Akt, IL-4, and STAT Proteins Play Distinct Roles in Prostaglandin Production in Human Follicular Dendritic Cell-like Cells.

This study aimed to explore the role of Akt protein in the induction and inhibition of prostaglandin (PG) in human follicular dendritic cell (FDC)-like cells. FDC-like cells and B cells were isolated from human tonsils. PG production was assessed using enzyme immunoassay, while the upstream cyclooxygenase-2 (COX-2) protein levels were measured using immunoblotting with FDC-like cells transfected with Akt siRNA to analyze the impact of Akt knockdown. The COX-2 expression and PG production induced with IL-1ß were significantly increased by Akt knockdown. However, IL-1ß did not significantly alter either total or phosphorylated Akt protein levels. Akt knockdown resulted in the augmentation of COX-2 expression induced by B cells, although the addition of B cells did not significantly modulate both total and phosphorylated Akt proteins. In contrast, IL-4 specifically exhibited a potent inhibitory effect on COX-2 protein induction and PG production via STAT6. The inhibitory activity of IL-4 was not hampered by Akt knockdown. Interestingly, COX-2 expression levels induced with IL-1ß were markedly modulated with STAT1 and STAT3 knockdown. STAT1 silencing resulted in further augmentation of COX-2, whereas STAT3 silencing prohibited IL-1ß from stimulating COX-2 expression. The current results suggest that Akt, IL-4, and STAT1 play inhibitory roles in PG production in FDC-like cells and expand our knowledge of the immune inflammatory milieu.

NF-κB-dependent miR-31/155 biogenesis is essential for TNF-α-induced impairment of endothelial progenitor cell function.

Endothelial progenitor cell (EPC) dysfunction impairs vascular function and remodeling in inflammation-associated diseases, including preeclampsia. However, the underlying mechanism of this inflammation-induced dysfunction remains unclear. In the present study, we found increases in TNF-α and miR-31/155 levels and reduced numbers of circulating EPCs in patients with preeclampsia. Patient-derived mononuclear cells (MNCs) cultured in autologous serum had decreased endothelial nitric oxide synthase (eNOS) expression, nitric oxide production, and differentiation into EPCs with angiogenic potential, and these effects were inhibited by a TNF-α-neutralizing antibody and miR-31/155 inhibitors. Moreover, TNF-α treatment of normal MNCs increased miR-31/155 biogenesis, decreased eNOS expression, reduced EPC differentiation, and impaired angiogenic potential. The TNF-α-induced impairment of EPC differentiation and function was rescued by NF-κB p65 knockdown or miR-31/155 inhibitors. In addition, treatment of MNCs with synthetic miR-31/155 or an eNOS inhibitor mimicked the inhibitory effects of TNF-α on eNOS expression and EPC functions. Moreover, transplantation of EPCs that had been differentiated from TNF-α-treated MNCs decreased neovascularization and blood perfusion in ischemic mouse hindlimbs compared with those of normally differentiated EPCs. These findings suggest that NF-κB activation is required for TNF-α-induced impairment of EPC mobilization, differentiation, and function via miR-31/155 biogenesis and eNOS downregulation. Our data provide a new role for NF-κB-dependent miR-31/155 in EPC dysfunction under the pathogenic conditions of inflammation-associated vascular diseases, including preeclampsia.

Human Plasmablast Migration Toward CXCL12 Requires Glucose Oxidation by Enhanced Pyruvate Dehydrogenase Activity via AKT.

Migration of human plasmablast to the bone marrow is essential for the final differentiation of plasma cells and maintenance of effective humoral immunity. This migration is controlled by CXCL12/CXCR4-mediated activation of the protein kinase AKT. Herein, we show that the CXCL12-induced migration of human plasmablasts is dependent on glucose oxidation. Glucose depletion markedly inhibited plasmablast migration by 67%, and the glucose analog 2-deoxyglucose (2-DG) reduced the migration by 53%; conversely, glutamine depletion did not reduce the migration. CXCL12 boosted the oxygen consumption rate (OCR), and 2-DG treatment significantly reduced the levels of all measured tricarboxylic acid (TCA) cycle intermediates. AKT inhibitors blocked the CXCL12-mediated increase of OCR. CXCL12 enhanced the pyruvate dehydrogenase (PDH) activity by 13.5-fold in an AKT-dependent manner to promote mitochondrial oxidative phosphorylation. The knockdown and inhibition of PDH confirmed its indispensable role in CXCL12-induced migration. Cellular ATP levels fell by 91% upon exposure to 2-DG, and the mitochondrial ATP synthase inhibitor oligomycin inhibited CXCL12-induced migration by 85%. Low ATP levels inhibited the CXCL12-induced activation of AKT and phosphorylation of myosin light chains by 42%, which are required for cell migration. Thus, we have identified a mechanism that controls glucose oxidation via AKT signaling and PDH activation, which supports the migration of plasmablasts. This mechanism can provide insights into the proper development of long-lived plasma cells and is, therefore, essential for optimal humoral immunity. To our knowledge, this study is the first to investigate metabolic mechanisms underlying human plasmablast migration toward CXCL12.

TNF-α elicits phenotypic and functional alterations of vascular smooth muscle cells by miR-155-5p-dependent down-regulation of cGMP-dependent kinase 1.

cGMP-dependent protein kinase 1 (PKG1) plays an important role in nitric oxide (NO)/cGMP-mediated maintenance of vascular smooth muscle cell (VSMC) phenotype and vasorelaxation. Inflammatory cytokines, including tumor necrosis factor-α (TNFα), have long been understood to mediate several inflammatory vascular diseases. However, the underlying mechanism of TNFα-dependent inflammatory vascular disease is unclear. Here, we found that TNFα treatment decreased PKG1 expression in cultured VSMCs, which correlated with NF-κB-dependent biogenesis of miR-155-5p that targeted the 3'-UTR of PKG1 mRNA. TNFα induced VSMC phenotypic switching from a contractile to a synthetic state through the down-regulation of VSMC marker genes, suppression of actin polymerization, alteration of cell morphology, and elevation of cell proliferation and migration. All of these events were blocked by treatment with an inhibitor of miR-155-5p or PKG1, whereas transfection with miR-155-5p mimic or PKG1 siRNA promoted phenotypic modulation, similar to the response to TNFα. In addition, TNFα-induced miR-155-5p inhibited the vasorelaxant response of de-endothelialized mouse aortic vessels to 8-Br-cGMP by suppressing phosphorylation of myosin phosphatase and myosin light chain, both of which are downstream signal modulators of PKG1. Moreover, TNFα-induced VSMC phenotypic alteration and vasodilatory dysfunction were blocked by NF-κB inhibition. These results suggest that TNFα impairs NO/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by down-regulating PKG1 through NF-κB-dependent biogenesis of miR-155-5p. Thus, the NF-κB/miR-155-5p/PKG1 axis may be crucial in the pathogenesis of inflammatory vascular diseases, such as atherosclerotic intimal hyperplasia and preeclamptic hypertension.

Activated human B cells stimulate COX-2 expression in follicular dendritic cell-like cells via TNF-α.

In spite of the potential importance of cyclooxygenase (COX)-2 expression in the germinal center, its underlying cellular and molecular mechanisms are largely unknown. COX-2 is the key enzyme generating pleiotropic prostaglandins. Based on our previous findings, we hypothesized that lymphocytes would stimulate COX-2 expression in follicular dendritic cell (FDC) by liberating cytokines. In this study, we examined the effect of tonsillar lymphocytes on COX-2 expression in FDC-like cells by immunoblotting. B but not T cells induced COX-2 protein in a time- and dose-dependent manner. Sub-fractionation analysis of B cell subsets revealed that activated but not resting B cells were responsible for the COX-2 induction. Confocal microscopy of frozen tonsils demonstrated that FDCs indeed express COX-2 in situ, in line with the in vitro results. To identify the stimulating molecule, we added neutralizing antibodies to the coculture of FDC-like cells and B cells. COX-2 induction in FDC-like cells was markedly inhibited by TNF-α neutralizing antibody. Finally, the actual production of TNF-α by activated B cells was confirmed by an enzyme immunoassay. The current study implies an unrecognized cellular interaction between FDC and B cells leading to COX-2 expression during immune inflammatory responses.

miR-122-SOCS1-JAK2 axis regulates allergic inflammation and allergic inflammation-promoted cellular interactions.

The regulatory role of suppressor of cytokine signaling 1 (SOCS1) in inflammation has been reported. However, its role in allergic inflammation has not been previously reported. SOCS1 mediated in vitro and in vivo allergic inflammation. Histone deacetylase-3 (HDAC3), a mediator of allergic inflammation, interacted with SOCS1, and miR-384 inhibitor, a positive regulator of HDAC3, induced features of allergic inflammation in an SOCS1-dependent manner. miRNA array analysis showed that the expression of miR-122 was decreased by antigen-stimulation. TargetScan analysis predicted the binding of miR-122 to the 3'-UTR of SOCS1. miR-122 inhibitor induced in vitro and in vivo allergic features in SOCS1-dependent manner. SOCS1 was necessary for allergic inflammation-promoted enhanced tumorigenic and metastatic potential of cancer cells. SOCS1 and miR-122 regulated cellular interactions involving cancer cells, mast cells and macrophages during allergic inflammation. SOCS1 mimetic peptide, D-T-H-F-R-T-F-R-S-H-S-D-Y-R-R-I, inhibited in vitro and in vivo allergic inflammation, allergic inflammation-promoted enhanced tumorigenic and metastatic potential of cancer cells, and cellular interactions during allergic inflammation. Janus kinase 2 (JAK2) exhibited binding to SOCS1 mimetic peptide and mediated allergic inflammation. Transforming growth factor- Δ1 (TGF-Δ1) was decreased during allergic inflammation and showed an anti-allergic effect. SOCS1 and JAK2 regulated the production of anti-allergic TGF-Δ1. Taken together, our results show that miR-122-SOCS1 feedback loop can be employed as a target for the development of anti-allergic and anti-cancer drugs.

The caspase-8/Bid/cytochrome c axis links signals from death receptors to mitochondrial reactive oxygen species production.

Ligation of the death receptors for TNF-α, FasL, and TRAIL triggers two common pathways, caspase-dependent intrinsic apoptosis and intracellular reactive oxygen species (ROS) generation. The apoptotic pathway is well characterized; however, a signaling linker between the death receptor and ROS production has not been clearly elucidated. Here, we found that death receptor-induced ROS generation was strongly inhibited by mitochondrial complex I and II inhibitors, but not by inhibitors of NADPH oxidase, lipoxygenase, cyclooxygenase or xanthine oxidase, indicating that ROS are mostly generated by the impairment of the mitochondrial respiratory chain. ROS generation was accompanied by caspase-8 activation, Bid cleavage, and cytochrome c release; it was blocked in FADD- and caspase-8-deficient cells, as well as by caspase-8 knockdown and inhibitor. Moreover, Bid knockdown abrogated TNF-α- or TRAIL-induced ROS generation, whereas overexpression of truncated Bid (tBid) or knockdown of cytochrome c spontaneously elevated ROS production. In addition, p53-overexpressing cells accumulated intracellular ROS via cytochrome c release mediated by the BH3-only protein Noxa induction. In a cell-free reconstitution system, caspase-8-mediated Bid cleavage and recombinant tBid induced mitochondrial cytochrome c release and ROS generation, which were blocked by Bcl-xL and antioxidant enzymes. These data suggest that anti-apoptotic Bcl-2 proteins play an important role in mitochondrial ROS generation by preventing cytochrome c release. These data provide evidence that the FADD/caspase-8/Bid/cytochrome c axis is a crucial linker between death receptors and mitochondria, where they play a role in ROS generation and apoptosis.

A miRNA-101-3p/Bim axis as a determinant of serum deprivation-induced endothelial cell apoptosis.

Serum deprivation or withdrawal induces apoptosis in endothelial cells, resulting in endothelial cell dysfunction that is associated with cardiovascular disease. However, there is still limited information on the role of miRNA in serum deprivation-induced apoptosis. Here we found that serum deprivation increased caspase-dependent apoptosis through miRNA-101-3p downregulation, without altering expression of its host gene RNA 3'-terminal phosphate cyclase-like 1, which was highly correlated with suppressed expression levels of Dicer and Argonaute 2 (Ago2), indicating that miR-101-3p is post-transcriptionally elevated in serum-deprived conditions. The decreased miR-101-3p caused elevated Bim expression by targeting its 3'-untranslated region (3'-UTR). This resulted in activation of the intrinsic pathway of apoptosis via interaction with Bcl-2, decreased mitochondrial membrane potential, cytochrome c release, mitochondrial reactive oxygen species (ROS) production, and caspase activation. These events were abrogated by miR-101-3p mimic and the proapoptotic Bim siRNA, which suggest a determinant role of the miR-101-3p/Bim axis in serum deprivation-induced apoptosis. The apoptosis induced by miR-101-3p-mediated Bim expression is mediated by both caspase-3 and -1, which are activated by two distinct intrinsic mechanisms, cytochrome c release and ROS-induced inflammasome activation, respectively. In other words, the antioxidant inhibited endothelial cell death mediated by caspase-1 that activated caspase-7, but not caspase-3. These findings provide mechanistic insight into a novel function of miR-101-3p in serum withdrawal-induced apoptosis triggered by activating two different intrinsic or mitochondrial apoptosis pathways, implicating miR-101-3p as a therapeutic target that limits endothelial cell death associated with vascular disorders.

Positive feedback effect of PGE2 on cyclooxygenase-2 expression is mediated by inhibition of Akt phosphorylation in human follicular dendritic cell-like cells.

Prostaglandins (PGs) are bioactive lipid mediators generated from the phospholipids of cell membrane in response to various inflammatory signals. To understand the potential role of PGs in PG production itself during immune inflammatory responses, we examined the effect of PGE2, PGF2α, and beraprost on COX-2 expression using follicular dendritic cell (FDC)-like HK cells isolated from human tonsils. Those three PGs specifically augmented COX-2 protein expression in a dose-dependent manner after 4 or 8h of treatment. The enhancing effect was also reflected in the actual production of PGs and the viable cell recovery of germinal center B-cells. To investigate the underlying molecular mechanism, we examined the impact of PI3K inhibitors on PG-induced COX-2 expression. Interestingly, COX-2 induction by PGE2 and beraprost, but not PGF2α, was enhanced by wortmannin and LY294002. In line with this result, Akt phosphorylation was inhibited by PGE2 and beraprost but not by PGF2α. The distinct effect of PGE2 and beraprost from PGF2α was reproduced in Akt-knockdowned HK cells. Our current findings imply that PGE2 and PGI2 stimulate COX-2 expression in FDC by inhibiting Akt phosphorylation. Additional studies are warranted to determine the potential role of Akt as a therapeutic target in patients with inflammatory disorders.

The TLR7 agonist imiquimod induces anti-cancer effects via autophagic cell death and enhances anti-tumoral and systemic immunity during radiotherapy for melanoma.

Toll-like receptor (TLR) ligands are strongly considered immune-adjuvants for cancer immunotherapy and have been shown to exert direct anti-cancer effects. This study was performed to evaluate the synergistic anti-cancer and anti-metastatic effects of the TLR7 agonist imiquimod (IMQ) during radiotherapy for melanoma. The pretreatment of B16F10 or B16F1 cells with IMQ combined with γ-ionizing radiation (IR) led to enhanced cell death via autophagy, as demonstrated by increased expression levels of autophagy-related genes, and an increased number of autophagosomes in both cell lines. The results also confirmed that the autophagy process was accelerated via the reactive oxygen species (ROS)-mediated MAPK and NF-κB signaling pathway in the cells pretreated with IMQ combined with IR. Mice subcutaneously injected with melanoma cells showed a reduced tumor growth rate after treatment with IMQ and IR. Treatment with 3-methyladenine (3-MA), ameliorated the anti-cancer effect of IMQ combined with IR. Additionally, the combination therapy enhanced anti-cancer immunity, as demonstrated by an increased number of CD8+ T cells and decreased numbers of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) in the tumor lesions. Moreover, the combination therapy decreased the number of metastatic nodules in the lungs of mice that were injected with B16F10 cells via the tail vein. In addition, the combination therapy enhanced systemic anti-cancer immunity by increasing the abundances of T cell populations expressing IFN-γ and TNF-α. Therefore, these findings suggest that IMQ could serve as a radiosensitizer and immune booster during radiotherapy for melanoma patients.

Aspirin prevents TNF-α-induced endothelial cell dysfunction by regulating the NF-κB-dependent miR-155/eNOS pathway: Role of a miR-155/eNOS axis in preeclampsia.

Preeclampsia is an inflammatory disease with endothelial cell dysfunction that occurs via decreased endothelial nitric oxide synthase/nitric oxide (eNOS/NO) activity. Aspirin reduces the incidence of hypertensive pregnancy complications. However, the underlying mechanism has not been clearly explained. Here, we found that tumor necrosis factor (TNF)-α, microRNA (miR)-155, and eNOS levels as well as endothelial redox phenotype were differentially regulated in preeclamptic patients, implying the involvement of TNF-α- and redox signal-mediated miR-155 biogenesis and eNOS downregulation in the pathogenesis of preeclampsia. Aspirin prevented the TNF-α-mediated increase in miR-155 biogenesis and decreases in eNOS expression and NO/cGMP production in cultured human umbilical vein endothelial cells (HUVECs). Similar effects of aspirin were also observed in HUVECs treated with H2O2. The preventive effects of aspirin was associated with the inhibition of nuclear factor-κB (NF-κB)-dependent MIR155HG (miR-155 host gene) expression. Aspirin recovered the TNF-α-mediated decrease in wild-type, but not mutant, eNOS 3'-untranslated region reporter activity, whose effect was blocked by miR-155 mimic. Moreover, aspirin prevented TNF-α-mediated endothelial cell dysfunction associated with impaired vasorelaxation, angiogenesis, and trophoblast invasion, and the preventive effects were blocked by miR-155 mimic or an eNOS inhibitor. Aspirin rescued TNF-α-mediated eNOS downregulation coupled with endothelial dysfunction by inhibiting NF-κB-dependent transcriptional miR-155 biogenesis. Thus, the redox-sensitive NF-κB/miR-155/eNOS axis may be crucial in the pathogenesis of vascular disorders including preeclampsia.

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-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.

The tetrapeptide Arg-Leu-Tyr-Glu inhibits VEGF-induced angiogenesis.

Kringle 5, derived from plasminogen, is highly capable of inhibiting angiogenesis. Here, we have designed and synthesized 10 tetrapeptides, based on the amino acid properties of the core tetrapeptide Lys-Leu-Tyr-Asp (KLYD) originating from anti-angiogenic kringle 5 of human plasminogen. Of these, Arg-Leu-Tyr-Glu (RLYE) effectively inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation, migration and tube formation, with an IC50 of 0.06-0.08 nM, which was about ten-fold lower than that of the control peptide KLYD (0.79 nM), as well as suppressed developmental angiogenesis in a zebrafish model. Furthermore, this peptide effectively inhibited the cellular events that precede angiogenesis, such as ERK and eNOS phosphorylation and nitric oxide production, in endothelial cells stimulated with VEGF. Collectively, these data demonstrate that RLYE is a potent anti-angiogenic peptide that targets the VEGF signaling pathway.

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.

Hypoxia-responsive microRNA-101 promotes angiogenesis via heme oxygenase-1/vascular endothelial growth factor axis by targeting cullin 3.

AIMS: Hypoxia induces expression of various genes and microRNAs (miRs) that regulate angiogenesis and vascular function. In this study, we investigated a new functional role of new hypoxia-responsive miR-101 in angiogenesis and its underlying mechanism for regulating heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) expression. RESULTS: We found that hypoxia induced miR-101, which binds to the 3'untranslated region of cullin 3 (Cul3) and stabilizes nuclear factor erythroid-derived 2-related factor 2 (Nrf2) via inhibition of the proteasomal degradation pathway. miR-101 overexpression promoted Nrf2 nuclear accumulation, which was accompanied with increases in HO-1 induction, VEGF expression, and endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. The elevated NO-induced S-nitrosylation of Kelch-like ECH-associated protein 1 and subsequent induction of Nrf2-dependent HO-1 lead to further elevation of VEGF production via a positive feedback loop between the Nrf2/HO-1 and VEGF/eNOS axes. Moreover, miR-101 promoted angiogenic signals and angiogenesis both in vitro and in vivo, and these events were attenuated by inhibiting the biological activity of HO-1, VEGF, or eNOS. Moreover, these effects were also observed in aortic rings from HO-1(+/-) and eNOS(-/-) mice. Local overexpression of miR-101 improved therapeutic angiogenesis and perfusion recovery in the ischemic mouse hindlimb, whereas antagomiR-101 diminished regional blood flow. INNOVATION: Hypoxia-responsive miR-101 stimulates angiogenesis by activating the HO-1/VEGF/eNOS axis via Cul3 targeting. Thus, miR-101 is a novel angiomir. CONCLUSION: Our results provide new mechanistic insights into a functional role of miR-101 as a potential therapeutic target in angiogenesis and vascular remodeling.

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.

TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions.

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca(2+) elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.