Gloria Benítez-King

Melatonin as a cytoskeletal modulator: implications for cell physiology and disease

Abstract:  The cytoskeleton is a phylogenetically well‐preserved structure that plays a key role in cell physiology. Dynamic and differential changes in cytoskeletal organization occur in cellular processes according to the cell type and the specific function. In neurons, microtubules, microfilaments and intermediate filament (IF) rearrangements occur during axogenesis, and neurite formation which eventually differentiate into axons and dendrites to constitute synaptic patterns of connectivity. In epithelial cells, dynamic modifications occur in the three main cytoskeletal components and phosphorylation of cytoskeletal associated proteins takes place during the formation of the epithelial cell monolayer that eventually will transport water. In pathological processes such as neurodegenerative and psychiatric diseases an abnormal cytoskeletal organization occurs. Melatonin, the main product secreted by pineal gland during dark phase of the photoperiod, is capable of influencing microfilament, microtubule and IF organization by acting as a cytoskeletal modulator. In this paper we will summarize the evidence which provides the data that melatonin regulates cytoskeletal organization and we describe recent findings, which indicate that melatonin effects on microfilament rearrangements in stress fibers are involved in the mechanism by which the indole synchronizes water transport in kidney‐derived epithelial cells. In addition, we review recent data, which indicates that melatonin protects the neuro‐cytoskeletal organization from damage caused by free radicals contributing to cell survival, in addition to the already described mechanism elicited by the indole to prevent apoptosis and to scavenge free radicals. Moreover, we discuss the implications of an altered cytoskeletal organization for neurodegenerative and psychiatric illnesses and its re‐establishment by melatonin.

Melatonin Modulates Cell Survival of New Neurons in the Hippocampus of Adult Mice

Regulation of adult hippocampal neurogenesis is influenced by circadian rhythm, affected by the manipulation of sleep, and is disturbed in animal models of affective disorders. These observations and the link between dysregulation of the circadian production of melatonin and neuropsychiatric disorders prompted us to investigate the potential role of melatonin in controlling adult hippocampal neurogenesis. In vitro, melatonin increased the number of new neurons derived from adult hippocampal neural precursor cells in vitro by promoting cell survival. This effect was partially dependent on the activation of melatonin receptors as it could be blocked by the application of receptor antagonist luzindole. There was no effect of melatonin on cell proliferation. Similarly, in the dentate gyrus of adult C57BL/6 mice in vivo, exogenous melatonin (8 mg/kg) also increased the survival of neuronal progenitor cells and post-mitotic immature neurons. Melatonin did not affect precursor cell proliferation in vivo and also did not influence neuronal and glial cell maturation. Moreover, melatonin showed antidepressant-like effects in the Porsolt forced swim test. These results indicate that melatonin through its receptor can modulate the survival of newborn neurons in the adult hippocampus, making it the first known exogenously applicable substance with such specificity

Effects of melatonin on microtubule assembly depend on hormone concentration: role of melatonin as a calmodulin antagonist

Huerto‐Delgadillo L, Antón‐Tay F, Benítez‐King G. Effects of melatonin on microtubule assembly depend on hormone concentration: rate of melatonin as a calmodulin antagonist. J. Pineal Res. 1994: 17: 55–62. ©Munksgaard, 1994

In vitro inhibition of Ca2+/calmodulin-dependent kinase II activity by melatonin

Recent evidence suggests that a melatonin (MEL) mechanism of action may be through modulation of Ca2+-activated calmodulin (CaM). MEL binds to CaM with a high affinity, and has been shown to act as a CaM antagonist. Among the CaM-dependent enzymes, Ca2+/Calmodulin-dependent protein kinase II (CaM-kinase II) is a particularly abundant enzyme in the nervous system. In the brain it phosphorylates a broad spectrum of substrates, thus modulating important neuronal functions. We describe the MEL effect on CaM-kinase II activity in vitro. CaM-kinase II was purified from rat brain by column chromatography, and identified by Western immunoblotting. CaM-kinase II activity was assessed in the presence of Ca2+/CaM by the kinases ability to phosphorylate the synthetic substrate syntide-2 and by enzyme autophosphorylation. MEL inhibited CaM-kinase II activity, and enzyme autophosphorylation. Inhibition of the enzyme by 10(-9) M MEL was nearly of 30%. Trifluoperazine (10 microM), W7 (10 microM), and compound 48/80 (30 micrograms/ml), inhibited CaM-kinase II activity by 40%, 42%, and 93%, respectively. Both EGTA (5 mM) and MEL (10(-5) M) abolished autophosphorylation. The effect of MEL on CaM-kinase II activity was specific, since neither serotonin, N-acetylserotonin, nor 6-hydroxymelatonin inhibited its activity. Our results support the hypothesis that MEL acts as a CaM antagonist and cellular functions may be rhythmically regulated by MEL modulation of CaM-dependent protein phosphorylation.

Melatonin modifies calmodulin cell levels in MDCK and N1E-115 cell lines and inhibits phosphodiesterase activity in vitro

The interaction between melatonin and calmodulin was explored. Calmodulin cell levels in MDCK and N1E-115 cells cultured with 10(-9) M melatonin were increased after 3 days but decreased after 6 days. Melatonin inhibited calmodulin-dependent phosphodiesterase and when either melatonin or [3H]melatonin was preincubated with calmodulin and separated by electrophoresis, comigration of calmodulin with the radioactivity as well as modification of the Ca2+ calmodulin shift were observed. The results point out that one of the mechanisms of action of melatonin is a calmodulin-melatonin interaction.

Chronic treatment with melatonin stimulates dendrite maturation and complexity in adult hippocampal neurogenesis of mice

Abstract:  In the course of adult hippocampal neurogenesis, the postmitotic maturation and survival phase is associated with dendrite maturation. Melatonin modulates the survival of new neurons with relative specificity. During this phase, the new neurons express microtubule‐associated protein doublecortin (DCX). Here, we show that the entire population of cells expressing DCX is increased after 14 days of treatment with melatonin. As melatonin also affects microtubule polymerization which is important for neuritogenesis and dendritogenesis, we studied the consequences of chronic melatonin administration on dendrite maturation of DCX‐positive cells. Treatment with melatonin increased the number of DCX‐positive immature neurons with more complex dendrites. Sholl analysis revealed that melatonin treatment lead to greater complexity of the dendritic tree. In addition, melatonin increased the total volume of the granular cell layer. Besides its survival‐promoting effect, melatonin thus also increases dendritic maturation in adult neurogenesis. This might open the opportunity of using melatonin as an adjuvant in attempts to extrinsically stimulate adult hippocampal neurogenesis in neuropsychiatric disease, dementia or cognitive ageing.

Melatonin stimulates calmodulin phosphorylation by protein kinase C

Abstract:  Calmodulin (CaM)‐dependent processes can be modulated by the availability of Ca+2, the subcellular distribution of both CaM and its target proteins, CaM antagonism, and post‐translational modifications such as CaM phosphorylation. Melatonin, the pineal secretory product synthesized during the dark phase of the photoperiod is an endogenous CaM antagonist. This indolamine causes CaM subcellular redistribution in epithelial MDCK and MCF‐7 cells, and selectively activates protein kinase C alpha (PKC α) in neuronal N1E‐115 cells. In the present work we have characterized the phosphorylation of CaM mediated by PKC α and its stimulation by melatonin in an in vitro reconstituted enzyme system. Additionally, the participation of MAPK and ERKs, downstream kinases of the PKC signaling pathway, was explored utilizing MDCK cell extracts as source of these kinases. Phosphorylation of CaM was characterized in the whole cells by MDCK cell metabolic labeling with [32P]‐orthoposhospate, and CaM separation by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, as well as by immunocolocalization of phosphorylated threonine/serine residues and CaM in cultured cells incubated with melatonin. Our results show that melatonin increased CaM phosphorylation by PKC α with an EC50 of 10−8 m in the presence of the phorbol ester, phorbol‐12‐myristate‐13‐acetate (PMA) in the in vitro reconstituted enzyme system. An increase in phosphorylated CaM was also observed in cells cultured with melatonin, or PMA for 2 hr, while, PKC, MAPK, or ERK inhibitors abolished CaM phosphorylation elicited by melatonin in MDCK cell extracts. Our data show that melatonin can stimulate phosphorylation of CaM by PKC α in the in vitro reconstituted system and suggest that in MDCK cells this phosphorylation is accomplished by PKC. Modification of CaM by melatonin can be another route to inhibit CaM interaction with its target enzymes.

Melatonin effects on the cytoskeletal organization of MDCK and neuroblastoma N1E-115 cells.

Despite the fact that many physiological and pharmacological actions of melatonin (MEL) have been described, its mechanism of action at the subcellular level remains unclear. It has been suggested that MEL has effects on cellular processes that involve microfilaments and microtubules. In the present study MEL effects on the cytoskel‐eton were evaluated in MDCK and N1E‐115 cells in which the microfilaments have been shown to participate in cell morphology and dome formation (MDCK) and the microtubules in neurite outgrowths.

In vitro stimulation of protein kinase C by melatonin.

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 μM) and protein kinase C inhibitors (100 μM) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins.

ROCK‐regulated cytoskeletal dynamics participate in the inhibitory effect of melatonin on cancer cell migration

Abstract:  Cell movement is generated by a driving force provided by dynamic cytoskeletal organization. Two main cytoskeletal‐dependent features, essential for migration, are the highly cell polarized structure and focal adhesion complexes. Cell migration and substrate anchorage are finely regulated by external signaling exerted by growth factors and hormones. In particular, the serine threonine kinase activated by the small GTPase Rho, the Rho‐associated protein kinase (ROCK), participate in both processes through regulation of actin rearrangements in lamellipodia, filopodia, ruffles, and stress fibers. Melatonin, the main product secreted by the pineal gland has oncostatic properties. In MCF‐7 cells, 1 nm melatonin reduces migration and invasiveness through increased expression of two cell surface adhesion proteins, E‐cadherin and β1‐integrin. In this work, we studied the microfilament and microtubule rearrangements elicited by melatonin in migrating leader MCF‐7 cells by a wound‐healing assay. Additionally, cell anchorage was estimated by quantification of focal adhesions in MCF‐7 cells cultured with melatonin. ROCK participation in the indole effects on anchorage and migration was explored by inhibition of the kinase activity with the specific inhibitor of ROCK, the Y‐27632 compound. The results indicate that ROCK participates in the melatonin inhibitory effects on cell migration by changing cytoskeletal organization of leader MCF‐7 cells. Also, they indicated that indole increased the number of focal contacts through ROCK. These results support the notion that melatonin inhibits cancer cell invasion and metastasis formation via ROCK‐regulated microfilament and microtubule organization that converge in a migration/anchorage switch.