Effect of TRPM7 siRNA on viability and cytotoxicity in HG treated HUVECs.

<p>The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) Confluent field by light microscopy. <i>Scale bar, 100 µM</i>. (B) Cell viability was assessed by MTT assay. (C) Cytotoxicity was assessed by LDH release assay. **<i>p</i><0.01 vs. control; ^##<i>p</i><0.01 vs. control siRNA. n=5 for MTT and LDH assay.</p

Role of TRPM7 Channels in Hyperglycemia-Mediated Injury of Vascular Endothelial Cells

<div><p>This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.</p> </div

Effect of U0126 on viability and phospho-ERK1/2 expression in HG treated HUVECs.

<p>The cells were preincubated with U0126 (10 μM) for 18h, then treated with TRPM7 siRNA for 48h, and finally stimulated with HG for 72h. (A) Cell viability was assessed by MTT assay. (B) Representative immunoblots showing phospho-ERK1/2 expression level. (C) The corresponding bar graphs showing the relative expression of phospho-ERK1/2 protein normalized to beta-actin. **<i>p</i><0.01 vs. control; ^##<i>p</i><0.01 vs. control siRNA. n=5 for immunoblotting.</p

Effect of HG on TRPM7 protein expression in HUVECs.

<p>(A) Representative immunoblots showing TRPM7 protein expression in HUVECs with or without HG (30 mM) for 72h. (B) The corresponding bar graphs showing relative expression of TRPM7 protein normalized to beta-actin. (C) Representative TRPM7-like currents recorded in HUVECs cultured in control or HG (30 mM) for 72h. (D) TRPM7-like current density. **<i>p</i><0.01 vs. HG; *<i>p</i><0.05 vs. control. n=4 for immunoblotting and 17-18 cells patched for current recording. </p

Effect of TRPM7 siRNA on eNOS protein expression, NO and ROS generation in HG treated HUVECs.

<p>The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) Representative immunoblots showing eNOS protein expression level. (B) The corresponding bar graphs showing the relative expression of eNOS protein normalized to beta-actin. (C) The production of NO was determined by measurement of nitrite, a stable product of NO. (D) The production of intracellular ROS was assessed by the oxidation of 2’,7’-dichlorofluorescin diacetate to fluorescent 2’,7’-dichlorofluorescein. **<i>p</i><0.01 vs. control; ^##<i>p</i><0.01 vs. control siRNA. n=3 for immunoblotting , 5 for ROS generation assay and 6 for NO measurement.</p

Effect of TRPM7 siRNA on MAPK pathway in HG treated HUVECs.

<p>The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) Representative immunoblots showing phospho-ERK1/2, phospho-p38MAPK and phospho-JNK protein expression level. (B) The corresponding bar showing the relative optical density of phospho-ERK1/2 protein normalized to total ERK1/2. **<i>p</i><0.01 vs. control; ^##<i>p</i><0.01 vs. control siRNA. n=5 for immunoblotting (C) The corresponding bar graphs showing the relative optical density of phospho-p38MAPK protein normalized to beta-actin. (D) The corresponding bar graphs showing the relative expression of phospho-JNK protein normalized to beta-actin. n=4.</p

Effect of TRPM7 siRNA on TRPM7 mRNA and protein expression in high D-glucose (HG, 30 mM) treated HUVECs.

<p>The cells were preincubated with TRPM7 siRNA or control siRNA for 48h, and then stimulated with HG for 72h. (A) TRPM7 protein level measured by immunofluorescence. <i>Scale bar, 100 µm</i>. (B) TRPM7 mRNA level detected by RT-PCR. (C) TRPM7 mRNA level normalized to GAPDH. (D) Representative immunoblots showing the level of TRPM7 protein expression. (E) The corresponding bar graphs showing the relative expression of TRPM7 protein normalized to beta-actin. **<i>p</i><0.01 vs. control; ^#<i>p</i><0.05 vs. control siRNA, ^##<i>P</i><0.01 vs. control siRNA. n=4 for RT-PCR and immunoblotting. </p

Silencing TRPM7 in Mouse Cortical Astrocytes Impairs Cell Proliferation and Migration via ERK and JNK Signaling Pathways

<div><p>Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel, is highly expressed expressed in the brain and plays a critical role in ischemic neuronal death. Astrocyte, the most abundant cell type in central nervous system (CNS), exerts many essential functions in the physiological and pathological conditions. Here we investigated the expression and functions of the TRPM7 channel in mouse cortical astrocytes. Using reverse transcription (RT)-PCR, immunostaining, western blot and patch clamp recording, we showed that functional TRPM7 channel is expressed in cultured mouse cortical astrocytes. Knocking down TRPM7 with specific siRNA impairs the proliferation and migration of astrocytes by 40.2% ± 3.9% and 40.1% ± 11.5%, respectively. Consistently, inhibition of TRPM7 with 2-aminoethoxydiphenyl borate (2-APB) also decreases astrocyte proliferation and migration by 46.1% ± 2.5% and 64.2% ± 2.4%. MAPKs and Akt signaling pathways have been shown to be implicated in TRPM7-mediated responses including cell proliferation and migration. Our data show that suppression of TRPM7 in astrocytes reduces the phosphorylation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not p38 mitogen-activated protein kinase and Akt. In addition, TRPM7, as a cation channel, has been involved in the Ca²⁺ and Mg²⁺ homeostasis in several types of cells. In our study, we found that silencing TRPM7 decreases the intracellular basal Mg²⁺ concentration without affecting Ca²⁺ concentration in astrocytes. However, an addition of Mg²⁺ to the growth medium could not rescue the impaired proliferation of astrocytes. Together, our data suggest that TRPM7 channel may play a critical role in the proliferation and migration of astrocytes via the ERK and JNK pathways.</p></div

ERK and JNK inhibitors impair astrocyte proliferation and migration.

<p><b>A</b> and <b>B</b>. MEK inhibitor (U0126, 10 μM) and JNK inhibitor (sp600125, 20 μM) inhibit astrocyte proliferation by 36.3% ± 11.8% and 42.9% ± 7.8% respectively (*, <i>p</i> < 0.05, n = 4). <b>C</b> and <b>D</b>. MEK inhibitor (U0126, 10 μM) and JNK inhibitor (sp600125, 20 μM) inhibit astrocyte migration by 17.9% ± 3.7% and 60.0% ± 4.1% respectively (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01, n = 6).</p

TRPM7 regulates intracellular Ca²⁺ and Mg²⁺ concentration of cortical astrocytes.

<p><b>A</b>. Overexpression of TRPM7 in HEK/M7 cells induced by 1 μg/mL tetracycline drastically increases intracellular Mg²⁺ concentration by 73.7% ±7.23% (*, <i>p</i> < 0.05, n = 3). However, knockdown of TRPM7 in mouse cortical astrocytes significantly decreases intracellular Mg²⁺ by 43.5% ± 9.8% (*, <i>p</i> < 0.05, n = 4). <b>B</b>. Intracellular Ca²⁺ concentration of cortical astrocytes is analyzed by flow cytometry. All astrocytes loaded with Fluo-3 AM have a strong fluorescence signal compared to the negative control which has not been loaded with any dye. <b>C</b>. Quantification of intracellular Ca²⁺ concentration indicates significant difference between control siRNA and TRPM7 siRNA treatments (n = 5). <b>D.</b> increasing extracellular Ca²⁺ and Mg²⁺ has no effect on the proliferation of cortical astrocytes. Addition of Ca²⁺ and Mg²⁺ to growth medium has no effect on the proliferation of cortical astrocytes, and could not rescue the inhibition of astrocyte proliferation induced by silence of TRPM7 (*, <i>p</i> < 0.05, n = 4).</p