Jung Min Ryu

LuxR Homologue SmcR Is Essential for Vibrio vulnificus Pathogenesis and Biofilm Detachment, and Its Expression is Induced by Host Cells

ABSTRACT Quorum sensing is a cell-to-cell communication system known to control many bacterial processes. In the present study, the functions of quorum sensing in the pathogenesis of Vibrio vulnificus, a food-borne pathogen, were assessed by evaluating the virulence of a mutant deficient in SmcR, a quorum-sensing regulator and homologue of LuxR. When biofilms were used as an inoculum, the smcR mutant was impaired in virulence and colonization capacity in the infection of mice. The lack of SmcR also resulted in decreased histopathological damage in mouse jejunum tissue. These results indicated that SmcR is essential for V. vulnificus pathogenesis. Moreover, the smcR mutant exhibited significantly reduced biofilm detachment. Upon exposure to INT-407 host cells, the wild type, but not the smcR mutant, revealed accelerated biofilm detachment. The INT-407 cells increased smcR expression by activating the expression of LuxS, an autoinducer-2 synthase, indicating that host cells manipulate the cellular level of SmcR through the quorum-sensing signaling of V. vulnificus. A whole-genome microarray analysis revealed that the genes primarily involved in biofilm detachment and formation are up- and downregulated by SmcR, respectively. Among the SmcR-regulated genes, vvpE encoding an elastolytic protease was the most upregulated, and the purified VvpE appeared to dissolve established biofilms directly in a concentration-dependent manner in vitro. These results suggest that the host cell-induced SmcR enhances the detachment of V. vulnificus biofilms entering the host intestine and thereby may promote the dispersal of the pathogen to new colonization loci, which is crucial for pathogenesis.

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

Interaction between PGE2 and EGF receptor through MAPKs in mouse embryonic stem cell proliferation

Abstract.Identifying the small molecules that permit precise regulation of embryonic stem (ES) cell proliferation should further support our understanding of the underlying molecular mechanisms of self renewal. In the present study, we showed that PGE2 increased [3H]-thymidine incorporation in a time and dose dependent manner. In addition, PGE2 increased the expression of cell cycle regulatory proteins, the percentage of cells in S phase and the total number of cells. PGE2 obviously increased E-type prostaglandin (EP) receptor 1 mRNA expression level compare to 2, 3, 4 subtypes. EP1 antagonist also blocked PGE2-induced cell cycle regulatory protein expression and thymidine incorporation. PGE2 caused phosphorylation of protine kinase C, Src, epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation, and p44/42 mitogen-activated protein kinase (MAPK), which were blocked by each inhibitors. In conclusion, PGE2-stimulated proliferation is mediated by MAPK via EP1 receptor-dependent PKC and EGF receptor-dependent PI3K/Akt signaling pathways in mouse ES cells.

Fucoidan protects mesenchymal stem cells against oxidative stress and enhances vascular regeneration in a murine hindlimb ischemia model

BACKGROUND Mesenchymal stem cells (MSCs) have the potential to differentiate into multiple cell lineages. Given this potential for tissue regeneration, MSC-based therapeutic applications have been considered in recent years. However, ischemia-induced apoptosis has been reported to be one of the main causes of MSC death following transplantation. The primary objective of this study was to determine whether a natural antioxidant, fucoidan, could protect MSCs from ischemia-induced apoptosis in vitro and in vivo. Furthermore, we investigated the mechanism of action of fucoidans anti-ischemic effect in MSCs. METHODS AND RESULT Pre-treatment with fucoidan (10 μg/mL) suppressed the increase in H2O2-induced reactive oxygen species (ROS) levels and drastically reduced apoptotic cell death in MSCs. Fucoidan inhibited the activation of the pro-apoptotic proteins p38-mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and caspase-3, and augmented the expression of the anti-apoptosis protein cellular inhibitor of apoptosis (cIAP). Moreover, fucoidan significantly increased manganese superoxide dismutase (MnSOD) expression and decreased cellular ROS levels via the Akt pathway, resulting in enhanced cell survival. In a murine hindlimb ischemia model, transplanted fucoidan-treated MSCs showed significantly enhanced cell survival and proliferation in ischemic tissues. Functional recovery and limb salvage also remarkably improved in mice injected with fucoidan-stimulated MSCs compared with mice injected with non-stimulated MSCs. CONCLUSION Taken together, these results show that fucoidan protects MSCs from ischemia-induced cell death by modulation of apoptosis-associated proteins and cellular ROS levels through regulation of the MnSOD and Akt pathways, suggesting that fucoidan could be powerful therapeutic adjuvant for MSC-based therapy in ischemic diseases.

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.

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.

Fucoidan improves bioactivity and vasculogenic potential of mesenchymal stem cells in murine hind limb ischemia associated with chronic kidney disease

Chronic kidney disease (CKD) is a significant risk factor for cardiovascular and peripheral vascular disease. Although mesenchymal stem cell (MSC)-based therapy is a promising strategy for treatment of ischemic diseases associated with CKD, the associated pathophysiological conditions lead to low survival and proliferation of transplanted MSCs. To address these limitations, we investigated the effects of fucoidan, a sulfated polysaccharide, on the bioactivity of adipose tissue-derived MSCs and the potential of fucoidan-treated MSCs to improve neovascularization in ischemic tissues of CKD mice. Treatment of MSCs with fucoidan increased their proliferative potential and the expression of cell cycle-associated proteins, such as cyclin E, cyclin dependent kinase (CDK) 2, cyclin D1, and CDK4, via focal adhesion kinase and the phosphatidylinositol-4,5-bisphosphate 3-kinase-Akt axis. Moreover, fucoidan enhanced the immunomodulatory activity of MSCs through the ERK-IDO-1 signal cascade. Fucoidan was found to augment the proliferation, incorporation, and endothelial differentiation of transplanted MSCs at ischemic sites in CKD mice hind limbs. In addition, transplantation of fucoidan-treated MSCs enhanced the ratio of blood flow and limb salvage in CKD mice with hind limb ischemia. To our knowledge, our findings are the first to reveal that fucoidan enhances the bioactivity of MSCs and improves their neovascularization in ischemic injured tissues of CKD. In conclusion, fucoidan-treated MSCs may provide an important pathway toward therapeutic neovascularization in patients with CKD.

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.