J. Pineal Res. 2014; 57:385–392
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12177
Journal of Pineal Research
Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways Abstract: Osteoblastic differentiation and bone-forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3-E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time- and concentration-dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3-E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways.
Introduction Maintenance of bone homeostasis is tightly regulated by blood supply, cellular elements, cytokines, and growth factors and involves a number of molecular signaling pathways. Bone repair and regeneration are also well-regulated events, involving a number of cell types and cytokines [1, 2]. Moreover, cellular responses to oxygen play an important role in many aspects of physiologic homeostasis and tissue metabolism, and critical changes in the cellular microenvironment occur under hypoxic conditions, when the oxygen supply is inadequate [3, 4]. Hypoxia occurs when the blood supply to tissues is reduced or disrupted. This situation occurs in conditions such as aging [5], inflammation [6], bone fracture [7], and diabetes [8] and possibly also occurs at bone graft sites [9]. Hypoxia is an important factor in bone pathophysiology and regulates inhibition and activation of the expression of many genes in osteoblasts [10]. Bone homeostasis, bone healing and regeneration, and wound healing are all hampered under hypoxic conditions [4, 11, 12]. The cellular demand for oxygen varies considerably among different tissues, and the response to low oxygen can therefore also differ according to the type and characteristics of the cells [3, 13]. In bone, osteoblasts are crucial cells, and
Jang-Ho Son1, Yeong-Cheol Cho1, Iel-Yong Sung1, In-Ryoung Kim2, Bong-Soo Park2 and Yong-Deok Kim3,4 1
Department of Oral and Maxillofacial Surgery, Ulsan University Hospital, College of Medicine, Ulsan University, Ulsan, South Korea; 2 Department of Oral Anatomy, School of Dentistry, Pusan National University, Yangsan, South Korea; 3Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University, Yangsan, South Korea; 4Dental Research Institute and Institute of Translational Dental Sciences, Pusan National University Dental Hospital, Pusan National University, Yangsan, South Korea Key words: bone metabolism, hypoxia, pineal gland, signaling pathway Address reprint requests to Yong-Deok Kim, Department of Oral and Maxillofacial Surgery, Pusan National University, Mulgeum, Beomeo, Yangsan, Kyoungnam 626-770, Korea. E-mail: [email protected] Received April 20, 2014; Accepted September 12, 2014.
their differentiation tends to be inhibited under hypoxic conditions [14, 15]. A number of key molecules that are involved in and that promote the complex physiological processes involved in bone formation and metabolism have been identified to date. Melatonin, the concentrations of which vary among different bodily fluids, including saliva, has been shown to influence cell differentiation, where it can either stimulate or suppress cell division, depending on its concentration and the cell type targeted [16, 17]. A number of in vitro and in vivo studies have suggested that melatonin has beneficial effects on bone metabolism, including bone anabolic effects, where melatonin promotes osteoblastic differentiation [18–24]. Although many previous studies have investigated the effect of melatonin on osteoblastic differentiation, the precise mechanisms and the signaling pathways involved in this process, and particularly those that come into play under hypoxic conditions, remain unknown. This study therefore aimed to determine whether melatonin could promote osteoblastic differentiation and mineralization of mouse preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the signaling mechanisms mediating the actions of melatonin during this processes. 385
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Materials and methods Culture of MC3T3-E1 cells and their differentiation under hypoxic conditions MC3T3-E1 cells, a clonal mouse osteoblastic cell line, were purchased from the ATCC (Rockville, MD, USA). Cells were cultured in an alpha modification of Eagle’s medium (a-MEM without ascorbic acid; Gibco BL, Grand Island, NY, USA), with 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin, at 37°C. To induce differentiation, cells were seeded into a 12-well or 100-mm culture dish and allowed to reach confluence. At confluence (day 0), cells were transferred to a-MEM containing 10% FBS, 1% penicillin–streptomycin, 10 mM b-glycerophosphate, and 100 lg/mL ascorbic acid, gassed with 95% N2 and 5% CO2 (Anaerobic System PROOX model 110; BioSpherix, Lacona, NY, USA), and incubated at 37°C within the chamber for different time intervals (1, 3, 7, and 14 days). The cells were exposed to 1% oxygen tension. MTT assay To ensure that melatonin and hypoxia were not cytotoxic, cell proliferation was assessed using a MTT assay (0.5 mg/mL final concentration), according to the manufacturer’s instructions. Cells (1 9 104) were cultured in a 96well plate and incubated for 24 hr. Various concentrations of melatonin (0–250 lM) were then added to the cells, and, subsequently, the cells were incubated under 1% hypoxic conditions for different time intervals. Thereafter, 100 lL of MTT (0.5 mg/mL final concentration) was added and the cells were incubated at 37°C in the dark for an additional 4 hr, to induce the production of formazan crystals. The medium was aspirated, and the formed formazan crystals were dissolved in DMSO. Cell viability was monitored on an ELISA reader (Sunrise Remote Control; Tecan, Gr€ odig, Austria] by reading absorbance at 570 nm. Alkaline phosphatase (ALP) enzyme assay To evaluate whether melatonin affects osteoblast differentiation, the activity of ALP, a marker of osteoblast differentiation, was analyzed. Cells were plated in 6-well plates at a density of 2 9 105 cells/well. Following treatment with melatonin, the cells were washed twice with phosphate-buffered saline (PBS), scraped into 500 lL of 10 mM Tris-HCl buffer (pH 7.6) containing 0.1% Triton-X, placed on ice, and sonicated to lyse the cells. Protein concentrations in the lysates were determined using the Bradford protein assay. ALP activity in the cellular fraction was measured using a fluorometric detection kit (Nanjing Jiancheng Biotechnology Co. Ltd., Nanjing, China). A standard curve was created using p-nitrophenol as a standard, and ALP activity of each sample was normalized to the total protein concentration. Mineralization analysis Mineralization of MC3T3-E1 cells was determined in 12-well plates using Alizarin red S staining. The cells were 386
fixed with ice-cold 70% ethanol and stained with Alizarin red S to detect calcification. For quantitative analysis, cells were destained with ethylpyridium chloride and transferred to a 96-well plate, and the absorbance was then measured at 550 nm using a microplate reader. Western blot assay The effects of melatonin on the p38 Mapk and Prkd1 signaling pathways during the osteoblastic differentiation processes were assessed using Western blotting. Cells were plated at a density of 2 9 106 cells in 100-mm culture dishes. Cells were washed twice with ice-cold PBS and centrifuged at 400 g for 10 min. Total cell proteins were extracted by lysing cells with RIPA buffer (300 mM NaCl, 50 mM Tris-HCl [pH 7.6], 0.5% TritonX-100, 2 mM PMSF, 2 lg/mL aprotinin, and 2 lg/mL leupeptin), and the lysates were then incubated at 4°C for 1 hr. The lysates were centrifuged at 17,500 g for 15 min at 4 on, and sodium dodecyl sulfate (SDS) and sodium deoxycholic acid (0.2% final concentration) were added. Protein concentrations in the cell lysates were determined with a Bradford protein assay (Bio-Rad, Richmond, CA, USA), and BSA was used as a protein standard. Samples equivalent to 20 lg of total protein per well were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5– 15% gels and were then transferred to polyvinylidene fluoride membranes (Amersham GE Healthcare; Little Chalfont, UK). After incubating with antibodies, detection was performed using SuperSignal West Femto enhanced chemiluminescence substrate, and bands were then imaged with an Alpha Imager HP (Alpha Innotech, Santa Clara, CA, USA). Equivalent protein loading was confirmed by Ponceau S staining. Real-time RT-PCR Expression of osteogenic genes was analyzed with real-time reverse transcription-polymerase chain reaction (RTPCR). SYBR Green chemistry was used to quantitate mRNA levels for Alp, Osterix (Osx), type I collagen (Col1), osteocalcin (Ocn), and the housekeeping gene (Gapdh) according to an optimized protocol. Total RNA was extracted from cultured MC3T3-E1 cells using TRIzol reagent (Invitrogen, Life Technologies; Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (2 lg) was used to generate single-stranded cDNA, which was then used as template in real-time PCR. PCR was performed on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems 7500 System, Foster City, CA, USA; with Sequence Detection System software version 2.0.1) using SYBR Green PCR Master Mix (Applied Biosystems). The sense and antisense primers used were as follows: mouse Alp sense, 50 -GACTGGTACTCGGATA ACGAGA-30 and antisense, 50 -CTCATGATGTCCG TGGTCAATC-30 ; mouse Ocn sense, 50 -GCAGCTT GGTGCACACCTAG-30 and antisense, 50 -GGAGCT GCTGTGACATCCAT-30 ; mouse Col1 sense, 50 -ACCTC CCAGTGGCGGTTATGAC-30 and antisense, 50 -AGT TCTTCTGAGGCACAGACGG-30 ; mouse Rplp0 sense, 50 -AAGCGCGTCCTGGCATTGTCT-30 and antisense,
Melatonin promote hypoxic osteoblast p38 50 -CCGCAGGGGCAGCAGTGGT-30 . Real-time PCR was performed using 1 lL of cDNA in a 20-lL reaction volume with LightCycler Real-time PCR System [Roche Applied Science, Indianapolis, IN, USA]. The doublestranded DNA-specific dye SYBR Green I was incorporated into the PCR buffer provided in the SYBR Premix Ex TaqTM reagent. The temperature profile for the reaction was 95°C for 15 min, followed by 40 cycles each consisting of a denaturation step at 95°C for 30 s, annealing at 54°C (Alp), or 58°C (Ocn, Col1) for 30 s, and an extension step at 72°C for 1 min. Rplp0 was used to correct for differences in RNA isolation, RNA degradation, and the efficiency of reverse transcription. Statistical analysis Results presented are representative of at least 3 independent experiments and are expressed as the mean SEM. All data were analyzed using Statistical Product and Service Solution (v19; SPSS Inc., Chicago, IL, USA). Oneway and two-way ANOVA models were used to compare expression levels with Tukey’s studentized range test as post hoc analysis. The significance level was set at 0.05.
Results Melatonin-treated and nontreated cells showed timedependent (24, 48, 72 hr) increases in cell viability under hypoxic culturing conditions, as described previously. The viability of melatonin-treated cells was slightly higher than that of nontreated cells (but not statistically different). These results indicated that hypoxia does not reduce the viability of MC3T3-E1 cells significantly. MC3T3-E1 cells were incubated with or without melatonin, as indicated, during osteoblast differentiation, under hypoxic conditions. For elucidating the anabolic activity of melatonin in the process of osteoblast differentiation, ALP activity and gene expression profiles of osteogenic markers, such as Alp, Osx, Col1, and Ocn, were investigated after 1, 3, 7, and 14 days of treatment with 0–250 lM of melatonin (Figs 1 and 2). Melatonin promoted ALP activity and significantly increased the mRNA expression levels of Alp, Osx, Col1, and Ocn time- and concentration dependently. Melatonin gradually upregulated ALP activity and the expression of these genes until day 14. Mineralization of these cells was investigated using Alizarin red S staining. This staining showed that a (A)
mineralized matrix was formed in hypoxic MC3T3-E1 cells in the presence of melatonin, in a concentrationdependent manner, by day 14. This mineralization was increased in the melatonin-treated cells as compared with control cells, as seen by dense red staining in the melatonin-treated cells and a light red color in the untreated control cells (Fig. 3). More specifically, ALP activity and the mineralization of hypoxic MC3T3-E1 cells increased markedly in the presence of melatonin at concentrations higher than 50 lM. We investigated whether p38 Mapk is associated with melatonin-induced differentiation and mineralization in MC3T3-E1 cells. As shown in Fig. 4A, melatonin markedly stimulated the phosphorylation of p38 Mapk in a time-dependent manner. Next, to confirm that melatonininduced differentiation and mineralization was the result of p38 Mapk activation, we used a pharmacological approach, employing a p38 Mapk inhibitor, SB203580. Phosphorylation of activating transcription factor 2 (ATF2), a specific target protein of p38 Mapk, was markedly decreased when MC3T3-E1 cells were pretreated with SB203580 (Fig. 4B). In addition, melatonin-induced mineralization and stimulation of osteogenic gene expression were attenuated in the presence of SB203580 (Fig. 5). These results suggested that stress-activated protein kinase p38 mediates melatonin-induced differentiation and mineralization in osteoblastic MC3T3-E1 cells. Next, we examined whether Prkd1 is associated with melatonin-induced differentiation and mineralization of MC3T3-E1 cells during hypoxia. As shown in Fig. 6, melatonin markedly phosphorylated Prkd1 in a timedependent manner under hypoxic conditions. The phosphorylation of Prkd1 and p38 Mapk was suppressed in the presence of Go6976, a PKCt/PRKD inhibitor. Pretreatment of cells with 20 nM Go6976 significantly attenuated the phosphorylation of p38 Mapk induced by 100 lM melatonin. Moreover, melatonin induced activation of osteoblast marker genes and mineralized nodules were inhibited by 20 nM Go6976 (Fig. 6C,D). These results suggested that melatonin could induce differentiation and mineralization of osteoblastic MC3T3-E1 cells by activating Prkd1 and p38 Mapk signaling pathways.
Discussion Over the years, melatonin has been shown to influence cell differentiation. It can either stimulate or suppress cell (B)
Fig. 1. Effect of melatonin on ALP activity. MC3T3-E1 cells were cultured in osteogenic medium and treated with different concentrations of melatonin, as indicated, for 1, 3, 7, and 14 days, under hypoxic conditions. (A) Cells were cultured in osteogenic medium containing 100 lM melatonin for the indicated times and ALP activity in the lysates of these cells were then compared with those in control cells (0 lM melatonin). (B) Cells were cultured in medium containing various concentrations (0, 10, 50, 100, and 250 lM) of melatonin for 14 days, and ALP activity was determined. ALP activity was enhanced by melatonin at concentrations as low as 250 lM. Each value represents the mean SEM of the fold-change over the 1 day control. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control.
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Fig. 3. Melatonin promotes the mineralization in MC3T3-E1 cells under hypoxic conditions. Cells were cultured in osteogenic medium and treated with various concentrations of melatonin, after which mineralization deposits were identified by Alizarin red S staining. Quantification of Alizarin red S staining was performed after extraction with ethylpyridium chloride. *P < 0.01
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Fig. 2. mRNA expression levels of osteoblast marker genes (Alp, Osx, Col1, and Ocn) were determined in MC3T3-E1 cells by real-time reverse transcriptasepolymerase chain reaction. (A–D) Cells were treated with 100 lM melatonin and cultured for indicated times. Total mRNA was collected on days 1, 3, 7, and 14 from hypoxic cultured cells and gene expression determined. (E–H) Cells were treated with various concentrations (0, 10, 50, 100, and 250 lM) of melatonin and were then cultured for 14 days under hypoxic conditions, after which mRNA levels of these genes were determined. Melatonin significantly increased the mRNA expression levels of Alp, Osx, Col1, and Ocn time- and concentration dependently. Each value represents the mean SEM of the fold-change compared to the 1-day control. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with control.
division, depending on its concentration or the cell type targeted [17, 20, 25–27]. A number of in vitro and in vivo studies have suggested beneficial effects of melatonin on bone metabolism, including bone anabolic effects, and it has been hypothesized to have three principle effects on bone metabolism [28]. One of these effects is to promote osteoblastic differentiation [17, 28, 29]. Recently, Park et al. [29] reported that melatonin promoted differentiation of MC3T3-E1 cells, an osteoblast precursor cell line derived from mouse calvaria. Studies of human bone cells and human osteoblastic cell lines have shown that melatonin causes an increase in levels of markers of bone proliferation and differentiation, including increased synthesis of alkaline phosphatase, osteopontin, osteocalcin, and procollagen type I c-peptide [19–21, 28–30]. However, although many previous studies have focused on the effects of melatonin on osteoblastic differentiation, the precise mechanisms and the signaling pathways involved in this effect, especially those that are implemented under hypoxic conditions, remain unknown. Therefore, we evaluated the effects and mechanisms of melatonin on the differentiation of MC3T3-E1 cells under hypoxic conditions. To identify the effects of melatonin on osteoblastic differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions, ALP activity, as well as expression of
Melatonin promote hypoxic osteoblast p38 (A)
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Fig. 4. Melatonin induces p38 Mapk phosphorylation in MC3T3-E1 cells under hypoxic conditions. (A) Cells were treated with 100 lM melatonin for 120 min; this caused activation of p38 Mapk-mediated phosphorylation in a time-dependent manner. (B) Cells were pretreated with 10 or 20 lM of SB203580, a p38 Mapk inhibitor, for 2 hr and were then exposed to 100 lM melatonin for 1 hr, after which levels of the phosphorylated form of Atf2, a p38 target protein, were determined. Gapdh served as a reference protein.
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Fig. 5. MC3T3-E1 cells were incubated in osteogenic medium containing melatonin, in the absence or presence of SB203580, a p38 Mapk inhibitor, for 14 days. (A) Mineralized nodules in the cells were stained with Alizarin red S, which was then quantified after extraction with ethylpyridium chloride. (B) The effect of SB203580 on gene expression levels of osteoblastic biomarker-related genes was determined. Cells were incubated in osteogenic medium containing 0 or 100 lM of melatonin, in the absence or presence of SB203580, for 14 days. Each value represents the mean SEM of the fold-change increase over the control. * P < 0.01
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Fig. 6. Prkd1 is an upstream kinase for p38 Mapk, which is activated by melatonin in MC3T3-E1 cells. (A, B) Cells were pretreated with 10 or 20 nM of Go6976, a Prkd1 inhibitor, for 2 hr, as indicated, then exposed to 100 lM melatonin for 1 hr, after which phosphorylated forms of Prkd1 and p38 Mapk were determined. Gapdh served as a reference protein. (C, D) The effect of Go6976 on the expression of osteoblastic biomarker-related genes and the mineralization were determined. Cells were incubated in osteogenic medium containing 0 or 100 lM of melatonin, in the absence or presence of Go6976, for 14 days, after which mRNA levels of Alp, Osx, Col1, and Ocn were determined. Mineralized nodules in the cells were stained with Alizarin red S. * P < 0.01.
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osteogenic genes, such as Alp, Osx, Col1, and Ocn, and mineralization, was evaluated in hypoxic MC3T3-E1 cells. Pharmacologically effective doses of melatonin, which
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affect proliferation and differentiation of osteoblastic cells, vary in the range from nM to lM, depending on the cell line used [17, 19, 28]. Satomura et al. [19] reported that 389
Son et al. melatonin concentrations from 50 lM upwards promoted differentiation of human osteoblastic cells, while melatonin concentrations of 100 lM or more significantly overstimulated the formation of the mineralized matrix. On the other hand, treatment of hAMSCs with 50 nM melatonin for 10 days promoted the differentiation of these cells into osteoblast; the same was true for MC3T3-E1 cells treated for 12 days [17, 20]. There was no evidence of a peak in ALP activity, which is known to increase upon differentiation of osteoblasts and to decline thereafter, during the study period. Instead, ALP levels gradually increased throughout the experimental periods and reached its highest value on day 14. Moreover, ALP activity and mineralization reached significance by day 14 when cells had been treated with melatonin at concentrations of 50 lM or more. Therefore, it is possible that higher doses of melatonin are required to promote osteoblast differentiation under hypoxic conditions. In the present study, ALP activity, an early stage osteoblast differentiation marker, increased time- and concentration dependently upon exposure of cells, cultured under hypoxic conditions, to melatonin. Moreover, melatonin also markedly increased the mRNA expression levels of Alp, Osx, Col1, and Ocn mRNA levels in time- and concentration-dependent manners. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed by day 14 in a way that depended on the concentration of the melatonin. Overall, the results of the present study indicated that melatonin promotes the differentiation and mineralization of MC3T3-E1 cells, even under hypoxic condition. Recent studies have revealed that MAPKs regulate bone formation by osteoblasts; thus, it seems that MAPKs are key signal transducers in the regulation of bone mass [31– 33]. JNK, ERK,and p38 MAPK are MAPK members known to be involved in osteoblast differentiation [34, 35]. In a recent study, melatonin was found to promote the phosphorylation level of Erk and Jnk in MC3T3-E1 cells, but not p38 Mapk [29]. Similarly, it appears that p38 is probably not involved in melatonin-induced differentiation of hAMSC cells [30]. p38 MAPK is responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and is involved in differentiation of cells, including osteoblasts [35–37]. Therefore, we investigated whether p38 Mapk is associated with melatonin-induced differentiation and mineralization of MC3T3E1 cells under hypoxic conditions, using Western blotting. As shown in Figs 4 and 5, 100 lM of melatonin enhanced the phosphorylation level of p38 and Atf2 in MC3T3-E1 cells. Moreover, treatment of MC3T3-E1 cells with the p38 Mapk inhibitor SB203580 significantly inhibited the melatonin-enhanced expression of genes involved in mineralization and reduced the amount of mineralized crystal that was stained with Alizarin red S. Likewise, phosphorylation of Atf2 was markedly decreased when MC3T3-E1 cells were pretreated with SB203580. These results indicated that melatonin promotes the differentiation and the mineralization of MC3T3-E1 cells via the p38 Mapk pathway under hypoxic conditions. Prkd1 is known to be involved in a variety of cellular functions, including regulation of cell growth and 390
survival, cell motility, Golgi protein trafficking, nuclear factor-B-dependent gene regulation, and c-Jun N-terminal kinase activity [38–41]. More specific to the bone microenvironment, previous studies have shown that Prkd1 participates in osteoblast differentiation and upregulates Osx, a master osteoblastic transcription factor, during this process [42–45]. In the present study, we also observed that melatonin stimulated Prkd1 phosphorylation. Moreover, when cells were treated with Go6976, a Prkd1 inhibitor, levels of the phosphorylated forms of Prkd1 and p38 Mapk were decreased. Pretreatment with Go6976 also suppressed the melatonin-enhanced mineralization and expression of Alp, Osx, Col1, and Ocn. These results indicate that Prkd1 may be associated with melatonin-induced p38 Mapk signaling, as a kinase upstream from p38 Mapk, in hypoxic MC3E3-T1 cells. Additionally, we assessed the effect of melatonin receptors on the differentiation and mineralization of hypoxic MC3T3-E1 cells cultured in the presence of a melatonin receptor antagonist, luzindole, via Alizarin red S staining (Fig. 7). In this study, luzindole could counteract melatonin-induced anabolic effects. Thus, it appears that melatonin-induced effects are associated with the pathway of G-protein coupled melatonin receptors (MT1/MT2) under hypoxic conditions. In summary, the results reported here demonstrate that melatonin is capable of promoting differentiation and mineralization of preosteoblastic MC3T3-E1 cells grown under hypoxic culture conditions. Moreover, our results indicate that melatonin promotes osteoblastic differentiation of these cells via p38 Mapk and Prkd1 signaling pathways. Several in vivo studies have demonstrated
Fig. 7. MC3T3-E1 cells were incubated in the absence or presence of luzindole for 14 days. Luzindole supplemented with melatonin and osteogenic medium decreased mineralized nodules in the cells over that melatonin and osteogenic medium. (*P < 0.01)
Melatonin promote hypoxic osteoblast p38 an association between melatonin deficiency and bone disease. Considering that the O2 tension of normal bone tissues is lower than 6% and more hypoxic conditions are present in pathological states in pathologic conditions, the current findings suggest a potential role for melatonin in bone formation and support clinical applications for the use of melatonin in reversing damage to osteoblasts caused by pathological conditions and diseases. In addition, we suggest that melatonin could be used to accelerate bone formation at bone graft sites. Finally, continued in vivo researches would be very helpful and should be performed to elucidate these issues in the future.
Author contributions BSP and YDK contributed to study design. IYS, YCC, and IRK conducted the study. JHS, BSP, and YDK involved in data collection. JHS and YDK analyzed data and drafted the manuscript. BSP, IYS, YCC, and IRK helped to analyze data and revise the manuscript. All authors involved in approving final version of manuscript.
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12177
Journal of Pineal Research
Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways Abstract: Osteoblastic differentiation and bone-forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3-E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time- and concentration-dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3-E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways.
Introduction Maintenance of bone homeostasis is tightly regulated by blood supply, cellular elements, cytokines, and growth factors and involves a number of molecular signaling pathways. Bone repair and regeneration are also well-regulated events, involving a number of cell types and cytokines [1, 2]. Moreover, cellular responses to oxygen play an important role in many aspects of physiologic homeostasis and tissue metabolism, and critical changes in the cellular microenvironment occur under hypoxic conditions, when the oxygen supply is inadequate [3, 4]. Hypoxia occurs when the blood supply to tissues is reduced or disrupted. This situation occurs in conditions such as aging [5], inflammation [6], bone fracture [7], and diabetes [8] and possibly also occurs at bone graft sites [9]. Hypoxia is an important factor in bone pathophysiology and regulates inhibition and activation of the expression of many genes in osteoblasts [10]. Bone homeostasis, bone healing and regeneration, and wound healing are all hampered under hypoxic conditions [4, 11, 12]. The cellular demand for oxygen varies considerably among different tissues, and the response to low oxygen can therefore also differ according to the type and characteristics of the cells [3, 13]. In bone, osteoblasts are crucial cells, and
Jang-Ho Son1, Yeong-Cheol Cho1, Iel-Yong Sung1, In-Ryoung Kim2, Bong-Soo Park2 and Yong-Deok Kim3,4 1
Department of Oral and Maxillofacial Surgery, Ulsan University Hospital, College of Medicine, Ulsan University, Ulsan, South Korea; 2 Department of Oral Anatomy, School of Dentistry, Pusan National University, Yangsan, South Korea; 3Department of Oral and Maxillofacial Surgery, School of Dentistry, Pusan National University, Yangsan, South Korea; 4Dental Research Institute and Institute of Translational Dental Sciences, Pusan National University Dental Hospital, Pusan National University, Yangsan, South Korea Key words: bone metabolism, hypoxia, pineal gland, signaling pathway Address reprint requests to Yong-Deok Kim, Department of Oral and Maxillofacial Surgery, Pusan National University, Mulgeum, Beomeo, Yangsan, Kyoungnam 626-770, Korea. E-mail: [email protected] Received April 20, 2014; Accepted September 12, 2014.
their differentiation tends to be inhibited under hypoxic conditions [14, 15]. A number of key molecules that are involved in and that promote the complex physiological processes involved in bone formation and metabolism have been identified to date. Melatonin, the concentrations of which vary among different bodily fluids, including saliva, has been shown to influence cell differentiation, where it can either stimulate or suppress cell division, depending on its concentration and the cell type targeted [16, 17]. A number of in vitro and in vivo studies have suggested that melatonin has beneficial effects on bone metabolism, including bone anabolic effects, where melatonin promotes osteoblastic differentiation [18–24]. Although many previous studies have investigated the effect of melatonin on osteoblastic differentiation, the precise mechanisms and the signaling pathways involved in this process, and particularly those that come into play under hypoxic conditions, remain unknown. This study therefore aimed to determine whether melatonin could promote osteoblastic differentiation and mineralization of mouse preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the signaling mechanisms mediating the actions of melatonin during this processes. 385
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Materials and methods Culture of MC3T3-E1 cells and their differentiation under hypoxic conditions MC3T3-E1 cells, a clonal mouse osteoblastic cell line, were purchased from the ATCC (Rockville, MD, USA). Cells were cultured in an alpha modification of Eagle’s medium (a-MEM without ascorbic acid; Gibco BL, Grand Island, NY, USA), with 10% fetal bovine serum (FBS), and 1% penicillin–streptomycin, at 37°C. To induce differentiation, cells were seeded into a 12-well or 100-mm culture dish and allowed to reach confluence. At confluence (day 0), cells were transferred to a-MEM containing 10% FBS, 1% penicillin–streptomycin, 10 mM b-glycerophosphate, and 100 lg/mL ascorbic acid, gassed with 95% N2 and 5% CO2 (Anaerobic System PROOX model 110; BioSpherix, Lacona, NY, USA), and incubated at 37°C within the chamber for different time intervals (1, 3, 7, and 14 days). The cells were exposed to 1% oxygen tension. MTT assay To ensure that melatonin and hypoxia were not cytotoxic, cell proliferation was assessed using a MTT assay (0.5 mg/mL final concentration), according to the manufacturer’s instructions. Cells (1 9 104) were cultured in a 96well plate and incubated for 24 hr. Various concentrations of melatonin (0–250 lM) were then added to the cells, and, subsequently, the cells were incubated under 1% hypoxic conditions for different time intervals. Thereafter, 100 lL of MTT (0.5 mg/mL final concentration) was added and the cells were incubated at 37°C in the dark for an additional 4 hr, to induce the production of formazan crystals. The medium was aspirated, and the formed formazan crystals were dissolved in DMSO. Cell viability was monitored on an ELISA reader (Sunrise Remote Control; Tecan, Gr€ odig, Austria] by reading absorbance at 570 nm. Alkaline phosphatase (ALP) enzyme assay To evaluate whether melatonin affects osteoblast differentiation, the activity of ALP, a marker of osteoblast differentiation, was analyzed. Cells were plated in 6-well plates at a density of 2 9 105 cells/well. Following treatment with melatonin, the cells were washed twice with phosphate-buffered saline (PBS), scraped into 500 lL of 10 mM Tris-HCl buffer (pH 7.6) containing 0.1% Triton-X, placed on ice, and sonicated to lyse the cells. Protein concentrations in the lysates were determined using the Bradford protein assay. ALP activity in the cellular fraction was measured using a fluorometric detection kit (Nanjing Jiancheng Biotechnology Co. Ltd., Nanjing, China). A standard curve was created using p-nitrophenol as a standard, and ALP activity of each sample was normalized to the total protein concentration. Mineralization analysis Mineralization of MC3T3-E1 cells was determined in 12-well plates using Alizarin red S staining. The cells were 386
fixed with ice-cold 70% ethanol and stained with Alizarin red S to detect calcification. For quantitative analysis, cells were destained with ethylpyridium chloride and transferred to a 96-well plate, and the absorbance was then measured at 550 nm using a microplate reader. Western blot assay The effects of melatonin on the p38 Mapk and Prkd1 signaling pathways during the osteoblastic differentiation processes were assessed using Western blotting. Cells were plated at a density of 2 9 106 cells in 100-mm culture dishes. Cells were washed twice with ice-cold PBS and centrifuged at 400 g for 10 min. Total cell proteins were extracted by lysing cells with RIPA buffer (300 mM NaCl, 50 mM Tris-HCl [pH 7.6], 0.5% TritonX-100, 2 mM PMSF, 2 lg/mL aprotinin, and 2 lg/mL leupeptin), and the lysates were then incubated at 4°C for 1 hr. The lysates were centrifuged at 17,500 g for 15 min at 4 on, and sodium dodecyl sulfate (SDS) and sodium deoxycholic acid (0.2% final concentration) were added. Protein concentrations in the cell lysates were determined with a Bradford protein assay (Bio-Rad, Richmond, CA, USA), and BSA was used as a protein standard. Samples equivalent to 20 lg of total protein per well were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5– 15% gels and were then transferred to polyvinylidene fluoride membranes (Amersham GE Healthcare; Little Chalfont, UK). After incubating with antibodies, detection was performed using SuperSignal West Femto enhanced chemiluminescence substrate, and bands were then imaged with an Alpha Imager HP (Alpha Innotech, Santa Clara, CA, USA). Equivalent protein loading was confirmed by Ponceau S staining. Real-time RT-PCR Expression of osteogenic genes was analyzed with real-time reverse transcription-polymerase chain reaction (RTPCR). SYBR Green chemistry was used to quantitate mRNA levels for Alp, Osterix (Osx), type I collagen (Col1), osteocalcin (Ocn), and the housekeeping gene (Gapdh) according to an optimized protocol. Total RNA was extracted from cultured MC3T3-E1 cells using TRIzol reagent (Invitrogen, Life Technologies; Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (2 lg) was used to generate single-stranded cDNA, which was then used as template in real-time PCR. PCR was performed on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems 7500 System, Foster City, CA, USA; with Sequence Detection System software version 2.0.1) using SYBR Green PCR Master Mix (Applied Biosystems). The sense and antisense primers used were as follows: mouse Alp sense, 50 -GACTGGTACTCGGATA ACGAGA-30 and antisense, 50 -CTCATGATGTCCG TGGTCAATC-30 ; mouse Ocn sense, 50 -GCAGCTT GGTGCACACCTAG-30 and antisense, 50 -GGAGCT GCTGTGACATCCAT-30 ; mouse Col1 sense, 50 -ACCTC CCAGTGGCGGTTATGAC-30 and antisense, 50 -AGT TCTTCTGAGGCACAGACGG-30 ; mouse Rplp0 sense, 50 -AAGCGCGTCCTGGCATTGTCT-30 and antisense,
Melatonin promote hypoxic osteoblast p38 50 -CCGCAGGGGCAGCAGTGGT-30 . Real-time PCR was performed using 1 lL of cDNA in a 20-lL reaction volume with LightCycler Real-time PCR System [Roche Applied Science, Indianapolis, IN, USA]. The doublestranded DNA-specific dye SYBR Green I was incorporated into the PCR buffer provided in the SYBR Premix Ex TaqTM reagent. The temperature profile for the reaction was 95°C for 15 min, followed by 40 cycles each consisting of a denaturation step at 95°C for 30 s, annealing at 54°C (Alp), or 58°C (Ocn, Col1) for 30 s, and an extension step at 72°C for 1 min. Rplp0 was used to correct for differences in RNA isolation, RNA degradation, and the efficiency of reverse transcription. Statistical analysis Results presented are representative of at least 3 independent experiments and are expressed as the mean SEM. All data were analyzed using Statistical Product and Service Solution (v19; SPSS Inc., Chicago, IL, USA). Oneway and two-way ANOVA models were used to compare expression levels with Tukey’s studentized range test as post hoc analysis. The significance level was set at 0.05.
Results Melatonin-treated and nontreated cells showed timedependent (24, 48, 72 hr) increases in cell viability under hypoxic culturing conditions, as described previously. The viability of melatonin-treated cells was slightly higher than that of nontreated cells (but not statistically different). These results indicated that hypoxia does not reduce the viability of MC3T3-E1 cells significantly. MC3T3-E1 cells were incubated with or without melatonin, as indicated, during osteoblast differentiation, under hypoxic conditions. For elucidating the anabolic activity of melatonin in the process of osteoblast differentiation, ALP activity and gene expression profiles of osteogenic markers, such as Alp, Osx, Col1, and Ocn, were investigated after 1, 3, 7, and 14 days of treatment with 0–250 lM of melatonin (Figs 1 and 2). Melatonin promoted ALP activity and significantly increased the mRNA expression levels of Alp, Osx, Col1, and Ocn time- and concentration dependently. Melatonin gradually upregulated ALP activity and the expression of these genes until day 14. Mineralization of these cells was investigated using Alizarin red S staining. This staining showed that a (A)
mineralized matrix was formed in hypoxic MC3T3-E1 cells in the presence of melatonin, in a concentrationdependent manner, by day 14. This mineralization was increased in the melatonin-treated cells as compared with control cells, as seen by dense red staining in the melatonin-treated cells and a light red color in the untreated control cells (Fig. 3). More specifically, ALP activity and the mineralization of hypoxic MC3T3-E1 cells increased markedly in the presence of melatonin at concentrations higher than 50 lM. We investigated whether p38 Mapk is associated with melatonin-induced differentiation and mineralization in MC3T3-E1 cells. As shown in Fig. 4A, melatonin markedly stimulated the phosphorylation of p38 Mapk in a time-dependent manner. Next, to confirm that melatonininduced differentiation and mineralization was the result of p38 Mapk activation, we used a pharmacological approach, employing a p38 Mapk inhibitor, SB203580. Phosphorylation of activating transcription factor 2 (ATF2), a specific target protein of p38 Mapk, was markedly decreased when MC3T3-E1 cells were pretreated with SB203580 (Fig. 4B). In addition, melatonin-induced mineralization and stimulation of osteogenic gene expression were attenuated in the presence of SB203580 (Fig. 5). These results suggested that stress-activated protein kinase p38 mediates melatonin-induced differentiation and mineralization in osteoblastic MC3T3-E1 cells. Next, we examined whether Prkd1 is associated with melatonin-induced differentiation and mineralization of MC3T3-E1 cells during hypoxia. As shown in Fig. 6, melatonin markedly phosphorylated Prkd1 in a timedependent manner under hypoxic conditions. The phosphorylation of Prkd1 and p38 Mapk was suppressed in the presence of Go6976, a PKCt/PRKD inhibitor. Pretreatment of cells with 20 nM Go6976 significantly attenuated the phosphorylation of p38 Mapk induced by 100 lM melatonin. Moreover, melatonin induced activation of osteoblast marker genes and mineralized nodules were inhibited by 20 nM Go6976 (Fig. 6C,D). These results suggested that melatonin could induce differentiation and mineralization of osteoblastic MC3T3-E1 cells by activating Prkd1 and p38 Mapk signaling pathways.
Discussion Over the years, melatonin has been shown to influence cell differentiation. It can either stimulate or suppress cell (B)
Fig. 1. Effect of melatonin on ALP activity. MC3T3-E1 cells were cultured in osteogenic medium and treated with different concentrations of melatonin, as indicated, for 1, 3, 7, and 14 days, under hypoxic conditions. (A) Cells were cultured in osteogenic medium containing 100 lM melatonin for the indicated times and ALP activity in the lysates of these cells were then compared with those in control cells (0 lM melatonin). (B) Cells were cultured in medium containing various concentrations (0, 10, 50, 100, and 250 lM) of melatonin for 14 days, and ALP activity was determined. ALP activity was enhanced by melatonin at concentrations as low as 250 lM. Each value represents the mean SEM of the fold-change over the 1 day control. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control.
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Fig. 3. Melatonin promotes the mineralization in MC3T3-E1 cells under hypoxic conditions. Cells were cultured in osteogenic medium and treated with various concentrations of melatonin, after which mineralization deposits were identified by Alizarin red S staining. Quantification of Alizarin red S staining was performed after extraction with ethylpyridium chloride. *P < 0.01
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Fig. 2. mRNA expression levels of osteoblast marker genes (Alp, Osx, Col1, and Ocn) were determined in MC3T3-E1 cells by real-time reverse transcriptasepolymerase chain reaction. (A–D) Cells were treated with 100 lM melatonin and cultured for indicated times. Total mRNA was collected on days 1, 3, 7, and 14 from hypoxic cultured cells and gene expression determined. (E–H) Cells were treated with various concentrations (0, 10, 50, 100, and 250 lM) of melatonin and were then cultured for 14 days under hypoxic conditions, after which mRNA levels of these genes were determined. Melatonin significantly increased the mRNA expression levels of Alp, Osx, Col1, and Ocn time- and concentration dependently. Each value represents the mean SEM of the fold-change compared to the 1-day control. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with control.
division, depending on its concentration or the cell type targeted [17, 20, 25–27]. A number of in vitro and in vivo studies have suggested beneficial effects of melatonin on bone metabolism, including bone anabolic effects, and it has been hypothesized to have three principle effects on bone metabolism [28]. One of these effects is to promote osteoblastic differentiation [17, 28, 29]. Recently, Park et al. [29] reported that melatonin promoted differentiation of MC3T3-E1 cells, an osteoblast precursor cell line derived from mouse calvaria. Studies of human bone cells and human osteoblastic cell lines have shown that melatonin causes an increase in levels of markers of bone proliferation and differentiation, including increased synthesis of alkaline phosphatase, osteopontin, osteocalcin, and procollagen type I c-peptide [19–21, 28–30]. However, although many previous studies have focused on the effects of melatonin on osteoblastic differentiation, the precise mechanisms and the signaling pathways involved in this effect, especially those that are implemented under hypoxic conditions, remain unknown. Therefore, we evaluated the effects and mechanisms of melatonin on the differentiation of MC3T3-E1 cells under hypoxic conditions. To identify the effects of melatonin on osteoblastic differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions, ALP activity, as well as expression of
Melatonin promote hypoxic osteoblast p38 (A)
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Fig. 4. Melatonin induces p38 Mapk phosphorylation in MC3T3-E1 cells under hypoxic conditions. (A) Cells were treated with 100 lM melatonin for 120 min; this caused activation of p38 Mapk-mediated phosphorylation in a time-dependent manner. (B) Cells were pretreated with 10 or 20 lM of SB203580, a p38 Mapk inhibitor, for 2 hr and were then exposed to 100 lM melatonin for 1 hr, after which levels of the phosphorylated form of Atf2, a p38 target protein, were determined. Gapdh served as a reference protein.
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Fig. 5. MC3T3-E1 cells were incubated in osteogenic medium containing melatonin, in the absence or presence of SB203580, a p38 Mapk inhibitor, for 14 days. (A) Mineralized nodules in the cells were stained with Alizarin red S, which was then quantified after extraction with ethylpyridium chloride. (B) The effect of SB203580 on gene expression levels of osteoblastic biomarker-related genes was determined. Cells were incubated in osteogenic medium containing 0 or 100 lM of melatonin, in the absence or presence of SB203580, for 14 days. Each value represents the mean SEM of the fold-change increase over the control. * P < 0.01
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Fig. 6. Prkd1 is an upstream kinase for p38 Mapk, which is activated by melatonin in MC3T3-E1 cells. (A, B) Cells were pretreated with 10 or 20 nM of Go6976, a Prkd1 inhibitor, for 2 hr, as indicated, then exposed to 100 lM melatonin for 1 hr, after which phosphorylated forms of Prkd1 and p38 Mapk were determined. Gapdh served as a reference protein. (C, D) The effect of Go6976 on the expression of osteoblastic biomarker-related genes and the mineralization were determined. Cells were incubated in osteogenic medium containing 0 or 100 lM of melatonin, in the absence or presence of Go6976, for 14 days, after which mRNA levels of Alp, Osx, Col1, and Ocn were determined. Mineralized nodules in the cells were stained with Alizarin red S. * P < 0.01.
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osteogenic genes, such as Alp, Osx, Col1, and Ocn, and mineralization, was evaluated in hypoxic MC3T3-E1 cells. Pharmacologically effective doses of melatonin, which
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affect proliferation and differentiation of osteoblastic cells, vary in the range from nM to lM, depending on the cell line used [17, 19, 28]. Satomura et al. [19] reported that 389
Son et al. melatonin concentrations from 50 lM upwards promoted differentiation of human osteoblastic cells, while melatonin concentrations of 100 lM or more significantly overstimulated the formation of the mineralized matrix. On the other hand, treatment of hAMSCs with 50 nM melatonin for 10 days promoted the differentiation of these cells into osteoblast; the same was true for MC3T3-E1 cells treated for 12 days [17, 20]. There was no evidence of a peak in ALP activity, which is known to increase upon differentiation of osteoblasts and to decline thereafter, during the study period. Instead, ALP levels gradually increased throughout the experimental periods and reached its highest value on day 14. Moreover, ALP activity and mineralization reached significance by day 14 when cells had been treated with melatonin at concentrations of 50 lM or more. Therefore, it is possible that higher doses of melatonin are required to promote osteoblast differentiation under hypoxic conditions. In the present study, ALP activity, an early stage osteoblast differentiation marker, increased time- and concentration dependently upon exposure of cells, cultured under hypoxic conditions, to melatonin. Moreover, melatonin also markedly increased the mRNA expression levels of Alp, Osx, Col1, and Ocn mRNA levels in time- and concentration-dependent manners. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed by day 14 in a way that depended on the concentration of the melatonin. Overall, the results of the present study indicated that melatonin promotes the differentiation and mineralization of MC3T3-E1 cells, even under hypoxic condition. Recent studies have revealed that MAPKs regulate bone formation by osteoblasts; thus, it seems that MAPKs are key signal transducers in the regulation of bone mass [31– 33]. JNK, ERK,and p38 MAPK are MAPK members known to be involved in osteoblast differentiation [34, 35]. In a recent study, melatonin was found to promote the phosphorylation level of Erk and Jnk in MC3T3-E1 cells, but not p38 Mapk [29]. Similarly, it appears that p38 is probably not involved in melatonin-induced differentiation of hAMSC cells [30]. p38 MAPK is responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and is involved in differentiation of cells, including osteoblasts [35–37]. Therefore, we investigated whether p38 Mapk is associated with melatonin-induced differentiation and mineralization of MC3T3E1 cells under hypoxic conditions, using Western blotting. As shown in Figs 4 and 5, 100 lM of melatonin enhanced the phosphorylation level of p38 and Atf2 in MC3T3-E1 cells. Moreover, treatment of MC3T3-E1 cells with the p38 Mapk inhibitor SB203580 significantly inhibited the melatonin-enhanced expression of genes involved in mineralization and reduced the amount of mineralized crystal that was stained with Alizarin red S. Likewise, phosphorylation of Atf2 was markedly decreased when MC3T3-E1 cells were pretreated with SB203580. These results indicated that melatonin promotes the differentiation and the mineralization of MC3T3-E1 cells via the p38 Mapk pathway under hypoxic conditions. Prkd1 is known to be involved in a variety of cellular functions, including regulation of cell growth and 390
survival, cell motility, Golgi protein trafficking, nuclear factor-B-dependent gene regulation, and c-Jun N-terminal kinase activity [38–41]. More specific to the bone microenvironment, previous studies have shown that Prkd1 participates in osteoblast differentiation and upregulates Osx, a master osteoblastic transcription factor, during this process [42–45]. In the present study, we also observed that melatonin stimulated Prkd1 phosphorylation. Moreover, when cells were treated with Go6976, a Prkd1 inhibitor, levels of the phosphorylated forms of Prkd1 and p38 Mapk were decreased. Pretreatment with Go6976 also suppressed the melatonin-enhanced mineralization and expression of Alp, Osx, Col1, and Ocn. These results indicate that Prkd1 may be associated with melatonin-induced p38 Mapk signaling, as a kinase upstream from p38 Mapk, in hypoxic MC3E3-T1 cells. Additionally, we assessed the effect of melatonin receptors on the differentiation and mineralization of hypoxic MC3T3-E1 cells cultured in the presence of a melatonin receptor antagonist, luzindole, via Alizarin red S staining (Fig. 7). In this study, luzindole could counteract melatonin-induced anabolic effects. Thus, it appears that melatonin-induced effects are associated with the pathway of G-protein coupled melatonin receptors (MT1/MT2) under hypoxic conditions. In summary, the results reported here demonstrate that melatonin is capable of promoting differentiation and mineralization of preosteoblastic MC3T3-E1 cells grown under hypoxic culture conditions. Moreover, our results indicate that melatonin promotes osteoblastic differentiation of these cells via p38 Mapk and Prkd1 signaling pathways. Several in vivo studies have demonstrated
Fig. 7. MC3T3-E1 cells were incubated in the absence or presence of luzindole for 14 days. Luzindole supplemented with melatonin and osteogenic medium decreased mineralized nodules in the cells over that melatonin and osteogenic medium. (*P < 0.01)
Melatonin promote hypoxic osteoblast p38 an association between melatonin deficiency and bone disease. Considering that the O2 tension of normal bone tissues is lower than 6% and more hypoxic conditions are present in pathological states in pathologic conditions, the current findings suggest a potential role for melatonin in bone formation and support clinical applications for the use of melatonin in reversing damage to osteoblasts caused by pathological conditions and diseases. In addition, we suggest that melatonin could be used to accelerate bone formation at bone graft sites. Finally, continued in vivo researches would be very helpful and should be performed to elucidate these issues in the future.
Author contributions BSP and YDK contributed to study design. IYS, YCC, and IRK conducted the study. JHS, BSP, and YDK involved in data collection. JHS and YDK analyzed data and drafted the manuscript. BSP, IYS, YCC, and IRK helped to analyze data and revise the manuscript. All authors involved in approving final version of manuscript.
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