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the end of a 21-day treatment containing osteogenic (OS+) medium or at the beginning of the 21-day treatment and then withdrawn.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

The Elevation of Osteoblast Activity in Rat Bone Marrow Mesenchymal Stem Cells in Osteogenic Medium Exposed With Melatonin in Physiological Doses

“…The results of this study indicated different result from the one conducted by Radio et al (2006), Zaminy et al (2008) and Sethi et al (2010). These studies showed that the levels of ALP produced by osteoblast cells in the group provided with melatonin were higher than the group that were not provided with melatonin.…”

“…Study conducted by Feskanich et al (2008) states that low melatonin levels in more than 38,000 postmenopausal women who worked at night for more than 20 years significantly contribute to osteoporosis and the risk of suffering fractures in forearm and pelvis. Meanwhile, administration of melatonin is known to improve the mechanism of bone remodeling in BMMSCs (Radio et al 2006;Sethi et al 2010). Thus, this study was conducted to explain the elevation of osteoblast activity found in cultured BM-MSCs in the medium of osteogenic exposed with melatonin in physiological dosage, so it can be used as a scientific base to develop therapies for diseases that require increased osteoblast activity, such as osteoporosis.…”

Effect of melatonin on osseointegration of immediate loading implant supported mandibular over denture: Randomized clinical trail

“…[14] Melatonin increases pre-osteoblast/osteoblast/osteoblastlike cell proliferation, promotes the expression of type I collagen and bone marker proteins interthread bone is an intermediate situation of new bone in with the implant by stimulation of osteoblasts and the old bone in the total peri implant area by osteoclast inhibition, and melatonin continues its activity passing on from the 2 nd to the 5 th week and to the 8 th week. [14][15][16] Arat Bilhan et al (2015) they said that use of a splinted attachment can be preferred to reduce forces emerging around the implants. The use of 2 solitary attachments in cases with good bone quality and ideal sized implants are safe with the bilateral loading on over dentures.…”

An Early Development Budget Impact Model for the Use of Melatonin in the Treatment and Prevention of Osteoporosi

“…In support of this association, the Melatonin Osteoporosis Prevention Study (MOPS; NCT01152580) revealed that melatonin improved bone health in perimenopausal women by renormalizing bone marker turnover [17]. Previous work also from the Witt-Enderby group has shown a role for MT 2 melatonin receptors in melatonininduced mesenchymal stem cell differentiation into osteoblasts [18,19]. Exposure to artificial light at night affects melatonin production but may also affect bone physiology possibly through reduction in melatonin levels.…”

“…To demonstrate this association, a recent study has shown that female nightshift workers exposed to artificial light at night, a known suppressor of melatonin production, had an increased risk of hip and wrist fractures over the eight years of follow-up compared to cohorts that worked the day shift [20]. Moreover, melatonin may be a potential treatment option because pre-clinical research has shown it impacts bone metabolism by promoting osteoblast differentiation and activity [15,18,19,21,22] and by suppressing osteoclast activity via increasing osteoprotegrin and actions on receptor activator NF-κB ligand (RANKL) [15,23]. Also, when taken orally, melatonin is welltolerated and has a high safety profile.…”