Transient Receptor Potential Melastatin 7 Expression Research Articles

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.

Endotoxin Induces Fibrosis in Vascular Endothelial Cells through a Mechanism Dependent on Transient Receptor Protein Melastatin 7 Activity

The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial­to­mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.

Suppression of renal TRPM7 may alleviate kidney injury in the renal transplantation

The aim of this study was to investigate the effect of renal cortex transient receptor potential melastatin 7 (TRPM7) suppression on renal ischemia reperfusion injury induced by transplantation in mice. M7shRNA was used to decrease the expression of TRPM7 in NRK-52e cells. The mice were subjected to renal intra-parenchymal injection with lentivirus containing M7shRNA to produce hypo-expression of TRPM7 in renal cortex. Cell hypoxia mode and syngeneic renal transplantation in vivo were established. Then the effects of M7shRNA were measured by fluorescent probe for reactive oxygen species (ROS), intracellular calcium and magnesium; Western blot was applied for p38-MAPKs and Bax expression in cell studies. In vivo studies, mice were killed 24 h, 48 h, 72 h, 7 days and 21 days, respectively, after transplantation and the kidneys were dissected. Serum creatinine was measured, and the H&E, Masson's trichrome staining, TUNEL, Kim-1 and α-smooth muscle actin were used to evaluate pathological change. In cell model of hypoxia, the level of ROS in NRK-52e-M7shRNA was significantly lower than that in both NRK-52e and NRK-52e control cells, while the activation of p38-MAPKs was limited. In renal transplanted mice, renal function of M7shRNA group was conspicuously better than PBS- and vector-control-treated group. The histological examination showed that renal tubule injury and interstitial fibrosis were lower in M7shRNA-treated group compared with PBS and vector-control group. Suppression of renal cortex TRPM7 could alleviate kidney injury induced by transplantation in mice. The mechanism may involve reducing the early stage of ischemia reperfusion injury by inhibition of intracellular Ca(2+), Mg(2+) and ROS.

The interaction of transient receptor potential melastatin 7 with macrophages promotes vascular adventitial remodeling in transverse aortic constriction rats

Transient receptor potential melastatin 7 (TRPM7), a novel channel kinase, has been recently identified in the vasculature. However, its regulation and function in vascular diseases remain poorly understood. To address this lack of knowledge, we sought to examine whether TRPM7 can mediate the vascular remodeling process induced by pressure overload in the right common carotid artery proximal to the band (RCCA-B) in male Sprague-Dawley rats with transverse aortic constriction (TAC). The contribution of TRPM7 to amplified vascular remodeling after TAC was tested using morphometric and western blot analyses. Pressure overload-induced vascular wall thickening, especially in the adventitia, was readily detected in RCCA-B. The TRPM7 level was increased with a simultaneous accumulation of macrophages in the adventitia of RCCA-B, whereas the anti-inflammatory molecule annexin-1, a TRPM7 downstream target, was decreased. After the addition of the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB), significant reductions in macrophage accumulation as well as the expression of monocyte chemotactic protein-1, SM-22-α and collagen I were observed, whereas annexin-1 was rescued. Finally, in cultured vascular adventitial fibroblasts treated with macrophage-conditioned medium, there were marked increases in the expression of TRPM7 and SM-22-α with a concurrent reduction in annexin-1 expression; these effects were largely prevented by treatment with 2-APB and specific anti-TRPM7 small interfering RNA. Our findings provide the first demonstration of the potential regulatory roles of TRPM7 in the vascular inflammation, pressure overload-mediated vascular adventitial collagen accumulation and cell phenotypic transformation in TAC rats. The targeting of TRPM7 has potential therapeutic importance for vascular diseases.

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.

Transient receptor potential melastatin 7 is involved in oestrogen receptor-negative metastatic breast cancer cells migration through its kinase domain

Oestrogen receptor negative (ER−) invasive ductal carcinoma (IDC) represents a significant clinical challenge and therefore prompts the discovery of novel biomarkers. Transient receptor potential melastatin 7 (TRPM7), a channel protein that also contains a regulatory kinase domain, is overexpressed in IDC and regulates migration. However, the molecular mechanism remains poorly defined. Here, we examined whether TRPM7 regulates migration by its channel function or by its kinase domain. A Magnesium Inhibited Cation current was recorded in two ER− highly metastatic breast cancer cell lines. Down-regulation of TRPM7 neither affected Ca2+-, nor Mg2+-homoeostasis but significantly reduced cell migration via a Ca2+-independent pathway. Notably, the overexpression of the truncated kinase domain form of TRPM7 decreased cell migration, while the overexpression of the wild-type form strongly increased it. Concomitantly, TRPM7 silencing reduced the myosin IIA heavy chain phosphorylation. Furthermore, we found higher TRPM7 expression in ER− IDC tissues and lymph nodes than in the non-invasive tumoural samples. In conclusion, TRPM7 plays a critical role in breast cancer cell migration through its kinase domain, and our data support the consideration of using TRPM7 as a novel biomarker and a potential therapeutic target in the treatment of human ER− IDC.

Inhibition of cancer cell proliferation by midazolam by targeting transient receptor potential melastatin 7

Transient receptor potential melastatin 7 (TRPM7), a Ca2+-permeable channel, has been demonstrated to be present in cancer cells and involved in their growth and proliferation. The present study used midazolam, a benzodiazepine class anesthesic, to pharmacologically intervene in the expression of TRPM7 and to inhibit cancer cell proliferation. Midazolam significantly inhibited the growth and proliferation of FaDu human hypopharyngeal squamous cell carcinoma cells, concurring with the induction of G0/G1 cell cycle arrest and blockage of Rb activation. Central-type and peripheral-type benzodiazepine receptor antagonists did not abrogate proliferation inhibition by midazolam, while the specific TRPM7 agonist bradykinin reversed this effect. In addition, other benzodiazepines, diazepam and clonazepam also exhibited anti-proliferative activities. The inhibitory activity on cancer cell growth and proliferation, combined with the TRPM-dependent mechanism, reveals the anticancer potential of midazolam as a TRPM7 inhibitor and supports the suggestion that TRPM7 is a valuable target for pharmaceutical intervention.

Involvement of Transient Receptor Potential Melastatin 7 Channels in Sophorae Radix-induced Apoptosis in Cancer Cells: Sophorae Radix and TRPM7.

Sophorae Radix (SR) plays a role in a number of physiologic and pharmacologic functions in many organs.Objective:The aim of this study was to clarify the potential role for transient receptor potential melastatin 7 (TRPM7) channels in SR-inhibited growth and survival of AGS and MCF-7 cells, the most common human gastric and breast adenocarcinoma cell lines.Methods:The AGS and the MCF-7 cells were treated with varying concentrations of SR. Analyses of the caspase-3 and - 9 activity, the mitochondrial depolarization and the poly (ADPribose) polymerase (PARP) cleavage were conducted to determine if AGS and MCF-7 cell death occured by apoptosis. TRPM7 channel blockers (Gd3+ or 2-APB) and small interfering RNA (siRNA) were used in this study to confirm the role of TRPM7 channels. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in AGS and MCF-7 cell growth and survival.Results:The addition of SR to a culture medium inhibited AGS and MCF-7 cell growth and survival. Experimental results showed that the caspase-3 and -9 activity, the mitochondrial depolarization, and the degree of PARP cleavage was increased. TRPM7 channel blockade, either by Gd3+ or 2-APB or by suppressing TRPM7 expression with small interfering RNA, blocked the SR-induced inhibition of cell growth and survival. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SR-induced cell death.Conclusions:These findings indicate that SR inhibits the growth and survival of gastric and breast cancer cells due to a blockade of the TRPM7 channel activity. Therefore, TRPM7 channels may play an important role in the survival of patients with gastric and breast cancer.

Upregulation of TRPM7 Channels by Angiotensin II Triggers Phenotypic Switching of Vascular Smooth Muscle Cells of Ascending Aorta

Angiotensin II (Ang II) has pleiotropic effects on vascular smooth muscle cells (VSMCs). It has been demonstrated to promote the proliferative phenotype of VSMCs in mouse ascending aorta, but the underlying mechanisms remain incompletely understood. The present study was designed to explore whether the Ca(2+)-permeable transient receptor potential melastatin 7 (TRPM7) channel is involved in Ang II-induced phenotype switching of ascending aortic VSMCs and to dissect the molecular mechanisms by which TRPM7 modulates VSMC phenotype. As revealed by current recording, Ang II infusion increased TRPM7 whole-cell currents in ascending aortic VSMCs. The increase in TRPM7 currents was found to result from enhanced expression of TRPM7 protein rather than elevated single-channel activity (open probability and slope conductance) and/or reduced Mg(2+)-mediated channel block. Mechanistically, Ang II elevated TRPM7 expression via Ang II type 1 receptor-mediated ERK1/2 signaling. As indicated by the expression levels of VSMC differentiation marker genes, phenotypic switching of ascending aorta occurred during Ang II infusion. Meanwhile, ERK1/2-Elk-1 signaling pathway known to suppress VSMC differentiation was activated in Ang II-infused ascending aorta. Knockdown of TRPM7 with small interfering RNA established a causative role of TRPM7 in Ang II-induced phenotypic change and promotion of cell proliferation. Moreover, TRPM7 was shown to be required for Pyk2-ERK1/2-Elk-1 pathway activation by Ang II, which potentiated TRPM7 channel function and thus activated the Ca(2+)-sensitive kinase Pyk2. Finally, TRPM7 knockdown attenuated Ang II-induced displacement of myocardin from SM22 promoter, but the effects could be reversed by expression of constitutively active c-Src. Our data establish that upregulation of TRPM7 channels by Ang II contributes to the development of the proliferative phenotype of ascending aortic VSMCs, and TRPM7 channel suppresses VSMC gene expression via Ca(2+) influx-mediated activation of Pyk2-ERK1/2-Elk-1 pathway.

Transient Receptor Potential Melastatin 7 Channels are Involved in Ginsenoside Rg3-Induced Apoptosis in Gastric Cancer Cells

Ginsenosides play a role in a number of physiological and pharmacological functions in the gastrointestinal tract. The aim of this study was to clarify the potential role for transient receptor potential melastatin 7 (TRPM7) channels in ginsenoside Rg3-inhibited growth and survival of AGS cells, the most common human gastric adenocarcinoma cell line. The AGS cells were treated with varying concentrations of Rg3. Sub-G1 analysis, caspase-3 activity and poly(ADP-ribose) polymerase (PARP) cleavage analysis were conducted to determine whether AGS cell death occurs by apoptosis. TRPM7 channel blockers (La(3+) or 2-APB) and small interfering RNA (siRNA) were used in this study to confirm the role of TRPM7 channels. Furthermore, TRPM7 channels were over-expressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in AGS cell growth and survival. The addition of Rg3 to the culture medium inhibited AGS growth and survival. Experimental results showed sub-G1 was markedly increased, caspase-3 activity was elevated, and degree of PARP cleavage was increased. TRPM7 channel blockade, either by La(3+) or 2-APB or by suppressing TRPM7 expression with siRNA, blocked the Rg3-induced inhibition of cell growth and survival. Furthermore, TRPM7 channel over-expression in HEK 293 cells exacerbated Rg3-induced cell death. These findings indicate that ginsenoside Rg3 inhibits the growth and survival of gastric cancer cell which is because of the blockade of TRPM7 channel activity. Therefore, TRPM7 channels may play an important role in the survival of gastric cancer.

Suppression of transient receptor potential melastatin 7 channel induces cell death in gastric cancer

Ca2+ and Mg2+ have a fundamental role in many cellular processes and ion channels are involved in normal physiologic processes and in the pathology of various diseases. The aim here was to show that the presence and potential role of transient receptor potential melastatin 7 (TRPM7) channels in the growth and survival of AGS cells, the most common human gastric adenocarcinoma cell line. The patch-clamp technique for whole-cell recording was used in AGS cells. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Whole-cell voltage-clamp recordings revealed the TRPM7-like currents that activated spontaneously following loss of intracellular Mg2+. The current had a non-linear current-voltage relationship with the characteristic steep outward rectification associated with TRPM7 channels. Reverse transcription-polymerase chain reaction, western blotting, and immunoreactivity all showed abundant expression of TRPM7 messenger RNA and protein in AGS cells. Transfection of AGS cells with TRPM7 siRNA significantly reduced the expression of TRPM7 mRNA and protein as well as the amplitude of the TRPM7-like currents. Furthermore, we found that Mg2+ is critical for the growth and survival in AGS cells. Blockade of TRPM7 channels by La3+ and 2-APB or suppression of TRPM7 expression by siRNA inhibited the growth and survival of these cells. Human gastric adenocarcinoma cells express TRPM7 channel whose presence is essential for cell survival. The protein is a likely potential target for the pharmacological treatment of gastric cancer.

TrkA pathway(s) is involved in regulation of TRPM7 expression in hippocampal neurons subjected to ischemic-reperfusion and oxygen–glucose deprivation

The transient receptor potential melastatin 7 (TRPM7) is recently revealed playing a key role in anoxic neuronal death. Meanwhile, nerve growth factor (NGF), through activating TrkA pathway, has been widely accepted as a crucial factor for neuronal survival during cerebral ischemia. In this study, middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 5, 10, 20 and 30 h revealed an increasing up-regulation of TRPM7 expression in ipsilateral hippocampus after reperfusion and such a change reached a peak at 20 h, with high expression being maintained up to 30 h. Intracerebroventricular injection of NGF (500 ng) 30 min before MCAO, the expression of TRPM7 at 20 h after reperfusion was significantly reduced, and the effect of NGF was completely abolished by co-injection of TrkA inhibitor K252a. Furthermore, TRPM7 was found residing on hippocampal neuronal body and process, and TrkA was concurrently observed on the cell body by immunofluorescence staining. In agreement with in vivo, pre-incubation of cultured hippocampal neurons with NGF markedly down-regulated TRPM7 high expression at 20 h after oxygen–glucose deprivation (OGD), and this effect could be abrogated by K252a. In addition, when inhibitors wortmannin, U73122 and U0126 were introduced to block phosphatidylinositol-3 kinase (PI-3K), phospholipase C-γ (PLC-γ) and ras/mitogen-activated protein kinase (MAPK) pathways, respectively, only PI-3K inhibitor wortmannin substantially abolished NGF effects. These results suggest that TrkA, after being activated by NGF, can prevent up-regulation of TRPM7 expression in hippocampal neurons during reperfusion through PI-3K signal pathway. These findings open a new way for further investigations of the potential roles of TRPM7 and NGF in cerebral ischemia-reperfusion.

Transient Receptor Potential Melastatin 7–like Current in Human Head and Neck Carcinoma Cells: Role in Cell Proliferation

Ion channels are involved in normal physiologic processes and in the pathology of various diseases. In this study, we investigated the presence and potential function of transient receptor potential melastatin 7 (TRPM7) channels in the growth and proliferation of FaDu and SCC25 cells, two common human head and neck squamous carcinoma cell lines, using a combination of patch-clamp recording, Western blotting, immunocytochemistry, small interfering RNA (siRNA), fluorescent Ca(2+) imaging, and cell counting techniques. Although voltage-gated K(+) currents were recorded in all cells, none of FaDu cells express voltage-gated Na(+) or Ca(2+) currents. Perfusion of cells with NMDA or acidic solution did not activate inward currents, indicating a lack of NMDA receptor and acid-sensing channels. Lowering extracellular Ca(2+), however, induced a large nondesensitizing current reminiscent of Ca(2+)-sensing cation current or TRPM7 current previously described in other cells. This Ca(2+)-sensing current can be inhibited by Gd(3+), 2-aminoethoxydiphenyl borate (2-APB), or intracellular Mg(2+), consistent with the TRPM7 current being activated. Immunocytochemistry, Western blot, and reverse transcription-PCR detected the expression of TRPM7 protein and mRNA in these cells. Transfection of FaDu cells with TRPM7 siRNA significantly reduced the expression of TRPM7 mRNA and protein as well as the amplitude of the Ca(2+)-sensing current. Furthermore, we found that Ca(2+) is critical for the growth and proliferation of FaDu cells. Blockade of TRPM7 channels by Gd(3+) and 2-APB or suppression of TRPM7 expression by siRNA inhibited the growth and proliferation of these cells. Similar to FaDu cells, SCC25 cells also express TRPM7-like channels. Suppressing the function of these channels inhibited the proliferation of SCC25 cells.

SLC41A2 encodes a plasma-membrane Mg2+ transporter

The TRPM7 (transient receptor potential melastatin 7) ion channel has been implicated in the uptake of Mg2+ into vertebrate cells, as elimination of TRPM7 expression through gene targeting in DT40 B-lymphocytes renders them unable to grow in the absence of supplemental Mg2+. However, a residual capacity of TRPM7-deficient cells to accumulate Mg2+ and proliferate when provided with supplemental Mg2+ suggests the existence of Mg2+ uptake mechanism(s) other than TRPM7. Evaluation of the expression of several members of the SLC41 (solute carrier family 41) family, which exhibit homology with the MgtE class of prokaryotic putative bivalent-cation transporters, demonstrated that one, SLC41A2 (solute carrier family 41 member 2), is expressed in both wild-type and TRPM7-deficient DT40 cells. Characterization of heterologously expressed SLC41A2 protein indicated that it is a plasma-membrane protein with an N-terminus-outside/C-terminus-inside 11-TM (transmembrane)-span topology, consistent with its functioning as a trans-plasma-membrane transporter. In contrast with a previous report of ion-channel activity associated with SLC41A2 expression in oocytes, investigation of whole cell currents in SLC41A2-expressing DT40 cells revealed no novel currents of any type associated with SLC41A2 expression. However, expression of SLC41A2 in TRPM7-deficient cells under the control of a doxycycline-inducible promoter was able to conditionally enhance their net uptake of 26Mg2+ and conditionally and dose-dependently provide them with the capacity to grow in the absence of supplemental Mg2+, observations strongly supporting a model whereby SLC41A2 directly mediates trans-plasma-membrane Mg2+ transport. Overall, our results suggest that SLC41A2 functions as a plasma-membrane Mg2+ transporter in vertebrate cells.

Differential regulation of transient receptor potential melastatin 6 and 7 cation channels by ANG II in vascular smooth muscle cells from spontaneously hypertensive rats

Intracellular Mg2+ depletion has been implicated in vascular dysfunction in hypertension. We demonstrated that transient receptor potential melastatin 7 (TRPM7) cation channels mediate Mg2+ influx in VSMCs. Whether this plays a role in [Mg2+]i deficiency in hypertension is unclear. Here, we tested the hypothesis that downregulation of TRPM7 and its homologue TRPM6 is associated with reduced [Mg2+]i and that ANG II negatively regulates TRPM6/7 in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). Cultured VSMCs from Wistar Kyoto (WKY) and SHR were studied. mRNA and protein expression of TRPM6 and TRPM7 were assessed by RT-PCR and immunoblotting, respectively. Translocation of annexin-1, specific TRPM7 substrate, was measured as an index of TRPM7 activation. [Mg2+]i was determined using mag fura-2. VSMCs from WKY and SHR express TRPM6 and TRPM7. Basal TRPM6 expression was similar in WKY and SHR, but basal TRPM7 content was lower in VSMCs from SHR vs. WKY. This was associated with significantly reduced [Mg2+]i in SHR cells (P < 0.01). ANG II time-dependently increased TRPM6 expression, with similar responses in WKY and SHR. ANG II significantly increased TRPM7 expression in WKY (P < 0.05), but not in SHR. Annexin-1 translocation was reduced 1.5-2-fold in SHR vs. WKY. Our findings demonstrate that TRPM6 and TRPM7 are differentially regulated in VSMCs from SHR and WKY. Whereas TRPM6 is unaltered in SHR, expression of TRPM7 is blunted. This was associated with attenuated annexin-1 translocation and decreased VSMC [Mg2+]i in SHR. Downregulation of TRPM7, but not TRPM6, may play a role in altered Mg2+ homeostasis in VSMCs from SHR.