Epidermal growth factor signaling through transient receptor potential melastatin 7 cation channel regulates vascular smooth muscle cell function

Abstract Objective: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR–TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. Conclusions: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.


Immunoprecipitation
VSMCs were harvested and homogenized in HEPES buffer (130 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM d-glucose and 20 mM HEPES, pH 7.4), supplemented with PMSF 1 mM, pepstatin A 1 μg/ml, leupeptin 1 μg/ml, aprotinin 1 μg/ml (Sigma-Aldrich), sodium fluorate 10 mM (AnalaR Normapur; VWR International), and sodium orthovanadate 1 mM (Alfa Aesar). A total of 200 μl of VSMCs lysate containing 400 μg of protein was mixed with 2 μl of primary antibody (1:100) and the reaction mixture was rotated in a Stuart SB3 Rotator (Camlab Ltd) at a gentle speed overnight at 4 • C. The following day, 15 μl of Protein A and 15 μl of Protein G (Santa Cruz Biotechnology) were added to the reaction mixture followed by rotation at 4 • C for 4 h. The immunoprecipitation (IP) complex was then washed twice in lysis buffer followed by centrifugation at 14000 rpm for 30 s. The pellet was collected and prepared for immunoblotting.

Measurement of intracellular Mg 2+
Intracellular Mg 2+ levels in VSMCs were measured using the specific fluorescent probe Magnesium Green AM (Thermo Fisher, cat.#:M3735) as we previously described [11]. Briefly, VSMCs were kept in Ca 2+ /Mg 2+ -free HEPES buffer (150 mM NaCl, 5 mM KCl, 10 mM d-glucose and 20 mM HEPES) and incubated with 5 μM Magnesium Green for 30 min at 37 • C in dark and in constant gentle agitation to prevent cell adhesion. Subsequently, the stained cells were washed twice using phosphate-buffered saline (PBS) by centrifugation at 1200 rpm for 3 min at room temperature. The resulting cell pellet was resuspended in HEPES buffer with 1 mM Mg 2+ (150 mM NaCl, 5 mM KCl, 10 mM d-glucose, 20 mM HEPES and 1 mM MgCl 2 ). Cells were left at room temperature for 15 min to allow complete de-esterification of intracellular AM esters, followed by stimulation with EGF (50 ng/ml) for 5 min. In some studies, inhibitors such as gefitinib (1 μM), NS8593 (40 μM), apamin (1 μM), PP2 (10 μM) and 2-APB (30 μM) were added 30 min prior to EGF stimulation. Fluorescent signals of Magnesium Green were acquired using Flow Cytometry (FACS Canto II, BD Biosciences) and data were analyzed by FlowJo software (TreeStar, Ashland, U.S.A.).

Cell migration
VSMCs migration was assessed using the scratch-wound assay, a two-dimensional (2D) in vitro technique [40]. Confluent VSMCs cultured in 12-well plates were rendered quiescent overnight using DMEM with 0.5% FBS. The following day, a sterile pipette tip was used to scratch the cell monolayer, making a straight 'wound' with equal width. The medium was then replaced with fresh serum-free DMEM to remove floating cells and reduce the effects of cell proliferation. At zero-hour (0 h) time, three photos were taken from each well using a 10× objective (EVOS XL Core, Thermo Fisher). Cells were then incubated with EGF (50 ng/ml) in the presence and absence of inhibitors including gefitinib (1 μM), NS8593 (40 μM), 2-APB (30 μM) and PD98059 (20 μM). After 20 h, three photos were taken in the same areas for each well. Images were collected and analyzed using ImageJ software (National Institutes of Health, Bethesda, U.S.A.). Cell migration was calculated as the percentage of wound healing closure at 20 h relative to 0 h time point.

Proliferation assay
The cell tracking dye carboxyfluorescein succinimidyl ester (CFSE; cat.#: C34554, Thermo Fisher) was used in the proliferation assay. Following each cell division, the fluorescein-tagged cellular molecules are split evenly in two daughter cells, and cell proliferation can be assessed by measuring CFSE fluorescence. Briefly, isolated VSMCs were incubated with 5 μM CFSE in 1 ml of PBS containing 1% (v/v) FBS for 30 min at 37 • C. After incubation, CFSE-labeled cells were washed twice using DMEM, and then plated at ∼30% confluence in six-well plates using DMEM with 10% FBS. The following day, medium was replaced with DMEM with 5% FBS and cells were then cultured for 72 h. During this period, cells were treated with EGF (50 ng/ml) in the presence and absence of inhibitors including gefitinib (1 μM), NS8593 (40 μM), 2-APB (30 μM) and PD98059 (20 μM). After 72 h, cells were harvested in PBS containing 1% FBS and CFSE fluorescence was detected with an FITC channel, using Flow Cytometry (FACS Canto II, BD Biosciences). Data were analyzed using the FlowJo software (TreeStar, Ashland, U.S.A.).

Histology
Aortic segments from WT and TRPM7 +/ kinase mice were fixed in 10% buffered-formalin and embedded in paraffin. Sections (5-μm-thick) were stained with Hematoxylin and Eosin (HE). Briefly, aorta sections were deparaffinized and rehydrated in two 7-min changes of Histo-Clear (Fisher Scientific Ltd) and graded ethanol solutions (100, 95 and 70%, 7 min each), followed by 7 min in distilled water. Next, nuclei were stained with Harris' modified Hematoxylin (Cell Pathology Ltd) for 2 min and then rinsed in running tap water for 5 min followed by 30 s in 70% ethanol. Cytoplasmic structures were stained using Eosin Y solution (Sigma-Aldrich) for 2 min. Consequently, tissue sections were rinsed in graded ethanol solution (95% for 30 s, 100% for 1 and 7 min separately), followed by two 5-min changes in Histo-Clear. Tissue sections were then mounted with glass coverslips using DPX non-aqueous mounting medium (Merck Millipore). Images were acquired using a light microscope (ZEISS) with 20× and 40× objective and were analyzed using ImageJ software.

Live-cell imaging of intracellular TRPM7 movement
HEK-293 expressing yellow fluorescent protein (YFP)-tagged WT mouse TRPM7 (WT mTRPM7-YFP) [41]. The transient transfected HEK-293 cells were maintained in DMEM containing 10% FBS. After EGF (50 ng/ml) treatment, fluorescent signal of TRPM7 in HEK-293 cells were recorded for 15 min on an inverted epifluorescence microscope (Axio Observer Z1 Live-Cell imaging system, ZEISS) at excitatory wavelength of 490 nm and emission of 535 nm. Images were quired and analyzed by Zen Pro software (ZEISS).

Proximity ligation assays
VSMCs from WKY were seeded on cover glasses (13 mm, Thickness No.0; VWR) at a density of 5 × 10 4 cells per well in six-well plates. The following day, cells were stimulated with EGF (50 ng/ml) for 5 min with or without 30 min pre-treatment of inhibitors. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, and cell membranes were then stained by wheat germ agglutinin (WGA; cat.#: w6748; Invitrogen) for 25 min, followed by permeabilization using 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 10 min at room temperature. Cells were washed with Buffer A (0.01 M Tris-Base, 0.15 M NaCl, 0.05% Tween-20, pH 7.4) and blocked using Blocking Buffer for 1 h at 37 • C, followed incubation with primary antibodies EGFR (cat.#: 2646S; Cell Signaling; 1:100) and TRPM7 (cat.#: MA5-27620; Invitrogen; 1:100) overnight at 4 • C. The following day, proximity ligation assay (PLA) was performed according to the manufacturer's protocol using the Duolink Detection Kit (Sigma-Aldrich). Briefly, cells were washed using Buffer A and incubated with Duolink ® In Situ PLA ® Probe Anti-Mouse PLUS (cat.#: DUO92001) and Duolink ® In Situ PLA ® Probe Anti-Rabbit MINUS (cat.#:DUO92005) at 37 • C for 1 h to label TRPM7 and EGFR, respectively. Circular DNA molecules were formed in Ligation Buffer (working dilution 1:5) containing DNA Ligase (1:40) at 37 • C for 30 min, and were amplified in Amplification Buffer (1:5) containing the rolling circle polymerase (1:80) at 37 • C for 100 min. Coverslips were mounted with Duolink ® In Situ Mounting Medium with DAPI (cat.#: DUO82040; Sigma-Aldrich). Images were acquired using a laser-scanning confocal microscope (LSM 5 PASCAL ZEISS) under an oil immersion objective (63×/1.4 NA). In order to detect all PLA signals, a series of Z-stack images were collected and were analyzed by ImageJ software.

Statistical analysis
Results were expressed as mean + − standard error of the mean (SEM). Statistical analysis was conducted using Graph-Pad Pad 5.0 (GraphPad Software Inc, San Diego, CA). Unpaired and two-tailed t test or one-way analysis of variance (ANOVA) with Dunnett's or Tukey's post-test were used as appropriate. Results of statistical tests with P-value less than 0.05 were considered significant.

EGF regulates TRPM7 but not TRPM6 and SLC41A1 through EGFR and c-Src dependent mechanisms in rVSMCs
rVSMCs express EGFR and TRPM7 ( Figure 1, Supplementary Figure S1A,B). EGF stimulation for 24 h increased TRPM7 expression in rVSMCs, an effect that was attenuated in cells pretreated with gefitinib and PP2 ( Figure 1A). EGF had no effect on expression of other Mg 2+ channels and transporters like, TRPM6 and SLC41A1 (Supplementary Figure S1C,D).
To determine whether EGF influences the activity of TRPM7-kinase, we assessed the autophosphorylation site at the Ser 1511 domain, which is critically involved in TRPM7-kinase activation and signaling [39,42]. As shown in Figure  1B, EGF induced a rapid increase (within 5 min) in phosphorylation of TRPM7. This was significantly blunted by gefitinib and PP2 ( Figure 1B).

EGF influences VSMC Mg 2+ but not Ca 2+ transients through TRPM7-dependent pathways in rVSMCs
As TRPM7 is a divalent cation channel important in the regulation of Mg 2+ and Ca 2+ in VSMC, we investigated its role in EGF-induced changes in intracellular levels of Ca 2+ and Mg 2+ . In rVSMCs, EGF stimulation increased Ca 2+ transients, which was reduced by gefitinib and 2-APB ( Figure 1C,D). NS8593, a more selective TRPM7 inhibitor [43], did not affect Ca 2+ influx induced by EGF. Intracellular Mg 2+ levels were increased in rVSMCs stimulated with EGF (5 min), an effect that was reduced by gefitinib, 2-APB and NS8593 ( Figure 1E). Since NS8593 is also an inhibitor of SK channels [43], we used the selective SK channel inhibitor apamin as a control, which failed to change EGF effects on [Mg 2+ ] i .

EGF-induced phosphorylation of ERK1/2 involves c-Src and TRPM7
MAPKs, such as ERK1/2 and c-Src signaling are important downstream targets of EGF. EGF-induced phosphorylation of c-Src in rVSMCs. To evaluate the role of c-Src and TRPM7 in this pathway, ERK1/2 phosphorylation was assessed in EGF-stimulated rVSMCs in the presence of PP2, gefitinib, and TRPM7 inhibitors NS8593 and 2-APB. As shown in Figure 1F, EGF significantly increased phosphorylation of ERK1/2 in rVSMCs. These effects were blunted when EGFR, c-Src and TRPM7 were inhibited.

EGF enhances interaction between EGFR and TRPM7 in a c-Src-dependent manner
As shown in Figure 2A, EGFR was detected in rVSMCs when TRPM7 was immunoprecipitated. The association between EGFR and TRPM7 was further confirmed when TRPM7 was detected after EGFR IP. TRPM7-EGFR interaction was also determined by confocal microscopy showing co-localization in the cell membrane ( Figure 2B) and by PLA, using specific primary antibodies to EGFR and TRPM7. Figure 2C,D show that in basal conditions, there are PLA positive signals, indicating interaction between EGFR and TRPM7 in the cell membrane. PLA positive signals were increased by EGF stimulation, and reduced by gefitinib and PP2 ( Figure 2C,D).

EGF/EGFR does not influence TRPM7 trafficking in HEK-293 cells
Previous studies in kidney cell lines demonstrated that EGF promotes TRPM6 trafficking from the cytosol to the cell membrane [20]. Having demonstrated that TRPM7 is cell membrane-associated, we questioned whether EGF influences TRPM7 trafficking, as reported for TRPM6 in kidney cells. Using YFP-TRPM7, HEK-293 cells we assessed effects of EGF stimulation on TRPM7 mobilization. As shown in Supplementary Figure S2A,B and Supplementary Video S1 EGF did not influence TRPM7 translocation to the cell membrane, at least for the time period studied (10 min).

EGF promotes VSMCs migration and proliferation through TRPM7 and ERK1/2
Considering that VSMC proliferation and migration are key processes involved in vascular physiology, we assessed the importance of TRPM7 in these functional effects mediated by EGF. Using the CFSE proliferation assay, we found that EGF induced proliferation of rVSMCs in an EGFR-TRPM7-ERK1/2-dependent manner, since EGF stimulated cell growth, effects that were inhibited by gefitinib, TRPM7 inhibitors and the ERK1/2 inhibitor, PD98059 ( Figure  3A). To further assess the functional significance of EGF-TRPM7-ERK1/2, we examined VSMC migration using the scratch-wound assay. As shown in Figure 3B, EGF significantly enhanced rVSMCs migration. This was inhibited by gefitinib, NS8593, 2-APB and PD98059.

EGFR and c-Src are regulated by TRPM7-dependent mechanisms
Having demonstrated a feedforward loop between EGF/EGFR/c-Src and TRPM7 where interactions between EGFR and TRPM7 may be crucial for EGF signaling, we questioned whether TRPM7 plays a role in regulating vascular EGFR and c-Src. To address this, we evaluated the expression and phosphorylation of EGFR, c-Src and ERK1/2 in VSMCs and intact vascular tissue from mice deficient in TRPM7 ( Figure 4A-C). TRPM7 +/ kinase mice, characterized by reduced expression and phosphorylation of TRPM7 (Supplementary Figure S3) had significantly reduced expression and phosphorylation of EGFR and c-Src.

Reduced phosphorylation of EGFR is associated with vascular remodeling in TRPM7-deficient mice
EGF-induced proliferation and migration of VSMCs are major factors involved in vascular morphology and structure. We investigated the effects of TRPM7 deficiency on EGFR expression and vessel morphology by studying aorta from TRPM7 +/ kinase mice. These processes were associated with significant changes in vascular morphology as evidenced by significantly reduced vascular thickness in TRPM7 +/ kinase mice ( Figure 4D,E). Further demonstrating a critical role for EGFR and TRPM7 in vascular homeostasis, we found that expression of PCNA, a pro-prolifeartive marker and Notch3, a cell surface receptor expressed in VSMCs critically involved in vascular structure and development [44], was reduced in mVSMCs and aortas from TRPM7 +/ kinase mice (Supplementary Figure S4A-C).
We also examined aortae from TRPM7 R/R mice, another model of TRPM7 deficiency. As shown in Supplemental Figure S5, aortae from TRPM7 R/R mice exhibited reduced phosphorylation of EGFR and ERK1/2.

EGF/EGFR signaling and TRPM7 in hVSMCs
Next, we investigated whether the novel EGF-TRPM7 pathway observed in cells and tissues from rodents is also evident in humans. We confirmed that primary hVSMCs express TRPM7 and EGFR ( Figure 5A). EGF stimulation (5 min) induced a significant increase in [Mg 2+ ] i and ERK1/2 phosphorylation, effects that were attenuated by gefitinib, NS8593 and 2-APB ( Figure 5B,C). Long-term stimulation (5 h) with EGF increased TRPM7 expression, which was abolished by gefitinib ( Figure 5D). To test the functional significance of these events, we assessed EGF-induced migration of hVSMCs. As shown in Figure 5E, EGF stimulation significantly increased migration of hVSMCs. These responses were reduced by gefitinib, PP2, NS8593, 2-APB and PD98059, demonstrating that EGF-induced migration of hVSMC involves c-Src-TRPM7-ERK1/2-dependent pathways.

Discussion
Our study identifies TRPM7 as a signaling target of EGF in VSMCs. In particular we demonstrate that (i) EGF/EGFR induces phosphorylation of TRPM7, important in VSMC [Mg 2+ ] i but not [Ca 2+ ] i homeostasis, (ii) EGFR and TRPM7 physically interact on the cell membrane in a c-Src-dependent manner, (iii) EGF/EGFR-mediated activation of TRPM7 involves c-Src and (iv) EGF/EGFR/c-Src signaling promotes VSMC proliferation and migration through TRPM7-regulated ERK1/2-dependent processes. Moreover, we demonstrate that TRPM7 is both downstream and upstream of EGFR, suggesting that EGFR itself is regulated by TRPM7 in a feedforward manner. Together our data define a novel EGF signaling pathway involving c-Src and TRPM7 and show functional interplay between EGFR and TRPM7, important in the regulation of vascular homeostasis.
Vascular effects of EGF are mediated via well-defined signaling pathways involving MAPKs, phosphoinositide-3 kinase (PI3K), PLC-γ, c-Src and transactivation by G protein-coupled receptors [16]. Here we advance the field by delineating a role for TRPM7. This phenomenon seems to be highly regulated and cell-specific, because in VSMCs EGF did not influence TRPM6 or the other Mg 2+ transporter SLC41A1, but increased activity of TRPM7, whereas in renal tubule epithelial cells, EGFR activation stimulates TRPM6 but not TRPM7 [20].
The molecular significance of TRPM7 as a downstream target of EGF in the vascular system is still unclear but likely relates to its role as a cation channel, since EGF influences cellular Ca 2+ and Mg 2+ homeostasis, critically involved in VSMC regulation [8,45,46]. EGF signaling through Ca 2+ , a pivotal second messenger in VSMCs, has been well described [16], but there is a paucity of information about EGF and [Mg 2+ ] i . The first evidence suggesting a link between EGF and Mg 2+ homeostasis derived from clinical studies. Individuals with EGF mutations were shown to have associated isolated recessive renal hypomagnesemia [29] and cancer patients treated with the EGFR inhibitor cetuximab have reduced serum Mg 2+ levels that normalize upon therapy withdrawal [25,26]. Our findings in rat and human VSMCs further support the notion that EGF influences intracellular cation homeostasis. This seems to be more important for Mg 2+ than Ca 2+ because the TRPM7 inhibitor NS8593 attenuated Mg 2+ but not Ca 2+ responses, whereas the non-specific inhibitor of TRP channels 2-APB [47] reduced both Ca 2+ and Mg 2+ transients. Corroborating our findings others showed that in cancer cells EGF influences Ca 2+ in a TRPM7-independent manner probably through other TRP channels [48]. To explore potential molecular mechanisms whereby EGF influences TRPM7, we focused on c-Src, a key downstream kinase of EGFR activation [49,50]. In particular, PP2, a selective Src inhibitor, decreased activity and expression of TRPM7 in VSMC.
To further explore the relationship between EGF signaling and TRPM7, we employed a multidisciplinary approach of co-IP, live-cell imaging, and PLAs in rodent and hVSMCs, vascular tissue from TRPM7-deficient mice and TRPM7-transfected HEK cells. PLA is a powerful technique to detect, localize and quantify protein-protein interaction, which in combination with co-IP and co-localization experiments, clearly indicated EGFR-TRPM7 interaction in VSMCs. Molecular processes regulating this interaction are unclear but may depend on phosphorylation of the respective partner proteins, involvement of adaptor molecules that promote physical association and localization in subcellular compartments. TRPM7 localizes in intracellular vesicles and traffics to the membrane [51]. In our study TRPM7 clearly co-localized with EGFR on the cell membrane in VSMCs. However, in TRPM7-expressing HEK cells TRPM7 was located primarily in intracellular membrane compartments, similar to what has previously been shown for renal TRPM6 [20]. Hence TRPM7 trafficking may be differentially regulated in a cell-specific manner.
The interplay between EGFR and TRPM7 seems to be circuitous because EGFR is both upstream and downstream of TRPM7. This is supported by the findings that gefitinib inhibited phosphorylation of TRPM7, while TRPM7 deficiency was associated with reduced phosphorylation of EGFR and its downstream target c-Src. Of significance, we focused on phosphorylation of c-Src at tyrosine 416 (Y416), which is associated with high kinase activity, and phosphorylation of EGFR at Tyr 845 (Y845), a c-Src-dependent phosphorylation site [49,50]. Hence EGF/EGFR-induced activation of TRPM7 influences EGFR phosphorylation and may represent a compensatory mechanism to maintain [Mg 2+ ] homeostasis and EGF signaling in VSMC, especially in pathological conditions associated with abnormal EGF and/or TRPM7 regulation.

Figure 6. Schematic diagram demonstrating the novel signaling pathway involving EGFR-TRPM7 interaction in VSMCs
EGFR activation by EGF results in c-Src activation, which is required for TRPM7 activity (phosphorylation and expression) contributing to Mg 2+ homeostasis and interaction of EGFR and TRPM7 in the cell membrane. This signaling pathway is required for activation of ERK1/2. Cross-talk between EGF-EGFR and TRPM7 in VSMCs regulates cell migration and proliferation and may play an important role in vascular homeostasis.
A unique feature of TRPM7 is that is has dual properties functioning as an ion channel and as a cytosolic kinase [1,2,4]. It is therefore not surprising that in addition to regulating Mg 2+ influx, it participates in downstream signaling and cell function. TRPM7 influences tyrosine kinases, including EGFR and c-Src, as we demonstrate here, as well as MAP kinases [1,4]. Of the MAP kinases, ERK1/2 is especially important in EGF signaling in VSMCs, since it mediates cell growth, migration and contraction [16]. Our study suggests that EGF/EGFR stimulates ERK1/2 signaling through TRPM7-dependent pathways, since pharmacological and genetic inhibition of TRPM7 was associated with reduced EGF-induced ERK1/2 phosphorylation. Functionally, these processes influence VSMC proliferation and migration, since pharmacological inhibition of EGF, c-Src, ERK1/2 and TRPM7 attenuated EGF-stimulated responses. Previous studies demonstrated a role for TRPM7-ERK1/2 in cell proliferation, with variable responses. In endothelial cells, TRPM7 silencing promoted cell growth via ERK1/2, while in mouse cortical astrocytes, TRPM7 knockdown inhibited proliferation through ERK1/2 and c-Jun N-terminal kinases (JNK) [52,53]. To explore the biological significance of our cell-based studies, we examined intact vessels from TRPM7 +/ kinase mice, which have EGFR down-regulation, and showed reduced vascular wall thickness with decreased expression of PCNA and Notch3, markers of VSMC proliferation and differentiation, respectively [44,54]. Our observations of decreased aortic phosphorylation of EGFR and ERK1/2 in TRPM7 R/R mice, a second model of TRPM7 deficiency, further validates the notion that EGFR signaling is down-regulated when TRPM7 activity is reduced. Together our findings indicate that EGF/EGFR through c-Src-regulated TRPM7 and ERK1/2 signaling, promotes VSMC proliferation and migration, processes that play a role in vascular remodeling.
In summary, utilizing a combination of pharmacological, biochemical, imaging and molecular biological approaches together with cells and tissues from rodent models we show important cross-talk between EGFR and TRPM7. We provide new evidence that EGFR directly interacts with TRPM7 in VSMCs and that EGF through c-Src regulates TRPM7 in a feedforward system. Functionally these phenomena regulate [Mg 2+ ] i homeostasis, ERK1/2 signaling and VSMC function ( Figure 6). Of significance our results in rodent VSMCs were recapitulated in hVSMCs, indicating clinical relevance. These findings define a novel pathway in VSMCs and highlight important links between EGF signaling and TRPM7, which may be particularly important in conditions associated with EGFR hyperactivation, such as in cardiovascular disease and cancer.