Shalini Sethi

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the end of a 21‐day treatment containing osteogenic (OS+) medium or at the beginning of the 21‐day treatment and then withdrawn.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.

Effects on bone by the light/dark cycle and chronic treatment with melatonin and/or hormone replacement therapy in intact female mice

Abstract:  In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV‐Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd−/−) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real‐time RT‐PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17β‐estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.

N‐Acetyl‐5‐arylalkoxytryptamine Analogs: Probing the Melatonin Receptors for MT1‐Selectivity

A series of melatonin analogs obtained by the replacement of the ether methyl group with larger arylalkyl and aryloxyalkyl substituents was prepared in order to probe the melatonin receptors for MT1‐selectivity. The most MT1‐selective agents 11 and 15 were substituted with a Ph(CH2)3 or a PhO(CH2)3 group. Compounds 11 and 15 displayed 11.5‐fold and 11‐fold higher affinity for the MT1 receptors than for the MT2 subtype. Interestingly, in our binding assay 11 and 15 have shown considerably higher MT1‐affinity and selectivity than the reference ligand, the dimeric agomelatine 1a.

Synthesis and pharmacological evaluation of 1,2,3,4-tetrahydropyrazino[1,2-a]indole and 2-[(phenylmethylamino)methyl]-1H-indole analogues as novel melatoninergic ligands.

Two novel series of melatonin-derived compounds have been synthesized and pharmacologically evaluated at the MT(1) and MT(2) subtypes of melatonin receptors. Compounds 12b-c are non-selective high-affinity MT(1) and MT(2) receptor ligands (K(i)=7-11 nM). Compound 12b had little intrinsic activity at the MT(1) receptor and no intrinsic activity at the MT(2) receptor. Compound 20d displayed the highest MT(2) binding affinity (K(i)=2 nM) and moderate selectivity toward the MT(2) subtype (K(i) MT(1)/MT(2) ratio=8) behaving as MT(2) antagonist and MT(1) agonist (IC(50)=112 pM). The findings help define SARs around the positions 1 and 2 of melatonin with respect to binding affinity, MT(2) selectivity, and intrinsic activity.

C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processes

Abstract:  Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G‐protein‐coupled melatonin receptors, MT1 and MT2. In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site‐directed mutagenesis. Two mutations were constructed in the cytoplasmic C‐terminal tail of each receptor subtype: (i) a cysteine residue in the C‐terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT1C7.72A and MT2C7.77A) and (ii) the C‐terminal tail in the MT1 and MT2 receptors was truncated, removing the putative phosphorylation and β‐arrestin binding sites (MT1Y7.64 and MT2Y7.64). These mutations did not alter the affinity of 2‐[125I]‐iodomelatonin binding to the MT1 or MT2 receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3′,5′‐cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C‐terminal tail of both receptors (MT1Y7.64 and MT2Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C‐terminal tail in these receptor functions.

2-[(1,3-Dihydro-2H-isoindol-2-yl)methyl]melatonin – a novel MT2-selective melatonin receptor antagonist

A synthesis and pharmacological evaluation of new melatonin receptor ligands obtained by 2-substitution of melatonin with (indol-1-yl)methyl, (isoindolin-2-yl)methyl, and (tetrahydroiso-quinolin-2-yl)methyl groups is reported. The isoindoline analogue a displays high MT2 binding affinity (Ki = 2 nM) and high selectivity towards the MT2 subtype (Ki MT1/Ki MT2 = 124) behaving as an MT2-antagonist.