C-Abl, Estrogen Receptorβ Modulate Endothelial Nitric Oxide Synthase

PrefaceEstrogen 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.Methods1. 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 conclusion1. 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.DiscussionIn 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.