Analysis of the daily changes of melatonin receptors in the rat liver

Melatonin membrane (MT1 and MT2) and nuclear (RORα) receptors have been identified in several mammalian tissues, including the liver. The mechanisms regulating hepatic melatonin receptors are yet unknown. This study investigated whether these receptors exhibit daily changes and the effects of melatonin on their levels. Our results show that mRNAs for MT1/MT2 receptors exhibit circadian rhythms that were followed by rhythms in their respective protein levels; the acrophases for the two rhythms were reached at 04:00 and 05:00 hr, respectively. Pinealectomy blunted the rhythms in both mRNAs and protein levels. In contrast, mRNA and protein levels of nuclear receptor RORα increased significantly after pinealectomy. The cycles of the latter receptor also exhibited circadian rhythms which peaked at 03:00 and 03:45 hr, respectively. Melatonin administration (10–200 mg/kg) increased in a dose‐dependent manner the protein content of MT1/MT2 receptors, with no effects on RORα. Lunzindole treatment, however, did not affect melatonin receptor expression or content of either the membrane or nuclear receptors. Together with previously published findings which demonstrated the intracellular distribution of melatonin in rat liver, the current results support the conclusion that the circadian rhythms of MT1/MT2 and RORα receptors are under the control of the serum and intracellular melatonin levels. Moreover, the induction of MT1/MT2 receptors after the administration of high doses of melatonin further suggests that the therapeutic value of melatonin may not be restricted to only low doses of the indoleamine.     

Coexpression of MT1 and RORalpha1 melatonin receptors in the Syrian hamster Harderian gland

Melatonin acts through several specific receptors, including membrane receptors (MT(1) and MT(2)) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT(1) and RORalpha(1) in this gland was determined by nested RT-PCR followed by amplicon sequencing and Western-blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT(1) receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal-synthesized melatonin. On the contrary, although a RORalpha(1)-immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORalpha(1) on the role of this indoleamine against oxidative stress is discussed.     

Melatonin modulation of intracellular signaling pathways in hepatocarcinoma HepG2 cell line: role of the MT1 receptor

Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in hepatocarcinoma are limited. We have previously demonstrated that melatonin administration induces cycle arrest, apoptosis, and changes in the expression of its specific receptors in HepG2 human hepatocarcinoma cells. In this study, we used the receptor antagonist luzindole to assess the contribution of MT1 melatonin membrane receptor to melatonin effects on cell viability, mitogen-activated protein kinase (MAPKs) activation, and cAMP levels. Additionally, effects of MT1 inhibition on mRNA levels of cytosolic quinone reductase type-2 (NQO2) receptor and nuclear retinoic acid-related orphan receptor alpha (RORα) were tested. Melatonin, at 1000 and 2500 μm, significantly reduced cell viability. Pre-incubation with luzindole partially inhibited the effects of melatonin on cell viability. Melatonin at 2500 μm significantly reduced cAMP levels, and this effect was partially blocked by luzindole. Both melatonin concentrations increased the expression of phosphorylated p38, ERK, and JNK. ERK activation was completely abolished in the presence of luzindole. NQO2 but not RORα mRNA level significantly increased in luzindole-treated cells. Results obtained provide evidence that the melatonin effects on cell viability and proliferation in HepG2 cells are partially mediated through the MT1 membrane receptor, which seems to be related also with melatonin modulation of cAMP and ERK activation. This study also highlights a possible interplay between MT1 and NQO2 melatonin receptors in liver cancer cells.     

Activation of melatonin receptor 2 but not melatonin receptor 1 mediates melatonin‐conferred cardioprotection against myocardial ischemia/reperfusion injury

Accumulated pieces of evidence have proved the beneficial effects of melatonin on myocardial ischemia/reperfusion (MI/R) injury, and these effects were largely dependent on melatonin membrane receptor activation. In humans and other mammals, there are two types of melatonin receptors, including the melatonin receptor 1 (MT1, melatonin receptor 1a or MTNR1A) and melatonin receptor 1 (MT2, melatonin receptor 1b or MTNR1B) receptor subtypes. However, which receptor mediates melatonin‐conferred cardioprotection remains unclear. In this study, we employed both loss‐of‐function and gain‐of‐function approaches to reveal the answer. Mice (wild‐type; MT1 or MT2 silencing by in vivo minicircle vector; and those overexpressing MT1 or MT2 by in vivo AAV9 vector) were exposed to MI/R injury. Both MT1 and MT2 were present in wild‐type myocardium. MT2, but not MT1, was essentially upregulated after MI/R Melatonin administration significantly reduced myocardial injury and improved cardiac function after MI/R Mechanistically, melatonin treatment suppressed MI/R‐initiated myocardial oxidative stress and nitrative stress, alleviated endoplasmic reticulum stress and mitochondrial injury, and inhibited myocardial apoptosis. These beneficial actions of melatonin were absent in MT2‐silenced heart, but not the MT1 subtype. Furthermore, AAV9‐mediated cardiomyocyte‐specific overexpression of MT2, but not MT1, mitigated MI/R injury and improved cardiac dysfunction, which was accompanied by significant amelioration of oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Mechanistically, MT2 protected primary cardiomyocytes against hypoxia/reoxygenation injury via MT2/Notch1/Hes1/RORα signaling. Our study presents the first direct evidence that the MT2 subtype, but not MT1, is a novel endogenous cardiac protective receptor against MI/R injury. Medications specifically targeting MT2 may hold promise in fighting ischemic heart disease.     

Inverse agonist exposure enhances ligand binding and G protein activation of the human MT1 melatonin receptor, but leads to receptor down-regulation

Melatonin binds and activates G protein-coupled melatonin receptors. The density and affinity of the endogenous melatonin receptors change throughout the 24-hr day, and the exposure of recombinant melatonin receptors to melatonin often results in desensitization of the receptors. Receptor density, G protein activation and expression level were analyzed in CHO cell lines stably expressing the human MT1 receptors after 1 or 72 hr of exposure to melatonin (agonist, 10 nm) and luzindole (antagonist/inverse agonist, 10 microm). The 72-hr exposure to luzindole significantly increased the apparent receptor density in cell lines with both high and low MT1 receptor expression levels (MT1(high) and MT1(low) cells, respectively). In the constitutively active MT1(high) cells, luzindole pretreatment also stimulated the functional response to melatonin in [(35)S]GTPgammaS binding assays, whereas melatonin pretreatment attenuated the functional response at both time points. Receptor ELISA was used to analyze the cell membrane and total expression level of the MT1 receptor in intact and permeabilized cells, respectively. Luzindole pretreatment decreased the total cellular level of MT1 receptor in the MT1(high) cells at both time points but increased the cell surface expression of MT1 receptor at 72 hr. Melatonin significantly decreased MT1 receptor cell surface expression only in MT1(high) cells after a 1-hr treatment. These results indicate that melatonin treatment desensitizes MT1 receptors, whereas luzindole increases ligand binding and G-protein activation. Luzindole also stimulates downregulation of the MT1 receptor protein, interfering with the synthesis and/or degradation of the receptor.     

Distribution of melatonin receptors in murine pancreatic islets

Melatonin has multiple receptor-dependent and receptor-independent functions. At the cell membrane, melatonin interacts with its receptors MT1 and MT2, which are expressed in numerous tissues. Genome-wide association studies have recently shown that the MTNR1B/MT2 receptor may be involved in the pathogenesis of type 2 diabetes mellitus. In line with these findings, expression of melatonin receptors has been shown in mouse, rat, and human pancreatic islets. MT1 and MT2 are G-protein-coupled receptors and are proposed to exert inhibitory effects on insulin secretion. Here, we show by immunocytochemistry that these membrane melatonin receptors have distinct locations in the mouse islet. MT1 is expressed in α-cells while MT2 is located to the β-cells. These findings help to unravel the complex machinery underlying melatonin's role in the regulation of islet function.     

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines

The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.     

Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells

Among the non-neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin-specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT(1), MT(2)); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonin's antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca(2+) influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole.     

MT1 melatonin receptor internalization underlies melatonin-induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation

Abstract:  Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi_α 2,3, Gq_α, β-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with β-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.     

Abnormal melatonin receptor 1B expression in osteoblasts from girls with adolescent idiopathic scoliosis

Abstract: Melatonin signaling dysfunction has been associated with the etiology of adolescent idiopathic scoliosis (AIS). Genetic analysis has also associated the occurrence of AIS with the MT2 gene. Thus, we determined whether there is abnormality in the protein expression of melatonin receptors (MT) in AIS osteoblasts. In this study, we recruited 11 girls with severe AIS and eight normal subjects for intraoperative bone biopsies. MT1 and MT2 receptor protein expressions in the isolated osteoblasts were detected. Also, cell proliferation assay using different melatonin concentrations (0, 10^?9, 10^?5, 10^?4 m ) was carried out. The results showed that both MT1 and MT2 receptors are expressed in osteoblasts of the controls. While MT1 receptors were expressed in osteoblasts of all AIS subjects, osteoblasts of only 7 of 11 AIS showed expression of MT2 receptors. Melatonin stimulated control osteoblasts to proliferate. However, proliferation of AIS osteoblasts without expression of MT2 receptor, after treatment with melatonin, was minimal when compared with control and AIS osteoblasts with MT2 receptor expression. The proliferation of AIS osteoblasts with MT2 receptor was greater than those without. This is the first report demonstrating a difference between AIS and normal osteoblasts in the protein expression of MT2 receptor. The results suggest that there is a possible functional effect of MT2 receptor on osteoblast proliferation. AIS osteoblasts without expression of MT2 receptor showed the lowest percentage of viable cells after melatonin treatment. This possibly indicates the modulating role of melatonin through MT2 receptor on the proliferation of osteoblasts.     

Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

Abstract: Melatonin has shown particular promise as a neuroprotective agent to prevent motoneuron death in animal models of both amyotrophic lateral sclerosis (ALS) and spinal cord injuries (SCI). However, an understanding of the roles of endogenous melatonin receptors including MT1, MT2, and orphan G‐protein receptor 50 (GPR50) in neuroprotection is lacking. To address this deficiency, we utilized plasmids for transfection and overexpression of individual melatonin receptors in the ventral spinal cord 4.1 (VSC4.1) motoneuron cell line. Receptor‐mediated cytoprotection following exposure to glutamate at a toxic level (25 μm ) was determined by assessing cell viability, apoptosis, and intracellular free Ca²⁺ levels. Our findings indicate a novel role for MT1 and MT2 for increasing expression of the calcium‐binding proteins calbindin D28K and parvalbumin. Increased levels of calbindin D28K and parvalbumin in VSC4.1 cells overexpressing MT1 and MT2 were associated with cytoprotective effects including inhibition of proapoptotic signaling, downregulation of inflammatory factors, and expression of prosurvival markers. Interestingly, the neuroprotective effects conferred by overexpression of MT1 and/or MT2 were also associated with increases in the estrogen receptor β (ERβ): estrogen receptor α (ERα) ratio and upregulation of angiogenic factors. GPR50 did not exhibit cytoprotective effects. To further confirm the involvement of the melatonin receptors, we silenced both MT1 and MT2 in VSC4.1 cells using RNA interference technology. Knockdown of MT1 and MT2 led to an increase in glutamate toxicity, which was only partially reversed by melatonin treatment. Taken together, our findings suggest that the neuroprotection against glutamate toxicity exhibited by melatonin may depend on MT1 and MT2 but not GPR50.     

The role of proline residues in the structure and function of human MT2 melatonin receptor

Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTPgammaS-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.     

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.