Aims Transient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg2+ homeostasis and vascular signalling. However, the pathophysiological significance of TRPM7 in the cardiovascular system is unclear. The aim of this study was to investigate the role of this chanzyme in the cardiovascular system focusing on inflammation and fibrosis. Methods and results TRPM7-deficient mice with deletion of the kinase domain (TRPM7+/Δkinase) were studied and molecular mechanisms investigated in TRPM7+/Δkinase bone marrow-derived macrophages (BMDM) and co-culture systems with cardiac fibroblasts. TRPM7-deficient mice had significant cardiac hypertrophy, fibrosis, and inflammation. Cardiac collagen and fibronectin content, expression of pro-inflammatory mediators (SMAD3, TGFβ) and cytokines [interleukin (IL)-6, IL-10, IL-12, tumour necrosis factor-α] and phosphorylation of the pro-inflammatory signalling molecule Stat1, were increased in TRPM7+/Δkinase mice. These processes were associated with infiltration of inflammatory cells (F4/80+CD206+ cardiac macrophages) and increased galectin-3 expression. Cardiac [Mg2+]i, but not [Ca2+]i, was reduced in TRPM7+/Δkinase mice. Calpain, a downstream TRPM7 target, was upregulated (increased expression and activation) in TRPM7+/Δkinase hearts. Vascular functional and inflammatory responses, assessed in vivo by intra-vital microscopy, demonstrated impaired neutrophil rolling, increased neutrophil: endothelial attachment and transmigration of leucocytes in TRPM7+/Δkinase mice. TRPM7+/Δkinase BMDMs had increased levels of galectin-3, IL-10, and IL-6. In co-culture systems, TRPM7+/Δkinase macrophages increased expression of fibronectin, proliferating cell nuclear antigen, and TGFβ in cardiac fibroblasts from wild-type mice, effects ameliorated by MgCl2 treatment. Conclusions We identify a novel anti-inflammatory and anti-fibrotic role for TRPM7 and suggest that its protective effects are mediated, in part, through Mg2+-sensitive processes.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed chanzyme that possesses an ion channel permeable to the divalent cations Mg2+, Ca2+, and Zn2+, and an α-kinase that phosphorylates downstream substrates. TRPM7 and its homologue TRPM6 have been implicated in a variety of cellular functions and is critically associated with intracellular signaling, including receptor tyrosine kinase (RTK)-mediated pathways. Emerging evidence indicates that growth factors, such as EGF and VEGF, signal through their RTKs, which regulate activity of TRPM6 and TRPM7. TRPM6 is primarily an epithelial-associated channel, while TRPM7 is more ubiquitous. In this review we focus on TRPM7 and its association with growth factors, RTKs, and downstream kinase signaling. We also highlight how interplay between TRPM7, Mg2+ and signaling kinases influences cell function in physiological and pathological conditions, such as cancer and preeclampsia.

Aims NOX-derived reactive oxygen species (ROS) are mediators of signaling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. Methods and Results VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signaling molecules, actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were downregulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs NOX5 was upregulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1) and hyperphosphorylation (PKC, ERK1/2, MLC20) of signaling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. Conclusions We define NOX5/ROS/c-Src as a novel feedforward signaling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury. Translational Perspective Oxidative stress is a major factor contributing to vascular damage in hypertension. We corroborate experimental evidence that NOX-derived ROS generation is increased in human vascular smooth muscle cells (VSMC) and demonstrate that in human hypertension NOX5 upregulation is a major trigger of VSMC dysfunction. We uncover new regulatory molecular mechanisms of NOX5 and define NOX5/ROS/c-Src as a novel signaling pathway in human VSMCs. This system is augmented in hypertension contributing to abnormal VSMC redox signaling, cytoskeletal disorganization and vascular dysfunction. Modulating the NOX5/ROS/c-Src pathway may have therapeutic potential by targeting redox signaling pathways involved in vascular dysfunction associated with hypertension.

OBJECTIVE: Transient receptor potential 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 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 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 studies, proximity ligation assay 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.

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