TRPM7 regulates vascular endothelial cell adhesion and tube formation

Abstract

Transient receptor potential melastatin 7 (TRPM7) is a nonselective cation channel with an α-kinase domain in its COOH terminal, known to play a role in diverse physiological and pathological processes such as Mg2+ homeostasis, cell proliferation, and hypoxic neuronal injury. Increasing evidence suggests that TRPM7 contributes to the physiology/pathology of vascular systems. For example, we recently demonstrated that silencing TRPM7 promotes growth and proliferation and protects against hyperglycemia-induced injury in human umbilical vein endothelial cells (HUVECs). Here we investigated the potential effects of TRPM7 on morphology, adhesion, migration, and tube formation of vascular endothelial cells and the potential underlying mechanism. We showed that inhibition of TRPM7 function in HUVECs by silencing TRPM7 decreases the density of TRPM7-like current and cell surface area and inhibits cell adhesion to Matrigel. Silencing TRPM7 also promotes cell migration, wound healing, and tube formation. Further studies showed that the extracellular signal-regulated kinase (ERK) pathway is involved in the change of cell morphology and the increase in HUVEC migration induced by TRPM7 silencing. We also demonstrated that silencing TRPM7 enhances the phosphorylation of myosin light chain (MLC) in HUVECs, which might be involved in the enhancement of cell contractility and motility. Collectively, our data suggest that the TRPM7 channel negatively regulates the function of vascular endothelial cells. Further studies on the underlying mechanism may facilitate the development of the TRPM7 channel as a target for the therapeutic intervention of vascular diseases.

Keywords: HUVECs; TRPM7; adhesion; migration; tube formation.

Fig. 1.

Silencing transient receptor potential melastatin 7 (TRPM7) by specific siRNA in human umbilical vein endothelial cells (HUVECs). A: after TRPM7 siRNA or control siRNA was transfected to HUVECs for 72 h, total RNA was extracted and reverse transcribed to cDNA. Quantitative real-time PCR was performed to monitor mRNA level of TRPM7 in HUVECs. **P < 0.01 control vs. TRPM7 siRNA-treated cells (n = 3–6). B: representative currents elicited by voltage steps ranging from −100 to +100 mV in control siRNA- or TRPM7 siRNA1-transfected cells. C: current-voltage (I–V) relationship in control siRNA- or TRPM7 siRNA-transfected cells. D: densities of TRPM7-like current in cells transfected with control or TRPM7 siRNA cells. *P < 0.05, control siRNA- vs. TRPM7 siRNA1-treated cells (n = 18–32). E: bar graph showing intracellular Mg²⁺ levels evaluated at basal condition (0 mM Ca²⁺/1 mM Mg²⁺) by measuring the 340/380 nm ratio of Mag-Fura-2. HEK:TRPM7 cells were either untreated or treated with tetracycline (tet) for 48 h. HUVECs were treated with either control or TRPM7 siRNA1 for 72 h; n = 29–76 cells from 3–9 fields in 3–4 independent experiments. *P < 0.05 vs. control.

Fig. 2.

Silencing TRPM7 alters HUVEC morphology and adhesion to extracellular matrix. A: HUVECs were transfected with control siRNA or TRPM7 siRNA for 48–72 h and the representative images were captured to show the cell shape of HUVECs. Arrows indicate shape change of HUVECs with membrane filamentous extension (top right). Quantification of cell morphology change was calculated by measuring the average single cell surface area for 150 - 270 cells (bottom). Data were from at least 3–8 independent experiments. B: HUVECs transfected with siRNAs for 48–72 h were seeded to 96-well plates coated with Matrigel (1 mg/ml). Adhered cells were stained with crystal violet and the representative images were captured. Data were analyzed by measuring optical density of crystal violet extracted by 10% acetic acid at 595 nm (n = 3–5). *P < 0.05, **P < 0.01, control vs. TRPM7 siRNA1- or siRNA2-treated cells. Scale bar = 50 μm.

Fig. 3.

Silencing TRPM7 enhances HUVEC migration and tube formation. A: transwell assay was used for HUVEC migration. Representative images were captured and the migrated cells were counted. Data were from 4 independent experiments. Scale bar = 50 μm. *P < 0.05, control vs. TRPM7 siRNA1 or siRNA2-treated cells. B: wound healing assay was also used to measure HUVEC migration. The pictures of the same wound area were captured to show the wound area closure (scale bar = 250 μm). Wound healing rate was calculated by measuring closure area using NIH ImageJ software (n = 4–5). *P < 0.05, control vs. TRPM7 siRNA1 or siRNA2-treated cells. C: HUVECs were harvested after transfection with siRNAs for 48–72 h, seeded into 96-well plates (3 × 10⁴/well) precoated with Matrigel (5 mg/ml) and observed for 24 h. Representative images show the tube formation in control and TRPM7 siRNA group at 4-h time point (top). Tube length was quantified by NIH ImageJ software according to the pictures of tube formation (n = 3). Scale bar = 250 μm.*P < 0.05, control vs. TRPM7 siRNA1- or siRNA2-treated cells.

Fig. 4.

ERK signaling pathway was involved in morphology change and increased migration induced by silencing TRPM7. A: Western blotting was used to show MEK1/2 phosphorylation after transfection of control siRNA, TRPM7 siRNA1, or TRPM7 siRNA2 for 72 h (n = 4). B: morphology change of HUVECs treated with or without U0126 (a MEK inhibitor, 10 μM) for 72 h was observed. The representative images were captured, and the average single cell surface area for 150 cells was quantified. Data were from 4–10 independent experiments. C: migration of HUVECs treated with or without U0126 was investigated in transwell assay and the representative images were captured. Relative migration rate was calculated by normalization to control siRNA group without U0126 (n = 3–4). Scale bar = 100 μm. **P < 0.01, control vs. TRPM7 siRNA1- or siRNA2-treated cells. N.S., not significant vs. control. ##P < 0.01, DMSO vs. U0126.

Fig. 5.

Silencing TRPM7 does not change the expression of Orai1, stromal interaction molecule 1 (STIM1), and canonical TRP 3 (TRPC3). HUVECs were transfected with control siRNA or TRPM7 siRNA for 72 h. A: total RNA was extracted and reverse transcribed to cDNA. Quantitative real-time PCR was performed to monitor mRNA level of the indicated genes in HUVECs (n = 3). B: Western blotting was used to show protein levels of the indicated genes. Histogram indicates fold change of the indicated proteins normalized to β-actin (n = 3).

Fig. 6.

Silencing TRPM7 enhances phosphorylation of myosin light chain (MLC), which involves ERK pathway. A: Western blotting was used to show MLC phosphorylation after treatment with TRPM7 siRNA or control siRNA or untreated for 72 h. The phosphorylation of MLC was normalized to total MLC (n = 6). B: HUVECs transfected with control siRNA or TRPM7 siRNA were treated with U0126 (10 μM) for 72 h. Histogram indicates fold change of MLC phosphorylation normalized to total MLC (n = 5). *P < 0.05, control vs. TRPM7 siRNA1- or siRNA2-treated cells. N.S., not significant vs. control. #P < 0.05, DMSO vs. U0126.