Yong-Kyung Choe

Expression and regulation of NDRG2 (N-myc downstream regulated gene 2) during the differentiation of dendritic cells

We searched for genes with expressions specific to human monocyte‐derived dendritic cells (DCs) using differential display reverse transcription‐polymerase chain reaction, and found that N‐myc downstream regulated gene 2 (NDRG2), a member of a new family of differentiation‐related genes, was expressed in DCs. While DCs derived from CD34+ progenitor cells also showed strong NDRG2 expression, the corresponding mRNA expression was absent in other cell lines including monocytes, B cells, and NK cells. The inhibition of DC differentiation by dexamethasone or vitamin D3 treatment down‐regulated the expression of the NDRG2 gene in DCs. In addition, gene expression was induced in a myelomonocytic leukemia cell line, which is capable of differentiating into DCs in cytokine‐conditioned culture. The level of NDRG2 gene expression in DCs was significantly higher than that of other members of the NDRG gene family. Finally, in contrast to the stable NDRG2 expression in CD40‐stimulated DCs, the induction of DC maturation by lipopolysaccharide (LPS) resulted in the down‐regulation of NDRG2 gene expression. This down‐regulation is likely to be due to a modification and subsequent destabilization of NDRG2 mRNA, because co‐treating with actinomycin D and LPS significantly blocked this LPS effect. Taken together, our results indicate that NDRG2 is expressed during the differentiation of DCs, and that NDRG2 gene expression is differentially regulated by maturation‐inducing stimuli.

Down modulation of IL-18 expression by human papillomavirus type 16 E6 oncogene via binding to IL-18.

To understand modulation of a novel immune‐related cytokine, interleukin‐18, by human papillomavirus type (HPV) 16 oncogenes, HaCaT, normal keratinocyte cell line, and C‐33A, HPV‐negative cervical cancer cell line, were prepared to establish stable cell lines expressing E6, E6 mutant (E6m), E6E7, or E7 constitutively. Expressions of various HPV oncogene transcripts were identified by RT‐PCR. Expression of HPV oncogene E6 was reversely correlated to the expression of interleukin‐18, a novel pro‐inflammatory cytokine. The expression of E6 in C‐33A, independent of E6 splicing, resulted in decreased IL‐18 expression and that of IL‐18 was also significantly reduced in HaCaT cells expressing E6. The level of p53 was reduced in C‐33A cells expressing E6 whereas not altered in HaCaT cells expressing E6, suggesting that E6 downregulated IL‐18 expression via an independent pathway of p53 degradation in HaCaT cells which have a mutated p53 form. However, E7 did not affect IL‐18 expression significantly in both C‐33A and HaCaT cells. Cotransfection experiments showed that E6 oncogene did not inhibit the activities of IL‐18 promoter P1 and P2, suggesting that E6 oncogene indirectly inhibited IL‐18 expression. Taken together, E6, E6m and E6/E7 inhibited IL‐18 expression with some variation, assuming that cells expressing E6 oncogene can evade immune surveillance by downregulating the expression of immune stimulating cytokine gene, IL‐18, and inhibiting the cascade of downstream effects that follow activation of the IL‐18 receptor.

Dendritic cell-tumor coculturing vaccine can induce antitumor immunity through both NK and CTL interaction.

Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. We show here that immunization with bone marrow-derived DC cocultured with tumor cells can induce a protective immunity against challenges to viable tumor cells. In this study, we further investigated the mechanism by which the antitumor activity was induced. Immunization of mice with DC cocultured with murine colon carcinoma. CT-26 cells, augmented CTL activity against the tumor cells. Concomitantly, an increase in natural killer (NK) cell activity was also detected in the same mice. When DC were fixed with paraformaldehyde prior to coculturing with tumor cells, most of the CTL and NK cell activity diminished, indicating that DC are involved in the process of presenting the tumor antigen(s) to CTL. NK cell depletion in vivo produced markedly low tumor-specific CTL activity responsible for tumor prevention. In addition, RT-PCR analysis confirmed the high expression of INF-gamma mRNA in splenocytes after vaccination with DC cocultured with tumors, but low expression in splenocytes from NK-depleted mice. Most importantly, the tumor protective effect rendered to DC by the coculturing with CT-26 cells was not observed in NK-depleted mice, which suggests that DC can induce an antitumor immune response by enhancing NK cell-dependent CTL activation. Collectively, our results indicate that NK cells are required during the priming of cytotoxic T-cell response by DC-based tumor vaccine and seem to delineate a mechanism by which DC vaccine can provide the desired immunity.

Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation

Glucocorticoids (GC) are potent anti‐inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti‐inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)‐12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose‐ and time‐dependent manner as it was measured by determining annexin V‐positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex‐mediated apoptosis. The Dex‐induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z‐VAD‐fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex‐induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC‐mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.

Signaling pathways implicated in α‐melanocyte stimulating hormone‐induced lipolysis in 3T3‐L1 adipocytes

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying α‐melanocyte‐stimulating hormone (MSH)‐induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3‐L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone‐sensitive lipase, a rate‐limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal‐regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH‐induced glycerol release and HSL activity, indicating that MSH‐induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH‐induced lipolytic process via the regulation of HSL in 3T3‐L1 adipocytes. J. Cell. Biochem.

Activated natural killer cell-mediated immunity is required for the inhibition of tumor metastasis by dendritic cell vaccination

Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-g production, and IFN-γ- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8+ T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.

NDRG2 is one of novel intrinsic factors for regulation of IL-10 production in human myeloid cell

N-myc downstream-regulated gene 2 (NDRG2) implicated in cellular growth and differentiation was previously reported as it is specifically expressed in primary and in vitro-differentiated dendritic cells (DCs) from monocytes and CD34(+) progenitor cells. However, its function has yet to be investigated in DCs. Here, the novel NDRG2 function about modulation of cytokines in DC was observed in this study. The secretion of IL-10 was not found in the monocyte-derived DC cells with high level of NDRG2 expression, but IL-10 was abundantly secreted up to 1ng/ml in the monocyte-derived macrophages with low level of NDRG2 expression, and further confirmed that the expression of IL-10 was dramatically increased in NDRG2-silenced DCs under presence of LPS, and significantly reduced in the NDRG2-overexpressed U937 cells under stimulation of PMA. The secretion of IL-12p70 was significantly reduced in the siNDRG2 introduced DC cells. The intracellular signaling of IL-10 secretion was markedly inhibited by SB203580, inhibitor of p38 MAPK, in the LPS-activated DCs and phosphorylation of p38 MAPK was decreased in the NDRG2 introduced U937 cells under PMA-stimulation. Taken together, NDRG2 might have a pivotal role as one of intrinsic factors for the modulation of p38 MAPK phosphorylation, and subsequently involve in controlling of IL-10 production.

Epigenetic modification of TLR4 promotes activation of NF-κB by regulating methyl-CpG-binding domain protein 2 and Sp1 in gastric cancer

Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells.

A Simple ELISA for Screening Ligands of Peroxisome Proliferator-activated Receptor γ

Peroxisome proliferator-activated receptors (PPARs) are orphan nuclear hormone receptors that are known to control the expression of genes that are involved in lipid homeostasis and energy balance. PPARs activate gene transcription in response to a variety of compounds, including hypolipidemic drugs. Most of these compounds have high affinity to the ligand-binding domain (LBD) of PPARs and cause a conformational change within PPARs. As a result, the receptor is converted to an activated mode that promotes the recruitment of co-activators such as the steroid receptor co-activator-1 (SRC-1). Based on the activation mechanism of PPARs (the ligand binding to PPAR gamma induces interactions of the receptor with transcriptional co-activators), we performed Western blot and ELISA. These showed that the indomethacin, a PPAR gamma ligand, increased the binding between PPAR gamma and SRC-1 in a ligand dose-dependent manner. These results suggested that the in vitro conformational change of PPAR gamma by ligands was also induced, and increased the levels of the ligand-dependent interaction with SRC-1. Collectively, we developed a novel and useful ELISA system for the mass screening of PPAR gamma ligands. This screening system (based on the interaction between PPAR gamma and SRC-1) may be a promising system in the development of drugs for metabolic disorders.

Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation

Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.