Transient Receptor Potential Melastatin 7 Channels Research Articles

TRPM7 channels regulate breathing during sleep in obesity by acting peripherally in the carotid bodies.

Sleep-disordered breathing (SDB) affects over 50% of obese individuals. Exaggerated hypoxic chemoreflex is a cardinal trait of SDB in obesity. We have shown that leptin acts in the carotid bodies (CB) to augment chemoreflex and that leptin activates the transient receptor potential melastatin 7 (TRPM7) channel. However, the effect of leptin-TRPM7signalling in CB on breathing and SDB has not been characterized in diet-induced obesity (DIO). We hypothesized that leptin acts via TRPM7 in the CB to increase chemoreflex leading to SDB in obesity. DIO mice were implanted with EEG/EMG electrodes and transfected with Leprb short hairpin RNA (shRNA) or Trpm7 shRNA vs. control shRNA in the CB area bilaterally. Mice underwent a full-polysomnography and metabolic studies at baseline and after transfection. Ventilatory responses to hypoxia and hypercapnia were assessed during wakefulness. Leprb and Trpm7 were upregulated and their promoters were demethylated in the CB of DIO mice. Leprb knockdown in the CB did not significantly affect ventilation. Trpm7 knockdown in the CB stimulated breathing during sleep in normoxia. These effects were not driven by changes in CB chemosensitivity or metabolism. Under sustained hypoxia, Trpm7 shRNA in the CB augmented ventilation during sleep, but decreased oxyhaemoglobin saturation. We conclude that the suppression of TRPM7 in the CB improved sleep-related hypoventilation and that the respiratory effects of CB TRPM7 channels in obesity are independent of leptin. TRPM7signalling in the CB could be a therapeutic target for the treatment of obesity-related SDB. KEY POINTS: The leptin-TRPM7 axis in the carotid bodies may play an important role in the pathogenesis of sleep-disordered breathing. TRPM7 channels regulate breathing during sleep by acting peripherally in the carotid bodies. Suppression of TRPM7signalling in the carotid bodies improves the obesity-induced hypoventilation in mice. Pharmacological blockade of TRPM7 channels in the carotid bodies could be a therapy for sleep-disordered breathing in obesity.

TRPM7 kinase inactivation results in macrophage alkalinization and increased phagocytic activity

Transient receptor potential melastatin 7 (TRPM7) is a unique protein that is both an ion channel and a cytosolic protein kinase. Normally, TRPM7 channels are tonically inhibited by cytoplasmic Mg2+, polyamines and acidic pH. Channel basal activity can be increased by alkalinization or Mg2+ depletion. We have used the TRPM7 kinase-dead (KD K1646R) mouse to investigate the consequences of kinase inactivation in immune cell function. It also serves as a convenient system to elucidate kinase-channel interactions at a functional level.

NSAIDs Naproxen, Ibuprofen, Salicylate, and Aspirin Inhibit TRPM7 Channels by Cytosolic Acidification.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used for relieving pain and inflammation accompanying numerous disease states. The primary therapeutic mechanism of these widely used drugs is the inhibition of cyclooxygenase 1 and 2 (COX1, 2) enzymes that catalyze the conversion of arachidonic acid into prostaglandins. At higher doses, NSAIDs are used for prevention of certain types of cancer and as experimental treatments for Alzheimer’s disease. In the immune system, various NSAIDs have been reported to influence neutrophil function and lymphocyte proliferation, and affect ion channels and cellular calcium homeostasis. Transient receptor potential melastatin 7 (TRPM7) cation channels are highly expressed in T lymphocytes and are inhibited by Mg2+, acidic pH, and polyamines. Here, we report a novel effect of naproxen, ibuprofen, salicylate, and acetylsalicylate on TRPM7. At concentrations of 3–30mM, they reversibly inhibited TRPM7 channel currents. By measuring intracellular pH with the ratiometric indicator BCECF, we found that at 300μM to 30mM, these NSAIDs reversibly acidified the cytoplasm in a concentration-dependent manner, and propose that TRPM7 channel inhibition is a consequence of cytosolic acidification, rather than direct. NSAID inhibition of TRPM7 channels was slow, voltage-independent, and displayed use-dependence, increasing in potency upon repeated drug applications. The extent of channel inhibition by salicylate strongly depended on cellular PI(4,5)P2 levels, as revealed when this phospholipid was depleted with voltage-sensitive lipid phosphatase (VSP). Salicylate inhibited heterologously expressed wildtype TRPM7 channels but not the S1107R variant, which is insensitive to cytosolic pH, Mg2+, and PI(4,5)P2 depletion. NSAID-induced acidification was also observed in Schneider 2 cells from Drosophila, an organism that lacks orthologous COX genes, suggesting that this effect is unrelated to COX enzyme activity. A 24-h exposure to 300μM–10mM naproxen resulted in a concentration-dependent reduction in cell viability. In addition to TRPM7, the described NSAID effect would be expected to apply to other ion channels and transporters sensitive to intracellular pH.

CCT128930 is a novel and potent antagonist of TRPM7 channel

Transient receptor potential melastatin 7 (TRPM7) channels represent a major magnesium (Mg2+)-uptake component in mammalian cells and are negatively modulated by internal Mg2+. However, few TRPM7 modulators were identified so far, which hindered the understanding of the TRPM7 channel functions. In this study, we identified that CCT128930, an ATP-competitive protein kinase B inhibitor reported previously, was a potent TRPM7 channel antagonist. The inhibition of CCT128930 on TRPM7 was independent of intracellular Mg2+. In the absence and presence of 300 μM Mg2+ in pipette solution, the IC50 values were 0.86 ± 0.11 μM and 0.63 ± 0.09 μM, respectively. Subtype selectivity data showed that CCT128930 preferentially inhibited TRPM7 channels compared to TRPM6 and TRPM8 isoforms. In addition, CCT128930 was found to be able to reduce the endogenous TRPM7-like currents in SH-SY5Y neuroblastoma cells. At last, multiple residues in the superficial part of the TRPM7 selectivity filter were identified to be critical for the inhibitory activity of CCT128930 which are different from the determinants of Mg2+ and reported TRPM7 antagonists. Our results indicated that CCT128930 is a novel and potent TRPM7 channel antagonist.

Phagocytic activity of splenic macrophages is enhanced and accompanied by cytosolic alkalinization in TRPM7 kinase-dead mice.

Transient receptor potential melastatin 7 (TRPM7) is a unique protein functioning as a cation channel as well as a serine/threonine kinase and is highly expressed in immune cells such as lymphocytes and macrophages. TRPM7 kinase-dead (KD) mouse model has been used to investigate the role of this protein in immune cells; these animals display moderate splenomegaly and ectopic hemopoiesis. The basal TRPM7 current magnitudes in peritoneal macrophages isolated from KD mice were higher; however, the maximum currents, achieved after cytoplasmic Mg2+ washout, were not different. In the present study, we investigated the consequences of TRPM7 kinase inactivation in splenic and peritoneal macrophages. We measured the basal phagocytic activity of splenic macrophages using fluorescent latex beads, pHrodo zymosan bioparticles, and opsonized red blood cells. KD macrophages phagocytized more efficiently and had slightly higher baseline calcium levels compared to WT cells. We found no obvious differences in store-operated Ca2+ entry between WT and KD macrophages. By contrast, the resting cytosolic pH in KD macrophages was significantly more alkaline than in WT. Pharmacological blockade of sodium hydrogen exchanger 1 (NHE1) reversed the cytosolic alkalinization and reduced phagocytosis in KD macrophages. Basal TRPM7 channel activity in KD macrophages was also reduced after NHE1 blockade. Cytosolic Mg2+ sensitivity of TRPM7 channels measured in peritoneal macrophages was similar in WT and KD mice. The higher basal TRPM7 channel activity in KD macrophages is likely due to alkalinization. Our results identify a novel role for TRPM7 kinase as a suppressor of basal phagocytosis and a regulator of cellular pH.

Isoproterenol-Dependent Activation of TRPM7 Protects Against Neurotoxin-Induced Loss of Neuroblastoma Cells.

Neuronal function and their survival depend on the activation of ion channels. Loss of ion channel function is known to induce neurodegenerative diseases such as Parkinson’s that exhibit loss of dopaminergic neurons; however, mechanisms that could limit neuronal loss are not yet fully identified. Our data suggest that neurotoxin-mediated loss of neuroblastoma SH-SY5Y cells is inhibited by the addition of β-adrenergic receptor (β-AR) agonist isoproterenol. The addition of isoproterenol to SHSY-5Y cells showed increased Mg2+ influx and cell survival in the presence of neurotoxin especially at higher concentration of isoproterenol. Importantly, isoproterenol potentiated transient receptor potential melastatin-7 (TRPM7) channel activation that leads to an increase in intracellular Mg2+ levels. The addition of 2APB, which is a known TRPM7 channel blocker, significantly decreased the TRPM7 function and inhibited isoproterenol-mediated protection against neurotoxins. Moreover, neurotoxins inhibited TRPM7 expression and function, but the restoration of TRPM7 expression increased neuroblastoma cell survival. In contrast, TRPM7 silencing increased cell loss, decreased Mg2+ homeostasis, and inhibited mitochondrial function. Moreover, isoproterenol treatment prevented neurotoxin-mediated loss of TRPM7 expression and inhibited Bax expression that induces cell survival. These effects were dependent on the neurotoxin-induced increase in oxidative stress, which inhibits TRPM7 expression and function. Together, our results suggest a positive role for β-AR in activating TRPM7 channels that regulate Mg2+ homeostasis and are essential for the survival of SH-SY5Y cells from neurotoxin.

Magnesium-Induced Cell Survival Is Dependent on TRPM7 Expression and Function

Mg2+ homeostasis is essential for cell survival and the loss of this regulation has been associated with many neurodegenerative diseases, including loss of dopaminergic neurons. Although the neurotoxin-mediated loss of dopaminergic neurons in Parkinson disease models is extensively studied, the ion channel(s) that regulate Mg2+ homeostasis and thus could prevent neuronal cell death is not yet identified. Here, we show that TRPM7 (transient receptor potential melastatin 7) is involved in regulating Mg2+ homeostasis in dopaminergic cells. Importantly, transient loss of TRPM7 decreased intracellular Mg2+ levels and decreased dopaminergic cells/neurons survival. We provide further evidence that both increases in extracellular Mg2+ or transiently increasing TRPM7 levels protected dopaminergic SH-SY5Y cells against neurotoxin-mediated cell death. Neurotoxin treatment significantly decreased TRPM7 levels in both SH-SY5Y cells and the substantia nigra pars compacta region of mice, along with a decrease in Mg2+ influx. Moreover, Mg2+ supplementation showed a concentration-dependent decrease in caspase-3 activity, an increase in cell survival, restored mitochondrial membrane potential, and increase TRPM7 levels in neurotoxin-treated cells. In contrast, transient silencing of TRPM7 inhibited the positive effect of Mg2+ supplementation in protecting against neurotoxins. Whereas, TRPM7 overexpression not only maintained Mg2+ homeostasis but also inhibited caspase 3 activity that induced cell survival. Overall, these results suggest a significant role of TRPM7 channels in Mg2+ homeostasis and the survival of neurotoxin-induced loss of dopaminergic cells.

Abstract 774: Regulation of autophagy and epigenetic modulator micro RNA by transient receptor potential melastatin 7 (TRPM7) channel in glioma

Abstract High grade malignant gliomas are devastating, uniformly fatal cancers for which no effective therapies currently exist. Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. We have reported that TRPM7 channels, a subfamily member of the transient receptor potential (TRP), regulate glioma stem cell (GSC) growth/proliferation through STAT3 and Notch signaling pathways. Autophagy is a catabolic process that occurs during both physiological and pathological process which has been reported to have dual roles, functioning either as a tumor-suppressor or tumor-promoter in cancer, including glioma. In addition, because of the limited success of the traditional treatment in prolonging the overall survival in GBM patients, epigenetics and epigenetic modulators have been attracting an increasing amount of attention for their influence in many aspects of cancer. microRNAs (miRNA), which can be considered as epigenetic effectors, reversibly regulate transcription through binding to complementary sequences of mRNA and silencing its translation into proteins. Therefore, we first determined the role of autophagy process in regulating glioma by TRPM7 using real time RT2 Profile PCR arrays (SABioscience, PAHS-084Z). We found that ATG16L1, ATG9A, CDKN1B, CLN3, CXCR4, and RGS19 were upregulated in response to TRPM7 silencing with the fold change greater than 1.6 using the A172 cell line of human glioma. The gene changes were further validated in other three glioma cell lines U87MG, U373MG and SNB19, which demonstrated different glioma tumorigenicity. miroRNA microarray analysis were then were performed upon TRPM7 silencing in A172 cells (www.LCsciences.com). In-depth data analysis from miRNA microarray data showed a list of 16 downregulated and 10 upregulated miRNA whose transcripts are statistically significant with fold change greater than 2 by TRPM7 knock-down. Among these, the micro RNA of hsa-miR-26b-5p, has-miR4530, and has-miR-28-5p have shown to exert crucial influence on tumor growth and migration by modulating AKT, ERK and IGF-1 signaling pathways. Stem-loop RT-qPCR were then used to detect and quantify miRNAs identified in miRNA microarray data from U87MG, U373MG and SNB19 cell lines. Our data indicate that autophagy effects serve as tumor suppressors in gliomagenesis and are reversely regulated by TRPM7; while hsa-miR-26b-5p, has-miR4530, and has-miR-28-5p are regulated by TRPM7 and participate in glioma progression. Citation Format: Mingli Liu. Regulation of autophagy and epigenetic modulator micro RNA by transient receptor potential melastatin 7 (TRPM7) channel in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 774.

Carvacrol Attenuates Hippocampal Neuronal Death after Global Cerebral Ischemia via Inhibition of Transient Receptor Potential Melastatin 7.

Over the last two decades, evidence supporting the concept of zinc-induced neuronal death has been introduced, and several intervention strategies have been investigated. Vesicular zinc is released into the synaptic cleft, where it then translocates to the cytoplasm, which leads to the production of reactive oxygen species and neurodegeneration. Carvacrol inhibits transient receptor potential melastatin 7 (TRPM7), which regulates the homeostasis of extracellular metal ions, such as calcium and zinc. In the present study, we test whether carvacrol displays any neuroprotective effects after global cerebral ischemia (GCI), via a blockade of zinc influx. To test our hypothesis, we used eight-week-old male Sprague–Dawley rats, and a GCI model was induced by bilateral common carotid artery occlusion (CCAO), accompanied by blood withdrawal from the femoral artery. Ischemic duration was defined as a seven-minute electroencephalographic (EEG) isoelectric period. Carvacrol (50 mg/kg) was injected into the intraperitoneal space once per day for three days after the onset of GCI. The present study found that administration of carvacrol significantly decreased the number of degenerating neurons, microglial activation, oxidative damage, and zinc translocation after GCI, via downregulation of TRPM7 channels. These findings suggest that carvacrol, a TRPM7 inhibitor, may have therapeutic potential after GCI by reducing intracellular zinc translocation.

TRPM7 channels play a role in high glucose–induced endoplasmic reticulum stress and neuronal cell apoptosis

High-glucose (HG) levels and hyperglycemia associated with diabetes are known to cause neuronal damage. The detailed molecular mechanisms, however, remain to be elucidated. Here, we investigated the role of transient receptor potential melastatin 7 (TRPM7) channels in HG-mediated endoplasmic reticulum stress (ERS) and injury of NS20Y neuronal cells. The cells were incubated in the absence or presence of HG for 48 h. We found that mRNA and protein levels of TRPM7 and of ERS-associated proteins, such as C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and inducible nitric-oxide synthase (iNOS), increased in HG-treated cells, along with significantly increased TRPM7-associated currents in these cells. Similar results were obtained in cerebral cortical tissue from an insulin-deficiency model of diabetic mice. Moreover, HG treatment of cells activated ERS-associated proapoptotic caspase activity and induced cellular injury. Interestingly, a NOS inhibitor, l-NAME, suppressed the HG-induced increase of TRPM7 expression and cellular injury. siRNA-mediated TRPM7 knockdown or chemical inhibition of TRPM7 activity also suppressed HG-induced ERS and decreased cleaved caspase-12/caspase-3 levels and cell injury. Of note, TRPM7 overexpression increased ERS and cell injury independently of its kinase activity. Taken together, our findings suggest that TRPM7 channel activities play a key role in HG-associated ERS and cytotoxicity through an apoptosis-inducing signaling cascade involving HG, iNOS, TRPM7, ERS proteins, and caspases.

TRPM7 channels mediate the functional changes in cardiac fibroblasts induced by angiotensin II.

Transient receptor potential melastatin7 (TRPM7), a bifunctional channel protein owning both cation permeability and kinase activity, plays an important role in the pathophysiological process of many cell types, such as vascular smooth muscle cells, human glioma cells and mouse cortical astrocytes. However, whether TRPM7 channels play a key role in the functional change of cardiac fibroblasts(CFs) induced by angiotensinII (AngII) remains unknown. Using Cell Counting Kit-8 (CCK-8) assay, immunofluorescence assay, western blot analysis, RT-qPCR, RNA interference(RNAi) and whole-cell patch-clamp techniques, the present study aimed to explore the role of TRPM7 channels in the proliferation, differentiation and collagen synthesis of CFs induced by AngII. Our data showed that AngII time-dependently increased TRPM7 expression and TRPM7 currents in the CFs. Downregulation of TRPM7 attenuated the TRPM7 current density, and inhibited the proliferation, differentiation and collagen synthesis of CFs induced by AngII. Our results identified the TRPM7 channel as a pivotal member associated with the functional change of CFs induced by AngII, and suggest that the TRPM7 channel may represent a promising therapeutic strategy for the treatment of fibrosis-related cardiac diseases.

TRPM7 channel regulates ox-LDL-induced proliferation and migration of vascular smooth muscle cells via MEK-ERK pathways.

Transient receptor potential melastatin 7 (TRPM7) plays a key role in the pathophysiological response of multiple cell types. However, the role of TRPM7 channels in ox-LDL-induced proliferation and migration of VSMC remains unclear. This study used the thoracic aorta VSMCs to explore the effects of ox-LDL on cell proliferation and migration and to investigate the underlying molecular mechanisms and signaling pathways. Data demonstrated that ox-LDL significantly increased TRPM7 activity, and induced VSMC proliferation and migration. VSMC proliferation and migration were inhibited by nonspecific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7. Furthermore, the phosphorylation of ERK1/2 and MEK1/2 associated with cell proliferation and migration decreased in TRPM7-deficient VSMC. Therefore, TRPM7 may constitute a useful target for the treatment of atherosclerosis.

Inhibition of TRPM7 Attenuates Rat Aortic Smooth Muscle Cell Proliferation Induced by Angiotensin II: Role of Genistein.

Transient receptor potential melastatin 7 (TRPM7) is a Ca, Mg permeable nonselective cation channel of the TRP channel superfamily and plays an important role in cell growth and proliferation. Compounds that alter the activity and expression of the channel protein might be of therapeutic interest. In this study, we investigated the effects of genistein on TRPM7 channels and the proliferation of rat aortic smooth muscle cells (RAoSMCs). In primary cultured RAoSMCs, acute genistein (50 μM) exposure inhibited native TRPM7 currents, whereas chronic expose to genistein (50 μM) downregulated TRPM7 protein expression. The downregulation of TRPM7 protein expression induced by genistein was mimicked by c-Src inhibitor (PP2), but not by epidermal growth factor receptor tyrosine kinase inhibitor (lavendustin A), or daidzein. Additionally, genistein (50 μM) attenuated angiotensin II-induced cell proliferation. This study is the first to demonstrate inhibition of TRPM7 by isoflavone genistein through c-Src tyrosine kinase inhibition in RAoSMCs. Our results not only provide a new modulation mechanism of TRPM7 but also suggest that TRPM7 may serve as a new therapeutic target of genistein in the treatment of vascular diseases.

TRPM7 Regulates Axonal Outgrowth and Maturation of Primary Hippocampal Neurons.

Transient receptor potential melastatin 7 (TRPM7) is a calcium-permeable divalent cation channel and mediates neuronal cell death under ischemic stresses. In this study, we investigated the contribution of TRPM7 to neuronal development in mouse primary hippocampal neurons. We demonstrated that TRPM7 channels are highly expressed in the tips of the growth cone. Either knockdown of TRPM7 with target-specific shRNA or blocking channel conductance by a specific blocker waixenicin A enhanced axonal outgrowth in culture. Blocking TRPM7 activity by waixenicin A reduced calcium influx and accelerated the polarization of the hippocampal neurons as characterized by the development of distinct axons and dendrites. Furthermore, TRPM7 coprecipitated and colocalized with F-actin and α-actinin-1 at the growth cone. We conclude that calcium influx through TRPM7 inhibits axonal outgrowth and maturation by regulating the F-actin and α-actinin-1 protein complex. Inhibition of TRPM7 channel promotes axonal outgrowth, suggesting its therapeutic potential in neurodegenerative disorders.

TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis.

Effects of Schisandra Chinensis on Human Breast Cancer Cells

Fruits of Schisandra chinensis (SC) Baill are considered a traditional herbal medicine for the treatment and alleviation of various diseases. The purpose of this study was to investigate the anti-cancer effects of SC extract in human breast adenocarcinoma cells (MCF-7). We used human breast adenocarcinoma cell line, MCF-7 cells. We examined cell death by MTT assay and caspase 3 and 9 assay with SC extract. To examine the inhibitory effects of SC extract, cell cycle (sub G1) analysis and mitochondrial membrane depolarization was done the MCF-7 cells after one day with SC extract. In addition, to investigate the transient receptor potential melastatin 7 (TRPM7) currents, we used the whole cell patch clamp techniques. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in MCF-7 cell growth and survival. SC extract inhibited the growth of MCF-7 cells in a dose-dependent fashion. Also we showed that SC extract induced apoptosis in MCF-7 cells by MTT assay, caspase 3 and 9 assay, sub-G1 analysis and mitochondrial membrane depolarization. SC extract inhibited the TRPM7 currents in MCF-7 cells and in TRPM7 overexpressed HEK 293 cells. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SC extract-induced cell death. Our findings provide insight into unraveling the effects of SC extract in human breast adenocarcinoma cells and developing therapeutic agents against breast cancer. 

Inhibition of transient receptor potential melastatin 7 (TRPM7) channel induces RA FLSs apoptosis through endoplasmic reticulum (ER) stress.

Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) apoptosis induced by thapsigargin in vitro. In this study, using a combination of Western blotting, RT-PCR, and nuclear morphology analysis, we investigated the influence and potential function of TRPM7 channels on the apoptosis induced by thapsigargin in RA FLSs. Chemical inhibitors (Gd(3+) and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in RA FLSs apoptosis. The expression of TRPM7 was significantly potentiated in RA FLSs. Co-incubation of RA FLSs with Gd(3+), 2-APB, or TRPM7-siRNA increased cell apoptosis. Furthermore, we found that suppression of TRPM7 channels also increased the expression CHOP and calpain and decreased the expression caspase-3. We conclude that suppression of TRPM7 channels may increase RA FLSs apoptosis in vitro, and this is associated with endoplasmic reticulum (ER) stress. Therefore, inhibition of TRPM7 could activate ER stress and induce RA FLSs apoptosis.

Transient Receptor Potential Melastatin 7 (TRPM7) Contributes to H2O2-Induced Cardiac Fibrosis via Mediating Ca2+ Influx and Extracellular Signal–Regulated Kinase 1/2 (ERK1/2) Activation in Cardiac Fibroblasts

Transient receptor potential melastatin 7 (TRPM7), a Ca(2+)-nonselective cation channel, plays a key role in the pathophysiological response of multiple cell types. However, the role of TRPM7 channels in hydrogen peroxide (H2O2)-induced cardiac fibrosis remains unclear. This study aimed to explore whether TRPM7 channels are involved in H2O2-induced cardiac fibrosis and the underlying mechanisms. Our results showed that 2-aminoethoxydiphenylborate (2-APB), which is commonly used to block TRPM7 channels, inhibited H2O2-induced cardiac fibrosis via attenuating the overexpression of important fibrogenic biomarkers and growth factors in cardiac fibroblasts, including collagen type I (Col I), fibronectin (FN), smooth muscle α-actin (α-SMA), connective tissue growth factor (CTGF), and transforming growth factor-β1 (TGF-β1). In addition, 2-APB also decreased H2O2-mediated elevation of the concentration of intracellular Ca(2+) ([Ca(2+)]i). Meanwhile, silencing TRPM7 channels by shRNA interference also impaired the increased [Ca(2+)]i and upregulation of Col I, FN, α-SMA, CTGF, and TGF-β1 induced by H2O2. Furthermore, we found that H2O2-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2) decreased in TRPM7-shRNA cells and Ca(2+)-free culture media. These results demonstrated that TRPM7 channels contributed to H2O2-induced cardiac fibrosis and suggested that this contribution may be through mediating Ca(2+) influx and phosphorylation of ERK1/2.

Role of TRPM7 Channels in Hyperglycemia-Mediated Injury of Vascular Endothelial Cells

This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.

Inhibition of TRPM7 channels prevents proliferation and differentiation of human lung fibroblasts

Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in human lung fibroblast (MRC5) proliferation and differentiation induced by transforming growth factor β1 (TGF-β1) in vitro. We determined the expression of TRPM7 in MRC5 cells in response to TGF-β1 treatment in vitro. Chemical inhibitors (Gd(3+) and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in MRC5 cell proliferation and differentiation. The phosphorylation of Akt was determined by Western blotting. The expression of TRPM7 was significantly potentiated in response to TGF-β1. Co-incubation of MRC5 cells with Gd(3+), 2-APB or TRPM7-siRNA decreased cell proliferation and differentiation. Furthermore, we found that suppression of TRPM7 channels also reduced the p-Akt in MRC5 cells induced by TGF-β1. We conclude that suppression of TRPM7 channels may decrease fibroblast proliferation and differentiation stimulated by TGF-β1 in vitro and this is associated with Akt phosphorylation.