Roles of Thermosensitive Transient Receptor Channels TRPV1 and TRPM8 in Paclitaxel-Induced Peripheral Neuropathic Pain.

Paclitaxel, a microtubule-stabilizing chemotherapy drug, can cause severe paclitaxel-induced peripheral neuropathic pain (PIPNP). The roles of transient receptor potential (TRP) ion channel vanilloid 1 (TRPV1, a nociceptor and heat sensor) and melastatin 8 (TRPM8, a cold sensor) in PIPNP remain controversial. In this study, Western blotting, immunofluorescence staining, and calcium imaging revealed that the expression and functional activity of TRPV1 were upregulated in rat dorsal root ganglion (DRG) neurons in PIPNP. Behavioral assessments using the von Frey and brush tests demonstrated that mechanical hyperalgesia in PIPNP was significantly inhibited by intraperitoneal or intrathecal administration of the TRPV1 antagonist capsazepine, indicating that TRPV1 played a key role in PIPNP. Conversely, the expression of TRPM8 protein decreased and its channel activity was reduced in DRG neurons. Furthermore, activation of TRPM8 via topical application of menthol or intrathecal injection of WS-12 attenuated the mechanical pain. Mechanistically, the TRPV1 activity triggered by capsaicin (a TRPV1 agonist) was reduced after menthol application in cultured DRG neurons, especially in the paclitaxel-treated group. These findings showed that upregulation of TRPV1 and inhibition of TRPM8 are involved in the generation of PIPNP, and they suggested that inhibition of TRPV1 function in DRG neurons via activation of TRPM8 might underlie the analgesic effects of menthol.

TRPM8 as the rapid testosterone signaling receptor: Implications in the regulation of dimorphic sexual and social behaviors.

Testosterone regulates dimorphic sexual behaviors in all vertebrates. However, the molecular mechanism underlying these behaviors remains unclear. Here, we report that a newly identified rapid testosterone signaling receptor, Transient Receptor Potential Melastatin 8 (TRPM8), regulates dimorphic sexual and social behaviors in mice. We found that, along with higher steroid levels in the circulation, TRPM8-/- male mice exhibit increased mounting frequency indiscriminate of sex, delayed sexual satiety, and increased aggression compared to wild-type controls, while TRPM8-/- females display an increased olfaction-exploratory behavior. Furthermore, neuronal responses to acute testosterone application onto the amygdala were attenuated in TRPM8-/- males but remained unchanged in females. Moreover, activation of dopaminergic neurons in the ventral tegmental area following mating was impaired in TRPM8-/- males. Together, these results demonstrate that TRPM8 regulates dimorphic sexual and social behaviors, and potentially constitutes a signalosome for mediation of sex-reward mechanism in males. Thus, deficiency of TRPM8 might lead to a delayed sexual satiety phenomenon.

Suppressive effects of a transient receptor potential melastatin 8 (TRPM8) agonist on hyperthermia-induced febrile seizures in infant mice.

Background: Febrile seizures (FSs) are the most frequent type of seizures in infancy and childhood. Epileptiform discharges (EDs) on electroencephalogram at the time of first FS recurrence can increase the risk of epilepsy development. Therefore, inhibition of EDs is important. Recently, WS-3, a transient receptor potential melastatin 8 (TRPM8) agonist, reportedly suppressed penicillin G-induced cortical-focal EDs. However, the effects of TRPM8 agonists on FSs remain unknown. In this study, we aimed to clarify the effects of the TRPM8 agonist, and the absence of TRPM8 channels, on hyperthermia-induced FS by analyzing the fast ripple band. Methods: Hyperthermia (43°C for 30 min) induced by a heating pad caused FSs in postnatal day 7 wild-type (WT) and TRPM8 knockout (TRPM8KO) mice. FSs were defined as EDs occurring during behavioral seizures involving hindlimb clonus and loss of the righting reflex. Mice were injected with 1% dimethyl sulfoxide or 1 mM WS-3 20 min before the onset of hyperthermia, and electroencephalograms; movies; and rectal, brain and heating pad temperatures were recorded. Results: In wild-type mice, WS-3 reduced the fast ripple amplitude in the first FS without changing rectal and brain temperature thresholds. In contrast, the anti-FS effect induced by the TRPM8 agonist was not observed in TRPM8KO mice and, compared with wild-type mice, TRPM8 deficiency lowered the rectal and brain temperature thresholds for FSs, exacerbated the fast ripple amplitude, and prolonged the duration of the initial FS induced by hyperthermia. Conclusion: Our findings suggest that TRPM8 agonists can be used to treat hyperthermia-induced FSs.

Cold-inducible RNA-binding protein migrates from the nucleus to the cytoplasm under cold stress in normal human bronchial epithelial cells via TRPM8-mediated mechanism.

BACKGROUND: Cold-inducible RNA-binding protein (CIRP or hnRNP A18) is a multifunctional stress-responsive protein. Our previous study demonstrated that cold stress increased CIRP expression and migrated from the nucleus to the cytoplasm in airway epithelial cells. However, the mechanism through which CIRP migrates from the nucleus to the cytoplasm upon cold stress remains unknown. METHODS: The expression of CIRP in the bronchial epithelium was examined using immunofluorescence, real-time polymerase chain reaction (RT-PCR), and Western blotting. The expression of inflammatory factors interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) were detected by ELISA and RT-PCR. Transient receptor potential melastatin 8 (TRPM8) receptor function was characterized by Ca2+ imaging. RESULTS: Cold stress upregulated the expression of CIRP, inflammatory factors and promoted the translocation of CIRP from the nucleus to the cytoplasm in normal human bronchial epithelial (NHBE) cells. Cold stress activated the TRPM8/(Ca2+)/PKCα/glycogen synthase kinase 3ß (GSK3ß) signaling cascade, and that inhibition of this signaling pathway attenuated the migration of CIRP from the nucleus to cytoplasm but did not decrease its overexpression induced by cold stress. Knocked down CIRP expression or blocked CIRP migration between the nucleus and cytoplasm significantly decreased inflammatory factor expression. CONCLUSIONS: These results indicate that cold stress leads to the migration of CIRP from the nucleus to the cytoplasm with alteration of expression, which are involved in the expression of inflammatory factors (IL-1ß, IL-6, IL-8 and TNF-α) induced by cold air, through TRPM8/Ca2+/PKCα/GSK3ß signaling cascade.

Transient Receptor Potential Melastatin 8 (TRPM8) Channel Regulates Proliferation and Migration of Breast Cancer Cells by Activating the AMPK-ULK1 Pathway to Enhance Basal Autophagy.

The calcium-permeable cation channel TRPM8 (transient receptor potential melastatin 8) is a member of the TRP superfamily of cation channels that is upregulated in various types of cancer with high levels of autophagy, including prostate, pancreatic, breast, lung, and colon cancers. Autophagy is closely regulated by AMP-activated protein kinase (AMPK) and plays an important role in tumor growth by generating nutrients through degradation of intracellular structures. Additionally, AMPK activity is regulated by intracellular Ca2+ concentration. Considering that TRPM8 is a non-selective Ca2+-permeable cation channel and plays a key role in calcium homoeostasis, we hypothesized that TRPM8 may control AMPK activity thus modulating cellular autophagy to regulate the proliferation and migration of breast cancer cells. In this study, overexpression of TRPM8 enhanced the level of basal autophagy, whereas TRPM8 knockdown reduced the level of basal autophagy in several types of mammalian cancer cells. Moreover, the activity of the TRPM8 channel modulated the level of basal autophagy. The mechanism of regulation of autophagy by TRPM8 involves autophagy-associated signaling pathways for activation of AMPK and ULK1 and phagophore formation. Impaired AMPK abolished TRPM8-dependent regulation of autophagy. TRPM8 interacts with AMPK in a protein complex, and cytoplasmic C-terminus of TRPM8 mediates the TRPM8-AMPK interaction. Finally, basal autophagy mediates the regulatory effects of TRPM8 on the proliferation and migration of breast cancer cells. Thus, this study identifies TRPM8 as a novel regulator of basal autophagy in cancer cells acting by interacting with AMPK, which in turn activates AMPK to activate ULK1 in a coordinated cascade of TRPM8-mediated breast cancer progression.

TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8. Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PMTRPM8 activity elicits Ca2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PMTRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation.