c-Abl，雌激素受体β调控内皮型一氧化氮合成酶的研究
摘要
前言
雌激素及雌激素受体(ER)与女性心血管疾病密切相关。ER主要存在ERa和ERp两种亚型。ERp在调节血脂代谢、抗氧化应激损伤、改善血管内皮功能、抑制动脉粥样斑块形成、舒缩血管、减少心脏缺血再灌注损伤等方面起重要作用。ERp在人体内主要分布于心血管系统、中枢神经系统、免疫系统、胃肠道,肾脏和肺组织。人血管平滑肌细胞及内皮细胞、心肌细胞均发现有功能性ERp存在,在动脉血管壁内层和中层细胞中都有ERp。多种心血管系统病理生理调控过程中都有ERp的参与。大量的实验室基础研究发现ERp在高血压、血管内皮功能障碍、左心室肥厚、心肌梗死、心力衰竭等心血管疾病的发生、发展过程中起重要作用。
NOS(一氧化氮合成酶)系统包括三种不同的亚型：神经型一氧化氮合成酶(nNOS)、诱导型一氧化氮合成酶(iNOS)、内皮型一氧化氮合成酶(eNOS)。NO通过激活eNOS在内皮细胞产生;而激活的内皮细胞、炎性细胞、巨噬细胞、心肌细胞、血管平滑肌细胞则通过iNOS产生NO。NO是一种气体信号分子,也是一种内皮源性的血管舒张因子,参与心血管系统的广泛病理生理过程,在心肌细胞、内皮细胞、血管平滑肌细胞和炎症细胞之间的相互作用中起重要作用。NO抑制缺血再灌注损伤,抑制炎症,抗氧化应激损伤,对抗心肌梗死损伤,阻止左心室重塑,但过量的NO和氧化应激活性氧(ROS)共存则对心血管系统存在损伤性作用。
c-Abl (cellular-Abelson gene)是Abelson鼠白血病病毒v-abl原癌基因在细胞内的同源基因,是一个高度保守的基因,c-Abl是非受体酪氨酸激酶。在正常生理状态下,c-Abl可定位于细胞核、细胞质、线粒体、内质网等多种亚细胞结构,并与胞内信号受体、激酶、磷酸酶、细胞周期调节因子、转录因子、细胞骨架蛋白、核孔蛋白、蛋白酶体亚基等多种分子发生相互作用。其主要的生物学功能是参与细胞周期和细胞凋亡调控,并在氧化应激和DNA损伤修复过程中发挥重要作用。
研究已证明ERa可使eNOS启动子活性增强从而上调eNOS表达活性。ERβ的DNA结合区域与ERa有96%的同源性,配体结合区有55%的同源性。c-Abl可通过其SH3、SH2结构域介导蛋白间的相互作用。其中SH3结构域能特异性识别富含脯氨酸的PXXP(P代表脯氨酸,X代表任意氨基酸)序列。对ERβ的序列进行了研究,发现ERβ存在PXXP模体,而具有PXXP模体的蛋白可能与c-Abl发生相互作用。据此我们假设c-Abl可与ERβ相结合,ERβ可以上调eNOS表达；c-Abl可促进ERβ对eNOS表达的上调作用,并通过实验初步证实这一假设。
实验方法
1、我们设计人ERβ, eNOS启动子报告基因引物,PCR扩增基因,酶切,连接载体,转化感受态大肠杆菌,PCR扩增后测序,构建Flag-ERβ载体及eNOS启动子报告基因载体。
2、转化GST-SH2\GST-SH3质粒到大肠杆菌感受态细胞,制备了GST\GST-SH2和GST-SH3交联的GSH珠子。转染Flag-ER(3于293T细胞,裂解细胞,与GST、GST-SH2和GST-SH3交联的GSH珠子孵育后行GST pull down实验。应用抗-Flag抗体行Western blot检测,明确c-Abl的SH3及SH2结构域能否与ERp在体外相互结合。Flag-ERβ, Myc-cAbl共转染于真核细胞,抗-Flag抗体交联的琼脂糖珠子行免疫共沉淀检测,Western blot检测,明确二者可否在真核细胞中结合。
3、Flag-ERβ、Myc-cAbl共转染后,用抗Tyr-P抗体交联的琼脂糖珠子行免疫共沉淀,应用抗Tyr-P抗体及抗Flag抗体免疫印迹检测ERβ可否被c-Abl磷酸化。
4、将PRL, ERE-Luc, Flag-ERp, Myc-c-Abl质粒共转染,按照Promega公司试剂盒说明书进行荧光素酶活性测定,探讨cAbl对ERp调节的下游基因转录的影响。
5、将PRL、pGL2-eNOS-Lu、Flag-ERp、Myc-c-Abl、贡粒共转染,荧光素酶活性测定来探讨ERβ对eNOS转录活性的影响,以及c-Abl对其的调控。
6、lag-ERβ、Myc-c-Abl质粒共转染于内皮细胞系EA.hy926中,RTPCR及Western blot检测ERβ对eNOS表达的影响及及c-Abl对其的调控。
结果和结论
1、成功构建了eNOS启动子报告基因载体及ERβ真核表达载体。
2、ERβ与c-Abl体外、真核细胞内存在相互作用而形成复合物。
3、c-Abl可使ERβ磷酸化。
4、在293T细胞中Myc-c-Ab1能够使ERE-Luc:报道基因的转录活性增强2.22倍。加入STI571, ERE-Luc报道基因的转录活性增强1.04倍。在EA.hy926细胞中Myc-c-Abl能够使ERE-Luc报道基因的转录活性增强1.83倍,加入STI571后ERE-Luc报道基因的转录活性增强1.01倍。说明c-Abl可上调ERp转录活性。
5、在293T细胞中结果示Flag-ERβ能够使pGL2-eNOS-Luc报道基因的转录活性增强2.76倍,Myc-cAbl与Flag-ERβ共转染组使pGL2-eNOS-Luc报道基因的转录活性增强4.89倍。Myc-cAbl与Flag-ERβ共转染组加入STI571使pGL2-eNOS-Luc报道基因的转录活性增强2.87倍。在内皮细胞系EA.hy926中重复了这一实验,得到了类似的结果,Flag-ERβ能够使pGL2-eNOS-Luc报道基因的转录活性增强3.13倍,Myc-cAbl与flag-ERβ共转染组使pGL2-eNOS-Luc报道基因的转录活性增强5.72倍,Myc-cAbl与Flag-ERβ共转染组加入STI571使pGL2-eNOS-Luc报道基因的转录活性增强2.95倍。ERβ可增加eNOS转录活性,c-Abl可促进ERβ增加eNOS转录活性的作用,这种作用可被STI571抑制。
6、ERp增加eNOS表达,c-Abl促进ERp增加eNOS表达。
讨论
在机体环境中,蛋白质分子发挥重要作用,其中蛋白-蛋白之间的相互作用在蛋白转运、信号传导、免疫识别、细胞调控等多种生命活动中起关键作用。我们的研究发现c-Abl与ERp在体外通过c-Abl的SH3结构域相结合,二者可在真核细胞内存在相互作用而形成复合物,ERp磷酸化并上调ERp转录活性,人内皮细胞系EA.hy926中ERp上调eNOS转录活性、增加eNOS表达,c-Abl促进这种作用。这提示内皮细胞内可能存在c-Abl—ERp—eNOS信号转导通路。这有助于更深入的认识cAbl, ERβ, eNOS在心血管系统中的相互作用；更有助于明确其与相关蛋白及信号转导通路的相互作用；对于心血管系统疾病发生中的血管张力调节、氧化应激、缺血再灌注损伤、内皮功能不良、心肌细胞凋亡和坏死等方面的研究具有一定意义；可能对于心血管疾病的基因靶向治疗提供思路。当然c-Abl与ERβ及eNOS相关作用的具体信号转导通路还未知,我们相信随着基础及临床研究的系统与深入,将有助于继续认识雌激素及其受体在心血管系统中的作用机制,为女性心血管疾病的防治进一步提供理论依据。
Preface
Estrogen and estrogen receptor (ER) are closely related with cardiovascular disease in women. ER involves ERa and ERα. ERβplay an important role in many aspects such as regulate lipid metabolism, anti-oxidative stress injury, improve endothelial function, inhibit the formation of arterial plaque, modulate relaxation and contraction of blood vessels and reduce cardiac ischemia-reperfusion injury. ERβin the human body mainly exist in the cardiovascular system, central nervous system, immune system, gastrointestinal tract, kidney and lung tissue. It was found to have functional ERβexist in human vascular smooth muscle cells, endothelial cells and cardiac cells. Researchers found that ERβplay an important role in hypertention, vascular endothelial dysfunction, left ventricular hypertrophy, myocardial infarction, heart failure and many other cardiovascular diseases in a large number of laboratory and clinical study.
NOS Includes three different subtypes:nNOS, eNOS and iNOS. NO is produced in endothelial cells by activating eNOS. The activated endothelial cells, inflammatory cells, macrophages, cardiac cells, vascular smooth muscle cells produce NO through iNOS. NO is a gaseous signal molecule, is also a vascular endothelium-derived relaxing factor involved in pathophysiology of the cardiovascular system. NO play an important role in the interaction of myocardial cells, endothelial cells, vascular smooth muscle cells and inflammatory cells. NO reduce ischemia-reperfusion injury, inhibit inflammation, reduce myocardial infarction injury, prevent left ventricular remodeling. However, excessive NO co-existing with reactive oxygen species (ROS) damage to the cardiovascular system.
c-Abl(cellular-Abelson gene) is the homologous gene of proto-oncogene v-abl. C-Abl is a highly conserved gene, and also a non-receptor tyrosine kinases. Under normal physiological conditions, c-Abl may be located in the nucleus, cytoplasm, mitochondria, endoplasmic reticulum and other subcellular structures.c-Abl can interact with intracellular signal receptor, kinase, phosphatase, cell cycle regulators, transcription factors, cytoskeletal proteins, nuclear pore proteins and proteasome subunits. Its main biological function is involved in cell cycle and apoptosis regulation, oxidative stress and DNA damage repair.
It has been demonstrated that ERa can enhance eNOS promoter activity and thus increase eNOS expression.ERβandERa have 96% homology in DNA-binding domain and 55% homology in ligand-binding domain.C-Abl mediated protein-protein interactions with its SH3, SH2 domain. Its SH3 domain can specifically recognize proline-rich PXXP sequence(P refers to proline, X refers to any amino acid).We studied the sequence of ERβand found there has PXXP motif. And protein with the PXXP motif may interact with c-Abl. Accordingly, we assume that c-Abl may combine with ERβ, ERβmay up-regulate eNOS expression, c-Abl may promote the up-regulation. We tried to confirm this preliminary hypothesis through experiments.
Methods
1. We designed ERβprimer or eNOS promoter reporter gene primer, amplified gene using PCR, cutted it with enzymeO, connected with vector, transformed it to competent E. coli, PCR again, and sequenced it. We constructed Flag-ERβvector and eNOS promoter reporter gene vector.
2. We transformed GST-SH2 and GST-SH3 plasmid into competent E. coli, prepared GST, GST-SH2 and GST-SH3 beads cross-linked GSH. We transfected Flag-ERβinto 293T cells, lysed cells, incubated it with GST, GST-SH2, and GST-SH3 cross-linked GSH beads,then completed GST pull down experiments. We used anti-Flag antibody in western blot test to identify whether SH3 or SH2 domains of c-Abl can combine with ERP in vitro. Then we co-transfected Flag-ERβand Myc-cAbl in ukaryotic cells, used anti-Flag cross-linked agarose beads in co-immunoprecipitation, did Western blot detection to detect whether it can combine with each other in eukaryotic cells.
3. We co-transfected Flag-ERβand Myc-cAbl,used anti-Tyr-P cross-linked agarose beads in co-immunoprecipitation, did Western blot detection with anti-Tyr-P or anti-Flag to detect whether ERβcan be c-Abl phosphorylated.
4. The PRL, ERE-Luc, Flag-ERβ, Myc-c-Abl plasmids were transfected, according to Promega's kit instructions to determine the luciferase activity to explore how cAbl regulate ERβdownstream gene transcription.
5. The PRL, pGL2-eNOS-Luc, Flag-ERp, Myc-c-Abl plasmids were transfected, according to Promega's kit instructions to determine the luciferase activity to explore how ERP regulate eNOS transcription and how cAbl regulate it.
6. We cotransfected Flag-ERβ, Myc-c-Abl plasmids into the endothelial cells, RTPCR and Western blot were used to detect how ERP regulate eNOS expression and how cAbl regulate it.
Result and conclusion
1. The eNOS promoter reporter gene vector and ERβeukaryotic expression vector were successfully constructed.
2. ERβinteract with c-Abl and combine it to form complex in vitro and in eukaryotic cells.
3. ERβcan be phosphorylated by c-Abl.
4. Myc-c-Abl can make ERE-Luc reporter gene transcription activity increase 2.22 fold in the 293T cells.After adding STI571, ERE-Luc reporter gene transcription activity increased 1.04 fold.
In EA.hy926 cells Myc-c-Abl can make ERE-Luc reporter gene transcription activity increase 1.83 fold. After adding STI571, ERE-Luc reporter gene transcription activity was increased 1.01 fold.
5. In 293T cells Flag-ERβcan make eNOS promter-Luc reporter gene transcription activity increase 2.76-fold. In Myc-cAbl and Flag-ERβcotransfected group, eNOS promter-Luc reporter gene transcription activity was enhanced 4.89 fold. After adding STI571, eNOS promter-Luc reporter gene transcription activity was increased 2.87 fold.
In EA.hy926 cells Flag-ERβcan make eNOS promter-Luc reporter gene transcription activity increase 3.13fold. In Myc-cAbl and Flag-ERβcotransfected group, eNOS promter-Luc reporter gene transcription activity was enhanced 5.72 fold. After adding STI571, eNOS promter-Luc reporter gene transcription activity was increased 2.95 fold.
6. ERP increased eNOS expression and c-Abl promotes this effect.
Discussion
In the body environment, protein molecules play an important role including protein-protein interactions in protein transport, signal transduction, immune recognition, cell regulation and other life activities.Our research found that ERP interact with SH3 of c-Abl in vitro and they combine to form complex in eukaryotic cells. ERP can be phosphorylated by c-Abl and c-Abl enhanced ERp transcriptional activity. ERP increases eNOS transcriptional activity and c-Abl promotes it. ERβincreased eNOS expression and c-Abl promotes this effect in EA.hy926 cells. This suggests that there may exist c-Abl-ERβ-eNOS signaling pathway in endothelial cells. It helps we have a better understanding of cAbl, ERβ, eNOS interactions in the cardiovascular system; and have a easier way to clear its interactions with related proteins and signal transduction pathway. Our research has a certain significance in cardiovascular diseases including vascular tone regulation, oxidative stress, ischemia-reperfusion injury, endothelial dysfunction, myocardial apoptosis and necrosis etc.It may be targeted for gene therapy of cardiovascular disease. Although the specific signal transduction pathways of c-Abl, ERβand eNOS are unknown. With the basic and clinical research progressing in depth, we believe that it will contribute to the understanding of estrogen and estrogen receptor in the mechanisms of cardiovascular diseases and provide further theoretical basis for prevention and treatment of cardiovascular disease in women.
雌激素及雌激素受体(ER)与女性心血管疾病密切相关。ER主要存在ERa和ERp两种亚型。ERp在调节血脂代谢、抗氧化应激损伤、改善血管内皮功能、抑制动脉粥样斑块形成、舒缩血管、减少心脏缺血再灌注损伤等方面起重要作用。ERp在人体内主要分布于心血管系统、中枢神经系统、免疫系统、胃肠道,肾脏和肺组织。人血管平滑肌细胞及内皮细胞、心肌细胞均发现有功能性ERp存在,在动脉血管壁内层和中层细胞中都有ERp。多种心血管系统病理生理调控过程中都有ERp的参与。大量的实验室基础研究发现ERp在高血压、血管内皮功能障碍、左心室肥厚、心肌梗死、心力衰竭等心血管疾病的发生、发展过程中起重要作用。
NOS(一氧化氮合成酶)系统包括三种不同的亚型：神经型一氧化氮合成酶(nNOS)、诱导型一氧化氮合成酶(iNOS)、内皮型一氧化氮合成酶(eNOS)。NO通过激活eNOS在内皮细胞产生;而激活的内皮细胞、炎性细胞、巨噬细胞、心肌细胞、血管平滑肌细胞则通过iNOS产生NO。NO是一种气体信号分子,也是一种内皮源性的血管舒张因子,参与心血管系统的广泛病理生理过程,在心肌细胞、内皮细胞、血管平滑肌细胞和炎症细胞之间的相互作用中起重要作用。NO抑制缺血再灌注损伤,抑制炎症,抗氧化应激损伤,对抗心肌梗死损伤,阻止左心室重塑,但过量的NO和氧化应激活性氧(ROS)共存则对心血管系统存在损伤性作用。
c-Abl (cellular-Abelson gene)是Abelson鼠白血病病毒v-abl原癌基因在细胞内的同源基因,是一个高度保守的基因,c-Abl是非受体酪氨酸激酶。在正常生理状态下,c-Abl可定位于细胞核、细胞质、线粒体、内质网等多种亚细胞结构,并与胞内信号受体、激酶、磷酸酶、细胞周期调节因子、转录因子、细胞骨架蛋白、核孔蛋白、蛋白酶体亚基等多种分子发生相互作用。其主要的生物学功能是参与细胞周期和细胞凋亡调控,并在氧化应激和DNA损伤修复过程中发挥重要作用。
研究已证明ERa可使eNOS启动子活性增强从而上调eNOS表达活性。ERβ的DNA结合区域与ERa有96%的同源性,配体结合区有55%的同源性。c-Abl可通过其SH3、SH2结构域介导蛋白间的相互作用。其中SH3结构域能特异性识别富含脯氨酸的PXXP(P代表脯氨酸,X代表任意氨基酸)序列。对ERβ的序列进行了研究,发现ERβ存在PXXP模体,而具有PXXP模体的蛋白可能与c-Abl发生相互作用。据此我们假设c-Abl可与ERβ相结合,ERβ可以上调eNOS表达；c-Abl可促进ERβ对eNOS表达的上调作用,并通过实验初步证实这一假设。
实验方法
1、我们设计人ERβ, eNOS启动子报告基因引物,PCR扩增基因,酶切,连接载体,转化感受态大肠杆菌,PCR扩增后测序,构建Flag-ERβ载体及eNOS启动子报告基因载体。
2、转化GST-SH2\GST-SH3质粒到大肠杆菌感受态细胞,制备了GST\GST-SH2和GST-SH3交联的GSH珠子。转染Flag-ER(3于293T细胞,裂解细胞,与GST、GST-SH2和GST-SH3交联的GSH珠子孵育后行GST pull down实验。应用抗-Flag抗体行Western blot检测,明确c-Abl的SH3及SH2结构域能否与ERp在体外相互结合。Flag-ERβ, Myc-cAbl共转染于真核细胞,抗-Flag抗体交联的琼脂糖珠子行免疫共沉淀检测,Western blot检测,明确二者可否在真核细胞中结合。
3、Flag-ERβ、Myc-cAbl共转染后,用抗Tyr-P抗体交联的琼脂糖珠子行免疫共沉淀,应用抗Tyr-P抗体及抗Flag抗体免疫印迹检测ERβ可否被c-Abl磷酸化。
4、将PRL, ERE-Luc, Flag-ERp, Myc-c-Abl质粒共转染,按照Promega公司试剂盒说明书进行荧光素酶活性测定,探讨cAbl对ERp调节的下游基因转录的影响。
5、将PRL、pGL2-eNOS-Lu、Flag-ERp、Myc-c-Abl、贡粒共转染,荧光素酶活性测定来探讨ERβ对eNOS转录活性的影响,以及c-Abl对其的调控。
6、lag-ERβ、Myc-c-Abl质粒共转染于内皮细胞系EA.hy926中,RTPCR及Western blot检测ERβ对eNOS表达的影响及及c-Abl对其的调控。
结果和结论
1、成功构建了eNOS启动子报告基因载体及ERβ真核表达载体。
2、ERβ与c-Abl体外、真核细胞内存在相互作用而形成复合物。
3、c-Abl可使ERβ磷酸化。
4、在293T细胞中Myc-c-Ab1能够使ERE-Luc:报道基因的转录活性增强2.22倍。加入STI571, ERE-Luc报道基因的转录活性增强1.04倍。在EA.hy926细胞中Myc-c-Abl能够使ERE-Luc报道基因的转录活性增强1.83倍,加入STI571后ERE-Luc报道基因的转录活性增强1.01倍。说明c-Abl可上调ERp转录活性。
5、在293T细胞中结果示Flag-ERβ能够使pGL2-eNOS-Luc报道基因的转录活性增强2.76倍,Myc-cAbl与Flag-ERβ共转染组使pGL2-eNOS-Luc报道基因的转录活性增强4.89倍。Myc-cAbl与Flag-ERβ共转染组加入STI571使pGL2-eNOS-Luc报道基因的转录活性增强2.87倍。在内皮细胞系EA.hy926中重复了这一实验,得到了类似的结果,Flag-ERβ能够使pGL2-eNOS-Luc报道基因的转录活性增强3.13倍,Myc-cAbl与flag-ERβ共转染组使pGL2-eNOS-Luc报道基因的转录活性增强5.72倍,Myc-cAbl与Flag-ERβ共转染组加入STI571使pGL2-eNOS-Luc报道基因的转录活性增强2.95倍。ERβ可增加eNOS转录活性,c-Abl可促进ERβ增加eNOS转录活性的作用,这种作用可被STI571抑制。
6、ERp增加eNOS表达,c-Abl促进ERp增加eNOS表达。
讨论
在机体环境中,蛋白质分子发挥重要作用,其中蛋白-蛋白之间的相互作用在蛋白转运、信号传导、免疫识别、细胞调控等多种生命活动中起关键作用。我们的研究发现c-Abl与ERp在体外通过c-Abl的SH3结构域相结合,二者可在真核细胞内存在相互作用而形成复合物,ERp磷酸化并上调ERp转录活性,人内皮细胞系EA.hy926中ERp上调eNOS转录活性、增加eNOS表达,c-Abl促进这种作用。这提示内皮细胞内可能存在c-Abl—ERp—eNOS信号转导通路。这有助于更深入的认识cAbl, ERβ, eNOS在心血管系统中的相互作用；更有助于明确其与相关蛋白及信号转导通路的相互作用；对于心血管系统疾病发生中的血管张力调节、氧化应激、缺血再灌注损伤、内皮功能不良、心肌细胞凋亡和坏死等方面的研究具有一定意义；可能对于心血管疾病的基因靶向治疗提供思路。当然c-Abl与ERβ及eNOS相关作用的具体信号转导通路还未知,我们相信随着基础及临床研究的系统与深入,将有助于继续认识雌激素及其受体在心血管系统中的作用机制,为女性心血管疾病的防治进一步提供理论依据。
Preface
Estrogen and estrogen receptor (ER) are closely related with cardiovascular disease in women. ER involves ERa and ERα. ERβplay an important role in many aspects such as regulate lipid metabolism, anti-oxidative stress injury, improve endothelial function, inhibit the formation of arterial plaque, modulate relaxation and contraction of blood vessels and reduce cardiac ischemia-reperfusion injury. ERβin the human body mainly exist in the cardiovascular system, central nervous system, immune system, gastrointestinal tract, kidney and lung tissue. It was found to have functional ERβexist in human vascular smooth muscle cells, endothelial cells and cardiac cells. Researchers found that ERβplay an important role in hypertention, vascular endothelial dysfunction, left ventricular hypertrophy, myocardial infarction, heart failure and many other cardiovascular diseases in a large number of laboratory and clinical study.
NOS Includes three different subtypes:nNOS, eNOS and iNOS. NO is produced in endothelial cells by activating eNOS. The activated endothelial cells, inflammatory cells, macrophages, cardiac cells, vascular smooth muscle cells produce NO through iNOS. NO is a gaseous signal molecule, is also a vascular endothelium-derived relaxing factor involved in pathophysiology of the cardiovascular system. NO play an important role in the interaction of myocardial cells, endothelial cells, vascular smooth muscle cells and inflammatory cells. NO reduce ischemia-reperfusion injury, inhibit inflammation, reduce myocardial infarction injury, prevent left ventricular remodeling. However, excessive NO co-existing with reactive oxygen species (ROS) damage to the cardiovascular system.
c-Abl(cellular-Abelson gene) is the homologous gene of proto-oncogene v-abl. C-Abl is a highly conserved gene, and also a non-receptor tyrosine kinases. Under normal physiological conditions, c-Abl may be located in the nucleus, cytoplasm, mitochondria, endoplasmic reticulum and other subcellular structures.c-Abl can interact with intracellular signal receptor, kinase, phosphatase, cell cycle regulators, transcription factors, cytoskeletal proteins, nuclear pore proteins and proteasome subunits. Its main biological function is involved in cell cycle and apoptosis regulation, oxidative stress and DNA damage repair.
It has been demonstrated that ERa can enhance eNOS promoter activity and thus increase eNOS expression.ERβandERa have 96% homology in DNA-binding domain and 55% homology in ligand-binding domain.C-Abl mediated protein-protein interactions with its SH3, SH2 domain. Its SH3 domain can specifically recognize proline-rich PXXP sequence(P refers to proline, X refers to any amino acid).We studied the sequence of ERβand found there has PXXP motif. And protein with the PXXP motif may interact with c-Abl. Accordingly, we assume that c-Abl may combine with ERβ, ERβmay up-regulate eNOS expression, c-Abl may promote the up-regulation. We tried to confirm this preliminary hypothesis through experiments.
Methods
1. We designed ERβprimer or eNOS promoter reporter gene primer, amplified gene using PCR, cutted it with enzymeO, connected with vector, transformed it to competent E. coli, PCR again, and sequenced it. We constructed Flag-ERβvector and eNOS promoter reporter gene vector.
2. We transformed GST-SH2 and GST-SH3 plasmid into competent E. coli, prepared GST, GST-SH2 and GST-SH3 beads cross-linked GSH. We transfected Flag-ERβinto 293T cells, lysed cells, incubated it with GST, GST-SH2, and GST-SH3 cross-linked GSH beads,then completed GST pull down experiments. We used anti-Flag antibody in western blot test to identify whether SH3 or SH2 domains of c-Abl can combine with ERP in vitro. Then we co-transfected Flag-ERβand Myc-cAbl in ukaryotic cells, used anti-Flag cross-linked agarose beads in co-immunoprecipitation, did Western blot detection to detect whether it can combine with each other in eukaryotic cells.
3. We co-transfected Flag-ERβand Myc-cAbl,used anti-Tyr-P cross-linked agarose beads in co-immunoprecipitation, did Western blot detection with anti-Tyr-P or anti-Flag to detect whether ERβcan be c-Abl phosphorylated.
4. The PRL, ERE-Luc, Flag-ERβ, Myc-c-Abl plasmids were transfected, according to Promega's kit instructions to determine the luciferase activity to explore how cAbl regulate ERβdownstream gene transcription.
5. The PRL, pGL2-eNOS-Luc, Flag-ERp, Myc-c-Abl plasmids were transfected, according to Promega's kit instructions to determine the luciferase activity to explore how ERP regulate eNOS transcription and how cAbl regulate it.
6. We cotransfected Flag-ERβ, Myc-c-Abl plasmids into the endothelial cells, RTPCR and Western blot were used to detect how ERP regulate eNOS expression and how cAbl regulate it.
Result and conclusion
1. The eNOS promoter reporter gene vector and ERβeukaryotic expression vector were successfully constructed.
2. ERβinteract with c-Abl and combine it to form complex in vitro and in eukaryotic cells.
3. ERβcan be phosphorylated by c-Abl.
4. Myc-c-Abl can make ERE-Luc reporter gene transcription activity increase 2.22 fold in the 293T cells.After adding STI571, ERE-Luc reporter gene transcription activity increased 1.04 fold.
In EA.hy926 cells Myc-c-Abl can make ERE-Luc reporter gene transcription activity increase 1.83 fold. After adding STI571, ERE-Luc reporter gene transcription activity was increased 1.01 fold.
5. In 293T cells Flag-ERβcan make eNOS promter-Luc reporter gene transcription activity increase 2.76-fold. In Myc-cAbl and Flag-ERβcotransfected group, eNOS promter-Luc reporter gene transcription activity was enhanced 4.89 fold. After adding STI571, eNOS promter-Luc reporter gene transcription activity was increased 2.87 fold.
In EA.hy926 cells Flag-ERβcan make eNOS promter-Luc reporter gene transcription activity increase 3.13fold. In Myc-cAbl and Flag-ERβcotransfected group, eNOS promter-Luc reporter gene transcription activity was enhanced 5.72 fold. After adding STI571, eNOS promter-Luc reporter gene transcription activity was increased 2.95 fold.
6. ERP increased eNOS expression and c-Abl promotes this effect.
Discussion
In the body environment, protein molecules play an important role including protein-protein interactions in protein transport, signal transduction, immune recognition, cell regulation and other life activities.Our research found that ERP interact with SH3 of c-Abl in vitro and they combine to form complex in eukaryotic cells. ERP can be phosphorylated by c-Abl and c-Abl enhanced ERp transcriptional activity. ERP increases eNOS transcriptional activity and c-Abl promotes it. ERβincreased eNOS expression and c-Abl promotes this effect in EA.hy926 cells. This suggests that there may exist c-Abl-ERβ-eNOS signaling pathway in endothelial cells. It helps we have a better understanding of cAbl, ERβ, eNOS interactions in the cardiovascular system; and have a easier way to clear its interactions with related proteins and signal transduction pathway. Our research has a certain significance in cardiovascular diseases including vascular tone regulation, oxidative stress, ischemia-reperfusion injury, endothelial dysfunction, myocardial apoptosis and necrosis etc.It may be targeted for gene therapy of cardiovascular disease. Although the specific signal transduction pathways of c-Abl, ERβand eNOS are unknown. With the basic and clinical research progressing in depth, we believe that it will contribute to the understanding of estrogen and estrogen receptor in the mechanisms of cardiovascular diseases and provide further theoretical basis for prevention and treatment of cardiovascular disease in women.
引文
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