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.

Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits.

The cold- and menthol-activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non-neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8-expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch-clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.

Menthol in electronic cigarettes: A contributor to respiratory disease?

Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL® cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL® cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease.

Reduced TRPM8 expression underpins reduced migraine risk and attenuated cold pain sensation in humans.

Multiple genome-wide association studies have identified non-coding single-nucleotide variants (SNVs) near (e.g., rs10166942[C]) or within (rs17862920[T]) the TRPM8 gene that encodes a cold thermosensor is associated with reduced migraine risk. Furthermore, rs10166942[C]) and rs10166942[T]) are more prevalent in populations that reside in hotter and colder climates, respectively. Here we assessed whether these alleles affect TRPM8 expression in humans and human physiologic responses to cold challenge. Here we show that TRPM8 expression is decreased from the chromosome harboring the rs10166942[C] allele in the human dorsal root ganglia. Moreover, carriers of rs10166942[C] required significantly lower temperatures and longer duration of exposure to reach a cold pain threshold (CPTh), which correlated with decreased TRPM8 expression expected in the carriers. This study provides evidence for a genotype-dependent influence on cold pain sensation suggesting that carriers of the reduced migraine risk allele have reduced sensitivity to cold stimuli and that TRPM8 acts as a cold thermosensor and cold pain transducer in humans. Reduced TRPM8 expression and function underpins the migraine protection in carriers of rs10166942[C]; thus, the evaluation of TRPM8 antagonists as migraine therapeutics is warranted. Furthermore, these results provide mechanistic insights for evolutionary positive selection of rs10166942[T] allele in adaptation along latitudinal cline to colder climates.

Expression Suppression and Activity Inhibition of TRPM7 Regulate Cytokine Production and Multiple Organ Dysfunction Syndrome During Endotoxemia: a New Target for Sepsis.

BACKGROUND: Main pathological features detected during sepsis and endotoxemia include over-secretion of pro-inflammatory cytokines and multiorgan dysfunction syndrome (MODS). Unfortunately, current clinical efforts to treat sepsis are unsatisfactory, and mortality remains high. Interestingly, transient receptor potential (TRP) melastatin 7 (TRPM7) ion channel controlling Ca2+ and Mg2+ permeability is involved in cytokine production and inflammatory response. Furthermore, TRPM7 downregulation has been shown to alleviate local symptoms in some models of sepsis, but its effects at a systemic level remain to be explored. OBJECTIVE: To test whether TRPM7 mediates cytokine production and MODS during endotoxemia. METHODS: Endotoxemic and sham-endotoxemic rats were subjected to pharmacological inhibition of TRPM7 using carvacrol, or to expression suppression by adenovirus delivery of shRNA (AdVshTRPM7). Then, cytokine and MODS levels in the blood were measured. RESULTS: Inhibition of TRPM7 with carvacrol and suppression with AdVshTRPM7 were both efficient in inhibiting the over-secretion of pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-12, in endotoxemic rats, without inducing downregulation in blood levels of antiinflammatory cytokines IL-10 and IL-4. Additionally, the use of carvacrol and AdVshTRPM7 significantly prevented liver and pancreas dysfunction, altered metabolic function, and hypoglycemia, induced by endotoxemia. Furthermore, muscle mass wasting and cardiac muscle damage were also significantly reduced by the use of carvacrol and AdVshTRPM7 in endotoxemic rats. CONCLUSION: Our results indicate TRPM7 ion channel as a key protein regulating inflammatory responses and MODS during sepsis. Moreover, TRPM7 appears as a novel molecular target for the management of sepsis.

The effects of TRPM2, TRPM6, TRPM7 and TRPM8 gene expression in hepatic ischemia reperfusion injury.

OBJECTIVE: Mammalian transient receptor potential melastatin (TRPM) channels are a form of calcium channels and they transport calcium and magnesium ions. TRPM has eight subclasses including TRPM1-8. TRPM2, TRPM6, TRPM7, TRPM8 are expressed especially in the liver cell. Therefore, we aim to investigate the effects of TRPM2, TRPM6, TRPM7, and TRPM8 gene expression and histopathologic changes after treatment of verapamil in the hepatic ischemia-reperfusion rat model. MATERIALS AND METHODS: Animals were randomly assigned to one or the other of the following groups including sham (n=8) group, verapamil (calcium entry blocker) (n=8) group, I/R group (n=8) and I/R- verapamil (n=8) group. TRPM 2, 6, 7, 8 gene expression level was were assessed by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) and histopathologic changes were determined by the hematoxylin and eosin (HE) examination. RESULTS: The expression level of TRPM 2, 6, 7, and 8 genes was were significantly higher in ischemia-reperfusion (I/R), verapamil, IR-verapamil groups compared to sham group. The p-values were 0.0024, < 0.0001, 0.0002, 0.006 for TRPM2, TRPM6, TRPM7, and TRPM8, respectively. Severe necrotic, degenerative differentiations and severe hemorrhagic areas were observed in hepatocytes from IR group. Also, moderate necrotic and degenerative differentiations and moderate hemorrhagic areas were observed in hepatocytes from IR-verapamil group. CONCLUSIONS: This is the first study reporting an association between the expression level of TRPM 2, 6, 7, 8 in a hepatic ischemia-reperfusion rat model. Moreover, TRPM 2, 6, 7, 8 affect hepatic ischemia-reperfusion.

Renal phospholipidosis and impaired magnesium handling in high-fat-diet-fed mice.

Hypomagnesemia (blood Mg2+ concentration <0.7 mM) is a common electrolyte disorder in patients with type 2 diabetes (T2D), but the etiology remains largely unknown. In patients with T2D, reduced blood Mg2+ levels are associated with an increased decline in renal function, independent of glycemic control and hypertension. To study the underlying mechanism of this phenomenon, we investigated the renal effects of hypomagnesemia in high-fat-diet (HFD)-fed mice. In mice fed a low dietary Mg2+, the HFD resulted in severe hypomagnesemia within 4 wk. Renal or intestinal Mg2+ wasting was not observed after 16 wk on the diets. Despite the absence of urinary or fecal Mg2+ loss, the HFD induced a reduction in the mRNA expression transient receptor potential melastatin type 6 in both the kidney and colon. mRNA expression of distal convoluted tubule (DCT)-specific genes was down-regulated by the LowMg-HFD, indicating atrophy of the DCT. The low dietary Mg2+ resulted in severe HFD-induced proximal tubule phospholipidosis, which was absent in mice on a NormalMg-HFD. This was accompanied by albuminuria, moderate renal damage, and alterations in renal energy metabolism, including enhanced gluconeogenesis and cholesterol synthesis. In conclusion, this study shows that hypomagnesemia is a consequence of diet-induced obesity and insulin resistance. Moreover, hypomagnesemia induces major structural changes in the diabetic kidney, including proximal tubular phospholipidosis, providing a novel mechanism for the increased renal decline in patients with hypomagnesemic T2D.-Kurstjens, S., Smeets, B., Overmars-Bos, C., Dijkman, H. B., den Braanker, D. J. W., de Bel, T., Bindels, R. J. M., Tack, C. J. J., Hoenderop, J. G. J., de Baaij, J. H. F. Renal phospholipidosis and impaired magnesium handling in high-fat-diet-fed mice.

Periprosthetic hypoxia as consequence of TRPM7 mediated cobalt influx in osteoblasts.

The reasons for the high number of loosened metal-on-metal (MoM) hip implants are still not fully understood. Hypoxia-inducible factor 1 (HIF-1) mediated signaling pathways, which normally modulate tissue metabolism under hypoxic circumstances, could be triggered by metallic wear debris and influence bone metabolism favoring osteolysis. This may lead to early loosening of the orthopedic implants. Immunhistochemical staining of periprosthetic tissues of failed artificial hip implants showed that the concentration of HIF-1α in the surrounding tissues of failed MoM hip implants was significantly higher in comparison to failed metal-on-polyethylene (MoP) hip implants and osteoarthritic tissues. Therefore, we examined the Co2+ -uptake mechanisms and the influence of Co2+ uptake on HIF-1α stabilization. Based on cobalt mediated quenching effects, calcium imaging experiments using fura-2 showed a concentration-dependent cobalt influx in MG-63 cells, which could be inhibited by the unspecific TRPM7 channel inhibitor 2-APB (20 µM) and TRPM7 specific siRNA. Western blots confirmed a dose dependent increase of HIF-1α upon stimulation with Co2+ . This effect could be abrogated by inhibition of cobalt influx using 2-APB. This study shows that chemical hypoxia originating from HIF-1α upregulation within the periprosthetic tissue is related to cobalt wear debris and highlights TRPM7 as an important key mediator in this context. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1806-1813, 2019.

Reactive astrocytes in multiple sclerosis impair neuronal outgrowth through TRPM7-mediated chondroitin sulfate proteoglycan production.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS), characterized by inflammation-mediated demyelination, axonal injury and neurodegeneration. The mechanisms underlying impaired neuronal function are not fully understood, but evidence is accumulating that the presence of the gliotic scar produced by reactive astrocytes play a critical role in these detrimental processes. Here, we identified astrocytic Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7), a Ca2+ -permeable nonselective cation channel, as a novel player in the formation of a gliotic scar. TRPM7 was found to be highly expressed in reactive astrocytes within well-characterized MS lesions and upregulated in primary astrocytes under chronic inflammatory conditions. TRPM7 overexpressing astrocytes impaired neuronal outgrowth in vitro by increasing the production of chondroitin sulfate proteoglycans, a key component of the gliotic scar. These findings indicate that astrocytic TRPM7 is a critical regulator of the formation of a gliotic scar and provide a novel mechanism by which reactive astrocytes affect neuronal outgrowth.

MicroRNA-150 suppresses epithelial-mesenchymal transition, invasion, and metastasis in prostate cancer through the TRPM4-mediated ß-catenin signaling pathway.

Prostate cancer (PCa) remains one of the leading causes of cancer-related deaths among males. The aim of the current study was to investigate the ability of microRNA-150 (miR-150) targeting transient receptor potential melastatin 4 (TRPM4) to mediate epithelial-mesenchymal transition (EMT), invasion, and metastasis through the ß-catenin signaling pathway in PCa. Microarray analysis was performed to identify PCa-related differentially expressed genes, after which both the mirDIP and TargetScan databases were employed in the prediction of the miRNAs regulating TRPM4. Immunohistochemistry and RT-qPCR were conducted to determine the expression pattern of miR-150 and TRPM4 in PCa. The relationship between miR-150 and TRPM4 expression was identified. By perturbing miR-150 and TRPM4 expression in PCa cells, cell proliferation, migration, invasion, cycle, and apoptosis as well as EMT markers were determined accordingly. Finally, tumor growth and metastasis were evaluated among nude mice. Higher TRPM4 expression and lower miR-150 expression and activation of the ß-catenin signaling pathway as well as EMT stimulation were detected in the PCa tissues. Our results confirmed TRPM4 as a target of miR-150. Upregulation of miR-150 resulted in inactivation of the ß-catenin signaling pathway. Furthermore, the upregulation of miR-150 or knockdown of TRPM4 was observed to suppress EMT, proliferation, migration, and invasion in vitro in addition to restrained tumor growth and metastasis in vivo. The evidence provided by our study highlights the involvement of miR-150 in the translational suppression of TRPM4 and the blockade of the ß-catenin signaling pathway, resulting in the inhibition of PCa progression.

Riluzole prevents oxaliplatin-induced cold allodynia via inhibition of overexpression of transient receptor potential melastatin 8 in rats.

Oxaliplatin causes acute cold hypersensitivity in most patients. We previously reported oxalate derived from oxaliplatin induced cold allodynia via overexpression of transient receptor potential melastatin 8 (TRPM8) in the dorsal root ganglion (DRG) in rats. In this study, we examined the effect of riluzole on oxaliplatin-induced cold allodynia. In cultured DRG neurons, riluzole suppressed oxalate-induced increase of the number of menthol (TRPM8 agonist)-sensitive cells. Moreover, riluzole prevented cold allodynia and increase in levels of TRPM8 mRNA in oxaliplatin-treated rats. These results suggest that riluzole prevents oxaliplatin-induced cold allodynia via inhibition of TRPM8 overexpression in the DRG.

TRPV1 and TRPM8 Channels and Nocifensive Behavior in a Rat Model for Dry Eye.

Purpose: Persistent ocular surface pain occurs in moderate to severe dry eye disease (DE); however, the mechanisms that underlie this symptom remain uncertain. The aim of this study was to determine if the transient receptor potential vanilloid ion channels play a role in hypertonic saline (HS)-evoked corneal reflexes in a model for aqueous tear deficient DE. Methods: Eye wipe behavior and orbicularis oculi muscle activity (OOemg) were measured after ocular instillation of HS, capsaicin, or menthol 14 days after exorbital gland removal. Total RNA and protein were measured from anterior eye segment and trigeminal ganglia of sham and DE rats. Results: Eye wipe behavior was enhanced in DE rats after HS and capsaicin instillation, but not after menthol when compared to sham rats. DE rats displayed greater OOemg activity after HS and capsaicin, but not after menthol, compared to sham rats. HS-evoked OOemg activity was reduced by selective TRPV1 antagonists and by coapplication of capsaicin plus QX-314, a charged lidocaine derivative. Menthol did not affect OOemg activity; however, selective antagonism of TRPM8 reduced HS-evoked OOemg activity. TRPV1 protein levels were increased in anterior eye segment and trigeminal ganglion samples from DE rats, whereas TRPM8 levels were not affected. Conclusions: These results suggest that TRPV1 plays a significant role in mediating enhanced nocifensive behavior in DE, while TRPM8 may play a lesser role. Strategies to target specific transducer molecules on corneal nerves may prove beneficial as adjunct therapies in managing ocular pain in moderate to severe cases of DE.

Ethanol's Effects on Transient Receptor Potential Channel Expression in Brain Microvascular Endothelial Cells.

Ethanol (EtOH), the main ingredient in alcoholic beverages, is well known for its behavioral, physiological, and immunosuppressive effects. There is evidence that EtOH acts through protein targets to exert its physiological effects; however, the mechanisms underlying EtOH's effects on inflammatory processes, particularly at the blood-brain barrier (BBB), are still poorly understood. Transient receptor potential (TRP) channels, the vanguards of human sensory systems, are novel molecular receptors significantly affected by EtOH, and are heavily expressed in brain microvascular endothelial cells (BMVECs), one of the cellular constituents of the BBB. EtOH's actions on endothelial TRP channels could affect intracellular Ca2+ and Mg2+ dynamics, which mediate leukocyte adhesion to endothelial cells and endothelial permeability at the BBB, thus altering immune and inflammatory responses. We examined the basal expression profiles of all 29 known mammalian TRP channels in mouse BMVECs and determined both EtOH concentration- and time-dependent effects on TRP expression using a PCR array. We also generated an in vitro BBB model to examine the involvement of a chosen TRP channel, TRP melastatin 7 (TRPM7), in EtOH-mediated alteration of BBB permeability. With the exception of the akyrin subfamily, members of five TRP subfamilies were expressed in mouse BMVECs, and their expression levels were modulated by EtOH in a concentration-dependent manner. In the in vitro BBB model, TRPM7 antagonists further enhanced EtOH-mediated alteration of BBB permeability. Because of the diversity of TRP channels in BMVECs that regulate cellular processes, EtOH can affect Ca2+/Mg2+ signaling, immune responses, lysosomal functions as well as BBB integrity.

The TRPM2 channel nexus from oxidative damage to Alzheimer's pathologies: An emerging novel intervention target for age-related dementia.

Alzheimer's disease (AD), an age-related neurodegenerative condition, is the most common cause of dementia among the elder people, but currently there is no treatment. A number of putative pathogenic events, particularly amyloid ß peptide (Aß) accumulation, are believed to be early triggers that initiate AD. However, thus far targeting Aß generation/aggregation as the mainstay strategy of drug development has not led to effective AD-modifying therapeutics. Oxidative damage is a conspicuous feature of AD, but this remains poorly defined phenomenon and mechanistically ill understood. The TRPM2 channel has emerged as a potentially ubiquitous molecular mechanism mediating oxidative damage and thus plays a vital role in the pathogenesis and progression of diverse neurodegenerative diseases. This article will review the emerging evidence from recent studies and propose a novel 'hypothesis' that multiple TRPM2-mediated cellular and molecular mechanisms cascade Aß and/or oxidative damage to AD pathologies. The 'hypothesis' based on these new findings discusses the prospect of considering the TRPM2 channel as a novel therapeutic target for intervening AD and age-related dementia.

Elevated Transient Receptor Potential Melastatin 8 (TRPM8) Expression Is Correlated with Poor Prognosis in Pancreatic Cancer.

BACKGROUND The transient receptor potential melastatin 8 (TRPM8) was found to be expressed abnormally in a variety of tumors and is associated with unfavorable prognosis in human cancers. However, its clinical significance in pancreatic cancer (PC) is mostly unknown. MATERIAL AND METHODS qRT-PCR was performed to measure the expression of TRPM8 in 110 pairs of PC tissues and the adjacent non-cancerous tissues. The association of TRPM8 expression with the clinical characters of PC patients was analyzed using the chi-square test. Furthermore, the prognostic value of TRPM8 was determined with Kaplan-Meier survival curve and Cox regression analysis. RESULTS We found that the expression level of TRPM8 was significantly elevated in PC tissues compared to the non-cancerous controls (P<0.001). In addition, a close relationship was observed between elevated TRPM8 expression with large tumor size (P=0.001), advanced TNM (P=0.013), and distant metastasis (P=0.034). Survival analysis suggested that patients with high TRPM8 expression has worse OS (P=0.001) and DFS (P<0.001) than those with low TRPM8 expression. Moreover, TRPM8 was confirmed as a valuable prognostic biomarker for OS (HR=1.913; 95% CI: 1.020-3.589; P=0.043) or DFS (HR=2.374; 95% CI: 1.269-4.443; P=0.007) of PC patients. CONCLUSIONS This study shows that TRPM8 expression is significantly up-regulated in PC and it might be a useful prognostic factor for patients with PC.

mRNA expression of transient receptor potential melastatin (TRPM) channels 2 and 7 in perinatal brain development.

TRPM7 and TRPM2 are non-specific cation channels of the Transient Receptor Potential channel superfamily. Each channel has gained attention for their potential to mediate oxidative and anoxic cell death (Rama and García, 2016; Naziroglu, 2011a; Abiria et al., 2017; Sun, 2017), however their physiological expression and roles in the developing brain remain poorly defined. We employed real-time reverse transcription PCR to examine mRNA expression of TRPM7 and TRPM2 in the developing rat brain and brain-specific cell types. We determined the temporal and spatial expression patterns at four developmental time points (postnatal day 7, 14, 21, and 90) in four critical regions of the brain (cortex, hippocampus, striatum, and cerebellum) and examined gene expression in neuronal, astrocytic, and microglial primary cell cultures. Our results revealed that TRPM7 mRNA expression peaks in the cortex at 2-weeks after birth, and thus correlates most closely with a period of rat brain development associated with neurite outgrowth, which is heightened at 2-weeks after birth. Our cell-specific gene expression assays revealed that TRPM7 was expressed at equivalent levels in neurons, astrocytes, and microglia. Conversely, TRPM2 was most highly expressed in microglia with little expression in neurons and astrocytes. In the hippocampus and striatum, the expression profile of TRPM2 parallels the perinatal expression timeline for microglial infiltration and maturation in the rat brain. Microglial maturation is highest from the time of birth, up to 7-days, but subsequently declines. The latter developmental expression profiles indicate a role for TRPM2 in microglial activation.

Morphological and functional changes in TRPM8-expressing corneal cold thermoreceptor neurons during aging and their impact on tearing in mice.

Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8-expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8BAC -EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 34°C and low-threshold, robust responses to cooling. The remaining TRPM8+ corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low-firing rate at 34°C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM8+ corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low-background activity and abnormal responsiveness to cooling pulses. These morpho-functional changes develop in parallel with an enhancement of tear's basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age-induced abnormal tearing and to the high incidence of DED in elderly people.

TRPM7 regulates angiotensin II-induced sinoatrial node fibrosis in sick sinus syndrome rats by mediating Smad signaling.

Sinoatrial node fibrosis is involved in the pathogenesis of sinus sick syndrome (SSS). Transient receptor potential (TRP) subfamily M member 7 (TRPM7) is implicated in cardiac fibrosis. However, the mechanisms underlying the regulation of sinoatrial node (SAN) fibrosis in SSS by TRPM7 remain unknown. The aim of this study was to investigate the role of angiotensin II (Ang II)/TRPM7/Smad pathway in the SAN fibrosis in rats with SSS. The rat SSS model was established with sodium hydroxide pinpoint pressing permeation. Forty-eight rats were randomly divided into six groups: normal control (ctrl), sham operation (sham), postoperative 1-, 2-, 3-, and 4-week SSS, respectively. The tissue explant culture method was used to culture cardiac fibroblasts (CFs) from rat SAN tissues. TRPM7 siRNA or encoding plasmids were used to knock down or overexpress TRPM7. Collagen (Col) distribution in SAN and atria was assessed using PASM-Masson staining. Ang II, Col I, and Col III levels in serum and tissues or in CFs were determined by ELISA. TRPM7, smad2 and p-smad2 levels were evaluated by real-time PCR, and/or western blot and immunohistochemistry. SAN and atria in rats of the SSS groups had more fibers and higher levels of Ang II, Col I and III than the sham rats. Similar findings were obtained for TRPM7 and pSmad2 expression. In vitro, Ang II promoted CFs collagen synthesis in a dose-dependent manner, and potentiated TRPM7 and p-Smad2 expression. TRPM7 depletion inhibited Ang II-induced p-Smad2 expression and collagen synthesis in CFs, whereas increased TRPM7 expression did the opposite. SAN fibrosis is regulated by the Ang II/TRPM7/Smad pathway in SSS, indicating that TRPM7 is a potential target for SAN fibrosis therapy in SSS.

TRPM7 Regulates AKT/FOXO1-Dependent Tumor Growth and Is an Independent Prognostic Indicator in Renal Cell Carcinoma.

Transient receptor potential melastatin 7 (TRPM7) is important for the tumorigenesis and progression of several cancers. However, little is known about TRPM7 expression and its clinical significance in clear cell renal cell carcinoma (ccRCC). The expression dynamics of TRPM7 was examined in a clinical cohort of RCC specimens by qPCR, immunoblotting, and IHC staining. A series of in vitro and in vivo assays were performed to elucidate the function of TRPM7 in RCC and the underlying mechanisms. For the first time, results demonstrate that TRPM7 expression is markedly higher in RCC cell lines and clinical samples and had a positive correlation with T status, tumor size, and poor patients' overall survival and progression-free survival. Preclinical studies using multiple RCC cells and a mouse model indicate that TRPM7 promotes cell proliferation and colony formation in vitro and tumor growth in vivo Mechanistically, TRPM7 promotes AKT phosphorylation, leading to repression of the FOXO1 expression and transcriptional activity. Moreover, luciferase reporter assays demonstrate that miR-129-3p directly targets the 3'-UTR of TRPM7 and acts as a negative regulator of TRPM7. These findings reveal an important role for TRPM7 in the regulation of RCC growth and represent a novel prognostic biomarker for this disease.Implications: TRPM7 is an independent prognostic indicator in RCC, and targeting the TRPM7 signaling pathway may be a novel therapeutic approach for the treatment of RCC. Mol Cancer Res; 16(6); 1013-23. ©2018 AACR.

Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology.

Background Recent genome-wide association studies have identified transient receptor potential M8 ( TRPM8) as a migraine susceptibility gene. TRPM8 is a nonselective cation channel that mediates cool perception. However, its precise role in migraine pathophysiology is elusive. Transient receptor potential V1 (TRPV1) is a nonselective cation channel activated by noxious heat. Both TRPM8 and TRPV1 are expressed in trigeminal ganglion (TG) neurons. Methods We investigated the functional roles of TRPM8 and TRPV1 in a meningeal inflammation-based migraine model by measuring the effects of facial TRPM8 activation on thermal allodynia and assessing receptor coexpression changes in TG neurons. We performed retrograde tracer labeling to identify TG neurons innervating the face and dura. Results We found that pharmacological TRPM8 activation reversed the meningeal inflammation-induced lowering of the facial heat pain threshold, an effect abolished by genetic ablation of TRPM8. No significant changes in the heat pain threshold were seen in sham-operated animals. Meningeal inflammation caused dynamic alterations in TRPM8/TRPV1 coexpression patterns in TG neurons, and colocalization was most pronounced when the ameliorating effect of TRPM8 activation on thermal allodynia was maximal. Our tracer assay disclosed the presence of dura-innervating TG neurons sending collaterals to the face. Approximately half of them were TRPV1-positive. We also demonstrated functional inhibition of TRPV1 by TRPM8 in a cell-based assay using c-Jun N-terminal kinase phosphorylation as a surrogate marker. Conclusions Our findings provide a plausible mechanism to explain how facial TRPM8 activation can relieve migraine by suppressing TRPV1 activity. Facial TRPM8 appears to be a promising therapeutic target for migraine.