Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with Gαq.

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.

Lysophosphatidic Acid Receptor 1 Is Important for Intestinal Epithelial Barrier Function and Susceptibility to Colitis.

Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1-/- (Lpa1-/-). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1-/- mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1-/- mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1-/- mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor-mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.

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.

Enhancement of high glucose-induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2 /Akt/NF-κB pathway.

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM-associated neuronal cell death. Previous investigators reported on a genome-wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose-induced neuronal cell death and the effect of melatonin against high glucose-induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN-induced putative kinase 1 (PINK1) and LC-3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker™ fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V-positive cells. In addition, high glucose-stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N-acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT2 receptor-specific inhibitor 4-P-PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin-regulated mitochondrial ROS production, cleaved caspase-3 and caspase-9 expressions, and the number of annexin V-positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT2 /Akt/NF-κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions.

Vibrio vulnificus VvpE Stimulates IL-1ß Production by the Hypomethylation of the IL-1ß Promoter and NF-κB Activation via Lipid Raft-Dependent ANXA2 Recruitment and Reactive Oxygen Species Signaling in Intestinal Epithelial Cells.

An inflammatory response is a hallmark of necrosis evoked by bacterial pathogens. Vibrio vulnificus, VvpE, is an elastase that is responsible for tissue necrosis and inflammation; however, the molecular mechanism by which it regulates host cell death has not been characterized. In the present study, we investigate the cellular mechanism of VvpE with regard to host cell death and the inflammatory response of human intestinal epithelial (INT-407) cells. The recombinant protein (r)VvpE (50 pg/ml) caused cytotoxicity mainly via necrosis coupled with IL-1ß production. The necrotic cell death induced by rVvpE is highly susceptible to the knockdown of annexin A (ANXA)2 and the sequestration of membrane cholesterol. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into membrane lipid rafts coupled with ANXA2 to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of redox-sensitive transcription factor NF-κB. The silencing of NF-κB inhibited IL-1ß production during necrosis. rVvpE induced hypomethylation and region-specific transcriptional occupancy by NF-κB in the IL-1ß promoter and has the ability to induce pyroptosis via NOD-, LRR-, and pyrin domain-containing 3 inflammasome. In a mouse model of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus negated the proinflammatory responses and maintained the physiological levels of the proliferation and migration of enterocytes. These results demonstrate that VvpE induces the hypomethylation of the IL-1ß promoter and the transcriptional regulation of NF-κB through lipid raft-dependent ANXA2 recruitment and ROS signaling to promote IL-1ß production in intestinal epithelial cells.

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 GSK3ß was followed by ß-catenin nuclear translocation in UCB-MSCs. Activation of ß-catenin was blocked by PKC inhibitors, and OA-induced EphB2 expression was suppressed by ß-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/GSK3ß/ß-catenin and Rac1 signaling pathways.

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 ß-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.

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.

The anti-allergic activity of Lactobacillus plantarum L67 and its application to yogurt.

Recently, interest in the beneficial role of probiotics in the protection and management of allergic diseases caused by immune disorders has been increasing. This study investigated the inhibitory effect of Lactobacillus plantarum L67 on induced allergic inflammatory response in bisphenol A-treated rat basophilic leukemia 2H3 (RBL-2H3) cells and mouse splenocytes. We also evaluated the applicability of L. plantarum L67 as a yogurt starter culture. We measured the ability of Lactobacillus strains to induce the production of IL-12 and IFN- γ in cultured splenocytes by ELISA. Bisphenol A (50µM)-treated RBL-2H3 cells were cotreated with a glycoprotein (18kDa) isolated from L. plantarum L67 (5-100µg/mL) for 30min. We measured the expression of mitogen-activated protein kinase (ERK and p38), AP-1 (c-Fos and c-Jun), T-bet, and GATA-binding protein 3 (GATA-3) using Western blotting to examine the differentiation of T helper cells. Furthermore, we evaluated the gene expression of IL-1ß, IL-6, and IL-10 using real-time quantitative PCR. Finally, we evaluated the applicability of L. plantarum L67 as a yogurt starter by measuring pH, enumeration of bacteria, and sensory scores. Our results showed that L67 protein inhibited the phosphorylation of ERK and p38 mitogen-activated protein kinase through the transcriptional activation of AP-1 in bisphenol A-treated RBL-2H3 cells. During differentiation of T helper cells, the expression of transcription factor GATA-3 was significantly suppressed by L67 protein (100µg/mL) treatment, whereas expression of transcription factor T-bet was increased. In addition, the L67 protein significantly attenuated the expression of T helper 2-linked cytokines IL-1ß, IL-6, and IL-10. These results indicate that L. plantarum L67, made available as yogurt starters and dietary supplements, has the potential to prevent allergy-related immune disorders.

Regulation of hypoxia-inducible factor 1α (HIF-1α) by lysophosphatidic acid is dependent on interplay between p53 and Krüppel-like factor 5.

Hypoxia-inducible factor 1α (HIF-1α) and p53 are pivotal regulators of tumor growth. Lysophosphatidic acid (LPA) is a lipid mediator that functions as a mitogen by acting through LPA receptors. We have shown previously that LPA stimulates HIF-1α expression in colon cancer cells. To determine the mechanism of HIF-1α induction by LPA, we compared the effect of LPA on HIF-1α in several colon cancer cell lines. LPA transcriptionally induced HIF-1α in colon cancer cells. HIF-1α induction was observed in cells expressing WT p53, where LPA decreased p53 expression. However, LPA failed to induce HIF-1α when the p53 gene was mutated. A decrease in p53 expression was dependent on induction of p53-specific E3 ubiquitin ligase Mdm2 by LPA. Krüppel-like factor 5 (KLF5) is an effector of LPA-induced proliferation of colon cancer cells. Because HIF-1α was necessary for LPA-induced growth of colon cancer cells, we determined the relationship between KLF5 and HIF-1α by a loss-of-function approach. Silencing of KLF5 inhibited LPA-induced HIF-1α induction, suggesting that KLF5 is an upstream regulator of HIF-1α. KLF5 and p53 binding to the Hif1α promoter was assessed by ChIP assay. LPA increased the occupancy of the Hif1α promoter by KLF5, while decreasing p53 binding. Transfection of HCT116 cells with KLF5 or p53 attenuated the binding of the other transcription factor. These results identify KLF5 as a transactivator of HIF-1α and show that LPA regulates HIF-1α by dynamically modulating its interaction with KLF5 and p53.

ZPDC glycoprotein (24 kDa) induces apoptosis and enhances activity of NK cells in N-nitrosodiethylamine-injected Balb/c.

Natural killer (NK) cells have anti-tumor activity in hepatocellular carcinoma (HCC) using secreting granules and cytotoxic ability. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC) has anti-oxidant effect and anti-cancer effect. The objective of this study was to determine whether ZPDC glycoprotein enhances activity of NK cells and induces apoptosis of liver cancer cells in diethylnitrosamine (DEN)-treated Balb/c mice. This study evaluated the secreting of perforin and granzyme B and cytotoxicity of NK cells, interleukin (IL)-2 and IL-12, apoptosis-related factors (bid, cytochrome c, and caspase-3) in liver tissue using Immunoblot and ELISA. The results demonstrated that ZPDC glycoprotein (20mg/kg, BW) induces secretion of perforin and granzyme B and NK cells activity. Also, it induces expression of apoptosis-related factors (bid, cytochrome c, and caspase-3) in liver tissues. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatocarcinogenesis without immunosuppression.

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.

Preventive effects of ZPDC glycoprotein (24 kDa) on hepatotoxicity induced by mercury chloride in vitro and in vivo.

Mercury is a potent environmental contaminant that exerts toxic effect on various vital organs in the human body. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC), which has antioxidant and anticancer effects. In the present study, we determined the preventive effects of ZPDC glycoprotein on hepatic damage induced by mercury chloride (HgCl2 ). We evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), cyclo-oxygenase (COX-2), inducible nitric oxide synthetase (iNOS), and activator protein (AP-1) and the quantitative expressions of nuclear factor E2-related factor (Nrf2), heme oxygenase (HO-1), metallothionein (MT) and reduced glutathione (GSH) in mercury-chloride-exposed (50 µM and 10 mg/kg body weight) primary cultured hepatocytes and ICR mice, using biochemical assays, radioactivity and immunoblot analysis. The results demonstrated that ZPDC glycoprotein decreased the levels of LDH, ALT, HO-1 and MT, whereas it increased the activities of hepatic antioxidant enzymes (SOD, CAT and GPx) and reduced GSH in mercury-chloride-exposed primary cultured hepatocytes. Also, it suppressed arachidonic acid release and expression of ERK, p38 MAPK, COX-2, iNOS, AP-1 and Nrf-2 in primary cultured hepatocytes and ICR mice exposed to mercury chloride. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatotoxicity induced by mercury chloride.

Broad-temperature-range mechanically tunable hydrogel microcapsules for controlled active release.

Here, we report PNIPAm-co-PEGDA hydrogel shelled microcapsules with a thin oil layer to achieve tunable thermo-responsive release of the encapsulated small hydrophilic actives. We use a microfluidic device integrated with a temperature-controlled chamber for consistent and reliable production of the microcapsules by utilizing triple emulsion drops (W/O/W/O) with a thin oil layer as capsule templates. The interstitial oil layer between the aqueous core and the PNIPAm-co-PEGDA shell provides a diffusion barrier for the encapsulated active until the temperature reaches a critical point above which the destabilization of interstitial oil layer occurs. We find that the destabilization of the oil layer with temperature increase is caused by outward expansion of the aqueous core due to volume increase and the radial inward compression from the deswelling of the thermo-responsive hydrogel shell. The copolymerization of NIPAm with PEGDA increases the biocompatibility of the resulting microcapsule while offering the ability to alter the compressive modulus in broad ranges by simply varying crosslinker concentrations thereby to precisely tune the onset release temperature. Based on this concept, we further demonstrate that the release temperature can be enhanced up to 62 °C by adjusting the shell thickness even without varying the chemical composition of the hydrogel shell. Moreover, we incorporate gold nanorods within the hydrogel shell to spatiotemporally regulate the active release from the microcapsules by illuminating with non-invasive near infrared (NIR) light.

MAGI-3 competes with NHERF-2 to negatively regulate LPA2 receptor signaling in colon cancer cells.

BACKGROUND & AIMS: Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA(2)) is overexpressed in colon tumors. LPA(2) interacts with membrane-associated guanylate kinase with inverted orientation-3 (MAGI-3) and the Na+/H+ exchanger regulatory factor 2 (NHERF-2), but the biological effects of these interactions are unknown. We investigated the roles of MAGI-3 and NHERF-2 in LPA(2)-mediated signaling in human colon cancer cells. METHODS: We overexpressed or knocked down MAGI-3 in HCT116 and SW480 cells. The effects of MAGI-3 and NHERF-2 in LPA-induced cell migration, invasion, inositol phosphate generation, and nuclear factor-κB activation were determined. Expression of MAGI-3 and NHERF-2 in human colon tumor tissues was analyzed using tissue microarray analysis. RESULTS: NHERF-2 promoted migration and invasion of colon cancer cells, whereas MAGI-3 inhibited these processes. MAGI-3 competed with NHERF-2 for binding to LPA(2) and phospholipase C-ß3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA(2)-induced phospholipase C activity. MAGI-3 increased the interaction of LPA(2) with Gα(12), whereas NHERF-2 preferentially promoted interaction between LPA(2) and Gα(q). MAGI-3 decreased the tumorigenic capacity of LPA(2) by attenuating the activities of nuclear factor-κB and c-Jun N-terminal kinase. MAGI-3 and NHERF-2 were expressed differentially in colon adenocarcinomas, consistent with their opposing effects. CONCLUSIONS: LPA(2) is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA(2) signaling.

Oral delivery of stem-cell-loaded hydrogel microcapsules restores gut inflammation and microbiota.

Mesenchymal stem cells (MSCs) are an attractive candidate for the treatment of inflammatory bowel disease (IBD), but their poor delivery rate to an inflamed colon is a major factor hampering the clinical potential of stem cell therapies. Moreover, there remains a formidable hurdle to overcome with regard to survival and homing in to injured sites. Here, we develop a strategy utilizing monodisperse hydrogel microcapsules with a thin intermediate oil layer prepared by a triple-emulsion drop-based microfluidic approach as an in-situ oral delivering carrier. The oral delivery of stem-cell-loaded hydrogel microcapsules (SC-HM) enhances MSC survival and retention in the hostile stomach environment due to the intermediate oil layer and low value of the overall stiffness, facilitating programmable cell release during gastrointestinal peristalsis. SC-HM is shown to induce tissue repair, reduce the colonic macrophage infiltration responsible for the secretion of the pro-inflammatory factors, and significantly mitigate the severity of IBD in a mouse model, where MSCs released by SC-HM successfully accumulate at the colonic crypt. Moreover, a metagenomics analysis reveals that SC-HM ameliorates the dysbiosis of specific bacterial genera, including Bacteroides acidifaciens, Lactobacillus (L.) gasseri, Lactobacillus reuteri, and L. intestinalis, implying optimization of the microorganism's composition and abundance. These findings demonstrate that SC-HM is a potential IBD treatment candidate.

Cell-Inspired Hydrogel Microcapsules with a Thin Oil Layer for Enhanced Retention of Highly Reactive Antioxidants.

In nature, individual cells are compartmentalized by a membrane that protects the cellular elements from the surrounding environment while simultaneously equipped with an antioxidant defense system to alleviate the oxidative stress resulting from light, oxygen, moisture, and temperature. However, this mechanism has not been realized in cellular mimics to effectively encapsulate and retain highly reactive antioxidants. Here, we report cell-inspired hydrogel microcapsules with an interstitial oil layer prepared by utilizing triple emulsion drops as templates to achieve enhanced retention of antioxidants. We employ ionic gelation for the hydrogel shell to prevent exposure of the encapsulated antioxidants to free radicals typically generated during photopolymerization. The interstitial oil layer in the microcapsule serves as an stimulus-responsive diffusion barrier, enabling efficient encapsulation and retention of antioxidants by providing an adequate pH microenvironment until osmotic pressure is applied to release the cargo on-demand. Moreover, addition of a lipophilic reducing agent in the oil layer induces a complementary reaction with the antioxidant, similar to the nonenzymatic antioxidant defense system in cells, leading to enhanced retention of the antioxidant activity. Furthermore, we show the complete recovery and even further enhancement in antioxidant activity by lowering the storage temperature, which decreases the oxidation rate while retaining the complementary reaction with the lipophilic reducing agent.

Grifola frondosa Extract Containing Bioactive Components Blocks Skin Fibroblastic Inflammation and Cytotoxicity Caused by Endocrine Disrupting Chemical, Bisphenol A.

Grifola frondosa (GF), a species of Basidiomycotina, is widely distributed across Asia and has been used as an immunomodulatory, anti-bacterial, and anti-cancer agent. In the present study, the pharmacological activity of the GF extract against an ecotoxicological industrial chemical, bisphenol A (BPA) in normal human dermal fibroblasts (NHDFs), was investigated. GF extract containing naringin, hesperidin, chlorogenic acid, and kaempferol showed an inhibitory effect on cell death and inflammation induced by BPA in the NHDFs. For the cell death caused by BPA, GF extract inhibited the production of reactive oxygen species responsible for the unique activation of the extracellular signal-regulated kinase. In addition, GF extract attenuated the expression of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) and the pro-inflammatory cytokine IL-1ß by the suppression of the redox-sensitive transcription factor, nuclear factor-kappa B (NF-κB) in BPA-treated NHDFs. For the inflammation triggered by BPA, GF extract blocked the inflammasome-mediated caspase-1 activation that leads to the secretion of IL-1ß protein. These results indicate that the GF extract is a functional antioxidant that prevents skin fibroblastic pyroptosis induced by BPA.

Lysophosphatidic acid 5 receptor induces activation of Na(+)/H(+) exchanger 3 via apical epidermal growth factor receptor in intestinal epithelial cells.

Na(+) absorption is a vital process present in all living organisms. We have reported previously that lysophosphatidic acid (LPA) acutely stimulates Na(+) and fluid absorption in human intestinal epithelial cells and mouse intestine by stimulation of Na(+)/H(+) exchanger 3 (NHE3) via LPA(5) receptor. In the current study, we investigated the mechanism of NHE3 activation by LPA(5) in Caco-2bbe cells. LPA(5)-dependent activation of NHE3 was blocked by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and U0126, but not by phosphatidylinositol 3-kinase inhibitor LY294002 or phospholipase C-ß inhibitor U73122. We found that LPA(5) transactivated the epidermal growth factor receptor (EGFR) and that inhibition of EGFR blocked LPA(5)-dependent activation of NHE3, suggesting an obligatory role of EGFR in the NHE3 regulation. Confocal immunofluorescence and surface biotinylation analyses showed that LPA(5) was located mostly in the apical membrane. EGFR, on the other hand, showed higher expression in the basolateral membrane. However, inhibition of apical EGFR, but not basolateral EGFR, abrogated LPA-induced regulation of MEK and NHE3, indicating that LPA(5) selectively activates apical EGFR. Furthermore, transactivation of EGFR independently activated the MEK-ERK pathway and proline-rich tyrosine kinase 2 (Pyk2). Similarly to MEK inhibition, knockdown of Pyk2 blocked activation of NHE3 by LPA. Furthermore, we showed that RhoA and Rho-associated kinase (ROCK) are involved in activation of Pyk2. Interestingly, LPA(5) did not directly activate RhoA but was required for transactivation of EGFR. Together, these results unveil a pivotal role of apical EGFR in NHE3 regulation by LPA and show that the RhoA-ROCK-Pyk2 and MEK-ERK pathways converge onto NHE3.

Nanospheres loaded with curcumin promote gut epithelial motility through F-actin-related migration signaling events.

Curcumin, a hydrophobic polyphenol of turmeric, has a variety of biological functions as an herbal supplement, but its poor gastric absorption rate is one of the major factors limiting its oral bioavailability. In the present study, we investigated the functional role of nanospheres loaded with curcumin (nCur) with regard to the motility of gut epithelial HCT116 cells and enterocyte migration along the crypt-villus axis. nCur significantly increased the motility of HCT116 cells and showed much higher migration efficacy than the curcumin. nCur stimulated the small GTPases Rac1 and the phosphorylation of protein kinase C, responsible for the distinctive activation of the mitogen-activated protein kinases. Interestingly, nCur significantly induced the expression of α-actinin, profilin-1, and filamentous (F)-actin as regulated by the phosphorylation of nuclear factor-kappa B during its promotion of cell migration. In mouse models of gut epithelial migration, treatment with nCur had an enhancing effect on the movement of enterocytes along the crypt-villus axis and the expression of cytoskeletal reorganization-related factors. These results indicate that nCur is a functional agent that promotes gut epithelial motility through F-actin-related migration signaling events.