Melatonin‐mediated inhibition of Cav3.2 T‐type Ca2+ channels induces sensory neuronal hypoexcitability through the novel protein kinase C‐eta isoform

Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T‐type Ca²⁺ channels (T‐type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T‐type channels in small TG neurons via the melatonin receptor 2 (MT₂ receptor) and a pertussis toxin‐sensitive G‐protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT₂ receptor coprecipitated with Gα_o. Both shRNA‐mediated knockdown of Gα_o and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin‐induced T‐type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC‐eta (PKC_η) while antagonism of PKC_η or shRNA targeting PKC_η prevented the melatonin‐mediated effects. In a heterologous expression system, activation of MT₂ receptor strongly inhibited Cav3.2 T‐type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKC_η dependent and was accompanied by a negative shift in the steady‐state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant‐induced inflammatory pain. These actions were inhibited by T‐type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T‐type channel activity through the MT₂ receptor coupled to novel G_βγ‐mediated PKC_η signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.     

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing

Melatonin, a circadian rhythm–promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)‐MSCs pretreated with melatonin enhanced wound closure, granulation, and re‐epithelialization at mouse skin wound sites, where relatively more UCB‐MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB‐MSCs. Melatonin (1 μm ) significantly increased the motility of UCB‐MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT₂). We found that Gαq coupled with MT₂ and that the binding of Gαq to MT₂ uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization‐related proteins such as profilin‐1, cofilin‐1, and F‐actin in UCB‐MSCs. Finally, a lack of MT₂ expression in UCB‐MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT₂ triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB‐MSCs motility.     

Detection of recombinant and endogenous mouse melatonin receptors by monoclonal antibodies targeting the C‐terminal domain

Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non‐24‐hour sleep‐wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT₁ and MT₂, belonging to the G protein‐coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT₁ and MT₂. Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT₁ and MT₂ by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT₁ or MT₂. None of the mABs cross‐reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR‐KO mice as control. MT₂ expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta‐cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.     

Molecular and cellular pharmacological properties of 5‐methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT): a nonspecific MT3 ligand

Abstract: 5‐Methoxycarbonylamino‐N‐acetyltryptamine (MCA‐NAT) has been initially described as a ligand at non MT₁, non MT₂ melatonin binding site (MT3) selective versus MT₁ and MT₂, two membrane melatonin receptors. MCA‐NAT activity has been reported by others in different models, in vivo, particularly in the intra‐ocular pressure (IOP) models in rabbits and monkeys. Its activity was systematically linked to either MT3 or to a new, yet unknown, melatonin receptor. In this article, the melatonin receptor pharmacology of MCA‐NAT is described. MCA‐NAT has micromolar range affinities at the melatonin receptors MT₁ and MT₂, while in functional studies, MCA‐NAT proved to be a powerful MT₁/MT₂ partial agonist in the sub‐micromolar range. These data strongly suggest that MCA‐NAT actions might be mediated by these receptors in vivo. Finally, as described by others, we show that MCA‐NAT is unable to elicit any type of receptor‐like functional responses from Chinese hamster ovary cells over‐expressing quinone reductase 2, the MT3.     

Functional MT1 and MT2 melatonin receptors in mammals

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT₁ and MT₂ proteinteoupled receptors viewed as potential targets for drug discovery. MT₁ melatonin receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation in cancer cells, and reproductive and metabolic functions. Ativation of MT₂ melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT₁ and MT₂ native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.     

Enhancement of high glucose‐induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2/Akt/NF‐κB pathway

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM‐associated neuronal cell death. Previous investigators reported on a genome‐wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose‐induced neuronal cell death and the effect of melatonin against high glucose‐induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN‐induced putative kinase 1 (PINK1) and LC‐3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker? fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V‐positive cells. In addition, high glucose‐stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N‐acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT₂ receptor‐specific inhibitor 4‐P‐PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin‐regulated mitochondrial ROS production, cleaved caspase‐3 and caspase‐9 expressions, and the number of annexin V‐positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT₂/Akt/NF‐κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions.     

Association of disease‐predisposition polymorphisms of the melatonin receptors and sunshine duration in the global human populations

Abstract: Melatonin is predominantly involved in signaling circadian and seasonal rhythms, and its synthesis is regulated by the environmental light/dark cycle. The selection pressure by geographically different environmental light/dark cycles, which is predominantly determined by sunshine duration, on the global distribution of genetic polymorphisms in the melatonin pathway is not well understood. Recent genetic association studies identified various disease‐predisposition polymorphisms in this pathway. We investigated the correlations between the prevalence of these clinically important single nucleotide polymorphisms (SNPs) and sunshine duration among worldwide human populations from twelve regions in the CEPH‐HGDP database rs4753426, a recently reported predisposition SNP for type 2 diabetes in the promoter of the MT₂ melatonin receptor gene (MTNR1B), which was not included in the CEPH‐HGDP genotyping array, was additionally genotyped. This SNP showed a marginally significant correlation in 760 CEPH‐HGDP DNA samples (r = ?0.5346, P = 0.0733), and it showed the most prominent association among the candidate melatonin pathway SNPs examined. To control for population structure, which may lead to a false positive correlation, we genotyped this SNP in a replication set of 1792 subjects from China. The correlation was confirmed among Chinese populations (r = ?0.8694, P = 0.0002), and was also statistically significant after correction of other climatic and geographical covariants in multiple regression analysis (β = ?0.907, P = 1.94 × 10^?5). Taken together, it suggests that the human melatonin signaling pathway, particularly MT₂ melatonin receptor may have undergone a selective pressure in response to global variation in sunshine duration.     

Melatonin influences somatostatin secretion from human pancreatic δ‐cells via MT1 and MT2 receptors

Melatonin is an effector of the diurnal clock on pancreatic islets. The membrane receptor‐transmitted inhibitory influence of melatonin on insulin secretion is well established and contrasts with the reported stimulation of glucagon release from α‐cells. Virtually, nothing is known concerning the melatonin‐mediated effects on islet δ‐cells. Analysis of a human pancreatic δ‐cell model, the cell line QGP‐1, and the use of a somatostatin‐specific radioimmunoassay showed that melatonin primarily has an inhibitory effect on somatostatin secretion in the physiological concentration range. In the pharmacological range, melatonin elicited slightly increased somatostatin release from δ‐cells. Cyclic adenosine monophosphate (cAMP) is the major second messenger dose‐dependently stimulating somatostatin secretion, in experiments employing the membrane‐permeable 8‐Br‐cAMP. 8‐Br‐cyclic guanosine monophosphate proved to be of only minor relevance to somatostatin release. As the inhibitory effect of 1 nm melatonin was reversed after incubation of QGP‐1 cells with the nonselective melatonin receptor antagonist luzindole, but not with the MT2‐selective antagonist 4‐P‐PDOT (4‐phenyl‐2‐propionamidotetraline), an involvement of the MT1 receptor can be assumed. Somatostatin release from the δ‐cells at low glucose concentrations was significantly inhibited during co‐incubation with 1 nm melatonin, an effect which was less pronounced at higher glucose levels. Transient expression experiments, overexpressing MT1, MT2, or a deletion variant as a control, indicated that the MT1 and not the MT2 receptor was the major transmitter of the inhibitory melatonin effect. These data point to a significant influence of melatonin on pancreatic δ‐cells and on somatostatin release.     

Melatonin is required for H2O2‐ and NO‐mediated defense signaling through MAPKKK3 and OXI1 in Arabidopsis thaliana

Melatonin influences plant innate immunity through the mitogen‐activated protein kinase (MAPK) pathway. However, the most upstream MAPK component in melatonin signaling and the dependence of generation of a reactive oxygen species (ROS) burst on melatonin synthesis and signaling remain unclear. In this study, treatment of several mekk (alias mapkkk)‐knockout Arabidopsis mutants with melatonin revealed that the MAPKKK3 and OXI1 (oxidative signal‐inducible1) kinases are responsible for triggering melatonin‐induced defense signaling pathways. In addition, melatonin induction upon infection with the avirulent pathogen Pseudomonas syringae DC3000 (avrRpt2) was independent of H₂O₂ and NO individually, but dependent on the combination of H₂O₂ and NO. Moreover, melatonin‐mediated induction of the expression of defense‐related genes, such as PR1 and ICS1, was not altered in the H₂O₂‐deficient rbohD/F‐knockout mutant cotreated with an NO scavenger, indicating that melatonin functions downstream of the ROS and NO burst. Collectively, the data indicate that melatonin‐mediated induction of an innate immune response requires multiple signaling molecules and activation of MAPKKK3 and OXI1, followed by triggering of downstream MAPK cascades, such as MAPK3 and MAPK6.     

Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT₁ and MT₂ receptors, belonging to the G protein‐coupled receptor (GPCR) super‐family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT₁ and MT₂ receptors, which comprises 378 individual proteins. Among the proteins interacting with MT₁, but not with MT₂, we identified several presynaptic proteins, suggesting a potential role of MT₁ in neurotransmission. Presynaptic localization of MT₁ receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT₁ physically interacts with the voltage‐gated calcium channel Ca_v2.2 and inhibits Ca_v2.2‐promoted Ca²⁺ entry in an agonist‐independent manner. In conclusion, we show that MT₁ is part of the presynaptic protein network and negatively regulates Ca_v2.2 activity, providing a first hint for potential synaptic functions of MT₁.     

Central role of PKCα in isoenzyme-selective regulation of cardiac transient outward current Ito and Kv4.3 channels

The transient outward current I_to is an important determinant of the early repolarization phase. I_to and its molecular basis Kv4.3 are regulated by adrenergic pathways including protein kinase C. However, the exact regulatory mechanisms have not been analyzed yet. We here analyzed isoenzyme specific regulation of Kv4.3 and I_to by PKC. Kv4.3 channels were expressed in Xenopus oocytes and currents were measured with double electrode voltage clamp technique. Patch clamp experiments were performed in isolated rat cardiomyocytes. Unspecific PKC stimulation with PMA resulted in a reduction of Kv4.3 current. Similar effects could be observed after activation of conventional PKC isoforms by TMX. Both effects were reversible by pharmacological inhibition of the conventional PKC isoenzymes (Gö6976). In contrast, activation of the novel PKC isoforms (ingenol) did not significantly affect Kv4.3 current. Whereas TMX-induced PKC activation was not attenuated inhibition of PKCβ, inhibition of PKCα with HBDDE prevented inhibitory effects of both PMA and TMX. Accordingly, stimulatory effects of PMA and TMX could be mimicked by the α-isoenzyme selective PKC activator iripallidal. Further evidence for the central role of PKCα was provided with the use of siRNAs. We found that PKCα siRNA but not PKCβ siRNA abolished the TMX induced effect. In isolated rat cardiomyocytes, PMA dependent I_to reduction could be completely abolished by pharmacologic inhibition of PKCα. In summary we show that PKCα plays a central role in protein kinase C dependent regulation of Kv4.3 current and native I_to. These results add to the current understanding of isoenzyme selective ion channel regulation by protein kinases.     

Melatonin sensitizes human breast cancer cells to ionizing radiation by downregulating proteins involved in double‐strand DNA break repair

Radiation and adjuvant endocrine therapy are nowadays considered a standard treatment option after surgery in breast cancer. Melatonin exerts oncostatic actions on human breast cancer cells. In the current study, we investigated the effects of a combination of radiotherapy and melatonin on human breast cancer cells. Melatonin (1 mm , 10 μm and 1 nm ) significantly inhibited the proliferation of MCF‐7 cells. Radiation alone inhibited the MCF‐7 cell proliferation in a dose‐dependent manner. Pretreatment of breast cancer cells with melatonin 1 wk before radiation led to a significantly greater decrease of MCF‐7 cell proliferation compared with radiation alone. Melatonin pretreatment before radiation also decreased G₂‐M phase arrest compared with irradiation alone, with a higher percentage of cells in the G₀‐G₁ phase and a lower percentage of cells in S phase. Radiation alone diminished RAD51 and DNA‐protein kinase (PKcs) mRNA expression, two main proteins involved in double‐strand DNA break repair. Treatment with melatonin for 7 days before radiation led to a significantly greater decrease in RAD51 and DNA?PKcs mRNA expression compared with radiation alone. Our findings suggest that melatonin pretreatment before radiation sensitizes breast cancer cells to the ionizing effects of radiation by decreasing cell proliferation, inducing cell cycle arrest and downregulating proteins involved in double‐strand DNA break repair. These findings may have implications for designing clinical trials using melatonin and radiotherapy.     

Evidence of the receptor‐mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein‐coupled and PI3K signaling pathways

Abstract: Melatonin has been shown to modulate glucose metabolism by influencing insulin secretion. Recent investigations have also indicated a regulatory function of melatonin on the pancreatic α‐cells. The present in vitro and in vivo studies evaluated whether melatonin mediates its effects via melatonin receptors and which signaling cascade is involved. Incubation experiments using the glucagon‐producing mouse pancreatic α‐cell line αTC1 clone 9 (αTC1.9) as well as isolated pancreatic islets of rats and mice revealed that melatonin increases glucagon secretion. Preincubation of αTC1.9 cells with the melatonin receptor antagonists luzindole and 4P‐PDOT abolished the glucagon‐stimulatory effect of melatonin. In addition, glucagon secretion was lower in the pancreatic islets of melatonin receptor knockout mice than in the islets of the wild‐type (WT) control animals. Investigations of melatonin receptor knockout mice revealed decreased plasma glucagon concentrations and elevated mRNA expression levels of the hepatic glucagon receptor when compared to WT mice. Furthermore, studies using pertussis toxin, as well as measurements of cAMP concentrations, ruled out the involvement of Gαi‐ and Gαs‐coupled signaling cascades in mediating the glucagon increase induced by melatonin. In contrast, inhibition of phospholipase C in αTC1.9 cells prevented the melatonin‐induced effect, indicating the physiological relevance of the Gαq‐coupled pathway. Our data point to the involvement of the phosphatidylinositol 3‐kinase signaling cascade in mediating melatonin effects in pancreatic α‐cells. In conclusion, these findings provide evidence that the glucagon‐stimulatory effect of melatonin in pancreatic α‐cells is melatonin receptor mediated, thus supporting the concept of melatonin‐modulated and diurnal glucagon release.