Transient Receptor Potential Melastatin 7 Expression Research Articles

F-actin microfilaments affect the LIPUS-promoted osteogenic differentiation of BMSCs through TRPM7.

The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, themolecular mechanisms responsible for LIPUS stimulation areunclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence wasperformed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown wasverifiedusingwesternblot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.

TRPM7 facilitates fibroblast-like synoviocyte proliferation, metastasis and inflammation through increasing IL-6 stability via the PKCα-HuR axis in rheumatoid arthritis

Transient receptor potential melastatin 7 (TRPM7) is a cation channel that plays a role in the progression of rheumatoid arthritis (RA), yet its involvement in synovial hyperplasia and inflammation has not been determined. We previously reported that TRPM7 affects the destruction of articular cartilage in RA. Herein, we further confirmed the involvement of TRPM7 in fibroblast-like synoviocyte (FLS) proliferation, metastasis and inflammation. We observed increased TRPM7 expression in FLSs derived from human RA patients. Pharmacological inhibition of TRPM7 protected primary RA-FLSs from proliferation, metastasis and inflammation. Furthermore, we found that TRPM7 contributes to RA-FLS proliferation, metastasis and inflammation by increasing the intracellular Ca2+ concentration. Mechanistically, the PKCα-HuR axis was demonstrated to respond to Ca2+ influx, leading to TRPM7-mediated RA-FLS proliferation, metastasis and inflammation. Moreover, HuR was shown to bind to IL-6 mRNA after nuclear translocation, which could be weakened by TRPM7 channel inhibition. Additionally, adeno-associated virus 9-mediated TRPM7 silencing is highly effective at alleviating synovial hyperplasia and inflammation in adjuvant-induced arthritis rats. In conclusion, our findings unveil a novel regulatory mechanism involved in the pathogenesis of RA and suggest that targeting TRPM7 might be a potential strategy for the prevention and treatment of RA.

LncRNA SNHG8 regulates the migration and angiogenesis of pHUVECs induced by high glucose via the TRPM7/ERK1/2 signaling axis

This study aimed to evaluate the regulatory effect and molecular mechanism of long noncoding RNA small nucleolus RNA host gene 8 (LncRNA SNHG8) in the migration and angiogenesis of primary human umbilical vein endothelial cells (pHUVECs) under high-glucose (HG) conditions. The HG-induced endothelial injury model was established in vitro.The cell model of silencing SNHG8, overexpressing SNHG8, and silencing TRPM7 was established by transfecting SNHG8-siRNA, SNHG8 plasmid and TRPM7-siRNA into cells with liposomes.The SNHG8 level was determined through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of transient receptor potential melastatin 7 (TRPM7), endothelial nitric oxide synthase (eNOS), p-eNOS, extracellular signal-regulated kinase 1/2(ERK1/2), and p-ERK1/2 were assessed through western blot. Nitric oxide (NO) levels were measured with DAF-FM. pHUVEC migration was examined through wound healing and Transwell assay, and pHUVEC angiogenesis was observed through a tube formation assay. Results showed that HG promoted the expression of lncRNA SNHG8 and TRPM7 and decreased the ratio of p-eNOS/eNOS and p-ERK1/2/ERK1/2 in pHUVECs . NO production, migration , and angiogenesis were inhibited in pHUVECs under HG conditions. Silencing lncRNA SNHG8 and TRPM7 could significantly reverse the HG-induced decrease in eNOS activation, NO production , migration, and angiogenesis . SNHG8 and U0126 (ERK pathway inhibitor) overexpression enhanced the HG effects, whereas using U0126 did not affect the TRPM7 expression. In conclusion, lncRNA SNHG8 participates in HG-induced endothelial cell injury and likely regulates NO production, migration, and angiogenesis of pHUVECs via the TRPM7/ERK1/2 signaling axis.

MiR135a administration ameliorates brain ischemic damage by preventing TRPM7 activation during brain ischemia.

miRNA-based strategies have recently emerged as a promising therapeutic approach in several neurodegenerative diseases. Unregulated cation influx is implicated in several cellular mechanisms underlying neural cell death during ischemia. The brain constitutively active isoform of transient receptor potential melastatin 7 (TRPM7) represents a glutamate excitotoxicity-independent pathway that significantly contributes to the pathological Ca2+ overload during ischemia. In the light of these premises, inhibition of TRPM7 may be a reasonable strategy to reduce ischemic injury. Since TRPM7 is a putative target of miRNA135a, the aim of the present paper was to evaluate the role played by miRNA135a in cerebral ischemia. Therefore, the specific objectives of the present paper were: (1) to evaluate miR135a expression in temporoparietal cortex of ischemic rats; (2) to investigate the effect of the intracerebroventricular (icv) infusion of miR135a on ischemic damage and neurological functions; and (3) to verify whether miR135a effects may be mediated by an alteration of TRPM7 expression. miR135a expression was evaluated by RT- PCR and FISH assay in temporoparietal cortex of ischemic rats. Ischemic volume and neurological functions were determined in rats subjected to transient middle cerebral artery occlusion (tMCAo) after miR135a intracerebroventricular perfusion. Target analysis was performed by Western blot. Our results demonstrated that, in brain cortex, 72 h after ischemia, miR135a expression increased, while TRPM7 expression was parallelly downregulated. Interestingly, miR135a icv perfusion strongly ameliorated the ischemic damage and improved neurological functions, and downregulated TRPM7 protein levels. The early prevention of TRPM7 activation is protective during brain ischemia.

Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway

Objective: To explore the regulatory mechanism of transient receptor potential melastatin-7 (TRPM7) in high glucose-induced renal tubular epithelial cell injury. Methods: The expression of TRPM7 in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells was detected by RT-qPCR. Then, the TRPM7 interference vector was constructed, and the downstream high mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4) signaling pathway proteins were detected. Next, in addition to interference with TRPM7 expression, overexpression of HMGB1 in high glucose-induced HK-2 cells was performed. Cell activity, apoptosis, oxidative stress levels, and inflammation levels were determined by CCK8, TUNEL, Western blotting, immunofluorescence and related kits. Results: TRPM7 expression was upregulated in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells. Interference with TRPM7 reduced cell damage, epithelial-mesenchymal transition, oxidative stress, and inflammatory response in high glucose-induced HK-2 cells via inhibiting the HMGB1/TLR4 signaling pathway. However, the effects induced by TRPM7 silencing were abrogated by HMGB1 overexpression. Conclusions: Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway. Further animal experiments and clinical trials are warranted to verify its effect.

Abstract 032: Downregulation Of Trpm7 Is Associated With Inflammation And Fibrosis In Aldosterone-induced Hypertension

Transient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg 2+ with an important role in cell homeostasis. Here, we investigated the importance of TRPM7-Mg 2+ in inflammatory response and oxidative stress in cardiac fibroblasts (CF) in the context of hypertension and organ damage induced by aldosterone and salt. Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600 μg/Kg/day) plus NaCl (1% drinking water), 4 weeks. Blood pressure (BP) was evaluated by tail cuff. Molecular mechanisms were investigated in primary culture of CF. Ca 2+ influx was assessed by fluorescence microscopy. Gene and protein expression was assessed by real-time PCR and immunoblotting respectively. M7+/Δ mice exhibited reduced TRPM7 expression (30%), phospho-TRPM7 (62%) and total tissue [Mg 2+ ] (28%). This was recapitulated in WT-aldo-salt. Aldo, salt and aldo-salt increased BP in M7+/Δ mice. In WT, only aldo-salt increased BP (134 mmHg). Cardiac collagen deposition (68%), expression of IL-6, TGFβ, p-Smad3 and p-ERK1/2 were increased (1.5-1.8 fold) in aldo-salt M7+/Δ versus WT. M7+/Δ CF exhibit increased mineralocorticoid target gene SGK1 (3-fold vs WT), which was increased by aldosterone (10-fold vs WT-aldo 5.4-fold). CF M7+/Δ had reduced aldosterone-induced calcium influx (peak-response 105±0.7 vs WT:149±10). CF M7+/Δ exhibited reduced proliferation (30%), and increases protein expression of TGFβ, IL-6, p-Smad3 and p-ERK1/2 (1.4-2.0-fold) vs WT. Nox2 (70%) and Nox4 (120%) expression was increased in CF M7+/Δ vs WT. Aldosterone increased Nox4 (54%) only in CF WT. These effects were associated with increased expression of oxidized peroxiredoxin-3 (76%). Increased gene expression for the antioxidant catalase (1.7-fold), NQO1 (2.6-fold) and Prdx1 (1.7-fold) was increased in M7+/Δ CF. Mg 2+ supplementation normalized intracellular [Mg 2+ ], cell proliferation, protein phosphorylation and Nox4 expression in CF TRPM7+/Δ (p<0.05). We identify a novel anti-inflammatory and anti-fibrotic role for TRPM7/Mg 2+ in cardiac fibroblasts. These processes are downregulated in aldo-induced hypertension.

TRPM7 is Involved in the Regulation of Proliferation, Migration and Osteogenic Differentiation of Human Dental Follicle Cells.

Dental follicle cells (DFCs) are promising candidates for tissue engineering. However, the molecular mechanisms that regulate the biological characteristics of DFCs are still unclear. Transient receptor potential melastatin 7 (TRPM7) is a Ca2+- and Mg2+-permeable cation channel. The aim of this study was to determine the impact of TRPM7 on the proliferation, migration and osteogenic differentiation of DFCs. PCR, Western blotting, Immunocytochemical staining and Patch clamp methods were used to identify the gene and protein expression of TRPM7 in DFCs. DFCs were infected with lentiviruses that expressed either TRPM7 specific shRNA or scrambled non-effective shRNA to investigate its functional role. Cell proliferation and migration were assessed using Cell Counting Kit-8 assays and transwell cell culture chambers separately. Cell osteogenic differentiation were determined by ALP assay kit and Alizarin Red staining. Gene and protein expression of TRPM7 were detected in DFCs, but not of TRPM6, which is a closely related channel with similar function. In the absence of Mg2+, typical whole cell TRPM7-like currents were recorded by patch clamp. These were inhibited by low concentrations of 2-APB, but activated by high concentrations of 2-APB. Functional studies demonstrated that suppression of TRPM7 expression inhibited the proliferation and migration of DFCs, and promoted their osteogenic differentiation. Furthermore, Mg2+ deficiency mimicked the effects of TRPM7 knockdown in terms of osteogenic differentiation of DFCs. These results demonstrate that TRPM7 is involved in regulating the proliferation, migration and osteogenic differentiation of DFCs.

Lianmei Qiwu Decoction relieves diabetic cardiac autonomic neuropathy by regulating AMPK/TrkA/TRPM7 signaling pathway

This study investigated the effect of Lianmei Qiwu Decoction(LMQWD) on the improvement of cardiac autonomic nerve remodeling in the diabetic rat model induced by the high-fat diet and explored the underlying mechanism of LMQWD through the AMP-activated protein kinase(AMPK)/tropomyosin receptor kinase A(TrkA)/transient receptor potential melastatin 7(TRPM7) signaling pathway. The diabetic rats were randomly divided into a model group, an LMQWD group, an AMPK agonist group, an unloaded TRPM7 adenovirus group(TRPM7-N), an overexpressed TRPM7 adenovirus group(TRPM7), an LMQWD + unloaded TRPM7 adenovirus group(LMQWD+TRPM7-N), an LMQWD + overexpressed TRPM7 adenovirus group(LMQWD+TRPM7), and a TRPM7 channel inhibitor group(TRPM7 inhibitor). After four weeks of treatment, programmed electrical stimulation(PES) was employed to detect the arrhythmia susceptibility of rats. The myocardial cell structure and myocardial tissue fibrosis of myocardial and ganglion samples in diabetic rats were observed by hematoxylin-eosin(HE) staining and Masson staining. The immunohistochemistry, immunofluorescence, real-time quantitative polymerase chain reaction(RT-PCR), and Western blot were adopted to detect the distribution and expression of TRPM7, tyrosine hydroxylase(TH), choline acetyltransferase(ChAT), growth associated protein-43(GAP-43), nerve growth factor(NGF), p-AMPK/AMPK, and other genes and related neural markers. The results showed that LMQWD could significantly reduce the arrhythmia susceptibility and the degree of fibrosis in myocardial tissues, decrease the levels of TH, ChAT, and GAP-43 in the myocardium and ganglion, increase NGF, inhibit the expression of TRPM7, and up-regulate p-AMPK/AMPK and p-TrkA/TrkA levels. This study indicated that LMQWD could attenuate cardiac autonomic nerve remodeling in the diabetic state, and its mechanism was associated with the activation of AMPK, further phosphorylation of TrkA, and inhibition of TRPM7 expression.

Involvement of TRPM7 in Alcohol-Induced Damage of the Blood-Brain Barrier in the Presence of HIV Viral Proteins.

Ethanol (EtOH) exerts its effects through various protein targets, including transient receptor potential melastatin 7 (TRPM7) channels, which play an essential role in cellular homeostasis. We demonstrated that TRPM7 is expressed in rat brain microvascular endothelial cells (rBMVECs), the major cellular component of the blood-brain barrier (BBB). Heavy alcohol drinking is often associated with HIV infection, however mechanisms underlying alcohol-induced BBB damage and HIV proteins, are not fully understood. We utilized the HIV-1 transgenic (HIV-1Tg) rat to mimic HIV-1 patients on combination anti-retroviral therapy (cART) and demonstrated TRPM7 expression in rBMVECs wass lower in adolescent HIV-1Tg rats compared to control animals, however control and HIV-1Tg rats expressed similar levels at 9 weeks, indicating persistent presence of HIV-1 proteins delayed TRPM7 expression. Binge exposure to EtOH (binge EtOH) decreased TRPM7 expression in control rBMVECs in a concentration-dependent manner, and abolished TRPM7 expression in HIV-1Tg rats. In human BMVECs (hBMVECs), TRPM7 expression was downregulated after treatment with EtOH, HIV-1 proteins, and in combination. Next, we constructed in vitro BBB models using BMVECs and found TRPM7 antagonists enhanced EtOH-mediated BBB integrity changes. Our study demonstrated alcohol decreased TRPM7 expression, whereby TRPM7 could be involved in the mechanisms underlying BBB alcohol-induced damage in HIV-1 patients on cART.

Effect of Static Pressure on Early Apoptosis of Condylar Chondrocytes by Activating Transient Receptor Potential Melastatin 7 (TRPM7) Channel

Background: This study is to detect the Ca2+ permeability of transient receptor potential melastatin 7 (TRPM7) channel under Static pressure, and to verify the effection of pressure on cell apoptosis through TRPM7 channel mediated Ca2+ influx. Materials and Methods: Rat condylar chondrocytes cells are divided into control group, pressure group and FTY720 group. TRPM7 protein content is detected by westernblot. Calcium current is detected by colorimetry, and flow cytometry is used to detect apoptotic cells. Results: The cell morphology is spindle or spindle shape with clear boundary. The nucleus is oval and located in the center of the cell. Compared with the control group, the protein content of TRPM7, the calcium concentration was significantly higher (P <0.05). apoptosis in the pressure group is significantly increased than that in the control group (P <0.05). Conclusion: Static pressure upregulates the expression of TRPM7 in chondrocytes, resulting in the increase of intracellular Ca2+ concentration, which leads to apoptosis through signal pathway.

Carvacrol Inhibits Expression of Transient Receptor Potential Melastatin 7 Channels and Alleviates Zinc Neurotoxicity Induced by Traumatic Brain Injury.

Carvacrol is a monoterpenoid phenol produced by aromatic plants such as oregano. Although the exact mechanism by which carvacrol acts has not yet been established, it appears to inhibit transient receptor potential melastatin 7 (TRPM7), which modulates the homeostasis of metal ions such as zinc and calcium. Several studies have demonstrated that carvacrol has protective effects against zinc neurotoxicity after ischemia and epilepsy. However, to date, no studies have investigated the effect of carvacrol on traumatic brain injury (TBI)-induced zinc neurotoxicity. In the present study, we investigated the therapeutic potential of carvacrol for the prevention of zinc-induced neuronal death after TBI. Rats were subjected to a controlled cortical impact, and carvacrol was injected at a dose of 50 mg/kg. Histological analysis was performed at 12 h, 24 h, and 7 days after TBI. We found that carvacrol reduced TBI-induced TRPM7 over-expression and free zinc accumulation. As a result, subsequent oxidative stress, dendritic damage, and neuronal degeneration were decreased. Moreover, carvacrol not only reduced microglial activation and delayed neuronal death but also improved neurological outcomes after TBI. Taken together, these findings suggest that carvacrol administration may have therapeutic potential after TBI by preventing neuronal death through the inhibition of TRPM7 expression and alleviation of zinc neurotoxicity.

Clinical and Prognostic Values of TRPM7 in Colon and Rectal Cancers.

Background and Objectives: Transient receptor potential melastatin 7 (TRPM7) is a unique channel protein, and functionally responsible for transportation of calcium and magnesium. Physiologically, the TRPM7 channel is involved in homeostasis of calcium and magnesium, and cell survival. TRPM7 expression is up-regulated in many cancers as malignant behaviors of cancer cells, and its deficiency suppresses their growth. Materials and Methods: In this study, we aimed to analyze clinical and prognostic characteristics of TRPM7 expression in colorectal cancers (CRC) using The Cancer Genome Atlas. Results: High expression of TRPM7 was observed in younger patients with rectal cancer (p = 0.0002). By quantitative correlation analysis, TRPM7 was negatively correlated with age (R = -0.239, p = 0.003) and p53 (R = -0.240, p = 0.002). Furthermore, it was positively correlated with APC expression (R = 0.534, p < 0.001) and KRAS expression (R = 0.319, p < 0.001). In colon cancer, there were no variables that showed a significant correlation with TRPM7. Survival analysis found that TRPM7 expression did not have any prognostic value in colon and rectal cancers. Conclusions: Our study highlights that TRPM7 expression in CRC, particularly in rectal cancer, may be a potential marker. Future studies are needed to provide deeper insights into the role of TRPM7 in rectal cancer.

TRPM7 channel inhibition attenuates rheumatoid arthritis articular chondrocyte ferroptosis by suppression of the PKCα-NOX4 axis

A role for ferroptosis in articular cartilage destruction associated with rheumatoid arthritis (RA) has not been identified. We previously reported transient receptor potential melastatin 7 (TRPM7) expression was correlated with RA cartilage destruction. Herein, we further characterized a role for TRPM7 in chondrocyte ferroptosis. The expression of TRPM7 was found to be elevated in articular chondrocytes derived from adjuvant arthritis (AA) rats, human RA patients, and cultured chondrocytes treated with the ferroptosis inducer, erastin. TRPM7 knockdown or pharmacological inhibition protected primary rat articular chondrocytes and human chondrocytes (C28/I2 cells) from ferroptosis. Moreover, TRPM7 channel activity was demonstrated to contribute to chondrocyte ferroptosis by elevation of intracellular Ca2+. Mechanistically, the PKCα-NOX4 axis was found to respond to stimulation with erastin, which resulted in TRPM7-mediated chondrocyte ferroptosis. Meanwhile, PKCα was shown to directly bind to NOX4, which could be reduced by TRPM7 channel inhibition. Adeno-associated virus 9-mediated TRPM7 silencing or TRPM7 blockade with 2-APB alleviated articular cartilage destruction in AA rats and inhibited chondrocyte ferroptosis. Collectively, both genetic and pharmacological inhibitions of TRPM7 attenuated articular cartilage damage and chondrocyte ferroptosis via the PKCα-NOX4 axis, suggesting that TRPM7-mediated chondrocyte ferroptosis is a promising target for the prevention and treatment of RA.

Potential Protective Role of TRPM7 and Involvement of PKC/ERK Pathway in Blue Light-Induced Apoptosis in Retinal Pigment Epithelium Cells in Vitro.

Purpose:Blue light triggers apoptosis of retinal pigment epithelium (RPE) cells and causes retinal damage. The aim of this study was to elucidate the protective role of transient receptor potential melastatin 7 (TRPM7) in photodamaged RPE cells.Methods:RPE cells were isolated from Sprague-Dawley (SD) rats and exposed to varying intensities of blue light (500–5000 lux) in vitro. Cell proliferation and metabolic activity were respectively assessed by bromodeoxyuridine (BrdU) incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to analyze the TRPM7, protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and Bcl2-associated x/B-cell lymphoma 2 (Bax/Bcl-2) messenger RNA (mRNA) and protein expression levels. The cells were transfected with TRPM7 small interfering RNA (siRNA) or transduced with TRPM7-overexpressing lentiviruses and cultured with or without the pigment epithelium-derived factor (PEDF).Results:Blue light inhibited the proliferation and metabolic activity of RPE cells in an intensity-dependent manner when compared to nonirradiated controls (P < 0.05). Compared to the control, photodamaged RPE cells showed decreased levels of TRPM7, PKC, ERK, and Bax, and an increase in Bcl-2 levels (P < 0.01). Forced expression of TRPM7 partially rescued the proliferative capacity of RPE cells (P < 0.01) and restored the levels of TRPM7, PKC, ERK, and Bax (P < 0.01), whereas TRPM7 knockdown had the opposite effects (P < 0.01). TRPM7 and PEDF synergistically alleviated the damaging effects of blue light.Conclusions:Blue light triggers apoptosis of RPE cells, and its deleterious effects can be partially attenuated by the synergistic action of TRPM7 and PEDF via the PKC/ERK signaling pathway.

Expression and prognostic value of TRPM7 in canine mammary tumours.

Canine mammary gland tumour (CMTs) are one of the most commonly found tumours in intact female dogs. A previous study on canine mammary glands demonstrated the presence of the transient receptor potential melastatin 7 (TRPM7) ion channels in healthy canine mammary tissues. However, the significance of TRPM7 in CMT is not yet known. TRPM7 is a Ca2+ and Mg2+ permeable cation channel that contains a protein kinase domain. The aim of this study was to determine TRPM7 expression in 57 benign and malignant CMT tissues of dogs using immunohistochemistry (IHC) and evaluate its correlation with clinicopathological features and explore the potential prognostic value of TRPM7 in a prospective survival study. IHC analysis shows that TRPM7 was expressed in the cytoplasm of neoplastic epithelial cells. Moreover, TRPM7 expression was significantly associated with tumour malignancy (P = .027), Ki‐67 index (P < .0001) and metastasis (P < .0001). Survival curve analysis indicates that high TRPM7 expression was significantly associated with poor disease‐free (P = .035) and overall survival (P = .011) in malignant CMTs. Our results demonstrate that TRPM7 is expressed in CMTs and that its expression is positively correlated with clinicopathological parameters. Thus, TRPM7 was assumed to be a potential prognostic factor for CMTs.

INTERACTION BETWEEN TRPM7 AND EPIDERMAL GROWTH FACTOR RECEPTOR MEDIATES VASCULAR SMOOTH MUSCLE CELL ACTIVATION AND PROLIFERATION

Objective: Epidermal growth factor (EGF) binds to EGF receptor (EGFR) and stimulates multiple vascular signaling pathways. Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein (channel+enzyme, termed chanzyme) playing an important role in vascular homeostasis. Previous studies in renal cells demonstrated interactions between EGFR and TRPM6, a homologue of TRPM7. Here we questioned the importance of TRPM7 in EGF-mediated effects on vascular smooth muscle cell (VSMC) function. Design and method: Primary cultured VSMCs were isolated from humans and rodent models. Rat VSMCs (rVSMCs) and human VSMCs (hVSMCs) were stimulated with EGF (50ng/ml) in the absence/presence of gefitinib (1 μM), PP2 (10 μM), 2APB (30 μM) and NS8593 (40 μM), inhibitors of EGFR, c-Src kinase and TRPM7 respectively. Aorta were isolated from wild type (WT), TRPM7 kinase-deficient (TRPM7+/-kinase), and TRPM7 kinase-dead (TRPM7R/R) mice. Protein expression was investigated by immunoblotting. Intracellular Ca2+ and Mg2+ were assessed using fluorescence microscopy and flow cytometry respectively. EGFR-TRPM7 interaction was explored by proximity ligation assay (PLA) and immunoprecipitation. VSMC migration and proliferation were assessed by wound-healing assay and CFSE proliferation studies. Results: In hVSMC and rVSMC, EGF increased TRPM7 expression (47%) and phosphorylation (21%), p &lt; 0.05 vs control; effects abolished by gefitinib and PP2. EGF-induced Mg2+ and Ca2+ influx was attenuated by gefitinib (4% and 8% respectively), NS8593 (5% for Mg2+) and 2-APB (6% and 13% respectively). EGF enhanced ERK1/2 phosphorylation (3-fold) in rVSMCs through c-Src, EGFR and TRPM7, p &lt; 0.05. Cell migration (26%) and proliferation (17%) were enhanced by EGF, and reduced by inhibitors of EGFR, TRPM7 and ERK1/2, p &lt; 0.05. EGF induced TRPM7-EGFR interaction, primarily in the cell membrane. VSMCs from TRPM7+/-kinase showed reduced EGFR expression (73%), phospho-c-Src (22%), and phospho-ERK1/2 (90%). Aortas from TRPM7R/R exhibited reduced phospho-EGFR (63%) and phospho-ERK1/2 (36%). Vessels from TRPM7+/-kinase showed reduced wall thickness. Conclusions: Our findings demonstrate important cross talk between EGFR and TRPM7. EGF/EGFR is both upstream and downstream of TRPM7. This interaction, which involves c-Src, regulates VSMC migration and proliferation. We identify a novel EGFR:TRPM7 pathway that may play an important role in vascular function and remodeling.

Carvacrol alleviates liver fibrosis by inhibiting TRPM7 and modulating the MAPK signaling pathway

Liver fibrosis is a compensatory response to the tissue repair process. The activation and proliferation of hepatic stellate cells (HSCs) are thought to be related to the occurrence of hepatic fibrosis. Therefore, inhibiting the activation and proliferation of HSCs is a key step in alleviating liver fibrosis. As a non-specific inhibitor of transient receptor potential melastatin 7 (TRPM7), carvacrol has anti-tumor, anti-inflammatory and anti-hepatic fibrosis activities. This study aimed to explore the protective effect of carvacrol on liver fibrosis and related molecular mechanisms. A CCl4-induced liver fibrosis mouse model and platelet-derived growth factor (PDGF-BB)-activated HSC-T6 cells (a rat hepatic stellate cell line) were employed for in vivo and in vitro experiments. C57BL/6J mice were orally administered different concentrations of carvacrol every day for 6 weeks during the development of CCl4-induced liver fibrosis. The results show that carvacrol could effectively reduce liver damage and the progression of liver fibrosis in mice, which are expressed as fibrotic markers levels were reduced and histopathological characteristics were improved. Moreover, carvacrol inhibited the proliferation and activation of HSC-T6 cells induced by PDGF-BB. In addition, it was found that carvacrol inhibits the expression of TRPM7 and mediated through mitogen-activated protein kinases (MAPK). Collectively, our study shows that carvacrol can reduce liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and the MAPK signaling pathway might be involved in this process.

Transient Receptor Potential Melastatin 7 Promotes Vascular Adventitial Fibroblasts Phenotypic Transformation and Inflammatory Reaction Induced by Mechanical Stretching Stress via p38 MAPK/JNK Pathway

Remodeling of the arteries is one of the pathological bases of hypertension. We have previously shown that transient receptor potential melastatin 7 (TRPM7) aggravates the vascular adventitial remodeling caused by pressure overload in the transverse aortic constriction (TAC) model. In this study, we sought to explore the functional expression and downstream signaling of TRPM7 in vascular adventitial fibroblasts (AFs) stimulated by mechanical stretching stress (MSS). The expression of TRPM7 was upregulated with a concomitant translocation to the cytoplasm in the AFs stimulated with 20% MSS. Meanwhile, the expression of α-smooth muscle actin (α-SMA), a marker of transformation from AFs to myofibroblasts (MFs) was also increased. Moreover, AF-conditioned medium caused a significant migration of macrophages after treatment with MSS and contained high levels of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Pharmacological and RNA interference approaches using the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB) and specific anti-TRPM7 small interfering RNA (si­RNA-TRPM7) abrogated these changes significantly. Further exploration uncloaked that inhibition of TRPM7 reduced the phosphorylation of p38 MAP kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) in the AFs stimulated with MSS. Furthermore, inhibition of the phosphorylation of p38MAPK or JNK could also alleviate the MSS-induced expression of α-SMA and secretion of inflammatory factors. These observations indicate that activated TRPM7 participates in the phenotypic transformation and inflammatory action of AFs in response to MSS through the p38MAPK/JNK pathway and suggest that TRPM7 may be a potential therapeutic target for vascular remodeling caused by hemodynamic changes in hypertension.

Abstract MP48: EGF Regulates VSMC Migration And Proliferation Through Crosstalk Between TRPM7 And EGFR

Epidermal growth factor (EGF), signals throught the EGF receptor (EGFR) and plays an important role in the pathogenesis of vascular remodeling. Transient receptor potential melastatin 7 (TRPM7) is a channel bound to a kinase domain important for Mg 2+ , Zn 2+ and Ca 2+ homeostasis. Cancer patients treated with EGFR inhibitors develop hypomagnesemia, suggesting a relationship between EGFR and TRPM7. Here we investigated the role of TRPM7 in EGF signaling in vascular smooth muscle cell (VSMC) from humans (hVSMC) and rats (rVSMC). VSMCs were stimulated with EGF (50ng/ml) for 5min and 24h with/without pretreatment of gefitinib (1μM), PP2 (10μM), 2APB (30μM) and NS8593 (40μM), inhibitors of EGFR, c-Src kinase and TRPM7 respectively. Aortas were isolated from wild type (WT), TRPM7-deficient (TRPM7 +/Δkinase ) and kinase-dead (TRPM7 R/R ) mice. Protein expression was assessed by immunoblotting. Ca 2+ and Mg 2+ were assessed using Cal-520 and Mg-green probes respectively. EGFR/TRPM7 interaction was investigated by proximity ligation assay (PLA), immunoprecipitation and confocal microscopy. VSMC migration and proliferation were examined by wound healing and CFSE proliferation assays. In hVSMC and rVSMC, EGF increased TRPM7 expression (47%) and phosphorylation (21%), (p&lt;0.05); effects abolished by gefitinib and PP2. EGF-induced Mg 2+ and Ca 2+ influx was attenuated by gefitinib (4% and 8% respectively), NS8593 (5% for Mg 2+ ) and 2-APB (6% and 13% respectively). EGF enhanced ERK1/2 phosphorylation (3-fold) through c-Src, EGFR and TRPM7, p&lt;0.05. Cell migration (26%) and proliferation (17%) were enhanced by EGF, and reduced by inhibitors of EGFR, TRPM7 and ERK1/2, p&lt;0.05. EGF induced TRPM7-EGFR interaction (51%), which was reduced by gefitinib (34%) and PP2 (25%). VSMC from TRPM7 +/Δkinase showed reduced EGFR expression (73%), phospho-c-Src (22%), and phospho-ERK1/2 (90%). Aortas from TRPM7 R/R exhibited reduced phospho-EGFR (63%) and phospho-ERK1/2 (36%). Vessels from TRPM7 +/Δkinase showed reduced wall thickness (35%). Our findings demonstrate that interaction between EGFR/TRPM7 is a key process underlying EGF-induced VSMC migration and growth. This novel EGF-c-Src-EGFR-TRPM7 pathway may play an important role in vascular remodeling.

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

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.