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Received Date : 15-Sep-2014 Revised Date

: 19-Sep-2014

Accepted Date : 19-Sep-2014 Article type

: Original Manuscript

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing

Sei-Jung Lee, Young Hyun Jung, Sang Yub Oh, Seung Pil Yun, Ho Jae Han*

Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute for Veterinary Science, and BK21 PLUS Creative Veterinary Research Center, Seoul National University, Seoul 151-741, Korea.

*Corresponding author: Ho Jae Han, D.V.M., Ph.D. Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul, 151-742, South Korea This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jpi.12179 This article is protected by copyright. All rights reserved.

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E-mail: [email protected] Tel: 82-2-880-1261 Fax: 82-2-885-2732

Running title: Effect of melatonin on human UCB-MSCs motility

Keywords: Melatonin; Melatonin receptor 2; Motility; Mouse skin wound; Umbilical cord blood derived mesenchymal stem cells

Abstract Melatonin, a circadian-rhythm–promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)-MSCs pre-treated with melatonin enhanced wound closure, granulation, and re-epithelialization at mouse skin wound sites, where relatively more UCBMSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB-MSCs. Melatonin (1 μM) significantly increased the motility of UCB-MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT2). We found that Gαq coupled with MT2 and that the binding of Gαq to MT2 uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently down-regulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization-related proteins such as profilin1, cofilin-1, and F-actin in UCB-MSCs. Finally, a lack of MT2 expression in UCB-MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT2 triggers FAK/paxillin phosphorylation to stimulate reorganization of the This article is protected by copyright. All rights reserved.

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actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB-MSCs motility.

Introduction Cell migration is a dynamic biological event that is regulated by a complex microenvironmental network and that plays an essential role in the skin wound healing processes including hemostasis, inflammation, new tissue formation, and finally tissue remodeling [1, 2]. Increasing evidence has suggested that stem cells improve the cutaneous tissue regeneration by promoting differentiation and angiogenesis [3, 4]. Although there are many factors that can enhance the therapeutic potential of stem cells, one of the most interesting questions centers on the mechanisms that increase the beneficial effects of stem cells. In fact, many studies have focused on the investigation of new extrinsic factors which activate stem cells in the processes of wound healing [5-8]. Therefore, identification of regulating factor to activate stem cells could provide a new therapeutic strategy for wound healing. However, extrinsic factors to efficiently stimulate stem cells after skin wounds are not fully understood.

Melatonin (5-methoxy-N-acetyltryptamine) is easily available, produces few side effects, and is a relatively inexpensive molecule. It is also a ubiquitously-acting substance produced by various locations in body, including the pineal gland, the skin, the lymphocytes and the gastrointestinal tract [9-13]. Melatonin mediates diverse effects through its cognate receptors, which include at least two members of the G protein-coupled receptor (GPCR) super-family, MT1-2, as well as putative MT3, while others may involve nuclear binding sites or may be receptor-independent [14-19]. In addition to the physiological role of melatonin such as circadian regulation, recent studies have shown that melatonin has a positive effect on the process of wound repair in dermal wounds [20] and that it can modulate the behavior of stem cells, including their differentiation and proliferation processes [21-26]. To date, however, the underlying cellular mechanisms of melatonin on stem cell motility and the receptor specificity of stem cells involved in this process remain largely unknown. There are no previous reports related to the molecular mechanisms of melatonin action which drive the migratory behavior of stem cells during the wound healing process. In this respect, we further This article is protected by copyright. All rights reserved.

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questioned by the mechanism underlying the beneficial effects of melatonin combined with stem cells in skin wounds. Human umbilical cord blood derived (UCB)-mesenchymal stem cells (MSCs) are considered one of the most abundant sources of non-embryonic stem cells [27]. They have a self-renewal capacity with the potential to differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes [28]. UCB-MSCs are easy to isolate and have low immunogenicity and multi-differentiation potential, while also remaining free from any ethical controversy [27-29]. Specifically, UCB-MSCs have been shown to improve wound healing in several studies [30, 31]. Due to the functional relevance of melatonin in improving placental efficiency [32-34], UCB-MSCs are likely to be important when attempting to define the role of melatonin in regulation of stem cell functions. In this study, therefore, we investigate the melatonin signaling pathway in promoting UCB-MSC motility and evaluate its potential therapeutic effect in a mouse cutaneous excisional wound model.

Materials and methods Materials Human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) were kindly provided by Medipost Co. (Seoul, Korea), which were isolated and expanded as reported previously [28]. These cells have been characterized to express CD105 (99.6%) and CD73 (96.3%), but not CD34 (0.1%), CD45 (0.2%) and CD14 (0.1%). They were positive for HLA-AB but generally not for HLA-DR [28]. The human UCB-derived MSCs differentiated into various cell types such as osteoblasts, chondrocytes, and adipocytes upon in vitro induction with the appropriate osteogenic, chondrogenic, and adipogenic differentiation stimuli [28]. Human adipose-derived mesenchymal stem cells (AD-MSCs) were kindly provided by Prof. Kyung-Sun Kang (Seoul National University, Korea). In present study, all the experiments were carried out with cells of 7 passage. Fetal bovine serum (FBS) was purchased from BioWhittaker Inc. (Walkersville, MO, USA). The following antibodies were purchased: F-actin, phospho-PKC, Gαq, Gαi, and Gα12 antibodies (Cell Signaling Technology, Danvers, MA, USA); Cdc42, Rac1, and RhoA antibodies (BD Biosciences, Franklin Lakes, NJ, USA); α-Actinin-1, α-Actinin-4, Arp2/3, β-actin, phospho-cofilin-1, This article is protected by copyright. All rights reserved.

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Cofilin-1, phospho-paxillin, paxillin, phospo-FAK, FAK, pan-cadherin, phospho-PKC, PKC, PKCα, PKCβ, PKCγ, PKCδ, PKCε, PKCθ, PKCζ, and profilin-1 antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); Horseradish peroxidase (HRP)-conjugated goat antirabbit and goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA). A23187, 5-bromo-2'-deoxyuridine (BrdU), bisindolylmaleimide I, staurosporine, melatonin (Mel, 5methoxy-N-acetyltryptamine), and mitomycin C were obtained from Sigma Chemical Company (St. Louis, MO, USA). All other reagents were of the highest purity commercially available and were used as received.

Culture of human UCB-MSCs and AD-MSCs Human UCB-MSCs and AD-MSCs were cultured without a feeder layer in the αminimum essential medium and (α-MEM; Thermo, MA, USA) and keratinocyte-serum free medium, respectively. The cells were grown in 1% penicillin and streptomycin, and 10% FBS. For each experiment, cells were grown in wells of 6- and 12-well plates, and in 35, 60, or 100-mm diameter culture dishes in an incubator maintained at 37 °C with 5% CO2. The medium was replaced with serum-free α-MEM at least 24 h before experiments. Following incubation, the cells were washed twice with phosphate-buffered saline (PBS) and then maintained in a serum-free α-MEM including all supplements and indicated agents.

Mouse skin wound healing model All animal procedures were performed following the National Institutes of Health Guidelines for the Humane Treatment of Animals, with approval from the Institutional Animal Care and Use Committee of Seoul National University (SNU-140123-6). Eight weekold age male ICR mice were used. All surgery was performed under anesthesia using a 2:1 mixture of ZoletilTM (20 mg/kg, Virbac Laboratories, Carros, France) and Xylazine HCl (10 mg/kg, Rompun®, Bayer, Germany) and all efforts were made to minimize suffering. In addition, six authors were Doctors of Veterinary Medicine with licenses granted from the Ministry of Agriculture and Forestry of Republic of Korea. Mouse skin wounding and stem This article is protected by copyright. All rights reserved.

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cell implantation were performed as described previously [35, 36]. The mouse skin wound healing model has been extensively used to study wound healing, cutaneous regeneration, stem cell transplantation, and immune rejection [35-37]. Although it previously has shown that CD4+/CD8+ lymphocytes and natural killer cells are involved in skin immune rejection through production of pro-inflammatory cytokines such as IFN-γ and TNF-α [38], human UCB-MSCs have been shown to retain low immunogenicity and did not trigger an immense immune reaction in unrelated donor transplantation [39]. Indeed, it reported human UCBMSCs maintained their immunophenotypes, multilineage differentiation potential, and did not form tumors when injected at a high dose into athymic nude mice [40]. These characteristics suggest that human UCB-MSCs have potential clinical use as effective delivery vehicles for therapeutic genes in the allogenic/xenogenic treatment of diseases. Briefly, after shaving backs and scrubbing with an organic iodine solution, circular fullthickness wound was surgically created by using a 6-mm-diameter sterile biopsy punch. UCB-MSCs were pre-treated with 1 μM melatonin as well as bromo-2'-deoxyuridine (BrdU, 2 μM) for 24 h prior to the skin transplantation. To test the functional role of UCB-MSCs pre-treated with melatonin, experimental animals were divided into four groups: wild type mice that were received vehicle (group 1, n=8) or 1 μM melatonin (group 2, n=8) without UCB-MSCs; and UCB-MSCs transplantation group mice that were given UCB-MSCs pretreated with vehicle (group 3, n=8) or 1 μM melatonin (group 4, n=8). To determine the role of MT2 in the mobilization of UCB-MSCs to skin wound area for re-epithelialization, UCB-MSCs transfected with MT2siRNA or non-targeting(nt)siRNA were pre-treated with 1 μM melatonin as well as bromo-2'-deoxyuridine (BrdU, 2 μM) for 24h prior to the skin transplantation and experimental animals and divided into four additional groups: UCB-MSCs/ntsiRNA transplantation group mice that were given UCBMSCs/ntsiRNA pre-treated with vehicle (group 5, n=8) or 1 μM melatonin (group 6, n=8); and UCB-MSCs/MT2siRNA transplantation group mice that were given UCBMSCs/MT2siRNA pre-treated with vehicle (group 7, n=8) or 1 μM melatonin (group 8, n=8). We have applied same volume of stem cells as described above. We injected 1 × 106 UCBThis article is protected by copyright. All rights reserved.

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MSCs in 70 μl PBS containing 50% growth factor–reduced Matrigel (BD Biosciences, NJ, USA) into the dermis at two sites around the wound and topically applied 0.3 × 106 UCBMSCs in 30 μl PBS containing the 50% Matrigel onto the wound bed at day 0 and 9. After that, the wounds were dressed with Tegaderm (3M, London, Canada). Images of wounds were made on days 0, 5, 9 and 12 with a digital camera system (D50, Nikon, Tokyo, Japan) at the same camera/subject distance (30 cm). The sizes of wound closure were determined by measuring wound resealing from the images captured at the wounded sites. The wound areas were measured planimetrically using the free-hand tool in Image J software (NIH, Bethesda, MD). Percent wound closure was calculated as the difference in wound size on a particular day compared to day 0 (time of wounding)/initial wound size. At day 12, the wound tissues were embed in O.C.T. compound (Sakura Finetek, CA, USA), stored at -70 °C, cut the samples to 6 μm thick frozen sections by using cryosectioning machine, and mounted on SuperFrost Plus slides (Thermo Fisher Scientific, IL, USA) for hematoxylin and eosin (H&E) staining and immunohistochemistry.

Small interfering (si)RNA transfection Cells were grown until 75% of the surface of the plate and transfected for 24 h with 200 pmol/L siRNAs specific for MT2, Gαq, Gαi, Gα12, FAK, paxillin, Cdc42 or 200 pmol/L non-targeting(nt) siRNA as a negative control (GenePharma, Shanghai, China) with HiPerFect Transfection Reagent (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. The sequences used are described in Supporting information Table S1 and determined siRNA efficacy for MT2, Gαq, Gαi, Gα12, FAK, and paxillin, respectively (Supporting information Fig. S1). Wound-healing migration assay Human UCB-MSCs were seeded at 4 x 104 cells on low 35-mm dishes with both silicone reservoirs, which are separated by a 500 μm thick wall (Ibidi, Martinsried, Germany) [41] This article is protected by copyright. All rights reserved.

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and incubated until the cell reached around 100% confluence in serum containing medium. After serum starvation for 24 h, the silicone reservoirs were removed with sterile forceps to create a wound field. The cells were incubated for an additional 24 h with melatonin (1 μM) and visualized with an Olympus FluoView™ 300 confocal microscope with 100x objective.

OrisTM cell migration assay Human UCB-MSCs were seeded at 3 x 102 cells/100μl in OrisTM well (Platypus Technologies, WI, USA) and incubated for 24 h to permit cell adhesion. Inserts were carefully removed when the cell reached around 70% confluence, and the wells were gently washed with culture medium. Cells were then incubated with melatonin (1 μM) and serumfree medium. Cell motility was observed microscopically after 24 h. Cell populations in endpoint assays were stained with 5 μM calcein AM for 30 min. Migrated cells were quantified through measurement of fluorescence signals by using a microplate reader at excitation and emission wavelengths of 485 and 515 nm, respectively [42].

Transwell migration assay In vitro transwell migration assay was performed in transwell permeable support with 8.0 μm pore size membrane (Corning Incorporated Life Sciences, Lowell, MA ) according to the manufacturer's instruction. UCB-MSCs suspensions (5 × 104 cells/mL) were placed into the upper chamber in 0.2 mL of serum-free medium. The lower compartment was filled with 0.6 mL serum-free medium containing 1 μM melatonin. After incubation for 24 h, cells that had migrated to the lower surface of the filters were fixed in acetone for 5 min at room temperature and visualized with H&E staining method.

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RNA isolation and Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was extracted using the RNeasy Plus Mini Kit (Quiagen, Valencia, CA, USA). Reverse transcription (RT) was carried out with 3 μg of RNA using a Maxime RT premix kit (iNtRON Biotechnology, Sungnam, Korea). The cDNA (5 µl) for MT1 and MT2 were amplified by using the following primer pairs: MT1, 5'- TCCTGGTCATCC TGTCGGTGTATC-3' and 5'-CTGCTGTACAGTTTGTCGTACTTG-3'; MT2, 5’-TCCTG GTGATCCTCTCCGTGCTCA-3’ and 5’-AGCCAGATGAGGCAGATGTGCAGA-3’; and

β-actin, 5’-AACCGCGAGAAGATGACC-3’ and 5’- AGCAGCCGTGGCCATCTC-3’.

Confocal microscopy Either UCB-MSCs or frozen wound sections were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.2% Triton X-100 in PBS for 5 min, and blocked in PBS containing 5% normal goat serum for 30 min at room temperature. Cells were then stained with primary antibody for overnight at 4°C. Following three washes with PBS, the cells were incubated with Alexa 488-conjugated goat anti-rabbit/mouse IgM, phalloidin, or BrdU (Invitrogen Co., Carlsbad, CA, USA), counterstained with PI in PBS containing 1% (v/v) BSA, and washed three times for 10 min each with PBS. Samples were mounted on slides and visualized with an Olympus FluoView™ 300 confocal microscope with 400x objective.

Western blot analysis Cells were harvested, washed twice with PBS, and lysed with buffer (20 mM Tris [pH 7.5], 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mg/mL aprotinin, and 1 mM phenylmethylsulfonylfluoride [PMSF]) for 30 min on ice. The lysates were then cleared by centrifugation (22,250 x g at 4°C for 30 min). Protein concentration was determined by the Bradford method [43]. Equal amounts of protein (20 μg) were resolved by 10% sodium This article is protected by copyright. All rights reserved.

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dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membranes. The membranes were washed with TBST solution (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, and 0.05% Tween-20), blocked with 5% skim milk for 1 h, and incubated with appropriate primary antibody at 4°C for overnight. The membrane was then washed and detected with a horseradish peroxidase-conjugated secondary antibody. The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Buckinghamshire, UK).

Measurement of calcium influx Changes in intracellular calcium concentrations were monitored using Fluo-3-AM that had initially been dissolved in dimethylsulfoxide (DMSO). Cells in 35 mm-diameter culture dishes were rinsed with a Bath Solution [140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, 5.5 mM HEPES (pH 7.4)] and were then incubated in a Bath Solution containing 3 μM Fluo-3-AM for 40 min, rinsed, mounted on a perfusion chamber, and scanned at 1 s intervals using Olympus FluoView™ 300 confocal microscope with 300x objective. The fluorescence was produced by excitation at 488 nm and the emitted light was observed at 515 nm. All analyses of calcium influx were processed in a single cell, and the results are expressed as the fluorescent intensity (F/F0%, arbitrary unit, where F is fluorescence captured at a particular time and F0 is initial fluorescence image captured).

Immunoprecipitation Interaction of MT2 with G proteins or F-actin with α-actinin-1/4 was analyzed by immunoprecipitation and Western blotting. Cells were lysed with lysis buffer (1% Triton X100 in 50 mM Tris–HCl pH 7.4 containing 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 2.5 mM Na4PO7, 100 mM NaF, 200 nM microcystin lysine–arginine, and protease inhibitors). This article is protected by copyright. All rights reserved.

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Cell lysates (400 mg) were mixed with 10 mg of each anti-bodies. The samples were incubated for 4 h, mixed with Protein A/G PLUS-agarose immunoprecipitation reagent (Pierce, Rockford, IL, USA) and then incubated for an additional 12 h. The beads were washed four times, and the bound proteins were released from the beads by boiling in SDS‐PAGE sample buffer for 5 min. Samples were analyzed by Western blotting.

Affinity precipitation of cellular GTP-Cdc42, -Rac1, and -RhoA Activation of Cdc42, Rac1, and RhoA activities were determined by using an affinity precipitation assay kits (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Cells starved for 24 h were stimulated with melatonin and lysed for 5 min in ice-cold cell lysis buffer. Four hundred micrograms of lysates were incubated for 1 h with agarose beads coupled with the Cdc42/Rac binding domain (GST-PAK-PBD) or with Rho-binding domain of rhotekin (GST-Rhotekin-RBD), and the bound Cdc42, Rac1, and RhoA proteins were eluted with 2 X laemmli sample buffer and subjected to Western blot using anti-Cdc42, anti-Rac1, and anti-RhoA antibodies, respectively.

Statistical analysis Results are expressed as means ± standard errors (SE). All experiments were analyzed by ANOVA, followed in some cases by a comparison of treatment means with the control using the Bonferroni-Dunn test. Differences were considered statistically significant at P < 0.05.

Results Because melatonin has a clinically beneficial effect on stem cell transplantation [44], we initially evaluated the effect of UCB-MSCs pre-treated with 1 μM melatonin on skin wound healing in mice. There were no significant differences between the wound sizes of mice treated with a vehicle or with 1 μM melatonin alone, although spontaneous wound healing was observed from day 9 (Fig. 1A), suggesting the exogenous melatonin at a concentration of This article is protected by copyright. All rights reserved.

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1 μM does not have a relevant effect on skin wound healing. On days 9 and 12, however, the percentages of the wound area were significantly decreased in the mice group that received UCB-MSCs pretreated with 1 μM melatonin during wound healing compared to UCB-MSCs pre-treated with a vehicle. It was noted that UCB-MSCs transplantation alone enhanced wound closure compared to mice treated with the vehicle from day 9. A histologic examination on day 12 showed that the wound bed was still not completely covered with epidermis in mice treated with the vehicle or 1 μM of melatonin alone (Fig. 1B). The UCBMSCs transplantation groups showed increased re-epithelialization from a mechanical skin wound, but the mice group that received UCB-MSCs pre-treated with 1 μM of melatonin showed enhanced levels of the wound closure, granulation, and re-epithelialization at mouse skin wound sites, showing nearly complete restoration of the epidermis. The effect of melatonin on skin wound healing was further visualized by staining stem cells harboring 5bromo-2'-deoxyuridine (BrdU) with the Alexa Fluor® 488-conjugated BrdU antibody. Fig. 1C shows that melatonin significantly increased the number of cells that migrated to the wound sites, suggesting that the wound healing-promoting effect of melatonin is caused by increased the motility of UCB-MSCs.

To evaluate the role of melatonin in the regulation of stem cell motility, UCB-MSCs were exposed to various concentrations (0~1 mM) of melatonin for 24 h. Melatonin significantly induced cell motility of UCB-MSCs from 100 nM to 1 mM compared to the vehicle alone (Fig. 2A). An increase in cell motility was observed after 8 h of incubation with 1 μM of melatonin (Fig. 2B). The results after the calcein AM staining of UCB-MSCs in an OrisTM cell migration assay also revealed that 1 μM of melatonin significantly induced the translocation of the cell bodies into the denuded area for wound healing (a 210% increase compared to the control; P < 0.05) (Fig. 2C). We further confirmed the migration-promoting effect of melatonin based on a Transwell migration assay. Melatonin significantly increased the number of cells that migrated to the lower surface of the membrane compared to the vehicle (Fig. 2D). melatonin (1 μM) also has the ability to stimulate the motility of human This article is protected by copyright. All rights reserved.

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adipose-derived mesenchymal stem cells (AD-MSCs) (Fig. 2E). This result suggests that the functional role of melatonin to induce motility is reproducible in different assays and with other types of MSCs. In order to examine the mechanism causing these effects by melatonin, we determined the expression levels of melatonin receptors in UCB-MSCs. As shown in Fig. 2F, we detected MT2 expression, but not MT1. Moreover, the melatonin-induced stem cell motility was inhibited by MT2siRNA as well as MT2 inhibitor 4-phenyl-2propionamidotetralin (4P-PDOT) (Fig. 2F). We further explored the ability of MT2 to regulate cell motility by staining a filamentous (F)-actin structure with Alexa Fluor® 488conjugated phalloidin in vitro via a wound-healing migration assay. In contrast to the control, 1 μM of melatonin evoked substantial migration of cells into the denuded area, whereas the cell migration-promoting activity of melatonin was attenuated by MT2siRNA and 4P-PDOT (Fig. 2G). Additionally, mitomycin C (1 μg/mL), a cell-cycle-arresting compound, did not inhibit melatonin-induced cell motility (Fig. 2H). These results suggest that the effect of melatonin via MT2 in promoting cell motility is an independent process of cell proliferation and that it triggers the MT2 receptor-mediated signaling pathway.

MT2 receptors interact with heterotrimeric G proteins to regulate specific cellular signaling pathways. To identify Gα proteins coupled with MT2, we co-immunoprecipitated MT2 and G proteins. Figure 3A shows that Gαq co-immunoprecipitated with MT2 and, importantly, that the interaction with MT2 was enhanced by the melatonin treatment. Despite the frequent involvement of Gαi and Gα12 in GPCR-mediated cell migration, Gαi and Gα12 did not interact with MT2. The silencing of Gαi and Gα12 with siRNAs did not affect melatonin-dependent UCB-MSC motility, ruling out the role of Gαi and Gα12 (Fig. 3B). However, the inhibition of Gαq by GαqsiRNA blocked melatonin-induced cell motility in an OrisTM cell migration assay (Fig. 3B, left panel) and in an in vitro wound-healing migration assay (Fig. 3B, right panel). We then asked whether Gαq is localized into the membrane during cell migration. In the basal condition, Gαq is expressed in both the cytoplasm and This article is protected by copyright. All rights reserved.

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nucleus (Fig. 3C). Importantly, melatonin increased the immunofluorescence signal of Gαq in the membranes of UCB-MSCs. These results indicate that MT2 regulates UCB-MSC motility by interacting with Gαq along the leading edges of the lamellipodia. Given that melatonin binding to MT2 regulates phospholipase C (PLC) signaling [45], we further assessed whether melatonin induces the phosphorylation and translocation of PKC as an important downstream intermediate of PLC in human UCB-MSCs. Melatonin increased PKC phosphorylation from 15 to 60 min (Fig. 4A). In addition, melatonin-induced cell motility was inhibited by the PKC inhibitors bisindolylmaleimide I and staurosporine (Fig. 4B). In an experiment to identify the specific PKC isotypes, the translocation of PKCζ, but not PKCα, PKCβ, PKCγ, PKCδ, PKCε, or PKCθ, from the cytosol to the membrane compartment was observed after cells were treated with 1 μM of melatonin for 15 min (Fig. 4C). The membrane translocation of PKCζ was further confirmed by immunofluorescence staining in melatonin-treated UCB-MSCs (Fig. 4D). However, melatonin did not stimulate calcium influx, which was enhanced by A23187, as a positive control (Fig. 4E). Interestingly, the silencing of Gαq with GαqsiRNA significantly blocked the melatonin-induced phosphorylation of PKC (Fig. 4F). These results indicate that MT2 coupling with Gαq uniquely stimulates atypical PKC activation to promote UCB-MSC motility.

The effect of melatonin on the phosphorylation of focal adhesion kinase (FAK) and paxillin, which are believed to be essential factors in cell migration signaling pathways, was also examined. Melatonin (1 μM) significantly induced the phosphorylation of FAK and paxillin from 15 to 60 min (Fig. 5A). In addition, melatonin-induced cell motility was inhibited by the silencing of FAK and paxillin with FAKsiRNA and paxillinsiRNA in an OrisTM cell migration assay (Fig. 5B, left panel) and in an in vitro wound-healing migration assay (Fig. 5B, right panel). Increased staining levels of p-FAK and p-paxillin were observed after cells were treated with 1 μM of melatonin for 30 min (Fig. 5C). Interestingly, a

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pretreatment with the PKC inhibitors bisindolylmaleimide I and staurosporine significantly blocked the melatonin-induced phosphorylation of FAK and paxillin (Fig. 5D).

To gain insight into how melatonin may take part in the initiation of cytoskeletal reorganization, we further examined whether melatonin regulates Rho GTPase activity. Affinity precipitation for small Rho GTPases revealed that Cdc42 activity, but not Rac1 or RhoA, was uniquely increased by a treatment with 1 μM of melatonin (Fig. 6A). Melatonin also induced GTP-mediated actin-related protein (Arp2/3) interaction at 30 min (Fig. 6A). FAKsiRNA and paxillinsiRNA significantly blocked the melatonin-induced activation of Cdc42 and Arp2/3 in promoting UCB-MSC motility (Fig. 6B). The translocation of Cdc42 and Arp2/3 to the leading edges of lamellipodia was observed after cells were treated with 1 μM of melatonin for 30 min (Fig. 6C). We also determined whether melatonin regulates the cytoskeletal reorganization-related proteins that are responsible for cell motility. Increases in profilin-1 expression and in cofilin-1 phosphorylation were observed after 12 h of incubation with 1 μM of melatonin (Fig. 6D, left panel). A treatment with melatonin also increased the expression of F-actin, but not α-actinin-1 or α-actinin-4 (Fig. 6D, right panel). However, the F-actin interaction levels with α-actinin-1 and α-actinin-4 were increased after a treatment with melatonin (Fig. 6E). We confirmed that increased staining levels of profilin-1 (Fig. 6F, left panel) and p-cofilin-1 were observed after cells were treated with 1 μM of melatonin for 12 h (Fig. 6F, right panel). Interestingly, the results according to confocal immunofluorescence microscopy revealed that melatonin significantly induced the colocalization of F-actin and α-actinin-1 (Fig. 6G, left panel), as well as that of F-actin and αactinin-4 (Fig. 6G, right panel), in UCB-MSCs. To verify the role of melatonin in promoting cell motility orchestrated by MT2, we further tested the effect of MT2 on skin wound healing in mouse. Analogous to the result of the preactivation of UCB-MSCs with melatonin (Fig. 1A), wound healing on days 9 and 12 was significantly accelerated in the mice group that received UCB-MSCs/ntsiRNA, which were pre-treated with melatonin, compared to the vehicle group. In contrast, the mice group that This article is protected by copyright. All rights reserved.

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received UCB-MSCs/MT2siRNA showed a significant delay in wound healing despite the melatonin pre-treatment (Fig. 7A). A histologic examination on day 12 showed that the wound bed was still not completely covered with epidermis and a cornified layer in mice treated with UCB-MSCs/MT2siRNA, which were pre-treated with melatonin. However, the transplantation of UCB-MSCs/ntsiRNA, pre-treated with melatonin, led to the nearly complete restoration of re-epithelialization from a mechanical skin wound (Fig. 7B). In addition, Fig.7C shows that the pre-treatment with melatonin significantly increased the number of cells that migrated to the wound sites in the mice group that received UCBMSCs/ntsiRNA, whereas it failed to enhance the engraftment of stem cells when mice were given UCB-MSCs/MT2siRNA.

Discussion Our data demonstrate that a pretreatment of melatonin onto UCB-MSCs enhances skin wound healing in the mouse and that MT2 coupling with Gαq facilitates human UCB-MSC motility by stimulating the Cdc42-mediated cytoskeletal reorganization process through the PKCζ-mediated FAK/paxillin pathway. We are the first to show that UCB-MSCs pre-treated with melatonin have the ability to enhance epidermal reorganization and restore the normal tissue microarchitecture by regulating stem cell migration at the wound site. Thus, UCBMSCs may be more useful if they are pre-activated by melatonin, as doing so can demonstrate the potential benefits of stem cell pre-activation. The pre-activation of UCBMSCs may offer a means of improving the potency of these cells without the need for additional cell numbers. Concerning the concentration of melatonin, it was previously reported that the physiological levels of melatonin are in the picomolar and low nanomolar range in the blood, whereas the melatonin concentrations of some tissues and cells can be exceptionally high. However, this was found to be physiological [46]. Thus, the concentration of melatonin in the experiment and the application time justify, in part, the discrepancies found among several studies. For example, previous reports have shown that melatonin at low doses (≤ 1 μM) induces the proliferation, survival, and differentiation of neural stem cells [22, 47], whereas

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at high doses (>100 mM), it inhibits the adipogenic differentiation of mesenchymal stem cells [48]. We believe that these broad effects of melatonin are in part due to the presence of multiple melatonin receptors in different types of cells, and outcomes may vary depending on the cellular concept. Therefore, our findings suggest that melatonin is a good candidate for cell transplantation as a pre-activation agent which induces stem cell motility. These results are supported by a previous report showing that melatonin is a functional agent that stimulates the differentiation and proliferation of neural stem cells [21, 22]. We subsequently showed that melatonin acts as a strong stimulator of UCB-MSC motility and that MT2 is the primary melatonin receptor regulating UCB-MSC motility. In contrast, previous result revealed that MT1 is the key functional receptor during the transplantation of neural stem cells and that it has neuroprotective effects on the nigrostriatal system [44]. Given that melatonin interaction is limited to MT2 in UCB-MSCs and the loss of the MT2 function inhibits melatonin-induced cell motility, our results indicate that MT2 is the major melatonin receptor that regulates UCB-MSC motility, providing the first evidence that melatonin may have unique MT2 receptor signaling pathways during UCB-MSC migration. The MT2 receptor initially couples with heterotrimeric Gα proteins. It was shown previously that MT2 couples with mainly Gαi to modulate circadian clock genes, whereas MT1 interacts with Gαi, Gαq, and Gαs [49, 50]. In UCB-MSCs, however, we found that MT2 interaction was coupled with only Gαq, and that MT2-mediated cell migration is selectively regulated by Gαq, but not by Gαi or Gα12. These results are further supported by a previous study in which Gαq subunit-linked signal transduction was found to be the critical step in the regulation of the proliferation and integrin signaling of stem cells [51]. To the best of our knowledge, this is the first study to show a unique association between MT2 and Gαq in the regulation of stem cell motility. The Gαq–dependent pathway requires the activation of PKC [52], which is an important component of the circadian effects of melatonin [45]. We observed that melatonin induces PKC translocation and phosphorylation, in that PKC is required for MT2 activation to promote UCB-MSC motility despite the lack of involvement in the influx of calcium on PKC activation. Although the discrepancy with

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regard to the calcium influx may be due to differences in species, cell types, or experimental conditions, our results indicate that UCB-MSC motility is completely abrogated by the inhibition of the PKC pathways, providing strong evidence that PKC plays an important role in the melatonin signaling pathway. Interestingly, melatonin uniquely activated atypical PKCζ in all PKC isoforms in UCB-MSCs. Although conventional PKCα activation was found to require the MT2-mediated circadian effects [53], many studies have established a critical role of PKCζ in directional migration [54, 55]. Particularly, PKCζ appears to play a major role in hematopoietic stem cell motility, compared to conventional or novel types of PKC [56]. Together, our results suggest that the effect of melatonin on PKCζ activation is likely to be compounded by the selective expression of MT2 coupling with Gαq in UCBMSCs. On the other hand, we found that melatonin induces the phosphorylation of FAK/paxillin through PKC and that the silencing of FAK/paxillin inhibits melatonin-induced UCB-MSC migration. Consistent with our results, PKC was shown to stimulate FAK/Src phosphorylation and thereby promote the activation of cytoskeletal-associated proteins during neuronal cell differentiation [57]. Evidence has indicated that the phosphorylation of FAK/paxillin is important for integrin-mediated stem cell motility [58]. FAK is a cytoplasmic protein tyrosine kinase that is involved in the integrin signaling pathway for cell migration [59]. Paxillin also plays a critical role as a scaffold protein in the recruitment of many signaling proteins, including FAK to the plasma membrane, and it contributes to the organization of the actin cytoskeleton at sites of integrin engagement with the extracellular matrix [60]. Importantly, FAK coupled with Src has been shown to phosphorylate paxillin at its tyrosine 118 site [61]. Therefore, our results strongly suggest that melatonin is a crucial microenvironmental cue that triggers PKCζ-dependent FAK/paxillin activation for UCBMSC motility. Next, we attempted to uncover the mechanism how FAK/paxillin links to other molecules that are important in the cell migration process. Rho GTPases, RhoA, Rac1, and Cdc42, have been implicated in the regulation of the assembly and organization of the actin cytoskeleton for cell motility [62]. However, the functional role of Rho GTPases in the melatonin signaling pathway is still under debate. A few reports have indicated that RhoA participates in the regulation of cancer cell metastasis and of urinary bladder contractile This article is protected by copyright. All rights reserved.

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impairment in response to melatonin [63, 64]. In the present study, however, we did not see any evidence of melatonin-mediated RhoA/Rac1 activation in UCB-MSCs. Instead, we found that melatonin uniquely activates Cdc42, which interacts with Arp2/3 in a FAK/paxillindependent manner, implying a functional role of melatonin in the determination of downstream Rho GTPases. Consistent with our results, evidence was found that the tyrosine phosphorylation of paxillin at tyrosine 31/118 sites is critical for the activation of the NWASP and Arp2/3 complex [65]. In addition, previous work has shown that Cdc42 stimulates actin cytoskeleton reorganization by directly recruiting and activating the NWASP and Arp2/3 complex for cell motility [66]. These results are further supported by other studies using hematopoietic stem cells and embryonic stem cells [67, 68]. Therefore, we suggest that the melatonin signaling pathway is crucially linked to Cdc42/Arp2/3 activation to promote UCB-MSC motility. Regarding the role of melatonin in Cdc42/Arp2/3 activation, we also determined the potential role of melatonin in the regulation of the cytoskeletal reorganization-related proteins profilin-1, cofilin-1, and F-actin, which are critical requirements for stem cell migration [68, 69]. Our results demonstrate that melatonin has ability to enhance cytoskeletal reorganization by regulating the levels of profilin-1, p- cofilin1, and F-actin in UCB-MSC motility. It is not clear whether this additional effect of melatonin in promoting cytoskeletal reorganization is a sequential result of the loss of cell cohesion or, alternatively, an independent process involving other cellular signaling events. However, it was clearly shown that profilin-1 and cofilin-1 play key roles in enhancing the actin assembly at the plasma membrane, thereby increasing F-actin expression, which drives cell motility and other actin-linked processes [70, 71]. Specifically, α-actinin cross-links Factin into bundles to form a cytoskeletal network [72]. Consistent with this, our results revealed that F-actin/α-actinin-1 and F-actin/α-actinin-4 interactions are increased by a melatonin treatment. Therefore, our results strongly suggest that MT2 triggers Gαqdependent PKCζ phosphorylation to regulate FAK/paxillin-mediated Cdc42/Arp2/3 activation, and that this signaling pathway governs the cytoskeletal reorganization process to promote melatonin -induced human UCB-MSC motility. Overall, these findings highlight the relevance of the MT2 receptor signaling pathway in promoting UCB-MSC motility. Moreover, we showed convincing in vivo proof that the

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silencing of the MT2 receptor in UCB-MSCs pre-activated with melatonin failed to regulate the repair of skin wounds. Therefore, our findings elucidate novel roles of melatonin and MT2 in UCB-MSC motility to enhance the re-epithelialization of injured tissue for skin wound healing. Although our study demonstrated that a pretreatment of melatonin onto UCB-MSCs leads to significantly improved wound healing while also defining the molecular mechanism by which melatonin acts in this process, further research is required to establish in greater detail in the effect of melatonin on the wound healing process.

Acknowledgements This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A120216), and a grant from the Next-Generation BioGreen 21 Program (No. PJ009090), Rural Development Administration, Republic of Korea.

Conflict of Interest The authors declare no conflict of interest.

Author Contributions S.L.: Study concept and design, acquisition of data, analysis and interpretation of data, statistical analysis, drafting of the manuscript Y.H.J.: Acquisition of data, analysis and interpretation of data S.Y.O.: Acquisition of data, analysis and interpretation of data S.P.Y.: Acquisition of data, analysis and interpretation of data H.J.H.: Study concept and design, analysis and interpretation of data, drafting of the manuscript, obtained funding, study supervision

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Figure legends Fig. 1. The role of melatonin on skin wound healing in vivo. (A) Representative gross images on skin wound healing at day 0, 5, 9, and 12 are shown (left panel). Mouse skin wounds were made by 6-mm-diameter biopsy punch and treated with vehicle, melatonin, UCB-MSCs + vehicle, and UCB-MSCs + melatonin, respectively. Quantifications of wound sizes relative to original wound size are shown (right panel). Error bars represent the mean ± S.E. n = 8. *P < 0.05 vs. vehicle alone, #P < 0.05 vs. UCB-MSCs + vehicle. (B) Representative wound tissues stained with H&E at day 12 are shown. n = 8. Scale bars, 100 μm. Abbreviations: Ep, epidermis; W, wound bed; CL, cornified layer. (C) BrdU-horboring UCB-MSCs were topically implanted onto the wound bed and/or injected into the dermis of the surrounding skin. Engraftment of UCB-MSCs on wound site at day 12 was determined by confocal microscopy using immunofluorescence staining of BrdU (green). Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, ×200). n = 4. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; Ep, epidermis; Mel, melatonin; PI, propidium iodide; W, wound bed; CL, cornified layer

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Fig. 2. The effect of melatonin on human UCB-MSCs motility. (A) Dose responses of melatonin for 24 h in OrisTM cell migration assay are shown. Data represent means ± S.E. of five independent experiments with triplicate dishes. *P < 0.05 vs. vehicle. (B) Time responses of 1 μM melatonin in OrisTM cell migration assay are shown. Error bars represent the mean ± S.E. n = 5. *P < 0.01 vs. vehicle. (C) Cells treated with 1 μM melatonin for 24 h were visualized by calcein AM (green) staining using OrisTM cell migration assay. n = 5. Scale bars represent 200 μm (magnification, ×40). (D) The migration capacity of UCBMSCs treated with melatonin (1 μM) for 24 h was assessed in transwell permeable support with 8.0 μm pore size membrane. Cells were removed from the upper chamber with a cotton swab, and the cells that migrated to the lower surface of the membrane were stained with H&E. Scale bars, 100 μm. (E) Human adipose-derived mesenchymal stem cells (AD-MSCs) treated with melatonin (1 μM) for 24 h were fixed and labeled with phalloidin-AlexaFluor 488 (green) to identify the migrating cells. n = 3. Scale bars represent 100 μm (magnification, ×100). (F) The effect of melatonin in UCB-MSCs transfected with MT2siRNA in OrisTM cell migration assay is shown. Cells were transfected for 24 h with specific siRNA for MT2 (200 pmol/L) using HiPerFect Transfection Reagent before melatonin (1 μM) exposure for 24 h. Non-targeting control siRNA was used as a negative control (200 l/L). Top panel shows the expression of MT1 and MT2 on total RNA of UCB-MSCs. On the other hand, cells were pretreated with MT2 inhibitors, 4P-PDOT (10 nM) for 30 min prior to melatonin (1 μM) exposure for 24h. Error bars represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle, #P< 0.05 vs. melatonin alone. (G) Wound-healing assay was performed and stained with phalloidinAlexaFluor 488 (green) to identify the migrating cells. n = 5. Scale bars represent 100 μm (magnification, ×100). (H) Effect of mitomycin C in melatonin-induced cell migration. Cells were pretreated with 1 μg/mL mitomycin C for 30 min prior to 1 μM melatonin exposure for 24 h. OrisTM cell migration assay was performed and quantified the value of fluorescence. This article is protected by copyright. All rights reserved.

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Data represent means ± SE of four independent experiments with triplicate dishes. *P < 0.05 vs. vehicle. Abbreviations: Mel, melatonin; MT2, melatonin receptor 2; RFU, Relative fluorescence units.

Fig. 3. MT2 coupling with Gαq regulates human UCB-MSCs motility. (A) The cells were incubated in the presence of melatonin (1 μM) for 15 min and then harvested. MT2 was immunoprecipitated with an anti-MT2 antibody, and co-immunoprecipitated Gα proteins were detected by using anti-Gαq, -Gαi, and -Gα12 antibodies (the left side). Expression of MT2, Gαq, Gαi, and Gα12 in total cell lysates is shown in the right side. Error bars represent the mean ± S.E. of four independent experiments for each condition determined from densitometry relative MT2 binding co-immunoprecipitated with MT2 antibody. *P < 0.01 vs. 0 min. (B) The effect of melatonin in UCB-MSCs transfected with GαqsiRNA, GαisiRNA, and Gα12siRNA in OrisTM cell migration assay (left panel) as well as in in vitro wound healing assay (right panel) is shown. Cells were transfected for 24 h with specific siRNA for Gα proteins before melatonin (1 μM) exposure for 24 h. Non-targeting control siRNA was used as a negative control. Error bars represent the mean ± SE. n = 4. *P < 0.05 vs. vehicle, #

P< 0.05 vs. melatonin alone. (C) Membrane translocation of Gαq (green) was determined by

confocal microscopy using immunofluorescence staining. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, ×400). n = 3. Abbreviations: Gα, G protein alpha; IP, immunoprecipitation; Mel, melatonin; MT2, melatonin receptor 2; PI, propidium iodide; RFU, Relative fluorescence units.

Fig. 4. Effect of melatonin on activation of protein kinase C (PKC). (A) Phosphorylation of PKC in cells treated with melatonin is shown. (B) Cells were pretreated with PKC inhibitors, This article is protected by copyright. All rights reserved.

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staurosporine (10 μM) and bisindolylmaleimide I (10 μM) for 30 min prior to melatonin (1 μM) exposure for 15 min. The effect of melatonin in UCB-MSCs treated with PKC inhibitors in OrisTM cell migration assay (left panel) as well as in in vitro wound healing assay (right panel) is shown. Error bars represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle, #P< 0.05 vs. melatonin alone. (C) Membrane translocation of PKC isoforms in cells treated with melatonin (1 μM) for 15 min was determined by Western blot analysis. The pan-cadherin was used as a plasma membrane control. Error bars represent the means ± S.E. from three independent experiments involving triplicates. *P < 0.01 vs. vehicle. (D) Membrane translocation of PKCζ (green) was determined by confocal microscopy using immunofluorescence staining. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, ×400). n = 3. (E) The cells were loaded with 2 μM Fluo-3/AM in serum-free medium for 40 min and treated with melatonin (1 μM). Cells were then treated A23187 (10 μM, Ca2+ ionophore) as a positive control. Changes in [Ca2+]i were monitored by confocal microscopy, and data are expressed as relative fluorescence intensity (RFI, F/F0 %, arbitrary unit). (F) Cells were transfected for 24 h with specific siRNA for Gαq before melatonin (1 μM) exposure for 15 min. Phosphorylation of PKC in cells treated with melatonin is shown. Error bars represent the mean ± S.E. n = 5. *P < 0.05 vs. vehicle, #

P< 0.01 vs. melatonin alone. Abbreviations: Mel, melatonin; Pan-cad, Pan-cadherin; PKC,

Protein kinase C; PI, propidium iodide; RFU, Relative fluorescence units; ROD, relative optical density.

Fig. 5. Effect of melatonin on phosphorylation of focal adhesion kinase (FAK) and paxillin. (A) Phosphorylation of FAK and paxillin in cell treated with melatonin was determined. Error bars represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle. (B) The effect of melatonin in UCB-MSCs transfected with FAKsiRNA and paxillinsiRNA in OrisTM cell migration assay (left panel) as well as in in vitro wound healing assay (right panel) is shown. Error bars This article is protected by copyright. All rights reserved.

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represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle, #P< 0.05 vs. melatonin alone. (C) The increased expressions of p-FAK (green, top panel) and p-paxillin (green, bottom panel) was determined by confocal microscopy using immunofluorescence staining. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, ×400). n = 3. (D) Cells were pretreated with bisindolylmaleimide I (10 μM) and staurosporine (10 μM) for 30 min, and then exposed to 1 μM melatonin for 30 min. Phosphorylation of FAK and paxillin is shown. Error bars represent the mean ± S.E. n = 5. *P < 0.01 vs. vehicle, #P< 0.01 vs. melatonin alone. Abbreviations: FAK, Focal adhesion kinase; Mel, melatonin; PI, propidium iodide; RFU, Relative fluorescence units; ROD, relative optical density.

Fig. 6. Effects of melatonin on Cdc 42 and Arp2/3 and cytoskeletal reorganization-related proteins. (A) Cells were treated with melatonin for 30 min, and the lysates (400 μg) were incubated with agarose beads coupled with GST-PAK-PBD or GST-Rhotekin-RBD. The bound activated GTP-Cdc42, GTP-Rac1, GTP-RhoA, and Arp2/3 were resolved by SDSPAGE, transferred, and blotted using an anti-Cdc42, anti-Rac1, anti-RhoA, and Arp2/3 antibodies to determine the extent of the activation of Cdc42, Rac1, RhoA, and Arp2/3. Total Cdc42, Rac1, RhoA, and Arp2/3 was determined using lysates (right panel). Error bars represent the mean ± S.E. n = 4. *P < 0.05 vs. vehicle. (B) The effect of melatonin in UCBMSCs transfected with FAKsiRNA and paxillinsiRNA in Cdc42 activation and Arp2/3 interaction is shown. Error bars represent the mean ± S.E. n = 4. *P < 0.01 vs. vehicle, #P< 0.01 vs. melatonin alone. (C) Membrane translocation of Cdc42 (green, top panel) and Arp2/3 (green, bottom panel) was determined by confocal microscopy using immunofluorescence staining. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, ×400). n = 3. (D) The cells were incubated in the presence of melatonin (1 μM) for 24 h and then harvested. Total protein was extracted and blotted with profilin-1, phospho-Cofilin-1, Cofilin-1, F-actin, α-actinin-1, and α-actinin-4

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antibodies. Error bars represent the mean ± S.E. of four independent experiments for each condition determined from densitometry relative to β-actin. *P < 0.05 vs. 0 h. (E) The cells were incubated in the presence of melatonin (1 μM) for 24 h and then harvested. F-actin was immunoprecipitated with an anti-F-actin antibody, and co-immunoprecipitated α-actinin-1/-4 were detected by using anti-α-actinin-1 and anti-α-actinin-4 antibodies (left side). Expression of α-actinin-1 and -4 in total cell lysates is shown in the right side. Error bars represent the mean ± S.E. n = 3. *P < 0.05 vs. 0 h. (F) The increased expressions of profilin-1 (green, left panel) and p-cofilin-1 (green, right panel) was determined by confocal microscopy using immunofluorescence staining. Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, ×400). n = 3. (G) The increased colocalization of F-actin (green) with α-actinin-1 and -4 (red) was determined by confocal microscopy using immunofluorescence staining. Scale bars, 100 μm (magnification, ×400). n = 3. Abbreviations: IP, immunoprecipitation; Arp2/3, actin-related protein 2/3; Mel, melatonin; PI, propidium iodide; ROD, relative optical density.

Fig. 7. The role of MT2 on skin wound healing in vivo. (A) Representative gross images on skin wound healing at day 0, 5, 9, and 12 are shown (left panel). Mouse skin wounds were made by 6-mm-diameter biopsy punch and treated with UCB-MSCs/ntsiRNA+vehicle, UCBMSCs/ntsiRNA+ melatonin, UCB-MSCs/MT2siRNA+ vehicle, and UCB-MSCs/MT2siRNA+ melatonin, respectively. Quantifications of wound sizes relative to original wound size are shown (right panel). Error bars represent the mean ± S.E. n = 8. *P < 0.05 vs. UCBMSCs/ntsiRNA+ melatonin. (B) Representative wound tissues stained with H&E at day 12 are shown. n = 8. Scale bars, 100 μm. Abbreviations: Ep, epidermis; W, wound bed; CL, cornified layer. (C) BrdU-horboring UCB-MSCs were topically implanted onto the wound bed and/or injected into the dermis of the surrounding skin. Engraftment of UCB-MSCs on wound site at day 12 was determined by confocal microscopy using immunofluorescence This article is protected by copyright. All rights reserved.

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staining of BrdU (green). Propidium iodide (PI) was used for nuclear counterstaining (red). Scale bars, 100 μm (magnification, ×200). n = 4. Abbreviations: BrdU, 5-bromo-2'deoxyuridine; Ep, epidermis; Mel, melatonin; PI, propidium iodide; W, wound bed; CL, cornified layer

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Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing.

Melatonin, a circadian rhythm-promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesen...
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