Silencing TRPM7 promotes growth/proliferation and nitric oxide production of vascular endothelial cells via the ERK pathway

Abstract

Aims: The presence and potential function of transient receptor potential melastatin 7 (TRPM7), a Ca2+-permeable non-selective cation channel of the TRP channel superfamily in human vascular endothelial cells, were examined.

Methods and results: Whole-cell patch-clamp recordings showed outward-rectifying currents in human umbilical vein endothelial cells (HUVECs), which was potentiated by removing the extracellular Ca2+ and Mg2+, but inhibited by non-specific TRPM7 blocker Gd3+ or 2-aminoethoxydiphenyl borate (2-APB). TRPM7 mRNA was detected in HUVECs by RT-PCR, but TRPM6, its closest homologue, was not. Silencing TRPM7 by small interfering RNA (siRNA) decreased the level of TRPM7 mRNA and the TRPM7-like current. Interestingly, knockdown of TRPM7 with siRNA or inhibition of TRPM7 function with 2-APB increased the phosphorylation of extracellular signal-regulated kinase (ERK) and enhanced growth/proliferation of HUVECs. This enhanced cell growth/proliferation was abolished by an inhibitor of the ERK signalling pathway. In addition to cell growth/proliferation, silencing TRPM7 also increased expression of nitric oxide synthase and nitric oxide production in an ERK pathway-dependent manner.

Conclusion: These observations suggest that TRPM7 channels may play an important role in the function of vascular endothelial cells.

Figure 1

TRPM7-like currents in HUVECs. (A) Representative currents elicited by voltage steps ranging from −100 to +100 mV with or without 8 mmol/L Mg-ATP in the pipette solution (n = 10 for 0 mmol/L Mg-ATP; n = 4 for 8 mmol/L Mg-ATP). (B) Time-dependent changes in the amplitude of TRPM7-like currents recorded at −80 and +80 mV in the absence or presence of Mg-ATP in the pipette solution (n = 7 for 0 mmol/L Mg-ATP; n = 4 for 8 mmol/L Mg-ATP). (C) Representative I–V relationship in the presence or absence of extracellular Ca²⁺/Mg²⁺, or following bath application of 10 µmol/L Gd³⁺. (D) Relative changes in the amplitude of TRPM7-like currents by Ca²⁺/Mg²⁺ removal, or by bath application of Gd³⁺ or 2-APB. n = 3–8. *P < 0.05, **P < 0.01.

Figure 2

TRPM7-siRNAs suppress the expression of TRPM7 and TRPM7-like current. (A) Total RNAs were isolated from indicated cells. Equal amounts of total RNA were reverse-transcribed and PCR-amplified using specific primers for TRPM6, TRPM7, or GAPDH. Expected molecular sizes of the fragments are 478, 399, and 723 bp, respectively. (B) RT–PCR analysis shows reduced expression of TRPM7 mRNA by TRPM7-siRNAs. HUVECs were untreated or treated with either control or TRPM7-siRNAs for 48 h. RNAs were extracted and then multiplex RT–PCR was performed. Bar chart shows densitometrically quantified relative ratio of TRPM7/GAPDH in HUVECs transfected with or without siRNAs (n = 3–4, **P < 0.01). (C) I–V relationship of TRPM7-like current in HUVECs transfected with control (left) or TRPM7-siRNA1 (right). Silencing TRPM7 inhibits TRPM7-like current and their potentiation by Ca²⁺/Mg²⁺ removal. Bar graph shows relative increase in the amplitude of TRPM7-like currents induced by Ca²⁺/Mg²⁺ removal in HUVECs transfected with either control (−) or TRPM7-siRNA1 (+). n = 8–9. **P < 0.01, control vs. TRPM7-siRNA1-treated cells.

Figure 3

TRPM7 silencing promotes growth/proliferation of HUVECs. (A) Photos taken at 72 h following transfection with control (left) or TRPM7-siRNA1 (right). Scale bar: 250 µm. (B and C) Normalized cell counting and LDH release for HUVECs treated with control or TRPM7-siRNAs for 72 h. n = 18–24. *P < 0.05, **P < 0.01 between control and siRNA-treated cells. (D) Cells were growth-arrested in 1% serum without supplement for 24 h and then allowed to grow in normal culture media for 72 h, followed by LDH assay. n = 12. **P < 0.01 between control and siRNA-treated cells. (E) Normalized LDH release for HMVECs treated with control or TRPM7-siRNA1 for 72 h. n = 20. *P < 0.05 between control and TRPM7-siRNA-treated cells.

Figure 4

ERK activation is involved in enhanced growth/proliferation of HUVECs by silencing TRPM7. (A) Representative immunoblots showing increased phospho-ERK (left) or phospho-MEK1/2 (right) in HUVECs 72 h following the transfection of the indicated siRNAs. Bar graphs show densitometrically quantified ratio of phospho-ERK/β-actin (left) and phospho-MEK1/2/β-actin (right) in HUVECs transfected with or without control or TRPM7-siRNAs (n=3, *P < 0.05, **P < 0.01, control siRNA vs. TRPM7-siRNA1-treated cells. (B) Immonoblotting showing the lack of effect on phospho-p38 MARK and phospho-JNK by TRPM7-siRNA1. Third lane shows control siRNA-treated cells incubated with 20 ng/ml TNFα (for 15 min) as a positive control. (C) Treatment of HUVECs with ERK inhibitor U0126 (10 or 30 µmol/L) attenuated or prevented enhancement of growth/proliferation by TRPM7-siRNA1. n = 7–11. **P < 0.01, control vs. TRPM7-siRNA1-treated cells, ^##P < 0.01, U0126 untreated vs. treated cells.

Figure 5

TRPM7 silencing enhances eNOS expression via ERK pathway. (A) RT–PCR (left) or immunoblotting (right) showing the effect of TRPM7-siRNA1 on the level of eNOS mRNA and protein in HUVECs. HUVECs were treated with control or TRPM7-siRNA1. 48 h after transfection, RT–PCR was performed using specific primers for TRPM7, eNOS, and GAPDH. For immunoblotting, eNOS protein level was examined 72 h after the transfection by immunoblotting with anti-eNOS antibody. (B) Treatment with ERK inhibitor U0126 reduced expression of eNOS and its enhancement by TRPM7-siRNA1. (C) TRPM7 silencing increased the production of NO as demonstrated by nitrite measurement. This increased production of nitrite was attenuated by 500 µmol/L l-NAME. n = 12–20. *P < 0.05, control vs. TRPM7-siRNA1-treated cells; ^#P < 0.05, l-NAME-treated vs. untreated cells. (D) Treatment of cells with l-NAME (100 and 1000 µmol/L) did not affect the enhancement of cell growth/proliferation by TRPM7-siRNA1. n = 9–11. **P < 0.01, control vs. TRPM7-siRNA1-treated cells.

Figure 6

Effect of 2-APB on ERK activity and growth/proliferation of HUVECs. (A) Treatment of HUVECs with 3 or 10 µmol/L 2-APB enhanced the growth/proliferation. n = 8–16. *P < 0.05, **P < 0.01, 2-APB-treated vs. untreated cells. Total LDH was analysed 72 h following the treatment with 2-APB. (B) Treatment of HUVECs with 10 µmol/L 2-APB for 48 h increased level of phospho-ERK protein. (C) Treatment of HUVECs with 2-APB (10 µmol/L) did not induce additional enhancement of the growth/proliferation in the presence of TRPM7-siRNA. n = 12. **P < 0.01, control vs. TRPM7-siRNA1-treated cells; ^##P < 0.01, 2-APB-treated vs. untreated cells.