TRPM8 Research Articles

<em>TRPM8</em> genetic variations associated with COPD risk in the Chinese Han population

TRPM8 plays a key role in COPD. The development of pulmonary hypertension (PH) in COPD adversely affects survival and exercise capacity. Thus, the aim of this study was to evaluate the possible association between single nucleotide polymorphisms (SNPs) in TRPM8 and COPD or PH in COPD among the Chinese Han population. A total of 513 COPD patients and 506 controls were enrolled in the study. Six tag SNPs (tSNPs) were genotyped. The relationship between COPD or PH in COPD and the six tSNPs was evaluated using the χ2 test and genetic model analysis. In the rs9789398 polymorphism, the T/C genotype was associated with an increased risk for COPD (P=0.005). Under the assumption of models of inheritance, there was an association between the rs9789398 polymorphism and COPD. In the rs9789675 polymorphism, the G/A genotype was associated with an increased risk for COPD (P=0.021). Furthermore, by the χ2 test, we found that the minor allele “A” of rs9789675 (odds ratio [OR] =0.63, 95% confidence interval [CI], 0.42–0.97, P=0.034) and the minor allele “C” of rs9789398 (OR =1.59, 95% CI, 1.03–2.44, P=0.034) were associated with a decreased risk of PH in COPD in allele models. In genetic models, the genotypes “GA” and “AA” of rs9789675 were associated with a decreased risk of PH in COPD. The genotypes “TC” and “CC” of rs9789398 were associated with a decreased risk of PH in COPD. Moreover, “CG” of rs1004478 was significantly associated with a decreased risk of PH in COPD. There was a significant association between the five SNPs (rs2362290, rs9789675, rs9789398, rs1003540, and rs104478) in the TRPM8 gene and the risk of PH in COPD. Our findings indicated that rs9789398 in the TRPM8 gene was significantly associated with the risk of COPD in the Chinese Han population. Moreover, rs9789675, rs9789398, and rs1004478 were significantly associated with the risk of PH in COPD. This study provides a novel insight into COPD and PH in the development of COPD.

TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection.

Calcium signaling in phagocytes is essential for cellular activation, migration and the potential resolution of infection or inflammation. The generation of reactive oxygen species (ROS) via activation of NADPH (nicotinamide adenine dinucleotide phosphate-) oxidase activity in macrophages has been linked to altered intracellular calcium concentrations. Because of its role as an oxidative stress sensor in phagocytes, we investigated the function of the cation channel transient receptor potential melastatin 2 (TRPM2) in macrophages during oxidative stress responses induced by Helicobacter pylori infection. We show that Trpm2−/− mice, when chronically infected with H. pylori, exhibit increased gastric inflammation and decreased bacterial colonization compared with WT mice. The absence of TRPM2 triggers greater macrophage production of inflammatory mediators and promotes classically activated macrophage M1 polarization in response to H. pylori. TRPM2-deficient macrophages upon H. pylori stimulation are unable to control intracellular calcium levels, which results in calcium overloading. Furthermore, increased intracellular calcium in TRPM2−/− macrophages enhanced MAPK and NADPH oxidase activities, compared to WT macrophages. Our data suggest that augmented production of ROS and inflammatory cytokines with TRPM2 deletion regulates oxidative stress in macrophages, and consequently, decreases H. pylori gastric colonization while increasing inflammation in the gastric mucosa.

Direct modulation of TRPM4 and TRPM3 channels by the phospholipase C inhibitor U73122.

Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122-sensitive ion channels. We performed detailed biophysical analysis of the U73122-induced currents in frequently used cell lines. At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca(2+) . U73122 also potentiated Ca(2) (+) -dependent TRPM4 currents in human Jurkat T-cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family. Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology.

Polymorphism in the TRP8 gene in Kyrgyz population: Putative association with highland adaptation

The human TRPM8 gene encodes a receptor mediating cold sensitivity, and this points to its putative role in cold adaptation. The structural variability of the TRPM8 gene for five single-nucleotide polymorphisms (SNPs) has been studied in the Kyrgyz population. The SNPs are located in the coding regions of the gene, and three of them are confined to a 20-bp segment in exon 7. The frequencies of minor SNP alleles are as follows: rs13004520 G/C = 0.06; rs28901637 A/T = 0.13; rs11562975 G/C = 0.27; rs7593557 G/A = 0.21; rs11563071 C/G = 0.12. The analyzed sample of Kyrgyz population includes 275 individuals living at different altitudes and under drastically different climatic conditions. The frequency of the minor rs11562975 allele in the highlanders (living above 3200 m ASL and higher) is lower by a factor of 1.5 than in the residents of lower regions (760–3000 m ASL; p < 0.01). This result indicates a selective role of rs11562975 in adapting to cold. A comparison of haplotype frequencies in the Kyrgyz population with Europeans, East Asians, and Africans shows a clear narrowing of genotype variation in Europeans in comparison to all the others. Probably, this phenomenon is related to a decline in the population size (bottleneck effect) during evolution. We consider the exon–intron structure of the TRPM8 gene. The epigenetic markers in the vicinity of the gene have been analyzed. Two strong binding sites for insulator ctcf proteins are present there. They are likely to be associated with chromatin conformation and alternative splicing regulation. A structural–functional characterization of genes for the TRP protein family is provided.

Transient Receptor Potential Melastatin 2:an Ion Channel for Oxidative Stress Sensing.

Transient receptor potential (TRP) channel is a superfamily of cation channels located on the cell membrane. TRP channels are classified into seven subfamilies based on the amino acid sequence homology,and transient receptor potential melastatin 2(TRPM2) is the second member of the TRPM subfamily. More evidences have revealed the important roles of TRPM2 in physiological and pathological events such as release of insulin from pancreatic Β-cells,inflammatory cytokines production from cells,and oxidative stress-induced cell death. As a cellular sensor for oxidative stress channel,TRPM2 is activated by a variety of factors. TRPM2 is a potential therapeutic target for oxidative stress-related diseases.

Congenital stationary night blindness with hypoplastic discs, negative electroretinogram and thinning of the inner nuclear layer.

To describe congenital stationary night blindness (CSNB) with negative electroretinogram, hypoplastic discs, nystagmus and thinning of the inner nuclear layer (INL). Retinal structure was analyzed qualitatively with spectral domain optical coherence tomography and wide field imaging. Retinal function was evaluated with full-field electroretinography (ffERG). Molecular genetic testing included next-generation sequencing (NGS) of the known genes involved in CSNB. Patients presented with CSNB presented with nystagmus, high myopia, hypoplastic discs and negative ffERG with no measurable rod response. The retinas appeared normal and automated segmentation of retinal layers demonstrated a relative reduction of thickness of the INL. There was no significant change in the ffERG after prolonged 2hour dark adaptation compared to standard 30minute dark adaptation. Affected family members harboured the homozygous 1-bp deletion c.2394delC in exon 18 of the TRPM1 gene, whereas their unaffected parents were heterozygous carriers. This data expands the genotype and phenotype spectrum of CSNB. The lack of improvement of rod responses after prolonged dark adaptation, together with thinning of the INL, is compatible with postreceptoral transmission dysfunction in the bipolar cells. Such knowledge may prove useful in future development of treatment for outer retinal dystrophies, using opsin genes to restore light responses in survivor neurons in the inner retina.

TRPM7 is down-regulated in both left atria and left ventricle of ischaemic cardiomyopathy patients and highly related to changes in ventricular function.

AimsThe kinase ion channel transient receptor potential melastatin 7 (TRPM7) is considered a modulator of cardiac fibrosis progression; nevertheless, we lack of studies analysing its role in human ischaemic cardiomyopathy (ICM). Our objective was to analyse the expression of genes encoding cardiac ion channels in human ICM, focusing on the alterations in mRNA levels of TRPM7 and its relationship with changes in the ventricular function.Methods and resultsRNA‐sequencing was carried out in 13 left ventricular (LV) samples of patients with ICM compared with a control group (n = 10). The analysis revealed a total of 25 ion channel genes differentially expressed. We performed an RTqPCR analysis of the TRPM7 mRNA in LV and left atrial samples and found that it was down‐regulated in both cavities (−1.43‐fold and −1.52‐fold, respectively). Atrial TRPM7 mRNA levels showed an excellent and inverse relationships with the depressed ejection fraction (r = −0.724, P = 0.042) and with the mitral A wave (r = −0.938, P = 0.006).ConclusionsWe report the down‐regulation of TRPM7 in tissue samples from both left atria and left ventricle in patients with ICM. We found an inverse relationship between both cardiac chambers mRNA levels with LV dysfunction, suggesting an important role of TRPM7 in the left atrial and LV functional depression found in this cardiomyopathy.

Association of TRPM3 Polymorphism (rs10780946) and Aspirin-Exacerbated Respiratory Disease (AERD).

Aspirin-exacerbated respiratory disease (AERD) refers to the combination of asthma rhinosinusitis and poliposis; ingestion of aspirin or other non-steroid anti-inflammatory drugs exacerbate asthma-like symptoms. The pathogenesis of AERD is unknown, and genetic and environmental factors contribute to the disease. Our objective is identifying polymorphisms associated with susceptibility in a Mexican mestizo population. Primarily we performed custom Illumina goldengate array-based genotyping of 1512 SNPs, carefully selected from a variety of acute/chronic inflammatory lung conditions previously reported. Four SNPs in TRPM3 gene showed the lowest p-values (rs10780946, rs7025694, rs1889915, and rs7047645). We further selected rs10780946 and rs7025694 for validation using Taqman genotyping (n=743; 288 AERD, 272 ATA, and 183 HC). rs10780946 showed association when compared between AERD and ATA groups under co-dominant (p=0.006), dominant (p=0.002), overdominant (p=0.01), and log-additive (p=0.03) genetic models. AERD showed increased heterozygous TC (rs10780946-rs7025694) haplotype compared to ATA and HC (p<0.05). We could not confirm any association between rs7025694 and AERD. rs10780946 TRPM3 polymorphism is associated with AERD susceptibility.

Shear stress activates monovalent cation channel transient receptor potential melastatin subfamily 4 in rat atrial myocytes via type 2 inositol 1,4,5-trisphosphate receptors and Ca(2+) release.

During each contraction and haemodynamic disturbance, cardiac myocytes are subjected to fluid shear stress as a result of blood flow and the relative movement of sheets of myocytes. The present study aimed to characterize the shear stress-sensitive membrane current in atrial myocytes using the whole-cell patch clamp technique, combined with pressurized fluid flow, as well as pharmacological and genetic interventions of specific proteins. The data obtained suggest that shear stress indirectly activates the monovalent cation current carried by transient receptor potential melastatin subfamily 4 channels via type 2 inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release in subsarcolemmal domains of atrial myocytes. Ca(2+) -mediated interactions between these two proteins under shear stress may be an important mechanism by which atrial cells measure mechanical stress and translate it to alter their excitability. Atrial myocytes are subjected to shear stress during the cardiac cycle under physiological or pathological conditions. The ionic currents regulated by shear stress remain poorly understood. We report the characteristics, molecular identity and activation mechanism of the shear stress-sensitive current (Ishear ) in rat atrial myocytes. A shear stress of ∼16dyn cm(-2) was applied to single myocytes using a pressurized microflow system, and the current was measured by whole-cell patch clamp. In symmetrical CsCl solutions with minimal concentrations of internal EGTA, Ishear showed an outwardly rectifying current-voltage relationship (reversal at -2mV). The current was conducted primarily (∼80%) by monovalent cations but not Ca(2+) . It was suppressed by intracellular Ca(2+) buffering at a fixed physiological level, inhibitors of transient receptor potential melastatin subfamily 4 (TRPM4), intracellular introduction of TRPM4 antibodies or knockdown of TRPM4 expression, suggesting that TRPM4 carries most of this current. A notable reduction in Ishear occurred upon inhibition of Ca(2+) release through the ryanodine receptors or inositol 1,4,5-trisphosphate receptors (IP3 R) and upon depletion of sarcoplasmic reticulum Ca(2+) . In type 2 IP3 R (IP3 R2) knockout atrial myocytes, Ishear was 10-20% of that in wild-type myocytes. Immunocytochemistry and proximity ligation assays revealed that TRPM4 and IP3 R2 were expressed at peripheral sites with co-localization, although they are not localized within 40nm. Peripheral localization of TRPM4 was intact in IP3 R2 knockout cells. The data obtained in the present study suggest that shear stress activates TRPM4 current by triggering Ca(2+) release from the IP3 R2 in the peripheral domains of atrial myocytes.

Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I

BackgroundProgressive cardiac conduction disease (PCCD) is one of the most common cardiac conduction disturbances. It has been causally related to rare mutations in several genes including SCN5A, SCN1B, TRPM4, LMNA and GJA5. Methods and resultsIn this study, by applying targeted next-generation sequencing (NGS) in 95 unrelated patients with PCCD, we have identified 13 rare variants in the TRPM4 gene, two of which are currently absent from public databases. This gene encodes a cardiac calcium-activated cationic channel which precise role and importance in cardiac conduction and disease is still debated. One novel variant, TRPM4-p.I376T, is carried by the proband of a large French 4-generation pedigree. Systematic familial screening showed that a total of 13 family members carry the mutation, including 10 out of the 11 tested affected individuals versus only 1 out of the 21 unaffected ones. Functional and biochemical analyses were performed using HEK293 cells, in whole-cell patch-clamp configuration and Western blotting. TRPM4-p.I376T results in an increased current density concomitant to an augmented TRPM4 channel expression at the cell surface. ConclusionsThis study is the first extensive NGS-based screening of TRPM4 coding variants in patients with PCCD. It reports the third largest pedigree diagnosed with isolated Progressive Familial Heart Block type I and confirms that this subtype of PCCD is caused by mutation-induced gain-of-expression and function of the TRPM4 ion channel.

Magnesium homeostasis goes awry in chemoresistance -TRPM6, TRPM7 and MagT1 in colon carcinoma LoVo cells

Chemoresistance is one of the most significant factors impeding the progress of cancer therapy (1). It is known that neoplastic cells accumulate magnesium and frequently upregulate one of its transporters, i.e.TRPM7 (2). We have investigated magnesium homeostasis in a model of chemoresistance i.e. colon carcinoma LoVo cells sensitive (LoVo-S) or resistant to doxorubicin (LoVo-R). We observed that LoVo-R have higher amount of total intracellular magnesium than LoVo-S. We studied the expression of some magnesium transporter (TRPM6, TRPM7 and MagT1) by Real Time PCR and Western Blot and found that TRPM6 and 7 are overexpressed in LoVo-S, while MagT1 is upregulated in LoVo-R. In LoVo-S, silencing TRPM7 retards cell growth and shifts the phenotype to one more similar to resistant cells. On the other hand, calpeptin, a calpain inhibitor, upregulates TRPM7, stimulated proliferation and enhances the sensitivity to doxorubicin of LoVo-R. Silencing MagT1 in LoVo-R markedly inhibited cell growth without affecting the response to doxorubicin. We conclude that alterations of magnesium homeostasis play a role in drug resistance.

Analysis of metabolic effects of menthol on WFS1-deficient mice.

In this study, we investigated the physiological regulation of energy metabolism in wild-type (WT) and WFS1-deficient (Wfs1KO) mice by measuring the effects of menthol treatment on the O2 consumption, CO2 production, rectal body temperature, and heat production. The basal metabolism and behavior was different between these genotypes as well as TRP family gene expressions. Wfs1KO mice had a shorter life span and weighed less than WT mice. The food and water intake of Wfs1KO mice was lower as well as the body temperature when compared to their WT littermates. Furthermore, Wfs1KO mice had higher basal O2 consumption, and CO2 and heat production than WT mice. In addition, Wfs1KO mice showed a higher response to menthol administration in comparison to WT mice. The strongest menthol effect was seen on different physiological measures 12 h after oral administration. The highest metabolic response of Wfs1KO mice was seen at the menthol dose of 10mg/kg. Menthol increased O2 consumption, and CO2 and heat production in Wfs1KO mice when compared to their WT littermates. In addition, the expression of Trpm8 gene was increased. In conclusion, our results show that the Wfs1KO mice develop a metabolic phenotype characterized with several physiological dysfunctions.

Hypomagnesemia in Type 2 Diabetes: A Vicious Circle?

Over the past decades, hypomagnesemia (serum Mg(2+) <0.7 mmol/L) has been strongly associated with type 2 diabetes mellitus (T2DM). Patients with hypomagnesemia show a more rapid disease progression and have an increased risk for diabetes complications. Clinical studies demonstrate that T2DM patients with hypomagnesemia have reduced pancreatic β-cell activity and are more insulin resistant. Moreover, dietary Mg(2+) supplementation for patients with T2DM improves glucose metabolism and insulin sensitivity. Intracellular Mg(2+) regulates glucokinase, KATP channels, and L-type Ca(2+) channels in pancreatic β-cells, preceding insulin secretion. Moreover, insulin receptor autophosphorylation is dependent on intracellular Mg(2+) concentrations, making Mg(2+) a direct factor in the development of insulin resistance. Conversely, insulin is an important regulator of Mg(2+) homeostasis. In the kidney, insulin activates the renal Mg(2+) channel transient receptor potential melastatin type 6 that determines the final urinary Mg(2+) excretion. Consequently, patients with T2DM and hypomagnesemia enter a vicious circle in which hypomagnesemia causes insulin resistance and insulin resistance reduces serum Mg(2+) concentrations. This Perspective provides a systematic overview of the molecular mechanisms underlying the effects of Mg(2+) on insulin secretion and insulin signaling. In addition to providing a review of current knowledge, we provide novel directions for future research and identify previously neglected contributors to hypomagnesemia in T2DM.

3-Iodothyronamine increases transient receptor potential melastatin channel 8 (TRPM8) activity in immortalized human corneal epithelial cells

3-Iodothyronamine (3T1AM) is an endogenous thyroid hormone metabolite that interacts with the human trace amine-associated receptor 1 (hTAAR1), a G-protein-coupled receptor, to induce numerous physiological responses including dose-dependent body temperature lowering in rodents. 3T1AM also directly activates cold-sensitive transient receptor potential melastatin 8 (TRPM8) channels in human conjunctival epithelial cells (HCjEC) at constant temperature as well as reducing rises in IL-6 release induced by transient receptor potential vanilloid 1 (TRPV1) activation by capsaicin (CAP). Here, we describe that 3T1AM-induced TRPM8 activation suppresses through crosstalk TRPV1 activation in immortalized human corneal epithelial cells (HCEC). RT-PCR and immunofluorescent staining identified TRPM8 gene and protein expression. Increases in Ca2+ influx induced by the TRPM8 agonists either 3T1AM (0.1–10μM), menthol (500μM), icilin (15–60μM) or temperature lowering (either <17°C or >17°C) were all blocked by 10–20μM BCTC, a mixed TRPV1/TRPM8 antagonist. BCTC blocked 3T1AM-induced recombinant TRPM8 activation of Ca2+ transients in an osteosarcoma heterologous expression system. The effects of BCTC in HCEC were attributable to selective TRPM8 inhibition since whole-cell patch-clamp currents underlying Ca2+ rises induced by 20μM CAP were BCTC insensitive. On the other hand, Ca2+ transients induced by activating TRPV1 with either CAP or a hyperosmolar medium were suppressed during exposure to either 1μM 3T1AM or 15μM icilin. All of these modulatory effects on intracellular Ca2+ regulation induced by the aforementioned agents were attributable to changes in underlying inward and outward current. Taken together, TRPM8 activation by 3T1AM markedly attenuates and even eliminates hyperosmolar and CAP induced TRPV1 activation through crosstalk.

Polymorphism in the TRP8 gene in Kyrgyz population: putative association with highland adaptation

The human TRPM8 gene encodes a receptor mediating cold sensitivity, and this fact points to its putative role in cold adaptation. The structural variability of the TRPM8 gene for five single-nucleotide polymorphisms (SNPs) has been studied in Kyrgyz population. The SNPs are located in coding regions of the gene, and three of them are confined to a segment of 20 bp in exon 7. The frequencies of minor SNP alleles are: rs13004520 G/C = 0.06; rs28901637 А/Т = 0.13; rs11562975 G/C = 0.27; rs7593557 G/A = 0.21; rs11563071 С/G = 0.12. The analyzed sample of Kyrgyz population includes 275 individuals living at different altitudes and under drastically different climatic conditions. The frequency of the minor rs11562975 allele in highlanders (living above 2 800 m A. S. L.) is one-third lower than in residents of lower regions (760–2 800 m A. S. L.; p &lt; 0.01). This result presumes a selective role of rs11562975 in cold adaptation. Comparison of haplotype frequencies in Kyrgyz people with Europeans, East Asians, and Africans shows a clear narrowing of genotype variation in Europeans in comparison to all others. Probably, this phenomenon is related to a population size decline (bottleneck effect) during the evolution. We consider the exon– intron structure of the TRPM8 gene. Epigenetic markers in the vicinity of the gene have been analyzed. Two strong binding sites for insulator ctcf proteins are present there. They are likely to be associated with chromatin conformation and alternative splicing regulation. A structure–functional characterization of genes for the TRP protein family is provided.

TRPM4-dependent post-synaptic depolarization is essential for the induction of NMDA receptor-dependent LTP in CA1 hippocampal neurons

TRPM4 is a calcium-activated but calcium-impermeable non-selective cation (CAN) channel. Previous studies have shown that TRPM4 is an important regulator of Ca2+-dependent changes in membrane potential in excitable and non-excitable cell types. However, its physiological significance in neurons of the central nervous system remained unclear. Here, we report that TRPM4 proteins form a CAN channel in CA1 neurons of the hippocampus and we show that TRPM4 is an essential co-activator of N-methyl-d-aspartate (NMDA) receptors (NMDAR) during the induction of long-term potentiation (LTP). Disrupting the Trpm4 gene in mice specifically eliminates NMDAR-dependent LTP, while basal synaptic transmission, short-term plasticity, and NMDAR-dependent long-term depression are unchanged. The induction of LTP in Trpm4−/− neurons was rescued by facilitating NMDA receptor activation or post-synaptic membrane depolarization. Accordingly, we obtained normal LTP in Trpm4−/− neurons in a pairing protocol, where post-synaptic depolarization was applied in parallel to pre-synaptic stimulation. Taken together, our data are consistent with a novel model of LTP induction in CA1 hippocampal neurons, in which TRPM4 is an essential player in a feed-forward loop that generates the post-synaptic membrane depolarization which is necessary to fully activate NMDA receptors during the induction of LTP but which is dispensable for the induction of long-term depression (LTD). These results have important implications for the understanding of the induction process of LTP and the development of nootropic medication.

MiR-211 and MITF modulation by Bcl-2 protein in melanoma cells.

Melanoma, the most lethal form of skin cancer, is frequently associated with alterations in several genes, among which the Bcl-2 oncogene plays an important role in progression, chemosensitivity and angiogenesis. Also microRNA (miRNA) are emerging as modulators of melanoma development and progression, and among them, miR-211, located within the melastatin-1/TRPM1 (transient receptor potential cation channel, subfamily M, member 1 protein) gene, is prevalently expressed in the melanocyte lineage and acts as oncosuppressor. Using several human melanoma cell lines and their Bcl-2 stably overexpressing derivatives, we evaluated whether there was a correlation between expression of Bcl-2 and miR-211. Western blot analysis and quantitative real-time polymerase chain reaction demonstrated reduced expression of pri-miR-211, miR-211, TRPM1, and MLANA levels, after Bcl-2 overexpression, associated with increased expression of well-known miR-211 target genes. Overexpression of mature miR-211 in Bcl-2 overexpressing cells rescued Bcl-2 ability to increase cell migration. A decreased nuclear localization of microphthalmia-associated transcription factor (MITF), a co-regulator of both miR-211 and TRPM1, and a reduced MITF recruitment at the TRPM1 and MLANA promoters were also evidenced in Bcl-2 overexpressing cells by immunofluorescence and chromatin immunoprecipitation experiments, respectively. Reduction of Bcl-2 expression by small interference RNA confirmed the ability of Bcl-2 to modulate miR-211 and TRPM1 expression. © 2015 Wiley Periodicals, Inc.

Involvement of neurogenomic regulation in maintenance of temperature homeostasis in the cold

We present data from several studies that indicate the important role of the genome in the regulation of temperature homeostasis. Long-term adaptation to cold causes specific changes in the expression of genes for thermosensitive TRP ion channels (TRPV3) and serotonin receptors (5HT-2A) in the hypothalamus. These changes are specific for the hypothalamus, the center of thermoregulation, and they are not observed in other parts of the brain. This may indicate that adaptive changes in the thermal sensitivity of hypothalamic neurons involve at least two adaptive genomic mechanisms, which regulate the ratio between thermosensitive TRP ion channels and mediator receptors located in a neuron. These two mechanisms can complement each other. With regard to the important role of ion channel TRPM8 in the formation of colddefense thermoregulatory, metabolic, and immune responses, it is reasonable to assume that variations in the functioning of the ion channel lead to the formation of diverse individual defense responses to environmental changes. Studies on humans provide evidence for the role of the rs11562975 single nucleotide polymorphism in the TRPM8 gene in the variability of temperature perception and, consequently, in the development of the cold defense response. Individuals with the heterozygous GC genotype are more sensitive to the cold and less sensitive to menthol (agonist of the TRPM8 ion channel) as compared to people with the homozygous GG genotype. In addition, this polymorphism results in a less energy-consuming hypometabolic response to cooling.

Expression of temperature-sensitive ion channel TRPM8 in sperm cells correlates with vertebrate evolution.

Transient Receptor Potential cation channel, subfamily Melastatin, member 8 (TRPM8) is involved in detection of cold temperature, different noxious compounds and in execution of thermo- as well as chemo-sensitive responses at cellular levels. Here we explored the molecular evolution of TRPM8 by analyzing sequences from various species. We elucidate that several regions of TRPM8 had different levels of selection pressure but the 4th–5th transmembrane regions remain highly conserved. Analysis of synteny suggests that since vertebrate origin, TRPM8 gene is linked with SPP2, a bone morphogen. TRPM8, especially the N-terminal region of it, seems to be highly variable in human population. We found 16,656 TRPM8 variants in 1092 human genomes with top variations being SNPs, insertions and deletions. A total of 692 missense mutations are also mapped to human TRPM8 protein of which 509 seem to be delateroiours in nature as supported by Polyphen V2, SIFT and Grantham deviation score. Using a highly specific antibody, we demonstrate that TRPM8 is expressed endogenously in the testis of rat and sperm cells of different vertebrates ranging from fish to higher mammals. We hypothesize that TRPM8 had emerged during vertebrate evolution (ca 450 MYA). We propose that expression of TRPM8 in sperm cell and its role in regulating sperm function are important factors that have guided its molecular evolution, and that these understandings may have medical importance.

Abstract A18: Personalized prevention of colorectal cancer trial

Abstract Background: High calcium or high magnesium consumption has been linked to reduced risks of colorectal adenoma and cancer. However, results are not consistent. In the Tennessee Colorectal Polyp Study (TCPS), the Calcium Polyp Prevention Study (a large-scale randomized trial), we found that high intake of supplemental calcium or total calcium was related to a reduced risk of colorectal adenoma or adenoma recurrence only among those with calcium/magnesium intake ratio below 2.8 or 2.6. This effect modification by the calcium/magnesium ratio cannot solely be attributed to the dietary intake in calcium or magnesium. Furthermore, we found in the TCPS that calcium/magnesium intake ratio significantly interacted with Thr1482Ile polymorphism (rs8042919, GàA) in the TRPM7 gene, in relation to both adenomas and hyperplastic polyps. The TRPM7 gene is involved in magnesium and calcium re(absorption) and homeostasis. Compared to those with the GG genotype, we found that people who carry A allele(s) were at 60% and 85% increased risks of adenoma and hyperplastic polyp, respectively, if they also consumed diets with calcium/magnesium intake ratio &amp;gt;2.8; and the corresponding risks increased by 345% and 1500% among those with the AA genotype. The risk was not increased among those who carried the A allele(s) if they consumed diets low in calcium/magnesium ratio (&amp;lt;2.8). Objectives: 1) Test the hypothesis whether reducing the calcium/magnesium through magnesium supplementation affects biomarkers related to colorectal carcinogenesis in rectal tissues; and 2) Test whether the effect differs by TRPM7 genotype. Methods: Personalized Prevention of Colorectal Cancer Trial (PPCCT, R01CA149633) is an ongoing double-blind, randomized trial of 12 week personalized magnesium supplementation. 288 participants with high calcium/magnesium intake ratio and at high risk of colorectal cancer are being recruited. The uniqueness of the trial includes 1) only target participants with a high calcium/magnesium ratio through nutritional screening using multiple 24-hour dietary recalls; 2) personalized doses are based on and used to reduce the current calcium/magnesium intake ratios; and finally, 3) the participants are also randomized by the TRPM7 genotype. Results: As of July 20, 2014, we have enrolled at least 170 participants. We have conducted immunohistochemical assays to measure expressions of carcinogenesis biomarkers, including apoptosis, cell proliferation, COX2 and TRPM7, in normal rectal tissues collected at baseline and the end of the intervention for 40 participants who first completed the trial. We are also assaying lipid profile and other novel biomarkers, including DNA methylation, and microbial markers. We have obtained some very promising preliminary findings. We will report updated findings in the presentation. Conclusions: The results from our study may ultimately help to develop personalized strategies to prevent colorectal cancer. Citation Format: Xiangzhu Zhu Zhu, Chang Yu, Martha J. Shrubsole, Xinqing Deng, Eugene Shubin, Jennifer M. Engle, Wei Zheng, Harvey J. Murff, Douglas L. Seidner, Reid M. Ness, Qi Dai. Personalized prevention of colorectal cancer trial. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A18.