Young Hyun Jung

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 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.

Novel Pathway for Hypoxia‐Induced Proliferation and Migration in Human Mesenchymal Stem Cells: Involvement of HIF‐1α, FASN, and mTORC1

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 UCB‐hMSCs. 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‐1α)/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 UCB‐hMSC proliferation and migration under hypoxia conditions via control of cell cycle and F‐actin organization modulating factors. In conclusion, the HIF‐1α/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

BNIP3 induction by hypoxia stimulates FASN-dependent free fatty acid production enhancing therapeutic potential of umbilical cord blood-derived human mesenchymal stem cells

Mitophagy under hypoxia is an important factor for maintaining and regulating stem cell functions. We previously demonstrated that fatty acid synthase (FASN) induced by hypoxia is a critical lipid metabolic factor determining the therapeutic efficacy of umbilical cord blood-derived human mesenchymal stem cells (UCB-hMSCs). Therefore, we investigated the mechanism of a major mitophagy regulator controlling lipid metabolism and therapeutic potential of UCB-hMSCs. This study revealed that Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)-dependent mitophagy is important for reducing mitochondrial reactive oxygen species accumulation, anti-apoptosis, and migration under hypoxia. And, BNIP3 expression was regulated by CREB binding protein-mediated transcriptional actions of HIF-1α and FOXO3. Silencing of BNIP3 suppressed free fatty acid (FFA) synthesis regulated by SREBP1/FASN pathway, which is involved in UCB-hMSC apoptosis via caspases cleavage and migration via cofilin-1-mediated F-actin reorganization in hypoxia. Moreover, reduced mouse skin wound-healing capacity of UCB-hMSC with hypoxia pretreatment by BNIP3 silencing was recovered by palmitic acid. Collectively, our findings suggest that BNIP3-mediated mitophagy under hypoxia leads to FASN-induced FFA synthesis, which is critical for therapeutic potential of UCB-hMSCs with hypoxia pretreatment.

High glucose upregulates BACE1-mediated Aβ production through ROS-dependent HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization in SK-N-MC cells

There is an accumulation of evidence indicating that the risk of Alzheimer’s disease is associated with diabetes mellitus, an indicator of high glucose concentrations in blood plasma. This study investigated the effect of high glucose on BACE1 expression and amyloidogenesis in vivo, and we present details of the mechanism associated with those effects. Our results, using ZLC and ZDF rat models, showed that ZDF rats have high levels of amyloid-beta (Aβ), phosphorylated tau, BACE1, and APP-C99. In vitro result with mouse hippocampal neuron and SK-N-MC, high glucose stimulated Aβ secretion and apoptosis in a dose-dependent manner. In addition, high glucose increased BACE1 and APP-C99 expressions, which were reversed by a reactive oxygen species (ROS) scavenger. Indeed, high glucose increased intracellular ROS levels and HIF-1α expression, associated with regulation of BACE1 and Liver X Receptor α (LXRα). In addition, high glucose induced ATP-binding cassette transporter A1 (ABCA1) down-regulation, was associated with LXR-induced lipid raft reorganization and BACE1 localization on the lipid raft. Furthermore, silencing of BACE1 expression was shown to regulate Aβ secretion and apoptosis of SK-N-MC. In conclusion, high glucose upregulates BACE1 expression and activity through HIF-1α and LXRα/ABCA1-regulated lipid raft reorganization, leading to Aβ production and apoptosis of SK-N-MC.

Galectin-1 stimulates motility of human umbilical cord blood-derived mesenchymal stem cells by downregulation of smad2/3-dependent collagen 3/5 and upregulation of NF-κB-dependent fibronectin/laminin 5 expression

Galectin-1 (Gal-1) belongs to a family of endogenous lectins with conserved carbohydrate recognition domains binding β-galactosidase sugars and plays a vital role in regulating stem cell functions including determination of cell fate. However, our understanding of the functional roles of Gal-1 in human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) is still fragmentary and incomplete. Gal-1 significantly increased motility after a 24-h incubation, and this effect was inhibited by β-lactose. We analyzed 17 extracellular matrix (ECM) genes in UCB-MSCs. Gal-1 decreased the expression of collagen genes COL3A1 (COL-3) and COL5A1 (COL-5) but increased the expression of fibronectin (FN) and laminin 5 (LM-5), that were reversed by β-lactose. Gal-1 increased protein kinase C (PKC), c-Src, and caveolin-1 (Cav-1) phosphorylation that was attenuated by β-lactose and the Src inhibitor PP2. In addition, pretreatment with the lipid raft disruptor Mβ-CD and the PKC inhibitors inhibited Gal-1-induced UCB-MSC motility. In addition, Gal-1 reduced smad2/3 phosphorylation and induced nuclear factor (NF)-κB phosphorylation. Pretreatment with Mβ-CD attenuated Gal-1-reduced smad2/3 phosphorylation, COL-3, and COL-5 expression but did not affect NF-κB phosphorylation, FN, or LM-5 expression. In contrast, PKC inhibitors only attenuated NF-κB phosphorylation, FN, and LM-5 expression. Reconstructing Gal-1-induced genetic changes by replacing it with siRNA specific for COL-3 or COL-5, or treatment of the cells with FN and LM-5 proteins, increased motility and its related proteins such as focal adhesion kinase, Akt, Erk, integrins, and matrix metalloproteinase-2. A combined treatment with COL-3/COL-5 siRNA or FN/LM-5 compared with that of single treatments was synergistic. However, a single Gal-1 treatment maximally stimulated motility and related protein phosphorylation/expression. These results demonstrate that Gal-1 stimulated human UCB-MSC motility by decreasing COL-3/COL-5 expression and increasing FN/LM-5 expression through a PKC-dependent NF-κB and c-Src/Cav-1-dependent smad2/3 pathway that was critical for governing the activation of FAK, Akt, Erk, integrins, and MMP2.

Netrin-1 Induces MMP-12-Dependent E-Cadherin Degradation Via the Distinct Activation of PKCα and FAK/Fyn in Promoting Mesenchymal Stem Cell Motility

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) α6β4. Ntn-1 in DCC stimulated protein kinase Cα (PKCα) activation, but not PKCɛ, PKCθ, and PKCζ, while Ntn-1 in INα6β4 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-κB), but they were concurrently downregulated by blocking the activities of PKCα, 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-κB 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, α-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 PKCα and FAK/Fyn, which is necessary to govern the activation of ERK, JNK, and NF-κB in promoting motility of UCB-MSCs.

Oleic acid enhances the motility of umbilical cord blood derived mesenchymal stem cells through EphB2-dependent F-actin formation.

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.

Reactive oxygen species induce MMP12-dependent degradation of collagen 5 and fibronectin to promote the motility of human umbilical cord-derived mesenchymal stem cells.

Reactive 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.

VvpE mediates the intestinal colonization of Vibrio vulnificus by the disruption of tight junctions.

The disruption of gastrointestinal tight junctions and their colonization evoked by enteric pathogens are hallmarks of the pathogenesis. Vibrio (V.) vulnificus, VvpE, is an elastase which is responsible for host surface adherence and vascular permeability; however, the functional roles of VvpE in the pathogenesis of V. vulnificus (WT) are poorly understood. In the present study, we have investigated the role of VvpE in regulation of intestinal tight junctions and the colonization of WT. We found that mutation of the vvpE gene from V. vulnificus (vvpE mutant) prevents intestinal tight/adherens junction dysregulation due to a WT infection and maintains the physiological level of the epithelial paracellular permeability. Interestingly, the vvpE mutant exhibited defective intestinal colonization abilities, whereas WT colonization was significantly elevated in the ileum in a time-dependent manner. Finally, the vvpE mutant negated the enterotoxicity, the breakdown of red blood cells, and pro-inflammatory responses, all of which are induced by the WT infection. In addition, the results of a LC-MS/MS analysis showed that VvpE contributes to WT pathogenesis in multiple ways by interacting with intestinal proteins, including β-globin, Annexin A2, Annexin A4, F-actin, and intelectin-1b. These results demonstrate that VvpE plays important role in promoting the tight junction disruption and intestinal colonization of V. vulnificus and that it also has the ability to interact with the intestinal proteins responsible for microbial pathogenesis.

Regulation of Stem Cell Fate by ROS-mediated Alteration of Metabolism

Stem cells have attracted much attention due to their distinct features that support infinite self-renewal and differentiation into the cellular derivatives of three lineages. Recent studies have suggested that many stem cells both embryonic and adult stem cells reside in a specialized niche defined by hypoxic condition. In this respect, distinguishing functional differences arising from the oxygen concentration is important in understanding the nature of stem cells and in controlling stem cell fate for therapeutic purposes. ROS act as cellular signaling molecules involved in the propagation of signaling and the translation of environmental cues into cellular responses to maintain cellular homeostasis, which is mediated by the coordination of various cellular processes, and to adapt cellular activity to available bioenergetic sources. Thus, in this review, we describe the physiological role of ROS in stem cell fate and its effect on the metabolic regulation of stem cells.