Author Correction: Succinate promotes stem cell migration through the GPR91-dependent regulation of DRP1-mediated mitochondrial fission.

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Modulation of sonic hedgehog-induced mouse embryonic stem cell behaviours through E-cadherin expression and integrin ß1-dependent F-actin formation.

BACKGROUND AND PURPOSE: The sonic hedgehog pathway (Shh) plays a central role in maintaining stem cell function and behaviour in various processes related to self-renewal and tissue regeneration. However, the therapeutic effect of Shh on mouse embryonic stem cells (mESCs) has not yet been clearly elucidated. Thus, we investigated the effect of Shh on the regulation of mESC behaviour as well as the effect of Shh-pretreated mESCs in skin wound healing. EXPERIMENTAL APPROACH: The underlying mechanisms of Shh signalling pathway in growth and motility of mESCs were investigated using Western blot analysis, a cell proliferation assay and cell migration assay. In addition, the effect of Shh-pretreated mESCs in skin wound healing was determined using a mouse excisional wound splinting model. KEY RESULTS: Shh disrupted the adherens junction through proteolysis by activating MMPs. In addition, the release of ß-catenin from adherens junctions mediated by Shh led to cell cycle-dependent mESC proliferation. Shh-mediated Gli1 expression led to integrin ß1 up-regulation, followed by FAK and Src phosphorylation. Furthermore, among the Rho-GTPases, Rac1 and Cdc42 were activated in a Shh-dependent manner while F-actin expression was suppressed by Rac1 and Cdc42 siRNA transfection. Consistent with the in vitro results, the skin wound healing assay revealed that Shh-treated mESCs increased angiogenesis and skin wound repair compared to that in Shh-treated mESCs transfected with integrin ß1 siRNA in vivo. CONCLUSIONS AND IMPLICATIONS: Our results imply that Shh induces adherens junction disruption and integrin ß1-dependent F-actin formation by a mechanism involving FAK/Src and Rac1/Cdc42 signalling pathways in mESCs.

High Glucose-Induced Reactive Oxygen Species Stimulates Human Mesenchymal Stem Cell Migration Through Snail and EZH2-Dependent E-Cadherin Repression.

BACKGROUND/AIMS: Glucose plays an important role in stem cell fate determination and behaviors. However, it is still not known how glucose contributes to the precise molecular mechanisms responsible for stem cell migration. Thus, we investigate the effect of glucose on the regulation of the human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) migration, and analyze the mechanism accompanied by this effect. METHODS: Western blot analysis, wound healing migration assays, immunoprecipitation, and chromatin immunoprecipitation assay were performed to investigate the effect of high glucose on hUCB-MSC migration. Additionally, hUCB-MSC transplantation was performed in the mouse excisional wound splinting model. RESULTS: High concentration glucose (25 mM) elicits hUCB-MSC migration compared to normal glucose and high glucose-pretreated hUCB-MSC transplantation into the wound sites in mice also accelerates skin wound repair. We therefore elucidated the detailed mechanisms how high glucose induces hUCB-MSC migration. We showed that high glucose regulates E-cadherin repression through increased Snail and EZH2 expressions. And, we found high glucose-induced reactive oxygen species (ROS) promotes two signaling; JNK which regulates γ-secretase leading to the cleavage of Notch proteins and PI3K/Akt signaling which enhances GSK-3ß phosphorylation. High glucose-mediated JNK/Notch pathway regulates the expression of EZH2, and PI3K/Akt/GSK-3ß pathway stimulates Snail stabilization, respectively. High glucose enhances the formation of EZH2/Snail/HDAC1 complex in the nucleus, which in turn causes E-cadherin repression. CONCLUSION: This study reveals that high glucose-induced ROS stimulates the migration of hUCB-MSC through E-cadherin repression via Snail and EZH2 signaling pathways.

Succinate promotes stem cell migration through the GPR91-dependent regulation of DRP1-mediated mitochondrial fission.

The role of metabolites produced from stem cell metabolism has been emerged as signaling molecules to regulate stem cell behaviors such as migration. The mitochondrial morphology is closely associated with the metabolic balance and stem cell function. However, the physiological role of succinate on human mesenchymal stem cell (hMSC) migration by regulating the mitochondrial morphology remains unclear. Here, we investigate the effect of succinate on hMSC migration via regulation of mitochondrial dynamics and its related signaling pathway. Succinate (50 µM) significantly accelerates hMSC migration. Succinate increases phosphorylation of pan-PKC, especially the atypical PKCζ level which was blocked by the knockdown of Gαq and Gα12. Activated PKCζ subsequently phosphorylates p38 MAPK. Cytosolic DRP1 is phosphorylated by p38 MAPK and results in DRP1 translocation to the mitochondria outer membrane, eventually inducing mitochondrial fragmentation. Mitochondrial fission-induced mitochondrial function elevates mitochondrial ROS (mtROS) levels and activates Rho GTPases, which then induces F-actin formation. Furthermore, in a skin excisional wound model, we found the effects of succinate-pretreated hMSC enhanced wound closure, vascularization and re-epithelialization and confirmed that DRP1 has a vital role in injured tissue regeneration. Overall, succinate promotes DRP1-mediated mitochondrial fission via GPR91, consequently stimulating the hMSC migration through mtROS-induced F-actin formation.

Membrane-Associated Effects of Glucocorticoid on BACE1 Upregulation and Aß Generation: Involvement of Lipid Raft-Mediated CREB Activation.

Glucocorticoid has been widely accepted to induce Alzheimer's disease, but the nongenomic effect of glucocorticoid on amyloid ß (Aß) generation has yet to be studied. Here, we investigated the effect of the nongenomic pathway induced by glucocorticoid on amyloid precursor protein processing enzymes as well as Aß production using male ICR mice and human neuroblastoma SK-N-MC cells. Mice groups exposed to restraint stress or intracerebroventricular injection of Aß showed impaired cognition, decreased intracellular glucocorticoid receptor (GR) level, but elevated level of membrane GR (mGR). In this respect, we identified the mGR-dependent pathway evoked by glucocorticoid using impermeable cortisol conjugated to BSA (cortisol-BSA) on SK-N-MC cells. Cortisol-BSA augmented the expression of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), the level of C-terminal fragment ß of amyloid precursor protein (C99) and Aß production, which were maintained even after blocking intracellular GR. We also found that cortisol-BSA enhanced the interaction between mGR and Gαs, which colocalized in the lipid raft. The subsequently activated CREB by cortisol-BSA bound to the CRE site of the BACE1 promoter increasing its expression, which was downregulated by inhibiting CBP. Consistently, blocking CBP attenuated cognitive impairment and Aß production induced by corticosterone treatment or intracerebroventricular injection of Aß more efficiently than inhibiting intracellular GR in mice. In conclusion, glucocorticoid couples mGR with Gαs and triggers cAMP-PKA-CREB axis dependent on the lipid raft to stimulate BACE1 upregulation and Aß generation.SIGNIFICANCE STATEMENT Patients with Alzheimer's disease (AD) have been growing sharply and stress is considered as the major environment factor of AD. Glucocorticoid is the primarily responsive factor to stress and is widely known to induce AD. However, most AD patients usually have impaired genomic pathway of glucocorticoid due to intracellular glucocorticoid receptor deficiency. In this respect, the genomic mechanism of glucocorticoid faces difficulties in explaining the consistent amyloid ß (Aß) production. Therefore, it is necessary to investigate the novel pathway of glucocorticoid on Aß generation to find a more selective therapeutic approach to AD patients. In this study, we revealed the importance of nongenomic pathway induced by glucocorticoid where membrane glucocorticoid receptor plays an important role in Aß formation.

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 19kDa 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.

Aß-Induced Drp1 phosphorylation through Akt activation promotes excessive mitochondrial fission leading to neuronal apoptosis.

Mitochondrial dysfunction is known as one of causative factors in Alzheimer's disease (AD), inducing neuronal cell death. Mitochondria regulate their functions through changing their morphology. The present work was undertaken to investigate whether Amyloid ß (Aß) affects mitochondrial morphology in neuronal cells to induce apoptosis. Aß treatment induced not only the fragmentation of mitochondria but also neuronal apoptosis in association with an increase in caspase-9 and -3 activity. Calcium influx induced by Aß up-regulated the activation of Akt through CaMKII resulting in changes to the phosphorylation level of Drp1 in a time-dependent manner. Translocation of Drp1 from the cytosol to mitochondria was blocked by CB-124005 (an Akt inhibitor). Recruitment of Drp1 to mitochondria led to ROS generation and mitochondrial fission, accompanied by dysfunction of mitochondria such as loss of membrane potential and ATP production. ROS generation and mitochondrial dysfunction by Aß were attenuated when treated with Mdivi-1, a selective Drp1 inhibitor. Furthermore, the sustained Akt activation induced not only the fragmentation of mitochondria but also the activation of mTOR, eventually suppressing autophagy. Inhibition of autophagic clearance of Aß led to increased ROS levels and aggravating mitochondrial defects, which were blocked by Rapamycin (an mTOR inhibitor). In conclusion, sustained phosphorylation of Akt by Aß directly activates Drp1 and inhibits autophagy through the mTOR pathway. Together, these changes elicit abundant mitochondrial fragmentation resulting in ROS-mediated neuronal apoptosis.

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.

Monodisperse polystyrene-silica core-shell particles and silica hollow spheres prepared by the Stöber method.

Monodisperse polystyrene (PS) bead particles (diameter approximately 750 nm) have been synthesized with an initiator, 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AIBA), using a surfactant-free emulsion polymerization. The PS particles charged positively by the cationic initiator exhibit uniform size with a narrow size distribution. The Stöber method was adopted to coat silica on the surface of the PS particles. The silica coatings were performed on different conditions; pH (10.0-12.5), tetraethylorthosilicate (TEOS) concentration (10-40 mM), and reaction time (1-5 h). The reaction rates are too low at low pH (10.0) so that no or little silica is coated on the PS bead particles. At high pH condition (approximately 12.5) relatively rough silica shell and heterogeneous nucleation of silica colloids were observed due to high reaction rate of TEOS hydrolysis and condensation reaction. Smooth surface with uniform thickness of silica shells were obtained at a pH value around 11. Also, the thickness of silica coated on the PS spheres increases with increase of TEOS concentration and reaction time. Silica hollow spheres were also obtained by calcination of the PS-silica core-shell particles.