The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation.

Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/ß-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/ß-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.

Genkwadaphnin promotes leukocyte migration by increasing CD44 expression via PKD1/NF-κB signaling pathway.

Genkwadaphnin (GD), an extract from the flower buds of Daphne genkwa Siebold & Zucc. (Thymelaeaceae) has been reported a significant anti-leukemic activity. However, its functional mechanism has not been defined well. To study the biological mechanism of GD function, we have investigated whether GD affects CD44 expression, which has a role in the regulation of immune cell motilities, and identified the related signaling pathways. GD treatment induced the increase of CD44 expression in a time- and concentration-dependent manner, which was specific for immune cells. GD activated PKD1/NF-κB signaling to induce CD44 expression, and resulted in the increased migration of K562 cells. In invasion assay, cell migratory ability was induced by GD and the transfection with CD44-specific short hairpin RNA resulted in reduction of its cell migration. GD treated human peripheral blood mononuclear cell (PBMC) were also shown the increased CD44 expression and migration. These data suggest that the induction of CD44 expression by GD treatment promotes immune cell transmigration resulting in the enhanced innate immunity.

PRDM1, a Tumor-Suppressor Gene, is Induced by Genkwadaphnin in Human Colon Cancer SW620 Cells.

Genkwadaphnin (GD-1) is isolated from the flower buds of Daphne genkwa Siebold et Zuccarini (Thymelaeaceae), and it has been used as a traditional Korean and Chinese medicine. In this study, the authors observe that GD-1 inhibits the growth of the colon cancer cell line, SW620, through the up-regulation of p21 expression in a PRDM1-dependent manner. After treatment with GD-1, the transcriptional repressor PRDM1 is prominently induced in SW620 cells. Furthermore, GD-1 induce the phosphorylation of PKD1 and MEK and subsequently provide PRDM1 enhancement, resulting in the suppression of c-Myc expression and the up-regulation of p21. PKD1 knockdown using siRNA abrogates PRDM1 expression by GD-1 and subsequently disrupts the regulation of c-Myc and p21 expression. Treating SW620 cells with GD-1 inhibits cell-cycle progression and is characterized by the down-regulation of c-Myc followed by the up-regulation of p21 expression. The up-regulation of p21 by GD-1 induces the growth arrest of the SW620 colon cancer cell line. Based on these data, the authors propose that GD-1 has tumor-suppressor activity that may contribute to the anti-tumor effects of PRDM1 in colon cancer.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (EC-18) Modulates Th2 Immunity through Attenuation of IL-4 Expression.

Controlling balance between T-helper type 1 (Th1) and T-helper type 2 (Th2) plays a pivotal role in maintaining the biological rhythm of Th1/Th2 and circumventing diseases caused by Th1/Th2 imbalance. Interleukin 4 (IL-4) is a Th2-type cytokine and often associated with hypersensitivity-related diseases such as atopic dermatitis and allergies when overexpressed. In this study, we have tried to elucidate the function of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (EC-18) as an essential modulator of Th1/Th2 balance. EC-18 has showed an inhibitory effect on the production of IL-4 in a dose-dependent manner. RT-PCR analysis has proved EC-18 affect the transcription of IL-4. By analyzing the phosphorylation status of Signal transducer and activator of transcription 6 (STAT6), which is a transcriptional activator of IL-4 expression, we discovered that EC-18 induced the decrease of STAT6 activity in several stimulated cell lines, which was also showed in STAT6 reporter analysis. Co-treatment of EC-18 significantly weakened atopy-like phenotypes in mice treated with an allergen. Collectively, our results suggest that EC-18 is a potent Th2 modulating factor by regulating the transcription of IL-4 via STAT6 modulation, and could be developed for immune-modulatory therapeutics.

Genkwadaphnin induces IFN-γ via PKD1/NF-κB/STAT1 dependent pathway in NK-92 cells.

The flower buds of Daphne genkwa Sieb. et Zucc. have been used as a traditional Chinese medicine although their functional mechanisms have not been discovered yet. We have studied the potential effects of the plant extracts on natural killer (NK) cell activation, and isolated an active fraction. Genkwadaphnin (GD-1) displayed a potent efficacy to induce IFN-γ transcription in NK cells with concentration- and time-dependent manners. GD-1 treatment triggered the phosphorylation of PKD1, a member of PKC family, MEK and ERK, resulting in IKK activation to induce IκB degradation, and the nuclear localization of p65, an NF-κB subunit, which regulates IFN-γ transcription. GD-1 effect on IFN-γ production was blocked by the addition of Rottlerin, a PKC inhibitor, CID 755673, a PKD inhibitor, or Bay11-7082, an IKKα inhibitor. The nuclear localization of p65 was also inhibited by the kinase inhibitors. Secreted IFN-γ activates STAT1 phosphorylation as autocrine-loops to sustain its secretion. GD-1 induced the phosphorylation of STAT1 probably through the increase of IFN-γ. STAT1 inhibitor also abrogated the sustained IFN-γ secretion. These results suggest that GD-1 is involved in the activation of PKD1 and/or ERK pathway, which activate NK-κB triggering IFN-γ production. As positive feedback loops, secreted IFN-γ activates STAT1 and elongates its production in NK-92 cells.

Genome-wide reorganization of histone H2AX toward particular fragile sites on cell activation.

γH2AX formation by phosphorylation of the histone variant H2AX is the key process in the repair of DNA lesions including those arising at fragile sites under replication stress. Here we demonstrate that H2AX is dynamically reorganized to preoccupy γH2AX hotspots on increased replication stress by activated cell proliferation and that H2AX is enriched in aphidicolin-induced replisome stalling sites in cycling cells. Interestingly, H2AX enrichment was particularly found in genomic regions that replicate in early S phase. High transcription activity, a hallmark of early replicating fragile sites, was a determinant of H2AX localization. Subtelomeric H2AX enrichment was also attributable to early replication and high gene density. In contrast, late replicating and infrequently transcribed regions, including common fragile sites and heterochromatin, lacked H2AX enrichment. In particular, heterochromatin was inaccessible to H2AX incorporation, maybe partly explaining the cause of mutation accumulation in cancer heterochromatin. Meanwhile, H2AX in actively dividing cells was intimately colocalized with INO80. INO80 silencing reduced H2AX levels, particularly at the INO80-enriched sites. Our findings suggest that active DNA replication is accompanied with the specific localization of H2AX and INO80 for efficient damage repair or replication-fork stabilization in actively transcribed regions.

NDRG2 positively regulates E-cadherin expression and prolongs overall survival in colon cancer patients.

To discover the molecular mechanism of N-Myc downstream-regulated gene 2 (NDRG2), a newly found differentiation-related tumor suppressor, the relationships between NDRG2 and E-cadherin were investigated in tumor cells and tissues. Positive correlations between the expression of E-cadherin and NDRG2 were shown in several colon cancer cell lines as well as in colon cancer tissues. According to the transcription assays using a reporter plasmid containing E-cadherin promoter region (-368~+51), NDRG2 introduction into colon cancer cell lines induced upregulation of E-cadherin promoter activity and its transcription. On the contrary, inhibition of NDRG2 expression by siRNA treatment caused the decrease of E-cadherin transcription. Snail, a zinc-finger transcriptional repressor, was shown to be a mediator of NDRG2-regulated E-cadherin expression. The enhancement of glycogen synthase kinase 3ß (GSK-3ß) activity by NDRG2 overexpression caused proteasomal degradation of Snail transcription factor followed by transcriptional de-repression of E-cadherin. We also found that NDRG2 could mediate cell density-regulated E-cadherin expression. The increase of NDRG2 expression with cell density preceded E-cadherin expression, and the regulation of Snail activity by GSK-3ß was also related to this process.

Collagen triple helix repeat containing-1 (CTHRC1) expression in invasive ductal carcinoma of the breast: the impact on prognosis and correlation to clinicopathologic features.

CTHRC1 has been known as a regulator of collagen expression and cell migration. The aim of this research was to clarify the clinicopathologic significance of CTHRC1 expression in human breast cancer. 22 cases of breast cancer tissues, randomly selected from clinically diagnosed patients, showed a significant increase of CTHRC1 mRNA expression compared to the normal tissue from the same patients using RT-PCR and real-time PCR. Additionally we investigated breast cancers from 189 patients by immunohistochemistry (IHC). A high level of CTHRC1 expression was observed in 111 (58.7 %) out of 189 breast cancer patients and the expression was significantly correlated with histologic grade (P = 0.026), nodal status (P < 0.001), and TNM pathologic stage (P = 0.002). High CTHRC1 expression was associated with a shorter recurrence free survival (P = 0.008). Taken together, the results showed that CTHRC1 over-expression was significantly associated with clinicopathological factors of poor prognosis in invasive ductal carcinoma. CTHRC1 could be used as a supplementary prognostic biomarker and a potential therapeutic target in breast cancer.

Enhancement of Transgene Expression by HDAC Inhibitors in Mouse Embryonic Stem Cells.

Embryonic stem (ES) cells can self-renew and differentiate to various cells depending on the culture condition. Although ES cells are a good model for cell type specification and can be useful for application in clinics in the future, studies on ES cells have many experimental restraints including low transfection efficiency and transgene expression. Here, we observed that transgene expression after transfection was enhanced by treatment with histone deacetylse (HDAC) inhibitors such as trichostatin A, sodium butyrate, and valproic acid. Transfection was performed using conventional transfection reagents with a retroviral vector encoding GFP under the control of CMV promoter as a reporter. Treatment of ES cells with HDAC inhibitors after transfection increased population of GFP positive cells up to 180% compared with untreated control. ES cells showed normal expression of stem cell markers after treatment with HDAC inhibitors. Transgene expression was further enhanced by modifying transfection procedure. GFP positive cells selected after transfection were proved to have the stem cell properties. Our improved protocol for enhanced gene delivery and expression in mouse ES cells without hampering ES cell properties will be useful for study and application of ES cells.

Expression of endothelial cell-specific molecule-1 regulated by hypoxia inducible factor-1α in human colon carcinoma: impact of ESM-1 on prognosis and its correlation with clinicopathological features.

Based on a previous finding that endothelial cell-specific molecule-1 (ESM-1) is a potential serum marker for colorectal cancer (CRC), the aim of this study was to clarify the clinicopathological significance of ESM-1 expression in CRC, and to explore the correlation between ESM-1 and HIF-1α in the tumorigenesis of CRC related to hypoxic conditions. ESM-1 mRNA expression was examined in CRC and corresponding normal mucosal tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. This experiment confirmed that ESM-1 levels were high in CRC. We screened the tissue samples of 143 CRC patients. By immunohistochemistry, we determined that the ESM-1 immunoreactivity was significantly correlated with the tumor size, depth of invasion, nodal status, distant metastasis and Dukes' stage, and was an independent prognostic factor for disease recurrence and worse survival outcome (P=0.001). The modulation of ESM-1 under hypoxia was investigated, and it was confirmed that ESM-1 expression was induced by HIF1-α and significantly attenuated by small interfering RNA (siRNA) targeting HIF-1α in CRC cells. These results showed that ESM-1 is significantly overexpressed, which is regulated by HIF-1α in CRC patients, and can be used as a potential biomarker and a therapeutic target for CRC.

Tigliane diterpene esters with IFN γ-inducing activity from the leaves of Aleurites fordii.

Bioactivity-guided fractionation on the leaves of Aleurites fordii led to the isolation of a new tigliane diterpene ester, 12-O-hexadecanoyl-7-oxo-5-ene-16-hydroxyphorbol-13-acetate (1) along with four known compounds, 12-O-hexadecanoyl-7-oxo-5-ene-phorbol-13-acetate (2), 12-O-hexadecanoyl-phorbol-13-acetate (3), 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (4), and 12-O-hexadecanoyl-4-deoxy-4α-16-hydroxyphorbol-13-acetate (5). The structures of these compounds were determined by interpretation of NMR (1D and 2D) spectroscopic data and MS data. All the isolates were evaluated for their effects on the induction of IFN-γ in NK92 cells. Compounds 3 and 4 exhibited the most potent responses in IFN-γ induction, comparable to the positive control, phorbol 12-myristate 13-acetate (PMA).

Genetic and metabolic characterization of insomnia.

Insomnia is reported to chronically affect 10∼15% of the adult population. However, very little is known about the genetics and metabolism of insomnia. Here we surveyed 10,038 Korean subjects whose genotypes have been previously profiled on a genome-wide scale. About 16.5% reported insomnia and displayed distinct metabolic changes reflecting an increase in insulin secretion, a higher risk of diabetes, and disrupted calcium signaling. Insomnia-associated genotypic differences were highly concentrated within genes involved in neural function. The most significant SNPs resided in ROR1 and PLCB1, genes known to be involved in bipolar disorder and schizophrenia, respectively. Putative enhancers, as indicated by the histone mark H3K4me1, were discovered within both genes near the significant SNPs. In neuronal cells, the enhancers were bound by PAX6, a neural transcription factor that is essential for central nervous system development. Open chromatin signatures were found on the enhancers in human pancreas, a tissue where PAX6 is known to play a role in insulin secretion. In PLCB1, CTCF was found to bind downstream of the enhancer and interact with PAX6, suggesting that it can probably inhibit gene activation by PAX6. PLCB4, a circadian gene that is closely located downstream of PLCB1, was identified as a candidate target gene. Hence, dysregulation of ROR1, PLCB1, or PLCB4 by PAX6 and CTCF may be one mechanism that links neural and pancreatic dysfunction not only in insomnia but also in the relevant psychiatric disorders that are accompanied with circadian rhythm disruption and metabolic syndrome.

Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor.

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/ß-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/ß-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.

Thymosin beta 4 enhances NK cell cytotoxicity mediated by ICAM-1.

Thymosin beta 4 (T beta 4), which is the major G-actin sequestering protein, has been shown to have ubiquitous distribution and multiple biological activities. However, T beta 4's functions in relation to natural killer(NK) cells are still unknown. In this study, we show that synthetic T beta 4 peptide increases NK cell cytotoxicity mediated by intercellular adhesion molecule-1 (ICAM-1) through the secretion of cytolytic granules to target cells. This suggests that T beta 4 is a key activator of NK cell cytotoxicity.

Upregulation of NF-kappaB upon differentiation of mouse embryonic stem cells.

NF-kappaB is a transcriptional regulator involved in many biological processes including proliferation, survival, and differentiation. Recently, we reported that expression and activity of NF-kappaB is comparatively low in undifferentiated human embryonic stem (ES) cells, but increases during differentiation. Here, we found a lower expression of NF-kappaB p65 protein in mouse ES cells when compared with mouse embryonic fibroblast cells. Protein levels of NF-kappaB p65 and relB were clearly enhanced during retinoic acid-induced differentiation. Furthermore, increased DNA binding activity of NF-kappaB in response to TNF-alpha, an agonist of NF-kappaB signaling, was seen in differentiated but not undifferentiated mouse ES cells. Taken together with our previous data in human ES cells, it is likely that NF-kappaB expression and activity of the NF-kappaB signaling pathway is comparatively low in undifferentiated ES cells, but increases during differentiation of ES cells in general.

Enhancement of NF-kappaB expression and activity upon differentiation of human embryonic stem cell line SNUhES3.

NF-kappaB is involved in many biological processes including proliferation, survival, and differentiation. Because human embryonic stem (ES) cells have the potential to differentiate to various lineages, understanding mechanisms involved in stemness and lineage differentiation is an important issue. We investigated expression of NF-kappaB in the human ES cell lines SNUhES3 and MizhES4 and found that expression of NF-kappaB mRNA and protein in these two cell lines was significantly lower compared to those of other adult cell lines. However, when SNUhES3 cells were induced to differentiate by retinoic acid, expression levels of NF-kappaB significantly increased compared to undifferentiated SNUhES3 cells. As the components of tumor necrosis factor-alpha (TNF-alpha) signaling are expressed comparably in undifferentiated and differentiated SNUhES3 cells, we examined the responsiveness of SNUhES3 cells to treatment with TNF-alpha, an agonist of NF-kappaB signaling. Nuclear localization of NF-kappaB in response to TNF-alpha was evident in differentiated, but not undifferentiated, SNUhES3 cells. In agreement with this observation, induction of interleukin-8 (IL-8) in response to TNF-alpha was seen only in differentiated SNUhES3 cells. On the basis of an IkappaB kinase (IKK) inhibitor study, expression of IL-8 induced by TNF-alpha was dependent on NF-kappaB activity. Taken together, our results suggest that expression and activity of NF-kappaB is comparatively low in undifferentiated human ES cells, but increases during differentiation of the ES cells.

Basic fibroblast growth factor activates ERK and induces c-fos in human embryonic stem cell line MizhES1.

Human embryonic stem (hES) cells can be maintained in a proliferative undifferentiated state in vitro by growing them on feeder layers of mouse embryonic fibroblast (MEF) cells along with basic fibroblast growth factor (bFGF/FGF-2). To understand the molecular mechanisms involved in the requirement of bFGF in human ES cells, we investigated expression of FGF receptors and intracellular signaling events in response to bFGF in human ES cell line MizhES1. On the basis of the results of RT-PCR, clear expression of FGF receptors FGFR1, FGR2, and FGFR3 was noticed. Because MAPK, PI3K, and PKC pathways are well-known pathways triggered by bFGF in other cells, these pathways were investigated after stimulation with bFGF. bFGF did not induce activation of PI3K or PKC, but induced activation of ERK (extracellular signal-regulated kinase). To monitor the consequences of ERK activation, we examined expression of the immediate early gene c-fos, one downstream target of the MEK1/ERK pathway. mRNA and protein levels of the c-fos gene were increased by bFGF. Induction of c-Fos was dependent on MEKl. Therefore, it is likely that bFGF contributes to maintenance of human ES cells, at least in part, through the MEK1/ERK pathway.

Enhancement of cell survival by stromal cell-derived factor-1/CXCL12 involves activation of CREB and induction of Mcl-1 and c-Fos in factor-dependent human cell line MO7e.

Stromal cell-derived factor-1 (SDF-1/CXCL12) enhances the survival of hematopoietic stem and progenitor cells in synergy with other cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), steel factor, and thrombopoietin (TPO), and both the PI3K/Akt and MAPK pathways have been linked to this survival. To further evaluate intracellular signaling involved in SDF-1/CXCL12 survival effects, we investigated modulation of downstream signaling molecules. The synergistic survival enhancement of SDF-1/CXCL12 plus other cytokines were directly linked to enhanced phosphorylation of p70/85S6K and cAMP responsive element binding protein (CREB), as well as enhanced induction of the Bcl-2 family member Mcl-1. Most prominently, c-Fos, a component of AP1 transcription factor, was synergistically induced by SDF-1/CXCL12 plus other cytokines. These results suggest that SDF-1/CXCL12 enhanced cell survival in synergy with other cytokines involves activation of CREB and induction of Mcl-1 and c-Fos.