TRPM8 modulates temperature regulation in a sex-dependent manner without affecting cold-induced bone loss.
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ABSTRACT:
SUBMITTER: Lelis Carvalho A
PROVIDER: S-EPMC8177490 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:TRPM8 is a member of the transient receptor potential ion channel superfamily, which is expressed in sensory neurons and is activated by cold and cooling compounds, such as menthol. Activation of TRPM8 by agonists takes place through shifts in its voltage activation curve, allowing channel opening at physiological membrane potentials. Here, we studied the role of the N-glycosylation occurring at the pore loop of TRPM8 on the function of the channel. Using heterologous expression of recombinant channels in HEK293 cells we found that the unglycosylated TRPM8 mutant (N934Q) displays marked functional differences compared with the wild type channel. These differences include a shift in the threshold of temperature activation and a reduced response to menthol and cold stimuli. Biophysical analysis indicated that these modifications are due to a shift in the voltage dependence of TRPM8 activation toward more positive potentials. By using tunicamycin, a drug that prevents N-glycosylation of proteins, we also evaluated the effect of the N-glycosylation on the responses of trigeminal sensory neurons expressing TRPM8. These experiments showed that the lack of N-glycosylation affects the function of native TRPM8 ion channels in a similar way to heterologously expressed ones, causing an important shift of the temperature threshold of cold-sensitive thermoreceptor neurons. Altogether, these results indicate that post-translational modification of TRPM8 is an important mechanism modulating cold thermoreceptor function, explaining the marked differences in temperature sensitivity observed between recombinant and native TRPM8 ion channels.
| S-EPMC3365712 | biostudies-literature
Project description:BACKGROUND AND PURPOSE:Gallium (Ga) has been shown to be effective in the treatment of disorders associated with accelerated bone loss, including cancer-related hypercalcemia and Paget's disease. These clinical applications suggest that Ga could reduce bone resorption. However, few studies have studied the effects of Ga on osteoclastic resorption. Here, we have explored the effects of Ga on bone cells in vitro. EXPERIMENTAL APPROACH:In different osteoclastic models [osteoclasts isolated from long bones of neonatal rabbits (RBC), murine RAW 264.7 cells and human CD14-positive cells], we have performed resorption activity tests, staining for tartrate resistant acid phosphatase (TRAP), real-time polymerase chain reaction analysis, viability and apoptotic assays. We also evaluated the effect of Ga on osteoblasts in terms of proliferation, viability and activity by using an osteoblastic cell line (MC3T3-E1) and primary mouse osteoblasts. KEY RESULTS:Gallium dose-dependently (0-100 microM) inhibited the in vitro resorption activity of RBC and induced a significant decrease in the expression level of transcripts coding for osteoclastic markers in RAW 264.7 cells. Ga also dramatically reduced the formation of TRAP-positive multinucleated cells. Ga down-regulated in a dose-dependant manner the expression of the transcription factor NFATc1. However, Ga did not affect the viability or activity of primary and MC3T3-E1 osteoblasts. CONCLUSIONS AND IMPLICATIONS:Gallium exhibits a dose-dependent anti-osteoclastic effect by reducing in vitro osteoclastic resorption, differentiation and formation without negatively affecting osteoblasts. We provide evidence that this inhibitory mechanism involves down-regulation of NFATc1 expression, a master regulator of RANK-induced osteoclastic differentiation.
| S-EPMC2925491 | biostudies-literature
Project description:Highlights • Tunica dartos smooth muscle (TDSM), which is responsible for scrotal thermoregulation, responds to cooling by enhancement of basal tension, and increase of the amplitude and duration of synaptically-evoked contractions.• TDSM cold-induced tension, but not synaptically-evoked contractions are sensitive to the blockers of TRPM8 cold/menthol receptor.• TRPM8 is localized to the TDSM cells' endoplasmic reticulum and its activation by cold causes intracellular calcium mobilization.• Chemical castration of male rats consequent to pharmacological blockade of androgen receptors eliminates the effects of cold on TDSM tension, but not on synaptically-evoked contractions, and abolishes TRPM8 protein expression.• TRPM8 is an important determinant of cold-dependent contractility of the tunica dartos muscle and scrotal thermoregulation which is crucial for maintaining normal spermatogenesis and male fertility. Tunica dartos smooth muscle (TDSM) lies beneath the scrotal skin, and its contraction leads to scrotum wrinkling upon cooling. However, neither the nature of TDSM cold-sensitivity nor the underlying molecular sensors are well understood. Here we have investigated the role of cold/menthol-sensitive TRPM8 channel in TDSM temperature-dependent contractility. The contraction of isolated male rat TDSM strips was studied by tensiometry. TRPM8 expression was assayed by RT-PCR and fluorescence immunochemistry. Isolated TDSM strips responded to cooling from 33 °C to 20 °C by enhancement of basal tension, and increase of the amplitude and duration of electric field stimulated (EFS) contractions. The effects of cold on basal tension, but not on EFS-contractions, could be 80% inhibited by TRPM8 blockers, capsazepine and BCTC [N-(4‑tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide], and could be partially mimicked by menthol. RT-PCR and immunolabeling showed TRPM8 mRNA and protein expression in TDSM cells with protein labelling being predominantly localized to intracellular compartments. Chemical castration of male rats consequent to the treatment with androgen receptor blocker, flutamide, led to the abrogation of cold effects on TDSM basal tension, but not on EFS-contractions, and to the disappearance of TRPM8 protein expression. We conclude that TRPM8 is involved in the maintenance of basal cold-induced TDSM tonus, but not in sympathetic nerve-mediated contractility, by acting as endoplasmic reticulum Ca2+ release channel whose expression in TDSM cells requires the presence of a functional androgen receptor. Thus, TRPM8 plays a crucial role in scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility.
| S-EPMC10074839 | biostudies-literature
Project description:Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.
| S-EPMC4491737 | biostudies-literature
Project description:Thermoregulation is a homeostatic process to maintain an organism's internal temperature within a physiological range compatible with life. In poikilotherms, body temperature fluctuates with that of the environment, with both physiological and behavioral responses employed to modify body temperature. Changing skin colour/reflectance and locomotor activity are both well-recognized temperature regulatory mechanisms, but little is known of the participating thermosensor/s. We find that Xenopus laevis tadpoles put in the cold exhibit a temperature-dependent, systemic, and rapid melanosome aggregation in melanophores, which lightens the skin. Cooling also induces a reduction in the locomotor performance. To identify the cold-sensor, we focus on transient receptor potential (trp) channel genes from a Trpm family. mRNAs for several Trpms are present in Xenopus tails, and Trpm8 protein is present in skin melanophores. Temperature-induced melanosome aggregation is mimicked by the Trpm8 agonist menthol (WS12) and blocked by a Trpm8 antagonist. The degree of skin lightening induced by cooling is correlated with locomotor performance, and both responses are rapidly regulated in a dose-dependent and correlated manner by the WS12 Trpm8 agonist. We propose that TRPM8 serves as a cool thermosensor in poikilotherms that helps coordinate skin lightening and behavioural locomotor performance as adaptive thermoregulatory responses to cold.
| S-EPMC9889708 | biostudies-literature
Project description:Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P(2)). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.
| S-EPMC2671608 | biostudies-literature
Project description:The hypoxia-inducible factor HIF-1 has recently been identified as an important modifier of longevity in the roundworm Caenorhabditis elegans. Studies have reported that HIF-1 can function as both a positive and negative regulator of life span, and several disparate models have been proposed for the role of HIF in aging. Here, we resolve many of the apparent discrepancies between these studies. We find that stabilization of HIF-1 increases life span robustly under all conditions tested; however, deletion of hif-1 increases life span in a temperature-dependent manner. Animals lacking HIF-1 are long lived at 25°C but not at 15°C. We further report that deletion or RNAi knockdown of hif-1 impairs healthspan at lower temperatures because of an age-dependent loss of vulval integrity. Deletion of hif-1 extends life span modestly at 20°C when animals displaying the vulval integrity defect are censored from the experimental data, but fails to extend life span if these animals are included. Knockdown of hif-1 results in nuclear relocalization of the FOXO transcription factor DAF-16, and DAF-16 is required for life span extension from deletion of hif-1 at all temperatures regardless of censoring.
| S-EPMC3980873 | biostudies-literature
Project description:It is not uncommon for patients chronically treated with opioids to exhibit opioid-induced hyperalgesia, and this has been widely reported clinically and experimentally. The molecular substrate for this hyperalgesia is multifaceted, and associated with a complex neural reorganization even in the periphery. For instance, we have recently shown that chronic morphine-induced heat hyperalgesia is associated with an increased expression of GluN2B containing N-methyl-D-aspartate receptors, as well as of the neuronal excitatory amino acid transporter 3/excitatory amino acid carrier 1, in small-diameter primary sensory neurons only. Cold allodynia is also a common complaint of patients chronically treated with opioids, yet its molecular mechanisms remain to be understood. Here we present evidence that the cold sensor TRPM8 channel is involved in opioid-induced hyperalgesia. After 7 days of morphine administration, we observed an upregulation of TRPM8 channels using patch clamp recording on sensory neurons and Western blot analysis on dorsal root ganglia. The selective TRPM8 antagonist RQ-00203078 blocked cold hyperalgesia in morphine-treated rats. Also, TRPM8 knockout mice failed to develop cold hyperalgesia after chronic administration of morphine. Our results show that chronic morphine upregulates TRPM8 channels, which is in contrast with the previous finding that acute morphine triggers TRPM8 internalization. PERSPECTIVE:Patients receiving chronic opioid are sensitive to cold. We show in mice and rats that sustained morphine administration induces cold hyperalgesia and an upregulation of TRPM8. Knockout or selectively blocking TRPM8 reduces morphine-induced cold hyperalgesia suggesting TRPM8 is regulated by opioids.
| S-EPMC5291755 | biostudies-literature
Project description:Pathological cardiac hypertrophy and cardiac fibrosis are remodeling events that result in mechanical stiffness and pathophysiological changes of the myocardium. Both humans and animal models display a sexual dimorphism where females are more protected from pathological remodeling. Fibroblast growth factor 2 (FGF2) mediates cardiac hypertrophy, cardiac fibrosis and protection against cardiac injury and is made in high molecular weight and low molecular weight isoforms (Hi FGF2 and Lo FGF2, respectively). Although some light has been shed on isoform-specific functions in cardiac pathophysiology, their roles in pathologic cardiac remodeling have yet to be determined. We tested the hypothesis that Lo FGF2 and Hi FGF2 modulate pathological cardiac remodeling in an isoform-specific manner. Young adult male and female mice between 8-12 weeks of age of mixed background that were deficient in either Hi FGF2 or Lo FGF2 (Hi KO or Lo KO, respectively) were subjected to daily injections of isoproterenol (Iso) for four days after which their hearts were compared to wildtype cohorts. Post-Iso treatment, female Lo KO hearts don't exhibit significant differences in their hypertrophic and fibrotic response, while female Hi KO hearts present with a blunted hypertrophic response. In male animals Lo KO hearts present with an exacerbated fibrotic response and increased alpha-smooth muscle actin protein expression while Hi KO hearts present with a blunted fibrotic response and increased atrial natriuretic factor protein expression Thus, in female hearts Hi FGF2 mediate cardiac hypertrophy while in male hearts Lo FGF2 and Hi FGF2 display an antithetical role in cardiac fibrosis where Lo FGF2 is protective while Hi FGF2 is damaging. In conclusion, cardiac remodeling following catecholamine overactivation is modulated by FGF2 in isoform- and sex-specific manners.
| S-EPMC3827774 | biostudies-literature
Project description:Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterol-rich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn(934) residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol- and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.
| S-EPMC2666574 | biostudies-literature