Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT₂melatonin 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 melatonin's 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.

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.…”

“…Research conducted by Radio et al (2006), Zaminy et al (2008) and Sethi et al (2010) carried out in serial manner while this study was only done once at day 21. Birminghamet al (2012) reported that the ALP expression was highest on days 5-14 while examination of ALP levels in this study was performed on day 21.…”

“…Melatonin in dosage of 50 nM binds to melatonin receptors in the BM-MSCs then increases the activity of WNT and ERK 1/2 and suppresses adipogenic transcription factor CCAAT enhancer binding protein  (C/EBP) and peroxisome proliferator-activated receptor  (PPAR) and induces Runt-related transcription factor (RUNX-2), distal-less homeobox 5 (Dlx5) and Osterix (OSX) which would trigger rat BM-MSCs differentiation into active osteoblasts which secretes several non-collagen protein, such as osteocalcin (Radio et al 2006;Rucci 2008). RUNX-2 increases the transcription of osteocalcin because it can bind directly the specific enhancer regions of genesencoding osteocalcin and activate other osteoblastic genes such as BMP-2.…”

“…A previous study reported that administration of melatonin can increase the activity of osteoblasts by increasing the osteogenic differentiation of BM-MSCs (Radio et al 2006;Sethiet al 2010). ALP and osteocalcin are the major protein used as marker of osteoblasts activity in the process of osteogenesis (Patti 2013 ).The study of this was limited to the examination of genes and have not yet reached the level of protein and thus the activity of osteoblasts found in cultured BMMSCs in medium osteogenic exposed with melatonin in physiological dosage remains unclear.…”

“…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.…”