Muramyl dipeptide potentiates a Bacillus anthracis poly-γ-d-glutamic acid capsule surrogate that induces maturation and activation of mouse dendritic cells.

Poly-γ-d-glutamic acid (PGA) of anthrax is an important pathogenic factor due to its anti-phagocytic activity. Additionally, PGA has the ability to activate mouse macrophages for the secretion of cytokines through Toll-like receptor (TLR) 2. Peptidoglycan (PGN), a major bacterial cell-wall component, induces inflammatory responses in the host. We assessed whether PGA can induce maturation and cytokine expression in immature mouse dendritic cells (DCs) in the existence of muramyl dipeptide (MDP), the minimum motif of PGN with immunostimulatory activity. Stimulation of immature DCs with PGA or MDP alone augmented expression of costimulatory molecules and MHC class II proteins, which are all cell surface markers indicative of maturation. The observed effects were further enhanced by costimulation of PGA and MDP. PGA alone was sufficient to induce expression of TNF-α, IL-6, MCP-1, and MIP1-α, whereas MDP alone did not under the same conditions. Treatment with MDP enhanced PGA-induced expression of the tested inflammatory mediators; however, the synergistic effect found for PGA and MDP was not observed in TLR2- or nucleotide-binding oligomerization domain (NOD) 2-knockout DCs. Additionally, MDP augmented PGA-induced MAP kinases and NF-κB activation, which is crucial for expression of cytokines. Furthermore, MAP kinase and NF-κB inhibitors attenuated MDP enhancement of PGA-induced cytokine production. In addition, co-culture of splenocytes and PGA/MDP-matured DCs induced higher expression of IL-2 and IFN-γ compared to that of splenocytes and PGA-matured DCs. Collectively, our results suggest that PGA and MDP cooperatively induce inflammatory responses in mouse DCs through TLR2 and NOD2 via MAP kinase and NF-κB pathways, subsequently leading to lymphocyte activation.

The pentapeptide Gly-Thr-Gly-Lys-Thr confers sensitivity to anti-cancer drugs by inhibition of CAGE binding to GSK3ß and decreasing the expression of cyclinD1.

We previously reported the role of cancer/testis antigen CAGE in the response to anti-cancer drugs. CAGE increased the expression of cyclinD1, and pGSK3ßSer9, an inactive GSK3ß, while decreasing the expression of phospho-cyclinD1Thr286. CAGE showed binding to GSK3ß and the domain of CAGE (amino acids 231-300) necessary for binding to GSK3ß and for the expression regulation of cyclinD1 was determined. 269GTGKT273 peptide, corresponding to the DEAD box helicase domain of CAGE, decreased the expression of cyclinD1 and pGSK3ßSer9 while increasing the expression of phospho-cyclinD1Thr286. GTGKT peptide showed the binding to CAGE and prevented CAGE from binding to GSK3ß. GTGKT peptide changed the localization of CAGE and inhibited the binding of CAGE to the promoter sequences of cyclin D1. GTGKT peptide enhanced the apoptotic effects of anti-cancer drugs and decreased the migration, invasion, angiogenic, tumorigenic and metastatic potential of anti-cancer drug-resistant cancer cells. We found that Lys272 of GTGKT peptide was necessary for conferring anti-cancer activity. Peptides corresponding to the DEAD box helicase domain of CAGE, such as AQTGTGKT, QTGTGKT and TGTGKT, also showed anti-cancer activity by preventing CAGE from binding to GSK3ß. GTGKT peptide showed ex vivo tumor homing potential. Thus, peptides corresponding to the DEAD box helicase domain of CAGE can be developed as anti-cancer drugs in cancer patients expressing CAGE.

miR-30a Regulates the Expression of CAGE and p53 and Regulates the Response to Anti-Cancer Drugs.

We have previously reported the role of miR-217 in anti-cancer drug-resistance. miRNA array and miRNA hybridization analysis predicted miR-30a-3p as a target of miR-217. miR-30a-3p and miR-217 formed a negative feedback loop and regulated the expression of each other. Ago1 immunoprecipitation and co-localization analysis revealed a possible interaction between miR-30a-3p and miR-217. miR-30a-3p conferred resistance to anti-cancer drugs and enhanced the invasion, migration, angiogenic, tumorigenic, and metastatic potential of cancer cells in CAGE-dependent manner. CAGE increased the expression of miR-30a-3p by binding to the promoter sequences of miR-30a-3p, suggesting a positive feedback loop between CAGE and miR-30a-3p. miR-30a-3p decreased the expression of p53, which showed the binding to the promoter sequences of miR-30a-3p and CAGE in anti-cancer drug-sensitive cancer cells. Luciferase activity assays showed that p53 serves as a target of miR-30a. Thus, the miR-30a-3p-CAGE-p53 feedback loop serves as a target for overcoming resistance to anti-cancer drugs.

miR-217 and CAGE form feedback loop and regulates the response to anti-cancer drugs through EGFR and HER2.

MicroRNA array analysis revealed that miR-217 expression was decreased in anti-cancer drug-resistant Malme3MR cancer cells. CAGE, a cancer/testis antigen, was predicted as a target of miR-217. Luciferase activity and ChIP assays revealed a negative feedback relationship between CAGE and miR-217. miR-217 and CAGE oppositely regulated the response to anti-cancer drugs such as taxol, gefitinib and trastuzumab, an inhibitor of HER2. miR-217 negatively regulated the tumorigenic, metastatic, angiogenic, migration and invasion potential of cancer cells. The xenograft of Malme3MR cells showed an increased expression of pEGFRY845. CAGE and miR-217 inhibitor regulated the expression of pEGFRY845. CAGE showed interactions with EGFR and HER2 and regulated the in vivo sensitivity to trastuzumab. The down-regulation of EGFR or HER2 enhanced the sensitivity to anti-cancer drugs. CAGE showed direct regulation of HER2 and was necessary for the interaction between EGFR and HER2 in Malme3MR cells. miR-217 inhibitor induced interactions of CAGE with EGFR and HER2 in Malme3M cells. The inhibition of EGFR by CAGE-binding GTGKT peptide enhanced the sensitivity to gefitinib and trastuzumab and prevented interactions of EGFR with CAGE and HER2. Our results show that miR-217-CAGE feedback loop serves as a target for overcoming resistance to various anti-cancer drugs, including EGFR and HER2 inhibitors.

Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015.

An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

The Hyaluronic Acid-HDAC3-miRNA Network in Allergic Inflammation.

We previously reported the anti-allergic effect of high molecular weight form of hyaluronic acid (HMW-HA). In doing so, HA targets CD44 and inhibits FcεRI signaling and cross-talk between epidermal growth factor receptor (EGFR) and FcεRI. We previously reported the role of histone deacetylases (HDACs) in allergic inflammation and allergic inflammation-promoted enhanced tumorigenic potential. We reported regulatory role of HA in the expression of HDAC3. In this review, we will discuss molecular mechanisms associated with anti-allergic effect of HA in relation with HDACs. The role of microRNAs (miRNAs) in allergic inflammation has been reported. We will also discuss the role of miRNAs in allergic inflammation in relation with HA-mediated anti-allergic effects.

miR-335 Targets SIAH2 and Confers Sensitivity to Anti-Cancer Drugs by Increasing the Expression of HDAC3.

We previously reported the role of histone deacetylase 3 (HDAC3) in response to anti-cancer drugs. The decreased expression of HDAC3 in anti-cancer drug-resistant cancer cell line is responsible for the resistance to anti-cancer drugs. In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression. MG132, an inhibitor of proteasomal degradation, induced the expression of HDAC3 in various anti-cancer drug-resistant cancer cell lines. Ubiquitination of HDAC3 was observed in various anti-cancer drug-resistant cancer cell lines. HDAC3 showed an interaction with SIAH2, an ubiquitin E3 ligase, that has increased expression in various anti-cancer drug-resistant cancer cell lines. miRNA array analysis showed the decreased expression of miR-335 in these cells. Targetscan analysis predicted the binding of miR-335 to the 3'-UTR of SIAH2. miR-335-mediated increased sensitivity to anti-cancer drugs was associated with its effect on HDAC3 and SIAH2 expression. miR-335 exerted apoptotic effects and inhibited ubiquitination of HDAC3 in anti-cancer drug-resistant cancer cell lines. miR-335 negatively regulated the invasion, migration, and growth rate of cancer cells. The mouse xenograft model showed that miR-335 negatively regulated the tumorigenic potential of cancer cells. The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs. The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.

Suppressor of cytokine signaling 1 is a positive regulator of TGF-ß-induced prostaglandin production in human follicular dendritic cell-like cells.

PGs are emerging as important immune modulators. Since our report on the expression of PG synthases in human follicular dendritic cells, we investigated the potential immunoregulatory function of PGs and their production mechanisms. In this study, we explored the intracellular signaling molecules mediating TGF-ß-induced cyclooxygenase (COX)-2 augmentation in follicular dendritic cell-like cells. TGF-ß triggered phosphorylation of Smad3 and ERK, which were essential for the increase in COX-2 protein. Interestingly, depletion of suppressor of cytokine signaling 1 (SOCS1) resulted in an almost complete inhibition of Smad3 phosphorylation and COX-2 induction. Nuclear translocation of Smad3 was inhibited in SOCS1-depleted cells. SOCS1 knockdown also downregulated TGF-ß-stimulated Snail expression and its binding to the Cox-2 promoter. In contrast, overexpression of SOCS1 gave rise to a significant increase in Snail and COX-2 proteins. SOCS1 was reported to be a negative regulator of cytokine signaling by various investigators. However, our current data suggest that SOCS1 promotes TGF-ß-induced COX-2 expression and PG production by facilitating Smad3 phosphorylation and Snail binding to the Cox-2 promoter. The complete understanding of the biological function of SOCS1 might be obtained via extensive studies with diverse cell types.

MicroRNA-26a/-26b-COX-2-MIP-2 Loop Regulates Allergic Inflammation and Allergic Inflammation-promoted Enhanced Tumorigenic and Metastatic Potential of Cancer Cells.

Cyclooxgenase-2 (COX-2) knock-out mouse experiments showed that COX-2 was necessary for in vivo allergic inflammation, such as passive cutaneous anaphylaxis, passive systemic anaphylaxis, and triphasic cutaneous allergic reaction. TargetScan analysis predicted COX-2 as a target of miR-26a and miR-26b. miR-26a/-26b decreased luciferase activity associated with COX-2-3'-UTR. miR-26a/-26b exerted negative effects on the features of in vitro and in vivo allergic inflammation by targeting COX-2. ChIP assays showed the binding of HDAC3 and SNAIL, but not COX-2, to the promoter sequences of miR-26a and miR-26b. Cytokine array analysis showed that the induction of chemokines, such as MIP-2, in the mouse passive systemic anaphylaxis model occurred in a COX-2-dependent manner. ChIP assays showed the binding of HDAC3 and COX-2 to the promoter sequences of MIP-2. In vitro and in vivo allergic inflammation was accompanied by the increased expression of MIP-2. miR-26a/-26b negatively regulated the expression of MIP-2. Allergic inflammation enhanced the tumorigenic and metastatic potential of cancer cells and induced positive feedback involving cancer cells and stromal cells, such as mast cells, macrophages, and endothelial cells. miR-26a mimic and miR-26b mimic negatively regulated the positive feedback between cancer cells and stromal cells and the positive feedback among stromal cells. miR-26a/-26b negatively regulated the enhanced tumorigenic potential by allergic inflammation. COX-2 was necessary for the enhanced metastatic potential of cancer cells by allergic inflammation. Taken together, our results indicate that the miR26a/-26b-COX-2-MIP-2 loop regulates allergic inflammation and the feedback relationship between allergic inflammation and the enhanced tumorigenic and metastatic potential.

Transglutaminase II/microRNA-218/-181a loop regulates positive feedback relationship between allergic inflammation and tumor metastasis.

The molecular mechanism of transglutaminase II (TGaseII)-mediated allergic inflammation remains largely unknown. TGaseII, induced by antigen stimulation, showed an interaction and co-localization with FcϵRI. TGaseII was necessary for in vivo allergic inflammation, such as triphasic cutaneous reaction, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. TGaseII was necessary for the enhanced metastatic potential of B16F1 melanoma cells by passive systemic anaphylaxis. TGaseII was shown to be a secreted protein. Recombinant TGaseII protein increased the histamine release and ß-hexosaminidase activity, and enhanced the metastatic potential of B16F1 mouse melanoma cells. Recombinant TGaseII protein induced the activation of EGF receptor and an interaction between EGF receptor and FcϵRI. Recombinant TGaseII protein displayed angiogenic potential accompanied by allergic inflammation. R2 peptide, an inhibitor of TGaseII, exerted negative effects on in vitro and in vivo allergic inflammation by regulating the expression of TGaseII and FcϵRI signaling. MicroRNA (miR)-218 and miR-181a, decreased during allergic inflammation, were predicted as negative regulators of TGaseII by microRNA array and TargetScan analysis. miR-218 and miR-181a formed a negative feedback loop with TGaseII and regulated the in vitro and in vivo allergic inflammation. TGaseII was necessary for the interaction between mast cells and macrophages during allergic inflammation. Mast cells and macrophages, activated during allergic inflammation, were responsible for the enhanced metastatic potential of tumor cells that are accompanied by allergic inflammation. In conclusion, the TGaseII/miR-218/-181a feedback loop can be employed for the development of anti-allergy therapeutics.

miR-326-histone deacetylase-3 feedback loop regulates the invasion and tumorigenic and angiogenic response to anti-cancer drugs.

Histone modification is known to be associated with multidrug resistance phenotypes. Cancer cell lines that are resistant or have been made resistant to anti-cancer drugs showed lower expression levels of histone deacetylase-3 (HDAC3), among the histone deacetylase(s), than cancer cell lines that were sensitive to anti-cancer drugs. Celastrol and Taxol decreased the expression of HDAC3 in cancer cell lines sensitive to anti-cancer drugs. HDAC3 negatively regulated the invasion, migration, and anchorage-independent growth of cancer cells. HDAC3 conferred sensitivity to anti-cancer drugs in vitro and in vivo. TargetScan analysis predicted miR-326 as a negative regulator of HDAC3. ChIP assays and luciferase assays showed a negative feedback loop between HDAC3 and miR-326. miR-326 decreased the apoptotic effect of anti-cancer drugs, and the miR-326 inhibitor increased the apoptotic effect of anti-cancer drugs. miR-326 enhanced the invasion and migration potential of cancer cells. The miR-326 inhibitor negatively regulated the tumorigenic, metastatic, and angiogenic potential of anti-cancer drug-resistant cancer cells. HDAC3 showed a positive feedback loop with miRNAs such as miR-200b, miR-217, and miR-335. miR-200b, miR-217, and miR-335 negatively regulated the expression of miR-326 and the invasion and migration potential of cancer cells while enhancing the apoptotic effect of anti-cancer drugs. TargetScan analysis predicted miR-200b and miR-217 as negative regulators of cancer-associated gene, a cancer/testis antigen, which is known to regulate the response to anti-cancer drugs. HDAC3 and miR-326 acted upstream of the cancer-associated gene. Thus, we show that the miR-326-HDAC3 feedback loop can be employed as a target for the development of anti-cancer therapeutics.

Histone deacetylase-3 mediates positive feedback relationship between anaphylaxis and tumor metastasis.

Allergic inflammation has been known to enhance the metastatic potential of tumor cells. The role of histone deacetylase-3 (HDAC3) in allergic skin inflammation was reported. We investigated HDAC3 involvement in the allergic inflammation-promotion of metastatic potential of tumor cells. Passive systemic anaphylaxis (PSA) induced HDAC3 expression and FcεRI signaling in BALB/c mice. PSA enhanced the tumorigenic and metastatic potential of mouse melanoma cells in HDAC3- and monocyte chemoattractant protein 1-(MCP1)-dependent manner. The PSA-mediated enhancement of metastatic potential involved the induction of HDAC3, MCP1, and CD11b (a macrophage marker) expression in the lung tumor tissues. We examined an interaction between anaphylaxis and tumor growth and metastasis at the molecular level. Conditioned medium from antigen-stimulated bone marrow-derived mouse mast cell cultures induced the expression of HDAC3, MCP1, and CCR2, a receptor for MCP1, in B16F1 mouse melanoma cells and enhanced migration and invasion potential of B16F1 cells. The conditioned medium from B16F10 cultures induced the activation of FcεRI signaling in lung mast cells in an HDAC3-dependent manner. FcεRI signaling was observed in lung tumors derived from B16F10 cells. Target scan analysis predicted HDAC3 to be as a target of miR-384, and miR-384 and HDAC3 were found to form a feedback regulatory loop. miR-384, which is decreased by PSA, negatively regulated HDAC3 expression, allergic inflammation, and the positive feedback regulatory loop between anaphylaxis and tumor metastasis. We show the miR-384/HDAC3 feedback loop to be a novel regulator of the positive feedback relationship between anaphylaxis and tumor metastasis.

HDAC3 acts as a negative regulator of angiogenesis.

Histone deacetylase-3 (HDAC3) is involved in cellular proliferation, apoptosis and transcriptional repression. However, the role of HDAC3 in angiogenesis remains unknown. HDAC3 negatively regulated the expression of angiogenic factors, such as VEGF and plasminogen activator inhibitor-1 (PAI-1). HDAC3 showed binding to promoter sequences of PAI-1. HDAC3 activity was necessary for the expression regulation of PAI-1 by HDAC3. VEGF decreased the expression of HDAC3, and the down-regulation of HDAC3 enhanced endothelial cell tube formation. HDAC3 negatively regulated tumor-induced angiogenic potential. We show the novel role of HDAC3 as a negative regulator of angiogenesis.

Nuclear localization signal domain of HDAC3 is necessary and sufficient for the expression regulation of MDR1.

Histone acetylation/deacetylation has been known to be associated with the transcriptional regulation of various genes. The role of histone deacetylase-3 in the expression regulation of MDR1 was investigated. The expression level of HDAC3 showed an inverse relationship with the expression level of MDR1. Wild-type HDAC3, but not catalytic mutant HDAC3(S424A), negatively regulated the expression of MDR1. Wild-type HDAC3, but not catalytic mutant HDAC3(S424A), showed binding to the promoter sequences of HDAC3. HDAC3 regulated the expression level, and the binding of Ac-H3(K9/14) and Ac-H4(K16) around the MDR1 promoter sequences. The nuclear localization signal domain of HDAC3 was necessary, and sufficient for the binding of HDAC3 to the MDR1 promoter sequences and for conferring sensitivity to microtubule-targeting drugs.

miR-200b and cancer/testis antigen CAGE form a feedback loop to regulate the invasion and tumorigenic and angiogenic responses of a cancer cell line to microtubule-targeting drugs.

Cancer/testis antigen cancer-associated gene (CAGE) is known to be involved in various cellular processes, such as proliferation, cell motility, and anti-cancer drug resistance. However, the mechanism of the expression regulation of CAGE remains unknown. Target scan analysis predicted the binding of microRNA-200b (miR-200b) to CAGE promoter sequences. The expression of CAGE showed an inverse relationship with miR-200b in various cancer cell lines. miR-200b was shown to bind to the 3'-UTR of CAGE and to regulate the expression of CAGE at the transcriptional level. miR-200b also enhanced the sensitivities to microtubule-targeting drugs in vitro. miR-200b and CAGE showed opposite regulations on invasion potential and responses to microtubule-targeting drugs. Xenograft experiments showed that miR-200b had negative effects on the tumorigenic and metastatic potential of cancer cells. The effect of miR-200b on metastatic potential involved the expression regulation of CAGE by miR-200b. miR-200b decreased the tumorigenic potential of a cancer cell line resistant to microtubule-targeting drugs in a manner associated with the down-regulation of CAGE. ChIP assays showed the direct regulation of miR-200b by CAGE. CAGE enhanced the invasion potential of a cancer cell line stably expressing miR-200b. miR-200b exerted a negative regulation on tumor-induced angiogenesis. The down-regulation of CAGE led to the decreased expression of plasminogen activator inhibitor-1, a TGFß-responsive protein involved in angiogenesis, and VEGF. CAGE mediated tumor-induced angiogenesis and was necessary for VEGF-promoted angiogenesis. Human recombinant CAGE protein displayed angiogenic potential. Thus, miR-200b and CAGE form a feedback regulatory loop and regulate the response to microtubule-targeting drugs, as well as the invasion, tumorigenic potential, and angiogenic potential.

DNA methyl transferase I acts as a negative regulator of allergic skin inflammation.

The role of DNA methyl transferase I (DNMT1) in allergic inflammation was investigated. Antigen stimulation decreased expression of DNMT1 in rat basophilic leukemia cells (RBL2H3). The down regulation of DNMT1 induced expression of histone deacetylase 3 (HDAC3). HDAC3 was necessary for allergic skin inflammation, such as such as triphasic cutaneous reaction and passive cutaneous anaphylaxis. The down regulation of DNMT1 resulted from activation of PKC and rac1 which were necessary for proteasome-dependent ubiquitination of DNMT1 by antigen stimulation. N-acetyl-L-cysteine, an inhibitor of reactive oxygen species production, exerted negative effects on allergic skin inflammation. Antigen stimulation led to increased expression of Tip60, a histone acetyl transferase. Wild type, but not mutant form, Tip60 decreased expression of DNMT1 while increasing expression of HDAC3, suggesting role for acetylation in ubiquitin-dependent proteasomal degradation of DNMT1. In vivo down regulation of DNMT1 increased ear thickness, typical of allergic skin inflammation, induced vascular leakage and promoted angiogenesis in BALB/c mouse. The down regulation of DNMT1 enhanced angiogenic potential of rat aortic endothelial cells (RAEC) accompanied by activation of VEGR-2 and induced interaction between VEGR-2 and syk in RAEC. The enhanced angiogenic potential of RAEC was associated with the induction of VEGF by down regulation of DNMT1 in RBL2H3 cells. The down regulation of DNMT1 induced leukocytes-endothelial cell interaction and expression of various adhesion molecules. Aspirin exerted a negative effect on allergic skin inflammation by indirect regulation on DNMT1 via Tip60. Taken together, these results suggest novel role for DNMT1 in allergic skin inflammation.

Histone deacetylase 3 mediates allergic skin inflammation by regulating expression of MCP1 protein.

We have shown the induction of histone deacetylase 3 (HDAC3) in antigen-stimulated rat basophilic leukemia cells via NF-κB. We investigated the role of HDAC3 in allergic skin inflammation. We used a BALB/c mouse model of triphasic cutaneous anaphylaxis (triphasic cutaneous reaction; TpCR) and passive cutaneous anaphylaxis (PCA) to examine the role of HDAC3 in allergic skin inflammation. Triphasic cutaneous reaction involved induction of HDAC3 and was mediated by HDAC3. HDAC3 showed an interaction with FcεRIß. Trichostatin A (TSA), an inhibitor of HDAC(s), disrupted this interaction. Cytokine array analysis showed that the down-regulation of HDAC3 led to the decreased secretion of monocyte chemoattractant protein 1 (MCP1). FcεRI was necessary for induction of HDAC3 and MCP1. ChIP assays showed that HDAC3, in association with Sp1 and c-Jun, was responsible for induction of MCP1 expression. TSA exerted a negative effect on induction of MCP1. HDAC3 exerted a negative regulation on expression of HDAC2 via interaction with Rac1. The down-regulation of HDAC3 or inactivation of Rac1 induced binding of HDAC2 to MCP1 promoter sequences. TSA exerted a negative effect on HDAC3-mediated TpCR. The BALB/c mouse model of PCA involved induction of HDAC3 and MCP1. HDAC3 and MCP1 were necessary for PCA that involved ear swelling, enhanced vascular permeability, and angiogenesis. Recombinant MCP1 enhanced ß-hexosaminidase activity and histamine release and also showed angiogenic potential. TSA exerted a negative effect on PCA. Our data show HDAC3 as a valuable target for the development of allergic skin inflammation therapeutics.

Hyaluronic acid promotes angiogenesis by inducing RHAMM-TGFß receptor interaction via CD44-PKCδ.

Hyaluronic acid (HA) has been shown to promote angiogenesis. However, the mechanism behind this effect remains largely unknown. Therefore, in this study, the mechanism of HA-induced angiogenesis was examined. CD44 and PKCδ were shown to be necessary for induction of the receptor for HA-mediated cell motility (RHAMM), a HA-binding protein. RHAMM was necessary for HA-promoted cellular invasion and endothelial cell tube formation. Cytokine arrays showed that HA induced the expression of plasminogen activator-inhibitor-1 (PAI), a downstream target of TGFß receptor signaling. The induction of PAI-1 was dependent on CD44 and PKCδ. HA also induced an interaction between RHAMM and TGFß receptor I, and induction of PAI-1 was dependent on RHAMM and TGFß receptor I. Histone deacetylase 3 (HDAC3), which is decreased by HA via rac1, reduced induction of plasminogen activator inhibitor-1 (PAI-1) by HA. ERK, which interacts with RHAMM, was necessary for induction of PAI-1 by HA. Snail, a downstream target of TGFß signaling, was also necessary for induction of PAI-1. The down regulation of PAI-1 prevented HA from enhancing endothelial cell tube formation and from inducing expression of angiogenic factors, such as ICAM-1, VCAM-1 and MMP-2. HDAC3 also exerted reduced expression of MMP-2. In this study, we provide a novel mechanism of HA-promoted angiogenesis, which involved RHAMM-TGFßRI signaling necessary for induction of PAI-1.

Integrin α(5) interacts with EGFR, is necessary for FcɛRI signaling and is necessary for allergic inflammation in relation with angiogenesis.

Recent reports have suggested role for epidermal growth factor receptor (EGFR) in asthma and skin inflammation. Integrin(s) are known to be necessary for the transactivation of EGFR. The roles of EGFR and integrin(s) in allergic inflammation were investigated. Antigen stimulation induced activation of EGFR and interaction between EGFR and integrin α(5) in Rat Basophilic Leukemia (RBL2H3) cells and bone marrow-derived mouse mast cells (BMMCs). Flow cytometry revealed increased phosphorylation of EGFR on cell surfaces. Antigen stimulation induced interaction between EGFR and FcɛRI in both RBL2H3 cells and BMMCs. Blocking of EGFR or integrin α exerted negative effects on rac1 activity and secretion of ß-hexosaminidase in both RBL2H3 cells and BMMCs. EGFR and integrin α(5) were found to be necessary for IgE-dependent cutaneous anaphylaxis. FAK (focal adhesion kinase), interacted with EGFR and with FcɛRI upon antigen stimulation, and it was necessary for the increased secretion of ß-hexosaminidase in both RBL2H3 cells and BMMCs. EGFR and integrin α(5) were necessary for interactions between activated RBL2H3 cells, BMMCs and rat aortic endothelial cells (RAECs). Conditioned medium of antigen-stimulated RBL2H3 cells promoted RAECs tube formation, rat aortic ring formation and blood vessel formation. Conditioned medium of antigen-stimulated BMMCs also had the same effects on RAECs. This enhanced angiogenic potential of RAECs was dependent on EGFR and integrin α(5). In conclusion, EGFR, via interaction with FcɛRI and integrin α(5), is necessary for allergic inflammation associated with cellular interaction.

Cancer/testis antigen CAGE exerts negative regulation on p53 expression through HDAC2 and confers resistance to anti-cancer drugs.

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M(R)) and the human hepatic cancer cell line SNU387 (SNU387(R)) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387(R) cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M(R) cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.