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.

Prostaglandin E2 stimulates COX-2 expression via mitogen-activated protein kinase p38 but not ERK in human follicular dendritic cell-like cells.

BACKGROUND: Prostaglandin E2 (PGE2) is an endogenous lipid mediator of inflammation. Its production is regulated by the rate-limiting upstream enzyme cyclooxygenase-2 (COX-2). We have recently demonstrated that the major cell type expressing COX-2 in the germinal center is follicular dendritic cell (FDC). In this study, to elucidate the molecular mechanism of PGE2 in COX-2 production, we asked whether mitogen-activated protein kinases ERK and p38 might regulate COX-2 expression. RESULTS: FDC-like cells were used to analyze the phosphorylation kinetics of ERK and p38 and the impact of genetic knockdown. PGE2 stimulation gave rise to a rapid increase of p38 but not ERK phosphorylation. In contrast, IL-1ß induced phosphorylation of both MAPKs. Knockdown of p38 resulted in a marked suppression of COX-2 expression induced by either PGE2 or IL-1ß. ERK knockdown did not significantly affect the effect of PGE2 and IL-1ß on COX-2 induction. The differential results of p38 and ERK siRNA transfection were reproduced in the production of prostaglandins and in experiments performed with pharmacologic inhibitors. CONCLUSIONS: Our data indicate that p38 is essentially required for PGE2 to induce COX-2 expression in FDC-like cells. The current study helps to expand our understanding of the biological function of FDC at the molecular level and provides a potential rationale for the pharmacologic or genetic approaches to regulate p38 MAPK in the treatment of various inflammatory disorders.

A paracrine effect of 15 (S)-hydroxyeicosatetraenoic acid revealed in prostaglandin production by human follicular dendritic cell-like cells.

Lipid mediators play active roles in each stage of inflammation under physiological and pathologic conditions. We have investigated the cellular source and functions of several prostanoids in the immune inflammatory responses using follicular dendritic cell (FDC)-like cells. In this study, we report a novel finding on the role of 15(S)- hydroxyeicosatetraenoic acid (HETE). Our observation of 15(S)-HETE uptake by FDC-like cells prompted to hypothesize that 15(S)-HETE might have a regulatory role in the other branch of eicosanoid production. The effects of 15(S)-HETE on COX-2 expression and prostaglandin (PG) production were analyzed by immunoblotting and specific enzyme immunoassays. The addition of 15(S)-HETE resulted in elevated levels of COX-2 expression and PG production. The enhanced PG production was not due to growth stimulation of FDC-like cells since 15(S)-HETE did not modulate FDC-like cell proliferation by the culture period of PG measurement. Peroxisome proliferator-activated receptor gamma (PPARγ) seems to mediate the augmenting activity as the antagonist GW9662 dose- dependently prevented 15(S)-HETE from increasing PG production. In addition, PPARγ protein expression was readily detected in FDC-like cells. These effects of 15(S)-HETE were displayed in the combined addition with IL-1ß. Based on these results, we suggest that 15(S)-HETE is an inflammatory costimulator of FDC acting in a paracrine fashion.

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.

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.

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.

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.

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.

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.

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.

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.

Wnt5a is secreted by follicular dendritic cells to protect germinal center B cells via Wnt/Ca2+/NFAT/NF-κB-B cell lymphoma 6 signaling.

Follicular dendritic cells (FDCs) protect germinal center (GC) B cells from rapid apoptosis to allow their survival and maturation. In this article, we show that FDCs normally produce and secrete Wnt5a to protect GC B cells. Wnt5a production is upregulated by polyI:C. Purified Wnt5a protects GC B cells from apoptosis in a dose-dependent manner. GC B cells are protected by FDC coculture or conditioned medium, and the protection is inhibited significantly by anti-Wnt5a Ab, suggesting a major role of Wnt5a in the FDC-mediated GC B cell protection. A calcium chelator BAPTA-AM blocks the Wnt5a-mediated GC B cell protection, implying a role of Wnt/Ca(2+) signaling in the GC B cell survival. Wnt5a and calcium ionophore activate NFATc1, NFATc2, NF-κB, and B cell lymphoma 6 (BCL-6) promptly and upregulate CD40 expression in GC B and Ramos cells, whereas p53 and JNK are not upregulated or activated. Cyclosporine A inhibits the Wnt5a and calcium-induced activation of NF-κB and BCL-6 in Ramos cells, supporting a role of ß-catenin-independent Wnt/Ca(2+)/NFAT/NF-κB-BCL-6 signaling. Our data support that Wnt5a is a novel survival factor for GC B cells and might be a potential target for the regulation of B cell immunity.

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.

Beraprost enhances the APC function of B cells by upregulating CD86 expression levels.

Lipid mediators are emerging as important regulators of the immune system. Based on our previous result that shows strong expression of prostacyclin synthase in the germinal center, we investigated whether prostacyclin would regulate the APC function of B cells. Owing to the very short half-life of prostacyclin in experimental conditions, we used a more stable analog, beraprost. Beraprost increased the amounts of the costimulatory molecule CD86 but not CD80 on the surface of activated B cells in time- and dose-dependent manners. However, the enhancing effect of beraprost was not observed on memory B cells, centroblasts, and centrocytes. Beraprost required BCR and CD40 signals to upregulate CD86 expression levels. Other prostanoids such as PGE(2), 6-keto-PGF(1α), and PGF(2α) failed to alter CD86 expression levels, whereas other prostacyclin analogs were as potent as beraprost. Results carried out with receptor antagonists revealed that beraprost enhanced CD86 levels by binding to prostacyclin receptor IP and by increasing intracellular cAMP concentrations. Beraprost-treated B cells potently stimulated allogeneic T cells, which was significantly abolished by CD86 neutralization. Our data imply an unrecognized cellular and molecular mechanism about the germinal center reactions.

Differential effects of substrate-analogue inhibitors on nitric oxide synthase dimerization.

Nitric oxide synthase (NOS) isoforms are hemoenzymes that are only active as homodimers. We have examined the effect of the substrate-analogue inhibitors, N(G)-monomethyl-L-arginine (L-NMA), N(G)-nitro-L-arginine (L-NNA), N(G)-nitro-L-arginine methyl ester (L-NAME), N(5)-(1-iminoethyl)-L-ornithine (L-NIO), and N(6)-(1-iminoethyl)-L-lysine (L-NIL), the guanidine-containing inhibitor aminoguanidine (AG), and the amidine moiety-containing iNOS-specific inhibitor 1400W, on the formation of NOS dimer. Of these inhibitors, L-NMA effectively not only inhibited iNOS dimerization, but also destabilized its dimeric form in RAW264.7 cells stimulated with lipopolysaccharide plus interferon-γ, but not eNOS dimerization in endothelial cells. Importantly, this inhibition was highly correlated with NO production. These inhibitory effects were significantly reversed by addition of L-arginine. However, L-NNA, L-NAME, and AG in part or significantly increased dimerization of iNOS and eNOS in intact cells, and the other inhibitors assessed did not alter dimerization of iNOS and eNOS. These data taken together suggest that substituted groups of an arginine guanidino moiety play an important role in NOS dimerization as well as its catalytic activity. Our results indicate that l-NMA can inhibit iNOS-dependent NO production by preventing iNOS dimerization and destabilizing its dimeric form.

Intratumoral electroporation of IL-12 cDNA eradicates established melanomas by Trp2(180-188)-specific CD8+ CTLs in a perforin/granzyme-mediated and IFN-γ-dependent manner: application of Trp2(180-188) peptides.

Intratumoral electroporation (IT-EP) with IL-12 cDNA (IT-EP/IL12) can lead to the eradication of established B16 melanoma tumors in mice. Here, we explore the immunological mechanism of the antitumor effects generated by this therapy. The results show that IT-EP/IL12 applied only once resulted in eradication in 70% animals with large established B16 tumors. Tumor eradication required the participation of CD8+ T cells, but not CD4+ T cells and NK cells. IT-EP/IL12 induced antigen-specific CD8+ T cell responses against the immunodominant Trp2(180-188) epitope and generated a systemic response, resulting in significant therapeutic effects against distal, untreated tumors. The therapeutic effect of IT-EP/IL12 was absent in perforin-deficient mice, indicating that tumor elimination occurred through conventional perforin/granzyme lysis by CTLs. Moreover, this therapy induced some degree of immunological memory that protected approximately one-third of the cured mice against a subsequent tumor challenge. Moreover, antitumor efficacy and long-term protection against B16 were significantly improved by concurrent Trp2 peptide immunization through more induction of Ag-specific CTL responses and more attraction of IFN-γ-expressing CD8+ T cells into tumor sites. The antitumor effect of IT-EP/IL12 required the participation of IFN-γ, which was shown to induce MHC class I expression on B16 cells and increase the lytic activity of the CD8+ CTL generated by IT-EP/IL12. The results from these animal studies may help in the development of IT-EP/IL12 for cancer patients.

Human follicular dendritic cells promote the APC capability of B cells by enhancing CD86 expression levels.

Follicular dendritic cells (FDCs) are an essential cellular component of the germinal center (GC) and are believed to exert regulatory effects on the various stages of GC reactions. According to our previous reports, human FDCs express prostacyclin synthase, and prostacyclin analogues augment adhesion and co-stimulatory molecules on the surface of activated B cells. These findings prompted us to investigate whether FDCs would contribute to the antigen-presenting capability of B cells by using the well-established FDC-like cells, HK cells, and tonsillar B cells. Our results show that HK cells significantly enhance the expression levels of CD54, CD80, and CD86 on the surface of activated B cells. The enhancing effect of HK cells on CD86 is impeded by indomethacin and an EP4 antagonist, implying that a certain prostaglandin is mediating the up-regulation. Prostacyclin indeed recapitulates the enhancing effect on CD86, which is inhibited by EP4 as well as IP antagonists. B cells co-cultured with HK cells exhibit an augmented APC activity, which is inhibited by CD86 neutralization. These results reveal another unrecognized function of human FDC.

Inducible costimulator promotes helper T-cell differentiation through phosphoinositide 3-kinase.

The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (T(FH)) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in T(FH) generation, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4(+) T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key T(FH) cytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of T(FH) and suggest that CD28 and ICOS play differential roles during a multistep process of T(FH) differentiation.

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.