Effect And Mechanism Of Estrogen On Mitigating Ionizing Radiation-induced Thrombocytopenia

With the development of science and technology,the utilization of nuclear energy and related technology becomes more and more popular in multiple fields,such as military,medicine and industry.Nevertheless,although nuclear energy makes our life much more convenient,the potential risk of ionizing radiation remarkably increases.Especially,nuclear accident and nuclear explosion will produce a large amount of patients with radiation injury.In addition,cancer patients receiving radiotherapy or bone marrow transplantation in clinic will also suffer from radiation injury.Bone marrow is a main target organ of radiation injury.Dramatic platelet reduction induced by radiation injury often further causes hemorrhage,infection and even death.Unfortuanately,at the moment,the drugs for treating thrombocytopenia are scarce in clinic and they are always toxic,expensive and less efficient.Therefore,it is of vital importance to seek a safe and effective avenue to prevent and treat ionizing radiation-induced thrombocytopenia.Platelets are produced by megakaryocytes in the bone marrow through a series of progresses involving the commited differentiation of hematopoietic stem cells into megakaryocytes,the proliferation of megakaryocyte progenitors and its differentiation into mature megakaryocytes,the release of platelets by mature megakaryocytes.The maturation of megakaryocytes is a key step.As reported,during the stage of megakaryocyte maturation,the cells firstly undergo endomitosis that involves DNA replication but not cytokinesis.Then,cells begin to synthesize a large quantity of protein and lipid necessary to create the demarcation membrane system.Subsequently,these mature megakaryocytes form proplatelets that protrude into the bone marrow sinusoids,where their terminal buds are released into the circulation.In vivo,the most important cytokine regulating thrombopoiesis is thrombopoietin(TPO).However,other research includind our own found that other cytokines also participate in the regulation of thrombopoiesis.Estrogen is the primary female sex hormone that mainly produced and secreted by the ovary in mammals and has an extensive biological effect in the body.More and more studies have reported that estrogen has distinct effects on the hematopoietic system.Interestingly,it is reported that estrogen can be synthesized by megakaryocytes themselves and a close relationship exists between estrogen and thrombopoiesis.Clinical data also suggest that gender differences is existed in platelet levels of human and this sex-based differences is related to the estrogen levels.Moreover,numerous studies reported that estrogen not only can promote megakaryocyte differentiation and proplatelet formation in vitro,but also can increase platelet numbers in vivo,while estrogen receptor(ER)antagonist tamoxifen or fulvestrant can suppress platelet production.All these suggested that estrogen signaling participates in the megakaryopoiesis and thrombopoiesis.However,the exact stage that estrogen affects and the underlying mechanisms remain to be clarified.Estrogen is a steroid hormone and estrogen signaling is primarily mediated through estrogen receptors ERa and ERβ,which are nuclear receptors.Normally,ERa and ERβ are located in the cell membrane.When binding with estrogen,they become a c t i v a t e d a n d f o r m h o m o d i m e r s o r h e t e r o d i m e r s.Then,they translocate into the nucleus,where they recruit transcription machinery and related co-factors to bind to the estrogen response element(ERE)in the promotor region of target genes.Thus,estrogen exerts biological effects through promoting or inhibiting the transcription of its target genes.Meanwhile,the distribution of ERa and ERβ displays obvious tissue specificity.Moreover,the structure and function of ERa and ERβ are distinct in many respects,such as ligand bond and recognition,receptor activation,co-activator or co-repressor recruitment and the regulated target genes of them.It has been reported that both ERa and ERβ are expressed in megakaryocytes.However,which receptor is responsible for the effect of estrogen on thrombopoiesis and the downstream target genes regulated by ERs and signal transduction pathways are poorly understood.On the other hand,estrogen is also a classical anti-radiation drug and has been demonstrated to have a distinct radiation protection effect in animal experiments.Administration of estrogen either before or after ionizing radiation can remarkably increase the survival rate.Meanwhile,giving estrogen to cancer patients before radiotherapy can attenuate the dramatic decrease of white blood cells after radiation.However,the effect of estrogen on preventing and treating ionizing radiation-induced thrombocytopenia still lack deep research.Therefore,to address the scientific problems raised above,by using mouse bone marrow(BM)-derived primary megakaryocytes,human cord blood(CB)-derived primary megakaryocytes,human megakaryocyte progenitor cell line M07 e,human megakaryocyte cell line Meg-01,c-Mpl-/-mice and ERβ-/-mice,we will firstly create sub-lethal total body irradiation mouse model by using normal C57BL/6J mice and giving them estrogen before or after ionizing radiation to evaluate the effect of estrogen on the recovery of platelet level.Then,we plan to investigate the role of estrogen in the proliferation,differentiation,polyploidization,proplatelet formation and platelet production to define the stage that estrogen affects by using cell activity analysis,flow cytometry analysis,cellular immunofluorescence,laser confocal microscopy and routine blood analysis.Finally,we will deeply analyze the molecular mechanism underlying the effects of estrogen by using western blot,RT-PCR,q RT-PCR,small interfering RNA(si RNA)knock-down technique,dual luciferase report system,chromatin immunocoprecipitation(ChIP).The primary results and conclusions are summarized as listed below:1.Using ionizing radiation-induced thrombocytopenia mouse model,we observe that acute radiation injury results in fast decrease of platelet level.Estrogen treatment before or after radiation c a n e f f i c i e n t l y a c c e l e r a t e t h e r e c o v e r y o f platelet level after irradiation and distinctly shorten the persistent time of t h r o m b o c y t o p e n i a c r i s i s,thereby increasing the survival of irradiated mice.These indicate that,as a traditional anti-radiation drug,estrogen can be used to prevent and treat ionizing radiation-induced thrombocytopenia.2.Although estrogen can not promote the proliferation of megakaryocyte progenitors,estrogen has a distinct ability to promote the differentiation,maturation,polyploidization,proplatelet formation and platelet production of megakaryocytes.In addition,estrogen can promote megakaryocyte maturation in bone marrow and ultimately elevate the platelet levels in mice.These suggest that estrogen may mainly affect the late stage of thrombopoiesis.3.Estrogen treatment can markedly activate ERβ in megakaryocytes but not ERa.Antagonize ERβ by fulvestrant or knock-down ERβ can significantly attenuate estrogen-induced megakaryocyte maturation and polyploidization,suggesting that ERβ is the main receptor responsible for the effects of estrogen.4.After the activation of ERβ induced by estrogen,among the several regulators involved in megakaryopoiesis,we uniquely find that the expression of GATA1 is significantly upregulated,while ERβ knock-down almost abrogate estrogen-induced GATA1 upregulation,suggesting that GATA1 may be the main target gene downstream of ERβ.We further verify that ERβ can bind the promotor of GATA1 by using dual luciferase assay and ChIP,and the potential ERE is located at-1574 to-1380.Moreover,estrogen fails to induce megakaryocyte polyploidization after GATA1 knock-down.All these indicate that GATA1 is the target gene downstream of ERβ.ERβ can bind the promotor of GATA1 after estrogen-induced activation to upregulate the expression of GATA1,thus promoting megakaryocyte polyploidization.5.Western blot analysis show that estrogen can enhance the expression and activation of STAT1,while estrogen fails to enhance the expression and activation of STAT1 after ERβ and GATA1 knock-down.Meanwhile,STAT1 knock-down almost abrogated estrogen-induced megakaryocyte polyploidization.All these indicate that STAT1 is the downstream target of GATA1 and mediates estrogen-induced megakaryocyte polyploidization.6.NF-E2 is a downstream target of GATA1.In vitro estrogen treatment can upregulate the expression of NF-E2,while GATA1 knock-down almost abrogate estrogeninduced NF-E2 upregulation.In addition,NF-E2 is a key regulator of proplatelet formation and platelet production.All these indicate that NF-E2 is the downstream target of GATA1 and mediates estrogen-induced proplatelet formation and platelet production.Conclusions: In the present study,we show that estrogen can effectively prevent and treat ionizing radiation-induced thrombocytopenia.We further demonstrate that estrogen has a distinct role in thrombopoiesis.We also for the first time deeply uncover the molecular mechanism underlying estrogen-induced megakaryocyte polyploidization and platelet production.Our findings provide a new insight into the precaution and treatment of thrombocytopenia in clinic.