Paula A. Witt-Enderby

Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade

Abstract:  The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10‐day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co‐exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P‐PDOT (MT2 receptor selective), inhibited the melatonin‐induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2‐[125I]‐iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2‐[125I]‐iodomelatonin binding as well as melatonin efficacy to inhibit forskolin‐induced cAMP accumulation. Further characterization of the mechanisms underlying melatonins effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin‐mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.

Therapeutic treatments potentially mediated by melatonin receptors: potential clinical uses in the prevention of osteoporosis, cancer and as an adjuvant therapy.

Abstract:  Melatonins therapeutic potential is grossly underestimated because its functional roles are diverse and its mechanism(s) of action are complex and varied. Melatonin produces cellular effects via a variety of mechanisms in a receptor independent and dependent manner. In addition, melatonin is a chronobiotic agent secreted from the pineal gland during the hours of darkness. This diurnal release of melatonin impacts the sensitivity of melatonin receptors throughout a 24‐hr period. This changing sensitivity probably contributes to the narrow therapeutic window for use of melatonin in treating sleep disorders, that is, at the light‐to‐dark (dusk) or dark‐to‐light (dawn) transition states. In addition to the cyclic changes in melatonin receptors, many genes cycle over the 24‐hr period, independent or dependent upon the light/dark cycle. Interestingly, many of these genes support a role for melatonin in modulating metabolic and cardiovascular physiology as well as bone metabolism and immune function and detoxification of chemical agents and cancer reduction. Melatonin also enhances the actions of a variety of drugs or hormones; however, the role of melatonin receptors in modulating these processes is not known. The goal of this review is to summarize the evidence related to the utility of melatonin as a therapeutic agent by focusing on its other potential uses besides sleep disorders. In particular, its use in cancer prevention, osteoporosis and, as an adjuvant to other therapies are discussed. Also, the role that melatonin and, particularly, its receptors play in these processes are highlighted.

Melatonin and breast cancer: cellular mechanisms, clinical studies and future perspectives

Recent studies have suggested that the pineal hormone melatonin may protect against breast cancer, and the mechanisms underlying its actions are becoming clearer. Melatonin works through receptors and distinct second messenger pathways to reduce cellular proliferation and to induce cellular differentiation. In addition, independently of receptors melatonin can modulate oestrogen-dependent pathways and reduce free-radical formation, thus preventing mutation and cellular toxicity. The fact that melatonin works through a myriad of signalling cascades that are protective to cells makes this hormone a good candidate for use in the clinic for the prevention and/or treatment of cancer. This review summarises cellular mechanisms governing the action of melatonin and then considers the potential use of melatonin in breast cancer prevention and treatment, with an emphasis on improving clinical outcomes.

Melatonin induction of filamentous structures in non-neuronal cells that is dependent on expression of the human mt1 melatonin receptor

Melatonin has gained recent popularity as a treatment for insomnia and other sleep disorders; however, its cellular effects are unknown. We report the effects of melatonin on the cellular morphology of Chinese hamster ovary (CHO) cells transformed to express the human melatonin receptors, mt1 and MT2. Our results show that melatonin exerts a strong influence on cellular shape and cytoskeletal organization in a receptor-dependent and possibly subtype-selective manner. The cell shape change that we see after a 5-h treatment of these non-neuronal cells with a pharmacological concentration of melatonin consists of the formation of long filamentous outgrowths that are reminiscent of the neurite processes produced by differentiating nerve cells. This morphological change occurs exclusively in cells expressing the mt1 receptor. We find that the microtubule and microfilament organization within these outgrowths is similar to that of neurites. Microtubules are required for the shape change to occur as Colcemid added in combination with melatonin completely blocks outgrowth formation. We demonstrate that the number of cells showing the altered cell shape is dependent on melatonin concentration, constant exposure to melatonin and that outgrowth frequencies increase when protein kinase A (PKA) is inhibited. Concomitant melatonin-dependent increases in MEK 1/2 and ERK 1/2 phosphorylation are noted in mt1-CHO cells only. The production of filamentous outgrowths is dependent on the translation of new protein but not the transcription of new mRNA. Outgrowth number is not controlled by centrosomes but is instead controlled by the polymerization state of the actin cytoskeleton. The results of this work show that the organization of the cytoskeleton is affected by processes specifically mediated or regulated by the mt1 receptor and may represent a novel alternative mechanism for the stimulation of process formation.

MT1 and MT2 Melatonin Receptors: Ligands, Models, Oligomers, and Therapeutic Potential

Numerous physiological functions of the pineal gland hormone melatonin are mediated via activation of two G-protein-coupled receptors, MT1 and MT2. The melatonergic drugs on the market, ramelteon and agomelatine, as well as the most advanced drug candidates under clinical evaluation, tasimelteon and TIK-301, are high-affinity nonselective MT1/MT2 agonists. A great number of MT2-selective ligands and, more recently, several MT1-selective agents have been reported to date. Herein, we review recent advances in the field focusing on high-affinity agonists and antagonists and those displaying selectivity toward MT1 and MT2 receptors. Moreover, the existing models of MT1 and MT2 receptors as well as the current status in the emerging field of melatonin receptor oligomerization are critically discussed. In addition to the already existing indications, such as insomnia, circadian sleep disorders, and depression, new potential therapeutic applications of melatonergic ligands including cardiovascular regulation, appetite control, tumor growth inhibition, and neurodegenerative diseases are presented.

Melatonin effects on bone: potential use for the prevention and treatment for osteopenia, osteoporosis, and periodontal disease and for use in bone-grafting procedures.

An important role for melatonin in bone formation and restructuring has emerged, and studies demonstrate the multiple mechanisms for these beneficial actions. Statistical analysis shows that even with existing osteoporotic therapies, bone‐related disease, and mortality are on the rise, creating a huge financial burden for societies worldwide. These findings suggest that novel alternatives need to be developed to either prevent or reverse bone loss to combat osteoporosis‐related fractures. The focus of this review describes melatonins role in bone physiology and discusses how disruption of melatonin rhythms by light exposure at night, shift work, and disease can adversely impact on bone. The signal transduction mechanisms underlying osteoblast and osteoclast differentiation and coupling with one another are discussed with a focus on how melatonin, through the regulation of RANKL and osteoprotegerin synthesis and release from osteoblasts, can induce osteoblastogenesis while inhibiting osteoclastogenesis. Also, melatonins free‐radical scavenging and antioxidant properties of this indoleamine are discussed as yet an additional mechanism by which melatonin can maintain ones bone health, especially oral health. The clinical use for melatonin in bone‐grafting procedures, in reversing bone loss due to osteopenia and osteoporosis, and in managing periodontal disease is discussed.

Determination of the minimal melatonin exposure required to induce osteoblast differentiation from human mesenchymal stem cells and these effects on downstream signaling pathways.

Abstract:  The purpose of this study was to determine the critical time periods of melatonin treatment required to induce human mesenchymal stem cells (hAMSCs) into osteoblasts and to determine which osteogenic genes are involved in the process. The study design consisted of adding melatonin for different times (2, 5, 10, 14 or 21 days) toward the end of a 21‐day treatment containing osteogenic (OS+) medium or at the beginning of the 21‐day treatment and then withdrawn. The results show that a 21‐day continuous melatonin treatment was required to induce both alkaline phosphatase (ALP) activity and calcium deposition and these effects were mediated through MT2Rs. Functional analysis revealed that peak ALP levels induced by melatonin were accompanied by attenuation of melatonin‐mediated inhibition of forskolin‐induced cAMP accumulation. Immunoprecipitation and western blot analyses, respectively, showed that MT2R/β‐arrestin scaffolds complexed to Gi, MEK1/2 and ERK1/2 formed in these differentiated hAMSCs (i.e., when ALP levels were highest) where ERK1/2 resided primarily in the cytosol. It is hypothesized that these complexes form to modulate the subcellular localization of ERK1/2 to affect osteogenic gene expression. Using real‐time RT‐PCR, chronic melatonin exposure induced the expression of osteogenic genes RUNX‐2, osteocalcin and BMP‐2, through MT2Rs. No melatonin‐mediated changes in the mRNA expression of ALP, BMP‐6 or in the oxidative enzymes MtTFA, PGC‐1α, Polγ, NRF‐1, PDH, PDK and LDH occurred. These data show that a continuous 21‐day melatonin exposure is required to induce osteoblast differentiation from hAMSCs through the formation of MT2R/Gi/β‐arrestin/MEK/ERK1/2 complexes to induce osteogenesis.

Melatonin osteoporosis prevention study (MOPS): a randomized, double-blind, placebo-controlled study examining the effects of melatonin on bone health and quality of life in perimenopausal women

Abstract:  The purpose of this double‐blind study was to assess the effects of nightly melatonin supplementation on bone health and quality of life in perimenopausal women. A total of 18 women (ages 45–54) were randomized to receive melatonin (3 mg, p.o., n = 13) or placebo (n = 5) nightly for 6 months. Bone density was measured by calcaneal ultrasound. Bone turnover marker (osteocalcin, OC for bone formation and NTX for bone resorption) levels were measured bimonthly in serum. Participants completed Menopause‐Specific Quality of Life‐Intervention (MENQOL) and Pittsburgh Sleep Quality Index (PSQI) questionnaires before and after treatment. Subjects also kept daily diaries recording menstrual cycling, well‐being, and sleep patterns. The results from this study showed no significant change (6‐month‐baseline) in bone density, NTX, or OC between groups; however, the ratio of NTX:OC trended downward over time toward a ratio of 1:1 in the melatonin group. Melatonin had no effect on vasomotor, psychosocial, or sexual MENQOL domain scores; however, it did improve physical domain scores compared to placebo (mean change melatonin: −0.6 versus placebo: 0.1, P < 0.05). Menstrual cycling was reduced in women taking melatonin (mean cycles melatonin: 4.3 versus placebo: 6.5, P < 0.05), and days between cycles were longer (mean days melatonin: 51.2 versus placebo: 24.1, P < 0.05). No differences in duration of menses occurred between groups. The overall PSQI score and average number of hours slept were similar between groups. These findings show that melatonin supplementation was well tolerated, improved physical symptoms associated with perimenopause, and may restore imbalances in bone remodeling to prevent bone loss. Further investigation is warranted.

Melatonin decreases cell proliferation and transformation in a melatonin receptor-dependent manner

There are conflicting claims for the role of melatonin in oncogenesis. In addition, the mechanism(s) underlying melatonins effects in oncogenic processes is (are) unknown. In this study, the effects of melatonin exposure on cell proliferation and transformation were assessed in NIH3T3 cells transfected with either the human mt(1) (NIH-mt1) or MT(2) (NIH-MT2) melatonin receptors. The effects of melatonin exposure on proliferation was assessed by direct cell counts and [(3)H]thymidine uptake assays. The effect of chronic melatonin pretreatment on transformation was assessed by focus assays. In both NIH-mt1 and NIH-MT2 cells, melatonin pretreatment decreased cell proliferation and transformation. Control (NIH-neo) cells did not show this effect. However, as revealed by the [(3)H]thymidine uptake assays, an increase in DNA synthesis occurred in NIH-mt1 cells, whereas no increase occurred in the NIH-MT2 or NIH-neo cells. Upon examination of melatonin receptors, a decrease in the function of both mt(1) and MT(2) receptors occurred. These data suggest that perhaps an attenuation of receptor-mediated processes are involved in the anti-proliferative and anti-transformation capabilities of melatonin in NIH3T3 cells. In addition, based on the [(3)H]thymidine assays, receptor mediated signal transduction mechanisms may slow the growth of cells via actions on the cell cycle. The results from this study shed new insight on the putative mechanisms underlying melatonins effects on cell proliferation and transformation and lends support for a protective role of melatonin in oncogenesis.

N1E-115 mouse neuroblastoma cells express mt1 melatonin receptors and produce neurites in response to melatonin

Melatonin, a pineal hormone that induces sleep, has become a popular over-the-counter drug. The cellular effects of melatonin, however, are only beginning to be studied. We have recently shown that stimulation of the MT1 melatonin receptor induces rapid and dramatic cytoskeletal rearrangements in transformed non-neuronal cells (Witt-Enderby et al., Cell. Motil. Cytoskel. 46 (2000) 28). These cytoskeletal changes result in the formation of structures that closely resemble neurites. In this work, we show that the N1E-115 mouse neuroblastoma cell line rapidly responds to melatonin stimulation and forms neurites within 24 h. We also demonstrate that these cells readily bind 2-[125I]iodomelatonin at levels consistent with what is noted for native tissues (B(max)=3.43+/-1.56 fmol/mg protein; K(d)=240 pM). Western analysis shows that these cells possess and express melatonin receptors of the MT1 subtype. Treatment with pertussis toxin eliminates neurite formation whereas treatment with the MT2 subtype-specific activator, BMNEP, does not induce neurite formation. We have previously shown that increases in MEK 1/2 and ERK 1/2 phosphorylation are correlated with the shape changes in transformed CHO cells. Western analysis of the MEK/ERK signaling pathway in N1E-115 cells shows that this pathway is most likely maximally and constitutively stimulated. This may account for the spontaneous production of neurites noted for this cell line after long culture periods. The results of this work show that melatonin receptor stimulation in a neuronal cell type results in the formation of neurites and that the receptors responsible for melatonin-induced neurite formation in N1E-115 cells are most likely of the MT1 subtype.