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 pretreated with melatonin enhanced wound closure, granulation, and re-epithelialization at mouse skin wound sites, where relatively more UCB-MSCs 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 downregulated 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 profilin-1, 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 actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB-MSCs motility.

The control of stem cells by oxygen signaling is an important way to improve various stem cell physiological functions and metabolic nutrient alteration. Lipid metabolism alteration via hypoxia is thought to be a key factor in controlling stem cell fate and function. However, the interaction between hypoxia and the metabolic and functional changes to stem cells is incompletely described. This study aimed to identify hypoxia-inducible lipid metabolic enzymes that can regulate umbilical cord blood (UCB)-derived human mesenchymal stem cell (hMSC) proliferation and migration and to demonstrate the signaling pathway that controls functional change in UCBhMSCs. Our results indicate that hypoxia treatment stimulates UCB-hMSC proliferation, and expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). FASN but not SCD1 is a key enzyme for regulation of UCB-hMSC proliferation and migration. Hypoxia-induced FASN expression was controlled by the hypoxia-inducible factor-1 alpha (HIF-1a)/SCAP/SREBP1 pathway. Mammalian target of rapamycin (mTOR) was phosphorylated by hypoxia, whereas inhibition of FASN by cerulenin suppressed hypoxia-induced mTOR phosphorylation as well as UCB-hMSC proliferation and migration. RAPTOR small interfering RNA transfection significantly inhibited hypoxia-induced proliferation and migration. Hypoxia-induced mTOR also regulated CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as that of c-myc, p-cofilin, profilin, and Rho GTPase. Taken together, the results suggest that mTORC1 mainly regulates UCBhMSC proliferation and migration under hypoxia conditions via control of cell cycle and F-actin organization modulating factors. In conclusion, the HIF-1a/FASN/mTORC1 axis is a key pathway linking hypoxia-induced lipid metabolism with proliferation and migration in UCB-hMSCs. STEM CELLS 2015;33:2182-2195

Netrin-1 (Ntn-1) is a potent inducer of neuronal cell migration; however, its molecular mechanism that guides the migratory behavior of stem cells has not been characterized. In this study, we investigate the role of Ntn-1 in promoting the motility of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and its related signaling pathways. Ntn-1 (50 ng/mL) significantly increased motility of UCB-MSCs, which was inhibited by blocking antibodies for deleted in colorectal cancer (DCC) and integrin (IN) a6b4. Ntn-1 in DCC stimulated protein kinase Ca (PKCa) activation, but not PKCe, PKCy, and PKCz, while Ntn-1 in INa6b4 induced the phosphorylation of focal adhesion kinase (FAK) and Fyn. Notably, Ntn-1 induced phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases ( JNK), and nuclear factor kappa-B (NF-kB), but they were concurrently downregulated by blocking the activities of PKCa, FAK, and Fyn. Ntn-1 uniquely increased the MMP-12 expression of all the matrix metalloproteinase (MMP) isoforms present in UCB-MSCs, though this was significantly blocked by an NF-kB inhibitor. Finally, Ntn-1 induced the MMP-12-dependent degradation of E-cadherin (E-cad), while Ntn-1 abrogated the interaction between E-cad and p120-catenin. In addition, Ntn-1 has the ability to stimulate cytoskeletal reorganization-related proteins, such as Cdc42, Rac1, Profilin-1, Cofilin-1, a-Actinin-4, and filamentous actin (F-actin) in UCB-MSCs. These results demonstrate that Ntn-1 induces MMP-12-dependent E-cad degradation via the distinct activation of PKCa and FAK/Fyn, which is necessary to govern the activation of ERK, JNK, and NF-kB in promoting motility of UCBMSCs.

The role of unsaturated fatty acids (UFAs) is essential for determining stem cell functions. Eph/Ephrin interactions are important for regulation of stem cell fate and localization within their niche, which is significant for a wide range of stem cell behavior. Although oleic acid (OA) and Ephrin receptors (Ephs) have critical roles in the maintenance of stem cell functions, interrelation between Ephs and OA has not been explored. Therefore, the present study investigated the effect of OA-pretreated UCB-MSCs in skin wound-healing and underlying mechanism of Eph expression. OA promoted the motility of UCB-MSCs via EphB2 expression. OA-mediated GPR40 activation leads to Gαq-dependent PKCα phosphorylation. In addition, OA-induced phosphorylation of GSK3β was followed by β-catenin nuclear translocation in UCB-MSCs. Activation of β-catenin was blocked by PKC inhibitors, and OA-induced EphB2 expression was suppressed by β-cateninsiRNA transfection. Of those Rho-GTPases, Rac1 was activated in an EphB2-dependent manner. Accordingly, knocking down EphB2 suppressed F-actin expression. In vivo skin wound-healing assay revealed that OA-treated UCB-MSCs enhanced skin wound repair compared to UCB-MSCs pretreated with EphB2siRNA and OA. In conclusion, we showed that OA enhances UCB-MSC motility through EphB2-dependent F-actin formation involving PKCα/GSK3β/β-catenin and Rac1 signaling pathways.

Background and PurposeReactive oxygen species (ROS) are potent regulators of stem cell behaviour; however, their physiological significance as regards MMP‐mediated regulation of the motility of human umbilical cord blood‐derived mesenchymal stem cells (UCB‐MSCs) has not been characterized. In the present study, we investigated the role of hydrogen peroxide (H2O2) and associated signalling pathways in promoting UCB‐MSCs motility.Experimental ApproachThe regulatory effects of H2O2 on the activation of PKC, MAPKs, NF‐κB and β‐catenin were determined. The expressions of MMP and extracellular matrix proteins were examined. Pharmacological inhibitors and gene‐specific siRNA were used to identify the signalling pathways of H2O2 that affect UCB‐MSCs motility. An experimental skin wound‐healing model was used to confirm the functional role of UCB‐MSCs treated with H2O2 in ICR mice.Key ResultsH2O2 increased the motility of UCB‐MSCs by activating PKCα via a calcium influx mechanism. H2O2 activated ERK and p38 MAPK, which are responsible for the distinct activation of transcription factors NF‐κB and β‐catenin. UCB‐MSCs expressed eight MMP genes, but only MMP12 expression was uniquely regulated by NF‐κB and β‐catenin activation. H2O2 increased the MMP12‐dependent degradation of collagen 5 (COL‐5) and fibronectin (FN) associated with UCB‐MSCs motility. Finally, topical transplantation of UCB‐MSCs treated with H2O2 enhanced skin wound healing in mice.Conclusions and ImplicationsH2O2 stimulated UCB‐MSCs motility by increasing MMP12‐dependent degradation of COL‐5 and FN through the activation of NF‐κB and glycogen synthase kinase‐3β/β‐catenin, which is critical for providing a suitable microenvironment for MSCs transplantation and re‐epithelialization of skin wounds in mice.