Articles in PresS. Am J Physiol Cell Physiol (December 3, 2014). doi:10.1152/ajpcell.00275.2013

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TRPM7 regulates vascular endothelial cell adhesion and tube formation

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Zhao Zeng1,2, Koichi Inoue2, Huawei Sun2, Tiandong Leng2, Xuechao Feng2, Li Zhu1*,

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Zhi-Gang Xiong2*

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Key Lab of Thrombosis and Hemostasis, Jiangsu Key Lab of Preventive and translational

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Medicine for Geriatric Diseases, Soochow University, Suzhou, China

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Cyrus Tang Hematology center, Collaborative Innovation Center of Hematology, MOH

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Neuroscience Institute, Morehouse School of Medicine, Atlanta, GA, USA

*Co-corresponding authors. Li Zhu. Tel.: +86 (512) 65880899 Ext 509; Fax: +86 (512) 65880929; Email: [email protected]. Zhi-Gang Xiong. Tel.: +1 (404) 752 -8683; Fax: +1 (404) 752 -1041; Email: [email protected]; Abstract:

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Transient receptor potential melastatin 7 (TRPM7) is a non-selective cation channel

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with an α-kinase domain in its C-terminal, known to play a role in diverse physiological

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and pathological processes such as Mg2+ homeostasis, cell proliferation, and hypoxic

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neuronal injury. Increasing evidence suggests that TRPM7 contributes to the

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physiology/pathology of vascular systems. For example, we recently demonstrated that

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silencing TRPM7 promotes growth and proliferation and protects against hyperglycemia-

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induced injury in human umbilical vein endothelial cells (HUVECs). Here we

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investigated the potential effects of TRPM7 on morphology, adhesion, migration, and

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tube formation of vascular endothelial cells and the potential underlying mechanism. We

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showed that inhibition of TRPM7 function in HUVECs by silencing TRPM7 decreases

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Copyright © 2014 by the American Physiological Society.

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the density of TRPM7-like current and cell surface area, and inhibits cell adhesion to

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Matrigel. Silencing TRPM7 also promotes cell migration, wound healing, and tube

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formation. Further studies showed that extracellular signal regulated kinase (ERK)

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pathway is involved in the change of cell morphology and the increase in HUVEC

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migration induced by TRPM7 silencing. We also demonstrated that silencing TRPM7

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enhances the phosphorylation of myosin light chain (MLC) in HUVECs, which might be

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involved in the enhancement of cell contractility and motility. Collectively, our data

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suggest that TRPM7 channel negatively regulate the function of vascular endothelial

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cells. Further studies on the underlying mechanism may facilitate the development of

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TRPM7 channel as a target for the therapeutic intervention of vascular diseases.

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Key words: TRPM7; HUVECs; adhesion; migration; angiogenesis

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Introduction

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Vascular endothelial cells play an important role in the regulation of vascular tone,

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vascular permeability, angiogenesis, and vascular inflammatory response (12, 53). Those

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cells express a variety of ion channels, including TRP channels that constitute a large

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family including canonical (TRPC), vanilloid (TRPV), and melastatin (TRPM) sub

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families (73). At least 19 members among the 28 mammalian TRP channel isoforms have

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been identified in vascular endothelial cells (38, 73). These channels appear to be

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involved in diverse physiological/pathological functions.

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Transient receptor potential melastatin 7 (TRPM7), a member of TRPM subfamily, is a

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Ca2+ and Mg2+-permeant channel protein in possession of its own kinase domain (49, 57-

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59). It has been reported to be expressed in many types of cells including tumor cells,

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neurons, hematopoietic cells, granulocytes, leukocytes, and microglia (22, 27, 33, 45, 57,

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63). As one of two unique channel-kinases along with TRPM6, TRPM7 not only

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possesses the ion permeability but also phosphorylates bio-molecules such as annexin I,

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myosin IIA heavy chain, and m-calpain (14, 16, 66). Recent studies have demonstrated

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that TRPM7 plays an important role in a large number of physiological and

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pathophysiological processes including cell growth/proliferation, adhesion and migration,

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motility and movements, oxidative stress and anoxic neuronal death (1, 13, 15, 30, 40,

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56, 63, 66).

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TRPM7 has been shown to be expressed in human vascular system and to play a role in

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vascular functions (3, 4, 6, 25, 31, 52, 68, 69, 74, 76). For example, in vascular smooth

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muscle cells, TRPM7 modulates Mg2+ homeostasis (6, 25). In vascular endothelial cells,

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TRPM7 modulates cell growth and proliferation (4, 31). It was also reported that the level

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of TRPM7 expression in endothelial cells can be up-regulated by high glucose, oxidative

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stress and laminar shear stress (3, 68, 69). All of these studies suggest that TRPM7 might

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be involved in cardiovascular diseases involving endothelial dysfunction. However, the

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role of TRPM7 on adhesion and angiogenic activity of vascular endothelial cells remains

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to be elucidated. Here we investigated the effect of TRPM7 on these processes in

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HUVECs and explored the potential underlying mechanisms.

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Materials and Methods

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Regents and antibodies

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STIM1 and anti-TRPC3 antibodies were from Alamone Labs (Jerusalem, Israel). Anti-

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phospho-MLC, anti-total MLC, anti-phospho-MEK1/2, anti-total MEK1/2 antibodies and

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U0126 were purchased from Cell Signaling (Beverly, MA). Growth factor-reduced

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Matrigel was purchased from BD Biosciences (Bedford, MA). Protease inhibitors

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cocktail were purchased from Sigma (St. Louis, MO). Phosphatase inhibitors cocktail

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was from Roche (Indianapolis, IN).

Anti-β-actin antibody was purchased from Abcam (Cambridge, MA). Anti-Orai1, anti-

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Cell culture

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HUVECs were purchased from ATCC and grown in EGM-2 medium containing 2% fetal

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bovine serum (FBS) and trophic factors (Lonza, Walkersville, MD), as described in our

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previous studies (31, 32). In some experiments, HUVECs were cultured in endothelial

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basal medium (EBM) containing 10% or 2% FBS without trophic factors.

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Human embryonic kidney (HEK-293) cells, with inducible expression of human TRPM7

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channels (HEK:TRPM7 cells), were cultured in DMEM supplemented with 10% FBS

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(30). For the induction of TRPM7, the cells were treated with 1 g/ml of tetracycline.

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RNA interference

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Knockdown of TRPM7 experiments were performed as described (31, 68). Briefly,

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dsRNAs 406-426 (TRPM7 siRNA1) and 450-470 (TRPM7 siRNA2) of TRPM7

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(NM_017672.4) were synthesized from Invitrogen. Cells were transfected with 50 nM

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siRNA using transfection reagent lipofectamineTM RNAiMAX (Invitrogen) according to

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the manufacturer’s instructions. Non-targeting siRNA (Invitrogen) was used as a negative

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control.

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Real time PCR

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Total RNAs were extracted with RNA purification kit (Qiagen, Valencia, CA) and

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transcribed to cDNA using superscript® First-strand synthesis system (Invitrogen). Real

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time PCR was performed using SYBR® Green supermix (Bio-Rad, Richmond, CA) in

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C1000TM Thermal cycler (Bio-Rad) as described (72). 1) 95 °C for 10 min, 2) 95 °C for

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10 s, 3) 61 °C for 10 s, 4) 72 °C for 15 s, plate read, 5) go to 2, 40 more times, 6) 95 °C

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for 10 s, 7) Melt curve 65 °C to 95 °C: increment 0.5 °C for 5 s, plate read. Primer sets

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were described in the “Table 1”.

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Electrophysiology

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Whole-cell voltage-clamp recordings were performed as described (31). Three to four

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days after transfection, cells were set on the stage of an inverted microscope (Ti-S;

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Nikon) and superfused at room temperature with an extracellular solution containing (in

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mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 10 glucose, and 20 HEPES (pH 7.4 with NaOH, 320–

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335 mOsm). Patch electrodes were fabricated from borosilicate capillary tubing of 1.5

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mm diameter (WPI) using a vertical puller (PP-83, Narishige). The electrode resistance

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ranged from 4 to 5 MΩ when filled with the intracellular solution (see below).

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For current–voltage (I–V) relationship, voltage steps ranged between -100 and +100 mV

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from a holding potential of -60 mV were applied. All recordings were performed at least

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5 min after establishing the whole-cell configuration. Membrane currents were recorded

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using an Axopatch 200B amplifier. Data were filtered at 2 kHz and digitized at 5 kHz by

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using a Digidata 1440A data-acquisition system and pClamp 10 software (Axon

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Instruments). Pipette solution contained (in mM) 140 Cs-methanesulfonate, 8 NaCl, 4.1

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CaCl2, 10 EGTA, 5 tetraethyl-ammonium chloride, 2 Na2-ATP and 10 HEPES (pH 7.3,

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with CsOH).

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Mg2+ imaging

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The intracellular Mg2+ levels were examined using Mag-Fura-2 (Invitrogen). Cells were

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incubated with 5 μM Mag-Fura-2-AM for 30 min at 37 °C in Ca2+-free extracellular

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solution, which contains (in mM) 140 NaCl, 5.4 KCl, 1 MgCl2, 10 glucose, and 20

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HEPES (pH 7.4 with NaOH, 320–335 mOsm), followed by de-esterification of the dye

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for another 30 min at room temperature. The coverslips containing dye-loaded cells were

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held in a recording chamber placed on the stage of an inverted microscope (Eclipse

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TE2000-U; Nikon). Mag-Fura-2 was excited at wavelengths of 340 nm and 380 nm.

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Fluorescence was detected with a 40× objective lens (Super Fluor ×40, numerical

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aperture = 0.90; Nikon) and a CCD camera (Cool- SNAP ES2; Photometrics). Digitized

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images were acquired, stored, and analyzed in a personal computer controlled by Axon

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Imaging Workbench software (INDEC Biosystems). The shutter and filter wheel

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(Lambda 10-3, Sutter Instrument) were also controlled by the software to allow timed

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illumination of cells at either 340 or 380 nm excitation wavelengths. Fluorescence was

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detected at an emission wavelength of 510 nm. Ratio images of 340/380 were analyzed

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by averaging pixel ratio values in circumscribed regions of randomly selected 7-10 cells

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within the field of view.

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Adhesion assay

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Ninety-six-well plates were coated with Matrigel (BD Biosciences, 1 mg/ml) overnight at

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4 °C and washed with sterilized H2O. HUVECs were harvested after transfection with

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siRNA for 48 h and seeded into 96-well plates (2-4 × 104 /well). After incubation for 1 h

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at 37 °C, the non-adherent cells were washed away with PBS. Adhered cells were fixed

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with 4% paraformaldehyde and stained with 0.5% crystal violet for 10 min. Three to five

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random fields were captured as representative images using light microscope (4×)

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connected with Nikon camera (D90). Crystal violet was extracted with 10% acetic acid,

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and optical density was measured at 595 nm.

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Transwell assay

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Transwell assays were performed using a modified Boyden chamber (8-μm pores,

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Corning 3422). After transfected with siRNA for 48 h and starved for 24 h, HUVECs (3-

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4 × 105 cells/ml) in 100 μl of serum-free medium were plated in the upper chamber,

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whereas 670 μl of medium containing 10% FBS were added to the lower chamber. After

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incubation for ~18 h in an incubator, cells that did not migrate through the pores were

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scraped off thoroughly with Q-Tips. Attached cells were fixed with 4%

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paraformaldehyde for 10 min and stained with 0.5% crystal violet/methanol for 10 min.

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Pictures of six to nine randomly picked fields for each group were taken by Nikon invert

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light microscope (10×) and the migrated cells were counted. Cells under different

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treatments were normalized to the control value and expressed as fold change in

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migration.

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Wound healing assay

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HUVECs transfected with siRNA for 48 h were seeded to 24-well plates at a density of 1

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× 105 cells/well. After adhesion to the plate, cells were starved with serum-free medium

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for 24 h and the cell monolayer was scratched with 1000-μl tip. After washing three times

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with serum-free medium, cells were incubated with EBM-2 medium containing 2% FBS

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for 24 h at 37 °C. Pictures were taken from the field of wound area with Nikon camera

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under light microscope (4×) and analyzed using NIH Image J software. Wound healing

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rate was calculated as: % wound closure = [(Area of original wound – Area of wound

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after healing)/Area of original wound] × 100%.

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Tube formation assay

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Tube formation on Matrigel was evaluated as a model of in vitro angiogenesis. Briefly,

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60 μl of EBM-2 containing 5 mg/ml Matrigel was added into 96-well plates and

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incubated at 37 °C for 30 min. HUVECs were harvested after tranfection with siRNA for

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48-72 h, and then seeded into the 96-well plates pre-coated with Matrigel at a density of 3

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× 104/well. Capillary tube structures were observed and the representative images were

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captured with an inverted microscope (4×) equipped with Nikon camera. Tube length was

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quantified using NIH Image J software.

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Immunoblotting

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Immunoblotting was performed as described (30, 31). Briefly, cells were lysed in RIPA

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buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium

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deoxycholate, 0.1% SDS, protease inhibitor and phosphatase inhibitor cocktail). After

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centrifugation at 13,000g at 4 °C for 10 min, the lysates were collected. The aliquots were

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mixed with laemmli sample buffer and boiled at 95 °C for 10 min. Proteins were

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separated by 10% SDS-PAGE, transfered to PVDF membranes, and probed with

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antibodies against phospho-MLC (1:500), total-MLC (1:500), β-actin (1:2000), TRPC3

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(1:200), Orai1 (1:200), STIM1 (1:500), total MEK1/2 (1:250) and phospho-MEK1/2 (1:

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1000) followed by HRP-conjugated secondary antibodies (1:2000). The signals were

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visualized by chemiluminescence using an ECL kit (Millipore).

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Statistical analysis

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All data were expressed as means ± SEM. Statistical analyses were done by Student t test

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or one-way analysis of variance on data collected from at least 3 independent

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experiments. Dunnett’s test was used to compensate for multiple experimental

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procedures. The differences were considered statistically significant when P < 0.05.

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Results:

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In order to investigate the effect of TRPM7 on HUVEC cytoskeleton reorganization,

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adhesion, migration, and tube formation, specific siRNAs targeting nucleotides 406-426

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and 450-470 of human TRPM7 (NM_017672.4) were synthesized according to our

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previous study (31). Data of quantitative real time PCR (Fig. 1A) showed that the level of

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TRPM7 mRNA in HUVECs transfected with TRPM7 siRNAs for 72 h was decreased as

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compared to cells transfected with non-targeting control siRNA (TRPM7 siRNA1; 39.67

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± 12.44 % and TRPM7 siRNA2; 29.49 ± 2.74 % of control, n=3-6, P