TRPM7 Expression Research Articles

Proton pump inhibitors and osteoporosis risk: exploring the role of TRPM7 channel.

Long-term use of proton pump inhibitors (PPIs) has been linked to an increased risk of osteoporosis, with various indirect mechanisms so far identified. Although no direct underlying mechanism for effect on bone cells have been investigated with the use of PPIs. Melastatin-like transient receptor potential 7 (TRPM7)channel has been engaged in the proliferation of bone cells. TRPM7 channel is regulated by extracellular Mg2+ and Ca2+ level, thatfurther encourages to analyse if anyimbalance with pantoprazole usage could alter bone remodelling process mediated by TRPM7. The present study was conducted to investigate the effect of pantoprazole on the calcium and magnesium level, the cations involved in the bone remodelling process, as well as role of the TRPM7 channel in the proliferation of bone cells. A cytotoxicity study was carried out to study the effect of pantoprazole on the bone cell using MC3T3-E1 cell line, together with the expression of TRPM7 was determined post-pantoprazole treatment. An in vivo study in rats was carried out for estimation of Ca2+, Mg2+ and Ca2+/Mg2+ ratio as well as bone strength was measured over a duration of 4weeks and 8weeks with the treatment of pantoprazole. A pilot-scale clinical study was carried out in patients with a fracture to support the evidence of preliminary findings from in-vitro and in vivo studies. MC3T3-E1 cell line treated with pantoprazole showed decreased cell viability in a dose-dependent manner and reduced expression of TRPM7 channel, evidencing interaction of TRPM7 and pantoprazole in the bone remodelling process. A pilot study conducted on 12 patients having major fractures showed changes in serum Mg2+ and Ca2+ levels over a period of 1month as well as the animal study also showed ionic imbalance over 8-week treatment with pantoprazole. Bone density measured for the patient at the end of the 1-month treatment was found to be in the osteopenic category, together with the animal study which showed a decrease in femur bone strength for the animal treated with pantoprazole over a period of 8weeks. The study findings proved a negative impact of pantoprazole use on Ca2+ and Mg2+ levels, which can impact TRPM7-mediated bone remodelling which serves to be a possible mechanism for osteoporosis upon pantoprazole use.

The expression and significance of TRPM8 among chronic rhinosinusitis with nasal polyps

Objective: To compare the expression and difference of melastatin-related transient receptor potential 8(TRPM8) among chronic rhinosinusitis, nasal polyps and normal mucosa tissues. And to explore the significant expression of TRPM8 among CRSwNP. Methods: Fifty-one patients underwent endoscopic sinus surgery in the Department of Otorhinolaryngology Head and Neck Surgery of Renmin Hospital of Wuhan University from February 2019 to January 2020 were recruited, including 33 males and 18 females, aged from 14 to 65 years old (34.55±1.689).Immunohistochemistry was used to detected the expression of TRPM8 protein among CRSsNP(17),CRSwNP (17) and control tissuses(17). In addition, the correlation between the expression of TRPM8 protein in CRSwNP patients and preoperative CT Lund-Mackay scores and preoperative VAS scores and sinonasal outcome test-20 scores was analyzed, respectively. The primary human nasal epithelial cells were cultured in vitro and the expression of TRPM8 was detected by quantitative real-time PCR and western blotting. The tissue in control group, chronic rhinosinusitis without nasal polyps (CRSsNP) group and the CRSwNP group were collected and grinded into tissue homogenized. The expression of TRPM8 protein was detected by western blotting after 24 h stimulation after homogenate was added into the medium of RPMI 2650 and primary nasal epithelial cells. Results: Compared with the control, the expression of TRPM8 was significantly up-regulated in nasal polyps (t=6.852, P<0.05). TRPM8 was mainly expressed in epithelial cells. The expression of TRPM8 in the epithelial cells of CRSsNP had no difference with the control group (t=1.980, P>0.05). In addition, the expression of TRPM8 in CRSwNP patients was positively correlated with the preoperative CT Lund-Mackay scores and VAS scores and SNOT-20 scores (r=0.512, P<0.05;r=0.853, P<0.01;r=0.814, P<0.01). After cultured primary epithelial cells in vitro, the expression level of TRPM8 in epithelial cells derived from nasal polyp was significantly higher than that in control group (t=8.845, P<0.05). By adding the homogenization of control and CRSsNP and CRSwNP tissues, the expression of TRPM8 in RPMI 2650 cells and primary nasal epithelial cells was changed and that was significantly increased after adding the homogenization of the group of CRSwNP. Conclusion: TRPM8 is highly expressed in nasal polyps epithelial cells, suggesting that TRPM8 may be involved in the pathogenesis of nasal polyps regulated by nasal epithelial cells.

Long Noncoding RNA ST7-AS1 Upregulates TRPM7 Expression by Sponging microRNA-543 to Promote Cervical Cancer Progression [Expression of Concern

Long Noncoding RNA ST7-AS1 Upregulates TRPM7 Expression by Sponging microRNA-543 to Promote Cervical Cancer Progression [Expression of Concern

Transient Receptor Potential Melastatin 2 Thermosensitive Neurons in the Preoptic Area Involved in Menopausal Hot Flashes in Ovariectomized Mice

Introduction: Menopausal hot flashes are related to hypothalamic preoptic area (POA) dysfunction. Thermosensitive transient receptor potential channels (ThermoTRPs) are involved in temperature sensing and regulation of thermosensitive neurons (TSNs) in the POA. Whether ThermoTRP-TSNs in the POA, particularly the non-noxious thermoreceptor, transient receptor potential melastatin 2 (TRPM2), are involved in the occurrence of hot flashes is still unclear. Methods: Twenty wild-type and 50 Trpm2-Cre adult female mice were randomly divided into sham (SHAM) and ovariectomy (OVX) groups. In the POA, ERα, ERβ, GPR30, TRPA1, TRPM8, TRPM2, and TRPV1 expression was detected by Western blot or/and quantitative real-time polymerase chain reaction and the number of TSNs expressing TRPM2 (TRPM2-TSNs) by immunofluorescence. Before and after TRPM2-TSN activation/inhibition, back (BST) and tail skin temperature (TST) and the proportion of glutamatergic and GABAergic neurons among TRPM2-TSNs were recorded. Results: Compared with SHAM, the expression of ERα, ERβ, TRPM2, and TRPM8 in the POA of the OVX group decreased, with a significantly larger change range for TRPM2 than TRPM8. In addition, the number of TRPM2-TSNs showing TRPA1, TRPM8, and TRPV1 expression in the OVX group decreased, and the proportion of glutamatergic and GABAergic neurons in TRPM2-TSNs decreased and increased, respectively. Meanwhile, BST and TST increased. After activating or inhibiting TRPM2-TSNs, the proportions of glutamatergic and GABAergic neurons in TRPM2-TSNs changed, along with the BST and TST. Conclusion: In menopause, the abnormal quantity and function of TRPM2-TSNs in the POA is key for the development of hot flashes, characterized by an imbalance in heat dissipation and production due to the corresponding imbalance in glutamatergic and GABAergic neurons.

Selected transient receptor potential channel genes' expression in peripheral blood mononuclear cells of multiple sclerosis.

Transient receptor potential channels have responsibilities in many cellular processes such as cytokine production, cell differentiation, and cytotoxicity by affecting intracellular cation levels or intracellular signal pathways. Multiple sclerosis is a chronic autoimmune central nervous system (CNS) disease caused by environmental and genetic factors. In this study, we aim to investigate TRPV1-TRPV4, TRPM2, TRPM4, TRPM7, TRPC6, and TRPA1 mRNA expression levels, which are associated with the inflammatory process, in the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RRMS) patients. Thirty-five healthy controls and age-gender matched thirty patients with RRMS were involved in the study. TRPC6, TRPA1, TRPM2, TRPM4, TRPM7, TRPV1, TRPV2, TRPV3, and TRPV4 PBMCs mRNA expression levels were determined by qPCR. In the present study, the TRPC6, TRPM7, TRPV1, TRPV3, and TRPV4 mRNA expressions of RRMS patients in PBMCs decreased at a significant level compared to the healthy control group (p = .000, p = .000, p = .044, p = .000, p = .004, respectively). The decreased expression of TRPC6, TRPM7, TRPV1, TRPV3, and TRPV4 in PBMCs may be associated with the pathogenesis of MS. Further studies are required to understand the mechanism of the relation between these TRP channels and MS and other autoimmune diseases.

Evidence for opposing selective forces operating on human-specific duplicated TCAF genes in Neanderthals and humans

TRP channel-associated factor 1/2 (TCAF1/TCAF2) proteins antagonistically regulate the cold-sensor protein TRPM8 in multiple human tissues. Understanding their significance has been complicated given the locus spans a gap-ridden region with complex segmental duplications in GRCh38. Using long-read sequencing, we sequence-resolve the locus, annotate full-length TCAF models in primate genomes, and show substantial human-specific TCAF copy number variation. We identify two human super haplogroups, H4 and H5, and establish that TCAF duplications originated ~1.7 million years ago but diversified only in Homo sapiens by recurrent structural mutations. Conversely, in all archaic-hominin samples the fixation for a specific H4 haplotype without duplication is likely due to positive selection. Here, our results of TCAF copy number expansion, selection signals in hominins, and differential TCAF2 expression between haplogroups and high TCAF2 and TRPM8 expression in liver and prostate in modern-day humans imply TCAF diversification among hominins potentially in response to cold or dietary adaptations.

Modulation of the Cardiac Myocyte Action Potential by the Magnesium-Sensitive TRPM6 and TRPM7-like Current.

The cardiac Mg2+-sensitive, TRPM6, and TRPM7-like channels remain undefined, especially with the uncertainty regarding TRPM6 expression in cardiomyocytes. Additionally, their contribution to the cardiac action potential (AP) profile is unclear. Immunofluorescence assays showed the expression of the TRPM6 and TRPM7 proteins in isolated pig atrial and ventricular cardiomyocytes, of which the expression was modulated by incubation in extracellular divalent cation-free conditions. In patch clamp studies of cells dialyzed with solutions containing zero intracellular Mg2+ concentration ([Mg2+]i) to activate the Mg2+-sensitive channels, raising extracellular [Mg2+] ([Mg2+]o) from the 0.9-mM baseline to 7.2 mM prolonged the AP duration (APD). In contrast, no such effect was observed in cells dialyzed with physiological [Mg2+]i. Under voltage clamp, in cells dialyzed with zero [Mg2+]i, depolarizing ramps induced an outward-rectifying current, which was suppressed by raising [Mg2+]o and was absent in cells dialyzed with physiological [Mg2+]i. In cells dialyzed with physiological [Mg2+]i, raising [Mg2+]o decreased the L-type Ca2+ current and the total delayed-rectifier current but had no effect on the APD. These results suggest a co-expression of the TRPM6 and TRPM7 proteins in cardiomyocytes, which are therefore the molecular candidates for the native cardiac Mg2+-sensitive channels, and also suggest that the cardiac Mg2+-sensitive current shortens the APD, with potential implications in arrhythmogenesis.

Evidence for the expression of TRPM6 and TRPM7 in cardiomyocytes from all four chamber walls of the human heart

The expression of the channels-enzymes TRPM6 and TRPM7 in the human heart remains poorly defined, and TRPM6 is generally considered not to be expressed in cardiomyocytes. We examined their expression at protein and mRNA levels using right atrial samples resected from patients (n = 72) with or without ischemic heart disease (IHD) and samples from all chamber walls of explanted human hearts (n = 9). TRPM6 and TRPM7 proteins were detected using immunofluorescence on isolated cardiomyocytes, ELISA on tissue homogenates, and immunostaining of cardiac tissue, whereas their mRNAs were detected by RT-qPCR. Both TRPM6 and TRPM7 were present in all chamber walls, with TRPM7 being more abundant. TRPM6 was co-expressed with TRPM7. The expression levels were dependent on cell incubation conditions (presence or absence of divalent cations, pH of the extracellular milieu, presence of TRP channel inhibitors 2-aminoethoxydiphenyl-borate and carvacrol). These drugs reduced TRPM7 immunofluorescence but increased that of TRPM6. TRPM6 and TRPM7 expression was increased in tissues from IHD patients. This is the first demonstration of the presence and co-expression of TRPM6 and TRPM7 in cardiomyocytes from all chamber walls of the human heart. The increased TRPM6 and TRPM7 expression in IHD suggests that the chanzymes are involved in the pathophysiology of the disease.

AUY922 induces retinal toxicity through attenuating TRPM1

BackgroundOcular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed.MethodsThe impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)‑based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches.ResultsHere, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922.ConclusionsOur study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Identification and characterization of Trpm4 gene involved in regulating Japanese flounder (Paralichthys olivaceus) inflammatory response.

The transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed Ca2+ -impermeable cation channel involved in modulating inflammatory and immune responses in mammals. However, the role of TRPM4 channel in fish immunity remains unclear. In this report, from a comparative immunological point of view, we identified and characterized a Trpm4 gene from Japanese flounder (Paralichthys olivaceus) and analysed its potential role in regulating the fish inflammatory response. The Japanese flounder Trpm4 gene is expressed in a wide range of tissues and encodes a 1264-amino acid protein which expresses on the cell surface and shares several conserved domains with its mammalian counterparts. In vitro inflammatory challenge and in vivo bacterial infection experiments revealed that Japanese flounder Trpm4 expression was significantly modulated following different immune challenges, indicating the implication of Trpm4 in the fish immune response. Overexpression of TRPM4 significantly attenuated LPS- and poly(I:C)-induced pro-inflammatory cytokine expression in Japanese flounder FG-9307 cells. In contrast, pharmacological inhibition of the endogenous TRPM4 channel activity in Japanese flounder head kidney macrophages resulted in increased pro-inflammatory cytokine expression following LPS and poly(I:C) stimulations. Taken together, these findings indicate that TRPM4 channels may play a conserved role in regulating inflammatory response(s) in fish.

Specific disruption of calcineurin-signaling in the distal convoluted tubule impacts the transcriptome and proteome, and causes hypomagnesemia and metabolic acidosis

Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria. Consistent with the hypomagnesemia, CnB1-KO mice showed a downregulation of proteins implicated in DCT magnesium transport, including TRPM6, CNNM2, SLC41A3 and parvalbumin but expression of calcium channel TRPV5 in the kidney was unchanged. The abundance of the chloride/bicarbonate exchanger pendrin was increased, likely explaining the acidosis. Plasma aldosterone levels, kidney renin expression, abundance of phosphorylated sodium chloride-cotransporter and abundance of the epithelial sodium channel were similar in control and CnB1-KO mice, consistent with a normal sodium balance. Long-term potassium homeostasis was maintained in CnB1-KO mice, but in-vivo and ex-vivo experiments indicated that CnB1 contributes to acute regulation of potassium balance and sodium chloride-cotransporter. Tacrolimus treatment of control and CnB1-KO mice demonstrated that CNI-related hypomagnesemia is linked to impaired calcineurin-signaling in DCT, while hypocalciuria and hyponatremia occur independently of CnB1 in DCT. Transcriptome and proteome analyses of isolated DCTs demonstrated that CnB1 deletion impacts the expression of several DCT-specific proteins and signaling pathways. Thus, our data support a critical role of calcineurin for DCT function and provide novel insights into the pathophysiology of CNI side effects and involved molecular players in the DCT.

Oridonin Promotes Apoptosis and Restrains the Viability and Migration of Bladder Cancer by Impeding TRPM7 Expression via the ERK and AKT Signaling Pathways.

Background Oridonin is a powerful anticancer compound found in Rabdosia rubescens. However, its potential impact on bladder cancer remains uninvestigated. In this work, we aimed to detect the anticancer effect of oridonin on bladder cancer and explore the molecular mechanisms involved. Methods The anticancer activity of oridonin was assessed in vitro with a CCK8 assay, an annexin V-FITC apoptosis analysis, and colony formation and Transwell migration assays which were performed with the human bladder cancer cell line T24. Levels of apoptosis-related proteins, melastatin transient receptor potential channel 7 (TRPM7), and signaling molecules were examined in oridonin-treated T24 cells by western blotting or RT-PCR. Oridonin anticancer efficacy was further validated in vivo with a T24 xenograft mouse model. Results Oridonin repressed the proliferative, colony-forming, and migratory capacities of T24 cells, triggered extensive apoptosis in vitro, and retarded tumor growth in vivo. Moreover, oridonin treatment significantly increased expression levels of p53 and cleaved caspase-3 and reduced expression of TRPM7, p-AKT, and p-ERK. Conclusion Oridonin exhibited outstanding antiproliferative and antimigratory effects on bladder cancer, and these effects were at least partially associated with targeting of TRPM7 through inactivation of the ERK and AKT signaling pathways. These findings provide insight for the clinical application of oridonin in bladder cancer prevention.

The Combined Influence of Magnesium and Insulin on Central Metabolic Functions and Expression of Genes Involved in Magnesium Homeostasis of Cultured Bovine Adipocytes.

At the onset of lactation, dairy cows suffer from insulin resistance, insulin deficiency or both, similar to human diabetes, resulting in lipolysis, ketosis and fatty liver. This work explored the combined effects of different levels of magnesium (0.1, 0.3, 1 and 3 mM) and insulin (25, 250 and 25,000 pM) on metabolic pathways and the expression of magnesium-responsive genes in a bovine adipocyte model. Magnesium starvation (0.1 mM) and low insulin (25 pM) independently decreased or tended to decrease the accumulation of non-polar lipids and uptake of the glucose analog 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG). Activity of glycerol 3-phosphate dehydrogenase (GPDH) was highest at 25 pM insulin and 3 mM magnesium. Expression of SLC41A1 and SLC41A3 was reduced at 0.1 mM magnesium either across insulin concentrations (SLC41A1) or at 250 pM insulin (SLC41A3). MAGT1 expression was reduced at 3 mM magnesium. NIPA1 expression was reduced at 3 mM and 0.1 mM magnesium at 25 and 250 pM insulin, respectively. Expression of SLC41A2, CNNM2, TRPM6 and TRPM7 was not affected. We conclude that magnesium promotes lipogenesis in adipocytes and inversely regulates the transcription of genes that increase vs. decrease cytosolic magnesium concentration. The induction of GAPDH activity by surplus magnesium at low insulin concentration can counteract excessive lipomobilization.

An in Vitro Study Elucidating the Effect of Oxidative Stress on Melanocytes

Oxidative stress plays a major role in melanocyte destruction in vitiligo, however the exact mechanism responsible for melanocyte death remains uncertain. We aimed to examine the effect of oxidative stress on melanocyte viability by MTT assay and expression of antioxidant genes (CAT, GPX1, G6PD and PRDX3), stress related genes (HSP60, HSP70, SERP1, SIRT1 and POLH) and melanocyte specific genes (MITF, TYR, TYRP1, TRPM1, EDN1, EZR and LAMP1) by real-time PCR upon exposing the normal human melanocytes (NHM), immortalized melanocytes derived from healthy human (PIG1) and from vitiligo patient (PIG3V) to cumene hydroperoxide (CHP). The transcript levels of selected genes were estimated by using real-time PCR. The NHM, PIG1 and PIG3V melanocytes showed significant decrease in viability under CHP (10-100μM) induced oxidative stress. PIG3V displayed significantly increased expression of PRDX3, HSP70, SERP1, POLH as well as decreased expression of CAT, MITF, TYR, TYRP1, TRPM1, EDN1 and LAMP1 under CHP (10 &amp; 20μM) treatment, as compared to NHM and/or PIG1 melanocytes. These results suggest that vitiligo melanocytes are more sensitive to CHP induced oxidative stress, as compared to normal melanocytes. The present study demonstrates that vitiligo may result from an insufficient response of melanocytes to oxidative stress induced by high H2O2 levels. Keywords: Vitiligo; melanocyte; PIG1; PIG3V; oxidative stress; cumene hydroperoxide (CHP).

TRPM7 is an essential regulator for volume-sensitive outwardly rectifying anion channel

Animal cells can regulate their volume after swelling by the regulatory volume decrease (RVD) mechanism. In epithelial cells, RVD is attained through KCl release mediated via volume-sensitive outwardly rectifying Cl− channels (VSOR) and Ca2+-activated K+ channels. Swelling-induced activation of TRPM7 cation channels leads to Ca2+ influx, thereby stimulating the K+ channels. Here, we examined whether TRPM7 plays any role in VSOR activation. When TRPM7 was knocked down in human HeLa cells or knocked out in chicken DT40 cells, not only TRPM7 activity and RVD efficacy but also VSOR activity were suppressed. Heterologous expression of TRPM7 in TRPM7-deficient DT40 cells rescued both VSOR activity and RVD, accompanied by an increase in the expression of LRRC8A, a core molecule of VSOR. TRPM7 exerts the facilitating action on VSOR activity first by enhancing molecular expression of LRRC8A mRNA through the mediation of steady-state Ca2+ influx and second by stabilizing the plasmalemmal expression of LRRC8A protein through the interaction between LRRC8A and the C-terminal domain of TRPM7. Therefore, TRPM7 functions as an essential regulator of VSOR activity and LRRC8A expression.

The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity.

The activity of the TRPM7 channel is negatively regulated by intracellular Mg2+. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg2+. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H2O2). Site-directed mutagenesis of full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. Mutation of C1809 or C1813 prevented expression of full-length TRPM7 on the plasma membrane. We therefore developed an assay to functionally reconstitute full-length TRPM7 by coexpressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than that of full-length TRPM7 and was insensitive to oxidative stress. Coexpression of M7cd and M7kd attenuated the inhibition by intracellular Mg2+ and restored sensitivity to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. We observed a similar effect when M7cd was coexpressed with the kinase-inactive mutant M7kd-K1645R, suggesting that the kinase activity is not essential for the reconstitution. However, coexpression of M7cd and M7kd carrying a mutation at either C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was observed when using M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by its kinase domain and that the cysteines in the zinc-binding motif play a role in the oxidative stress response of TRPM7.

TRPM7 is protective against hypertension, cardiovascular inflammation and fibrosis induced by aldosterone and salt

Introduction : TRPM7 is a channel permeable to Mg2+, Ca2+ and Zn2+ bound to an alpha-kinase domain with essential role in cation homeostasis and cell cycle and activation. Hyperaldosteronism, which induces hypertension and cardiovascular fibrosis, is associated with Mg2+ wasting. Here we investigate the importance of TRPM7 in hypertension and fibrosis induced by aldosterone and salt. Methods Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600µg/Kg/day) and/or 1% NaCl (drinking water) (aldo, salt or aldo-salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel structure was assessed by pressure myography. Inflammatory cell infiltrate was investigated by flow cytometry. Molecular mechanisms were investigated in cardiac fibroblasts (CF) from WT and M7+/Δ mice. Protein expression was assessed by western-blot and histology. Results M7+/Δ mice exhibited reduced TRPM7 expression (30%) and phosphorylation (62%), levels that were recapitulated in WT mice treated with aldo-salt. M7+/Δ exhibited increased BP by aldo, salt and aldo-salt (135-140mmHg) vs M7+/Δ veh (117mmHg) (p<0.05), whereas in WT, BP was increased only in the aldo-salt group (134mmHg). Mesenteric arteries from M7+/Δ aldo-salt exhibited thinner walls by reducing cross-sectional area (35%) vs WT aldo-salt (p<0.05). Aldo-salt induced greater collagen deposition in hearts (68%), kidneys (126%) and aortas (45%) from M7+/Δ vs WT. In kidneys from WT mice, aldo-salt increased frequency of macrophages (47% vs 32% WT veh) and CD4+T cells (33% vs 22% WT veh) (p<0.05), effects observed already in M7+/Δ veh (macrophages: 42%, CD4+T cells: 34%). Aldosterone increased CD8+T cells in kidneys from M7+/Δ (16% vs M7+/Δ-veh 9%). Hearts from M7+/Δ veh exhibited increased TGFβ, p-Smad3 and p-Stat1 (1.5-fold) whereas in WT these effects were only found after aldo-salt. Cardiac expression of PPM1A, a Mg2+-dependent phosphatase, was reduced (3-fold) only in M7+/Δ mice. CF from M7+/Δ mice showed reduced of proliferation (30%) and PPM1A (4-fold) and increased expression of TGFβ, IL-11, IL-6 (2-6-fold), p-Stat1 (2-fold), p-Smad3 (9-fold) and p-ERK1/2 (8-fold) compared with WT. Mg2+ supplementation normalized cell proliferation and reduced protein phosphorylation in M7+/Δ CF (p<0.05. Conclusions Our findings identify a protective role of TRPM7 in aldosterone-salt induced cardiovascular injury, which when downregulated, facilitates hypertension, vascular remodeling and cardiac fibrosis through Mg2+ -dependent mechanisms.

TRPM4 Expression During Postnatal Developmental of Mouse CA1 Pyramidal Neurons.

TRPM4 is a non-selective cation channel activated by intracellular calcium and permeable to monovalent cations. This channel participates in the control of neuronal firing, neuronal plasticity, and neuronal death. TRPM4 depolarizes dendritic spines and is critical for the induction of NMDA receptor-dependent long-term potentiation in CA1 pyramidal neurons. Despite its functional importance, no subcellular localization or expression during postnatal development has been described in this area. To examine the localization and expression of TRPM4, we performed duplex immunofluorescence and patch-clamp in brain slices at different postnatal ages in C57BL/6J mice. At P0 we found TRPM4 is expressed with a somatic pattern. At P7, P14, and P35, TRPM4 expression extended from the soma to the apical dendrites but was excluded from the axon initial segment. Patch-clamp recordings showed a TRPM4-like current active at the resting membrane potential from P0, which increased throughout the postnatal development. This current was dependent on intracellular Ca2+ (ICAN) and sensitive to 9-phenanthrol (9-Ph). Inhibiting TRPM4 with 9-Ph hyperpolarized the membrane potential at P14 and P35, with no effect in earlier stages. Together, these results show that TRPM4 is expressed in CA1 pyramidal neurons in the soma and apical dendrites and associated with a TRPM4-like current, which depolarizes the neurons. The expression, localization, and function of TRPM4 throughout postnatal development in the CA1 hippocampal may underlie an important mechanism of control of membrane potential and action potential firing during critical periods of neuronal development, particularly during the establishment of circuits.

Expression of Transient Receptor Potential Channel of Melastatin number 8 (TRPM8) in Non- Melanoma Skin Cancer: A Clinical and Immunohistochemical study

ABSTRACT Transient Receptor Potential Channel of Melastatin number 8 (TRPM8) is abnormally expressed in many cancers as lung, however little is known about TRPM8 expression in non-melanoma skin cancer (NMSC) including cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). This work aimed to study TRPM8 expression in NMSC. It included 100 skin biopsies (50 normal skin as control group, 15 BCC and 35 SCC). Immunohistochemical staining for TRPM8 was done and results were correlated with clinicopathological characters. There was significant higher TRPM8 H-score in NMSC than control skin. On comparing SCC cases to control, there was significant positive TRPM8 expression, strong intensity, diffuse pattern, cytoplasmic and nucleo-cytoplasmic localization and higher range of H-score in SCC. In contrast, BCC showed significant lower TRPM8 positive expression when compared to control skin. Higher TRPM8 H-score in SCC showed significant positive correlation with large tumor size and poor tumor differentiation.TRPM8 may be implicated in pathogenesis of NMSC. Its association with bad prognostic characters; potentiates its role as prognostic biomarker and open new chances for therapeutic intervention in NMSC. TRPM8 antagonists may share in decreasing tumor growth and progression and may serve as potential target for tumor immunotherapy.

Leptin Induces Epigenetic Regulation of Transient Receptor Potential Melastatin 7 in Rat Adrenal Pheochromocytoma Cells.

Obesity elevates the plasma level of leptin, which has been associated with hypertension. Our recent studies in mice demonstrated that leptin increases blood pressure by activating the carotid sinus nerve, which transmits the chemosensory input from carotid bodies (CBs) to the medullary centers, and that the effect of leptin is mediated via Trpm7 (TRP [transient receptor potential] melastatin 7) channels in CB glomus cells. We also found that Trpm7 overexpression and Trpm7 promoter demethylation in CBs correlate positively with the hyperleptinemia and leptin receptor overexpression in CBs. Hence, we postulated that leptin epigenetically regulates Trpm7 expression in CBs. We addressed our hypothesis by using rat adrenal pheochromocytoma (PC12) cells as a model of CB glomus cells. PC12 cells expressing LEPRb (long, active form of leptin receptor) showed dramatic induction of the promoter activity and expression of Trpm7 upon leptin treatment. The increased Trpm7 expression coincided with the reduction of CpG site-specific methylation and trimethylation of H3K27 (H3 [histone 3] K27 [lysine 27]) and the increase of acetylation of H3K27 and trimethylation of H3K4 (H3 lysine 4) at the Trpm7 promoter. The inhibitor of STAT3 (signal transducer and activator of transcription 3) signaling, SD1008, reversed the leptin-induced Trpm7 promoter activity via modulations of the binding of pSTAT3 (phosphorylated STAT3) and DNMT3B (DNA methyltransferase 3B) and modifications of H3K27 and H3K4 at the Trpm7 promoter. Our results suggest that leptin-activated pSTAT3 epigenetically regulates the transcription of Trpm7 through DNA methylation and histone modifications. Because epigenetic changes are reversible, targeting epigenetic modifications of Trpm7 may serve as a new therapeutic approach for the treatment of hypertension in obesity.