Regulation and function of TRPM7 in human endothelial cells: TRPM7 as a potential novel regulator of endothelial function

Abstract

TRPM7, a cation channel of the transient receptor potential channel family, has been identified as a ubiquitous magnesium transporter. We here show that TRPM7 is expressed in endothelial cells isolated from the umbilical vein (HUVEC), widely used as a model of macrovascular endothelium. Quiescence and senescence do not modulate TRPM7 amounts, whereas oxidative stress generated by the addition of hydrogen peroxide increases TRPM7 levels. Moreover, high extracellular magnesium decreases the levels of TRPM7 by activating calpains, while low extracellular magnesium, known to promote endothelial dysfunction, stimulates TRPM7 accumulation partly through the action of free radicals. Indeed, the antioxidant trolox prevents TRPM7 increase by low magnesium. We also demonstrate the unique behaviour of HUVEC in responding to pharmacological and genetic inhibition of TRPM7 with an increase of cell growth and migration. Our results indicate that TRPM7 modulates endothelial behavior and that any condition leading to TRPM7 upregulation might impair endothelial function.

Figure 1. TRPM7 amounts in quiescent, proliferating and senescent HUVEC.

(A) HUVEC were either cultured in complete growth medium (proliferating cells, P) or in starvation medium (quiescent cells, Q) for 2 days. (B) HUVEC were lyzed 2 days before (−2, preconfluent), 2 days after reaching confluence (+2, post-confluent) and on the day (T0) they reached confluence. (C) Subconfluent HUVEC were starved as described for 48 h. Then the cells were exposed for different times to complete growth medium (CGM) to induce the re-entry in the cell cycle. (D) High and low PD HUVEC were analyzed for TRPM7 levels. Cell extracts were utilized for Western blot using anti-TRPM7 antibodies. Actin was used to show that equal amounts of proteins were loaded per lane.

Figure 2. TRPM7 expression in endothelial cells cultured in different concentrations of Mg.

(A) RT-PCR was performed on RNA extracted from HUVEC exposed to different concentrations of Mg for 6 and 24 h using primers designed on TRPM7 sequence. (B) HUVEC were cultured in media containing different concentrations of Mg for 6 and 24 h. After lysis cell extracts were utilized for Western blot using anti-TRPM7 antibodies. In A and B actin was used as a control of loading.

Figure 3. TRPM7 amounts in HUVEC treated with protease inhibitors, H₂O₂ or the antioxidant trolox.

(A) Effects of bafilomycin, MG132 and calpeptin on TRPM7 total amounts. HUVEC were cultured in 5.0 mM Mg in the presence of MG132 (5 µM), bafilomycin (100 nM) or calpeptin (5 and 10 µg/ml) for 24 h. (B) Effects of H₂O₂ (100 µM) on TRPM7 total amounts in HUVEC. The cells were lysed 30 min or 24 h after adding H₂O₂. (C) Effects of trolox (10 µM) on TRPM7 total amounts in HUVEC. HUVEC cultured in 0.1 or 1.0 mM Mg were treated with trolox for 24 h. Western blot was performed as described above. Actin was used to show that equal amounts of proteins were loaded per lane.

Figure 4. Effect of TRPM7 inhibition on HUVEC proliferation.

(A) Western blot was performed on cell extracts of HUVEC transfected for 24, 48 and 72 h with shRNA against TRPM7. (B) HUVEC were treated with 2-APB and Co(III)hexaammine or transfected with a shRNA against TRPM7. After 72 h, HUVEC were harvested by digestion with trypsin, and viable cells were counted using a Burker chamber. Data refer to three separate experiments in triplicate ± standard deviation. **p<0.01, ***p<0.001.

Figure 5. Effect of TRPM7 inhibition on HUVEC migration and NO production.

HUVEC were treated with 2-APB and Co(III)hexaammine or transfected with a shRNA against TRPM7 for 24 h. (A) The cells were wounded and migration was allowed for 10 h. Microphotographs show the result of a representative assay (20×magnification). (B) The wound area was calculated by ImageJ software and expressed using an arbitrary value scale to quantify the results. The values are expressed as the mean of 3 different experiments ± standard deviation. *p<0.05, **p<0.01. (C) NOS activity was measured by using the Griess method.