The Role Of TRPM7 In Regulating Cell Physiological Processes, And Its Potential Mechanisms

Cardiovascular and cerebrovascular diseases have become the leading cause of human death. It is very urgent to deeply understand the potential pathogenesis of these diseases, and develop the effective pharmacy for therapies. Ion channels play an important role in the pathogenesis of cardio-and cerebrovascular disease. Recently, transient receptor potential (TRP) ion channels are acquired much more attention for playing an important role in the physiological and pathological processes in cardiovascular and cerebrovascular system. Most of 30 TRP channels are expressed in central nervous system (CNS), and at least 19 of them are expressed in endothelial cells. TRPM7, a member of melastatin subfamily of TRP channels, is acquired attention for its specific structure, widely expression, and diverse functions. Firstly, it TRPM7 possesses a-kinase domain in its c-terminal except for ion channel domain, so it is called chanzyme. The activity of TRPM7 is regulated by many factors such as autophosphorylation, PIP2, pH, oxidative stress, mechanical stress, and vasoactive, which suggests that TRPM7 has so many kinds of functions. Secondly, TRPM7 is widely expressed in the cardiovascular and cerebrovascular system including the heart, brain, lung, liver, kidney, vascular, and other tissues and organs. Thirdly, TRPM7 modulates diverse physiological and pathological processes such as proliferation, migration, adhesion, apoptosis, inflammation, ion homeostasis. Increasing evidence shows that TRPM7 is involved in modulation of physiological and pathological processes in brain and cardiovascular system including the early cardiogenesis, ischemic stroke, hypertension, atrial fibrillation, the proliferation and migration of vascular endothelial cells, the proliferation and magnesium homeostasis of smooth muscle cells. For example, TRPM7 can be activated by ROS which is produced by ischemic stroke or oxygen-glucose deprivation (OGD), resulting in the calcium influx and death of neurons, which can be inhibited by silencing or inhibiting TRPM7.Both vascular endothelial cells and astrocytes are a part of cardiovascular and central nervous system (CNS) and play an important role in physiological and pathological processes in cardiovascular system and CNS, respectively. Endothelial cells come from the inner lining of a blood vessel, which play an important role in vascular tone, coagulation, atherosclerosis, angiogenesis, immune response and so on. Previous studies show that silencing TRPM7 enhances human umbilical vein endothelial cell growth/proliferation and migration, and protects hyperglycemia induced injury, which suggests that TRPM7 play an important role in endothelial functions. Astrocytes, the most abundant cell type in CNS, are 6-10 times more than neurons. Astrocytes can associate with neuronal synapses and exert diverse functions including supporting and nourishing neurons, secreting and absorbing neurotransmitter, and regulating ion homeostasis and immune response. It also can adhere to adjacent microvascular endothelial cells and regulates the vascular tone and blood-brain barrier. TRPM7 is highly expressed in brain and involved in ischemia-induced neuronal death. However, the role of TRPM7 in the physiological processes of astrocytes has not been elucidated. We try to accomplish following aims by investigating the role of TRPM7 in modulating cell processes and their mechanisms.1. In order to study the role of TRPM7 in modulation of vascular endothelial dysfunctions, we investigated the effect of TRPM7 on human umbilical vein endothelial cell (HUVEC) morphology, adhesion, migration, and angiogenesis, and its mechanisms. It might provide some experimental evidence for targeting TRPM7 for therapies of cardiovascular diseases.2. In order to study the role of TRPM7 in modulation of glial cell functions in brain, we determined if TRPM7 is functional expressed and regulates cell proliferation and migration, and its potential mechanisms. It might provide some experimental evidence for targeting TRPM7 as a therapy of brain disorders.1. Small interfering RNA (siRNA) was used to specifically knock down the expression of messenger RNA (mRNA) and protein expression of TRPM7 channel in HUVECs. The efficiency of siRNA was analyzed through a combined technology with quantitative real time-PCR and electrophysiological methods.2. The change of HUVEC morphology and the ability of HUVEC adhesion to extracellular matrix (matrigel) were quantified by measuring average single cell area and adhesion assay, respectively.3. Transwell chamber assay and scratches assay were used to investigate the effect of TRPM7 on HUVEC migration.4. Tube formation on matrigel in vitro was used to investigate the effect of TRPM7 on HUVEC angiogenesis.5. The phosphorylation of the MEK1/2 and myosin light chain (MLC), and the expression of other calcium channels related genes such as Orail, Stim1, TRPC3 and TRPC1 were analyzed by Western blotting and quantitative RT-PCR.6. The functional expression of TRPM7 in astrocytes was determined by combined RT-PCR, western blotting and eletrophysiological methods.7. Specific siRNA was used to knock down the expression of TRPM7 in astrocytes. Some biological methods such as RT-PCR, real time PCR and western blotting were used to evaluate the efficiency of TRPM7 siRNAs.8. LDH assay and wound healing assay were used to study the role of TRPM7 in astrocyte proliferation and migration, respectively.9. The underlying mechanisms by which TRPM7 regulates astrocyte proliferation and migration were determined by western blotting.10. Intracellular Mg2+and Ca2+concentration were measured by magnesium assay kit and flow cytometry, respectively.11. The effect of extracellular Ca2+and Mg2+on astrocyte proliferation was investigated by LDH assay.1. Quantitative PCR results show the mRNA expression of TRPM7 in HUVECs was significantly down-regulated by～39.67% and～29.49% respectively, when transfected with either TRPM7 siRNA-1 or TRPM7 siRNA-272 h. Whole cell patch clamp results show that the density of TRPM7 current significantly reduced by～63.8% and ～45.5% respectively, when clamp at -80 mv and +80 mv. At the meaning time, magnesium assay show that intracellular Mg2+ concentration significantly reduced by～10% by silencing TRPM7 in HUVECs, comparing with control. All these data suggests that the expression and functions of TRPM7 were impaired by siRNA.2. Knock down of TRPM7 altered the morphology of HUVEC (from nearly discoid to elongated format). Statistics analysis shows that average single cell area is reduced by ～50%. Subsequently, the ability of HUVEC adhesion to extracellular matrix was decreased by ～45.67%.3. Transwell assay and scratch assay results show that HUVEC migration, wound healing and early angiogenesis (4 h) in vitro were enhanced by～300%,～56.64%, and ～39.5% respectively, after transfected with TRPM7 siRNA.4. Molecular mechanisms analysis showed that silencing TRPM7 in HUVEC increased the phosphorylation of MEK and MLC. The MEK selective inhibitor U0126 significantly inhibited increased MLC phosphorylation, the morphological change and enhanced migration of HUVEC induced by TRPM7 silence. It suggests that TRPM7 regulates the physiological processes of HUVECs at least in part via MEK/ERK/MLC signaling pathway.5. qPCR and western blotting results show knock down of TRPM7 in HUVEC did not alter the expression of TRPC3, Orai1, and Stim1, but increasing TRPC1 mRNA and protein expression.6. We successfully purified and cultured primary mouse cortical astrocytes, and immunofluorescence results show the density of astrocytes is higher than 95%.7. RT-PCR and immunofluorescence assay show that astrocytes express functional TRPM7 channels in the mRNA and protein level. At the meaning time, TRPM7-like current was recorded with whole cell patch clamp.8. The mRNA and protein expression of TRPM7 in astrocytes were significantly decreased by TRPM7 siRNA-1 and siRNA-2, but have no effect on the mRNA expression of TRPM4 and TRPM6. Further studies show that silencing TRPM7 in astrocytes reduced mRNA expression and TRPM7-like currents by TRPM7 siRNA-1.9. Silence or inhibition of TRPM7 in astrocytes significantly decreased the proliferation and migration. Western blotting results show that the phosphorylation of ERK1/2 and JNK in astrocytes is down-regulated by～24.37% and～36.64% respectively by silencing TRPM7, but not p38 and Akt. In addition, TRPM7 inhibitor 2-APB also impairs the ERK and JNK phosphorylation.10. Suppression of ERK1/2 and JNK by U0126 and SP600125 respectively mimics the inhibition of astrocyte proliferation and migration induced by silencing TRPM7. It suggests that TRPM7 regulates astrocyte proliferation and migration selectively through ERK and JNK pathway.11. Intracellular basal magnesium concentration is significantly increases in tetracycline-induced TRPM7 overexpression cell line HEK293. In addition, Mg2+ concentration decreased by knockdown of TRPM7 in astrocytes, but calcium level has no change in cytometry assay. However, the addition of extracellular magnesium has no effect on astrocytic growth inhibition caused by silencing TRPM7.1. Overall, TRPM7 presents a negative effect on the physiological processes in HUVECs. Targeted silence of TRPM7 alters cell shape, promotes cell migration and early angiogenesis, and up-regulates the expression of TRPC1, which indicates a protective effect on endothelium. MEK/ERK/MLC pathways might be involved in these processes. It suggests that TRPM7 might be new potential target for modulating the physiological functions of endothelial cells.2. Functional TRPM7 channel is expressed in mouse primary astrocytes. Silencing or inhibiting TRPM7 impairs ERK and JNK signaling pathways, and suppresses the proliferation and migration of astrocytes. TRPM7 also modulates the intracellular Mg2+ homeostasis in astrocytes, but addition of extracellular Mg2+ does not rescue the inhibition of TRPM7 siRNA-mediated proliferation. TRPM7 might be a new target for regulating astrocytic functions.3. Our study clarify that TRPM7 plays an important role in physiological processes in cardiovascular system and brain. It may provide some experimental data for diagnosis and treatment of vascular and cerebral disorders. The study of TRPM7 in crossover filed of blood vessels and nerves will provide some new ideas for prevention, diagnosis and treatment of some diseases.