title: 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the end of a 21-day treatment containing osteogenic (OS+) medium or at the beginning of the 21-day treatment and then withdrawn.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.
authors.affiliation: Duquesne University School of Pharmacy, Division of Pharmaceutical Sciences, Pittsburgh, PA 15282, USA.
authors.forename: Shalini
authors.initials: S
authors.lastname: Sethi
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Radio
authors.forename: Mary P
authors.initials: MP
authors.lastname: Kotlarczyk
authors.forename: Chien-Tsun
authors.initials: CT
authors.lastname: Chen
authors.forename: Yau-Huei
authors.initials: YH
authors.lastname: Wei
authors.forename: Ralf
authors.initials: R
authors.lastname: Jockers
authors.forename: Paula A
authors.initials: PA
authors.lastname: Witt-Enderby
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology*; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D055418:Bone Morphogenetic Protein 6 / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism; D008550:Melatonin / Q000494:pharmacology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects*; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D044123:Receptor, Melatonin, MT2 / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D000071557:beta-Arrestins
chemical_list: D000975:Antioxidants; D019390:Arrestins; D055418:Bone Morphogenetic Protein 6; D044123:Receptor, Melatonin, MT2; D014157:Transcription Factors; D000071557:beta-Arrestins; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D000469:Alkaline Phosphatase; D008550:Melatonin