and the ability of production cytokines was severely reducedafter irradiation. As a result, radiation damages on both hematopoietic cells andnon-hematopoietic stromal cells are the critical factors for treatment. So it is would be anew treatment strategy that find a suitable curing target involved in multiple ways couldexert regulative effects on both hematopoietic cell and stromal cells.Steroid receptor coactivator-3（SRC-3）, is a coactivator of nuclear receptors in SRCfamily. SRC-3can act as a coactivator and interact with nuclear receptors and othertranscription factors to enhance their effects on target gene transcription. SRC-3plays animportant role in physiological and pathological functions involved in cell proliferation,anti-apoptosis, oncogenesis, cancer development and so on. Recently, it has been reportedthat over-expression of SRC-3in malignant blood cells could promote cell survival throughinhibiting apoptosis. Although the mechanism of how SRC-3involves in hematopoieticregulation is still unclear, we propose that SRC-3could affect cell survival and apoptosis ofhematopoietic system in presence of an extrinsic stress. To validate the role of SRC-3mightplay in radiation-induced hematopoietic injury and elucidate the mechanism, we use theSRC-3^-/-mice and wild type （WT） as animal model to carry out the next experiments. Methods：The SRC-3knockout mice were introduced from Baylor College of Medicine （Huston,USA） to our laboratory and colonized. On the bases of successful reproduction andidentification, we obtained a number of SRC-3^-/-mice and SRC-3+/+mice suitable for ourexperiments. After dividing the two different gene type mice into two groups, SRC-3^-/-miceand WT mice were subjected to6.0Gy and4.5Gy total body irradiation （TBI） by60Coγ-radiation gamma ray.To validate the role of SRC-3might play in radiation-induced hematopoietic injuryand elucidate the mechanism, we carried out two parts of experiments in vivo and vitro. Inthe first part of experiments, the role of SRC-3in radiation-induced hematopoietic injurywas studied in vivo. We tested many situations in mice after TBI, including whole bodyeffect （general conditions, survival rates, mice weights）, hematopoietic damages onperipheral blood, bone marrow, spleen and thymus and changed levels of IGF-1，IL-3，IL-6,TPO in mice serum. In another part of experiments, the regulative and protectivemechanisms of SRC-3in radiation-induced hematopoietic injury were studied as following:1) We studied the bone marrow nucleated cells （BMNCs） from both SRC-3^-/-mice and WTmice on normal and post-radiation conditions respectively. We tested roles of SRC-3on cellproliferation, cell cycle and apoptosis in mice BMNCs, studied the regulative effects ofSRC-3on expression of cyclinA, cyclinE, cyclinD1, CDK2, CDK4, p21, p53and p-AKT.2)We studied BMSCs cultured from both SRC-3^-/-mice and WT mice on normal andpost-radiation conditions respectively. We tested roles of SRC-3on ability of cellproliferation, counts of colony-forming unit of fibroblast （CFU-F）, expression of p-AKTprotein and levels of VCAM-1, IGF-1, IL-3, IL-6and TPO secreted from BMSCssupernatant.Main results：1. A number of off-springs mice of three gene type as SRC-3+/+, SRC-3+/-and SRC-3^-/-mice were reproduction from SRC-3+/-parents. We used PCR and Western blot tocharacterize the off-springs on both gene and protein level. After identification, SRC-3^-/-mice and SRC-3+/+mice were divided into groups for following experiments.The SRC-3gene was knockout and SRC-3protein was not detected in bone marrow, spleenand thymus of SRC-3^-/-mice. 2. SRC-3^-/-mice has different characters from WT mice, such as light weight, smallsize, growth retardation and female sex immaturity. Under normal condition, the weight andIGF-1serum levels were significantly lower in SRC-3^-/-mice than WT mice （P<0.05）.Although the counts of WBC, RBC and PLT in peripheral blood and of bone marrownucleated cells in SRC-3^-/-mice were lower and the index of spleen and thymus were higherthan that in WT mice, but there were no statistically differences between two types of mice.3. After a lethal dose of6Gy TBI, SRC-3^-/-mice suffered a severer radiation-inducedhematopoietic injury in whole body, displaying worse situations, lower survival rates andless mice weights compared with WT mice （P<0.05）. It suggested that SRC-3^-/-mice hassignificantly weaker ability to radiation resistance.4. After a sublethal dose of4.5Gy TBI, the counts of WBC, RBC and PLT inperipheral blood, numbers of bone marrow nucleated cells and CFUs were statisticallylower in SRC-3^-/-mice compared with WT mice （P<0.05）. Noticeably, the effects of SRC-3exerted on megakaryocytic lineage were very obvious. It suggested that the hematopoieticcells, especially megakaryocytes in peripheral blood and bone marrow of SRC-3^-/-micesuffered severer radiation injury.5. After a sublethal dose of4.5Gy TBI, the index of spleen and thymus were higherthan that in WT mice, but there were no statistically differences between two types of mice.It suggested that lymphoid tissue has different radiation reaction from bone marrow inSRC-3^-/-mice.6. At all of the test time points after TBI, serum level of IGF-1was statistically lowerin SRC-3^-/-mice than that in WT mice （P<0.05）. On7th day post-irradiation, serum level ofIL-3and IL-6was statistically lower in SRC-3^-/-mice than that in WT mice （P<0.05）. Butthere were no statistically differences of TPO serum level between two types of mice.7. Under normal conditions, the apoptosis rate and Sca-1+cell number of BMNCs intwo types of mice were close. After radiation, the apoptosis rate and Sca-1+cell number ofBMNCs in SRC-3^-/-mice were statistically lower than that in WT mice （P<0.05）.Meanwhile, apoptosis protein of p53and Bax were significantly increased andanti-apoptosis protein NF-κB （p65） and Bcl-2significantly decreased in SRC-3^-/-mice. Itsuggested that BMNCs of SRC-3^-/-mice has significantly higher level of apoptosis rate andapoptosis protein expression after radiation. 8. Under normal conditions, the cell cycle and proliferation index of BMNCs in twotypes of mice were close. After radiation, the G0/G1phase was increased and S phaseblocked and the proliferation index of BMNCs in SRC-3^-/-mice were statistically lowerthan that in WT mice （P<0.05）. Meanwhile, cell cycle regulator such as CyclinD1, CyclinE,CyclinA,CDK2,CDK4, p53and p21were differently expressed in BMNCs of SRC-3^-/-micecompared with WT mice （P<0.05）. To detect the AKT and ER proliferation signalingpathways which SRC-3are both involved in, we found that p-AKT protein in AKTsignaling pathway in BMNCs of SRC-3^-/-mice was statistically lower than that in WT mice（P<0.05）; while ER protein has no statistically difference between two types of mice.9. Under normal conditions, the proliferation ability of BMSCs and counts of CFU-Fwas statistically lower in SRC-3^-/-mice than that in WT mice （P<0.05） and the differenceswere amplified after radiation （P<0.01）. Moreover, p-AKT protein in BMSCs of both miceafter radiation was tested to indicate the activation of AKT proliferation signaling pathway.We found that p-AKT protein in BMSCs of SRC-3^-/-mice was statistically lower than thatin WT mice after radiation （P<0.05）.10. Under normal conditions, levels of VCAM-1and IGF-1secreted from BMSCssupernatant of SRC-3^-/-mice was statistically lower than WT mice （P<0.05）. Moreover,after radiation, not only levels of VCAM-1and IGF-1, but also IL-3and IL-6from BMSCssupernatant of SRC-3^-/-mice were much lower than WT mice （P<0.05）. It suggested thatability of production cytokines of BMSCs in SRC-3^-/-mice is much lower than WT mice.Conclusion：1. Depletion of SRC-3in mice could increase the mortality after a lethal dose of TBI.It suggested that SRC-3could improve the survival rate and the ability to radiation resistanton whole body level.2. Depletion of SRC-3in mice could lead to severer radiation damages on bonemarrow hematopoietic cells. It suggested that SRC-3could exert effects on mitigatingradiation-induced hematopoietic injury and promoting the proliferation of hematopoieticcells, especially on megakaryocytes.3. SRC-3is related with maintain the serum level of hematopoietic cytokines of IGF-1,IL-3and IL-6. Depletion of SRC-3protein could depress the serum level of IGF-1and decrease the serum level of IL-3and IL-6after radiation.4. SRC-3could protect Sca-1+cell and decreased apoptosis cell after radiation. Thefurther mechanism could be decreased expression of apoptosis protein of p53and Bax andincreased expression of anti-apoptosis protein NF-κB （p65） and Bcl-2by SRC-3in miceBMNCs.5. SRC-3could promote the progress of cell cycle and cell proliferation after radiation.The further mechanism could be related with the regulative function of SRC-3in cell cycleregulator and AKT proliferation signaling pathway.6. SRC-3is necessary for normal proliferation and secreting function of mice BMSCs.Depletion of SRC-3in mice could lead to reduced proliferation ability, less CFU-F andlower levels of VCAM-1and IGF-1secreted from BMSCs supernatant. After radiation,p-AKT protein was lower depressed and levels of VCAM-1, IGF-1, IL-3and IL-6fromBMSCs supernatant were all decreased without SRC-3.