Cheol Yi Hong

Molecular Mechanism of Suppression of Testicular Steroidogenesis by Proinflammatory Cytokine Tumor Necrosis Factor Alpha

ABSTRACT Tumor necrosis factor alpha (TNF-α) has been demonstrated to inhibit steroidogenesis in Leydig cells at the transcriptional level of steroidogenic enzymes. However, the molecular mechanism of this observed gene repression is not well understood. We now demonstrate that nuclear factor κB (NF-κB) activated by TNF-α inhibits the transactivation of orphan nuclear receptors, which regulate the expression of steroidogenic-enzyme genes. TNF-α treatment suppressed the luteinizing-hormone-induced or Nur77/SF-1-stimulated promoter activity of steroidogenic-enzyme genes in Leydig cells. The TNF-α-mediated gene suppression was blocked by treatment with an inhibitor of NF-κB. In addition, overexpression of the p65 (RelA) subunit of NF-κB showed the same effect as TNF-α and inhibited Nur77 transactivation, suggesting the involvement of NF-κB activation in the observed gene repression. Physical association of Nur77 with p65 was revealed by mammalian two-hybrid, GST pull-down, and coimmunoprecipitation analyses. The NF-κB inhibition of Nur77 transactivation was likely due to the competition of p65 for Nur77 binding with coactivators. Finally, chromatin immunoprecipitation assays revealed that TNF-α treatment caused the recruitment of NF-κB to the promoter of the steroidogenic-enzyme P450c17 gene, supporting the hypothesis that the TNF-α-mediated gene repression involves NF-κB inhibition of the transcriptional activity of Nur77 and other orphan nuclear receptors. These findings provide a molecular mechanism underlying the inhibition of testicular steroidogenesis by proinflammatory cytokines.

Expression of MIS in the Testis Is Downregulated by Tumor Necrosis Factor Alpha through the Negative Regulation of SF-1 Transactivation by NF-κB

ABSTRACT The expression of Mullerian inhibiting substance (MIS), a key molecule in sex differentiation and reproduction, is tightly regulated. It has been suggested that meiotic germ cells repress MIS expression in testicular Sertoli cells, although the substance responsible for this cell-cell communication remains unknown. Here, we present the cytokine tumor necrosis factor alpha (TNF-α) as a strong candidate for such a substance and its downstream molecular events. TNF-α inhibited MIS expression in testis organ cultures, and TNF-α−/− testes showed high and prolonged MIS expression. Furthermore, in transient-transfection assays TNF-α suppressed the MIS promoter that was activated by steroidogenic factor 1 (SF-1), one of the major transcription factors that regulate MIS expression. The modulation of SF-1 transactivation by TNF-α is through the activation of NF-κB, which subsequently interacts with SF-1 and represses its transactivation. The physical association of NF-κB with SF-1 was shown by yeast two-hybrid protein interaction, glutathione S-transferase pull-down, and coimmunoprecipitation (ChIP) analyses. ChIP assays also revealed that endogenous NF-κB, as well as SF-1, is recruited to the MIS promoter upon TNF-α signaling. SF-1-bound NF-κB subsequently recruits histone deacetylases to inhibit the SF-1-activated gene expression. These results may identify, for the first time, the responsible substance and its action mechanism underlying the repression of MIS expression by meiotic germ cells in the testis.

ROS inhibit the expression of testicular steroidogenic enzyme genes via the suppression of Nur77 transactivation.

Steroidogenesis decreases with aging in the testis, whereas the levels of reactive oxygen species (ROS) increase. In addition, ROS have been reported to inhibit testicular steroidogenesis. Here, we investigated the effects of ROS on the transcriptional activity of Nur77, one of the major transcription factors that regulate the expression of steroidogenic enzyme genes. ROS signaling inhibited Nur77 transactivation, which was diminished by either treatment with c-Jun N-terminal kinase (JNK) inhibitor or the expression of a dominant negative form of JNK. This suggests the involvement of JNK signaling, which elevates the expression of c-Jun as well as its phosphorylation in Leydig cells. In transient transfection assays, c-Jun suppressed Nur77 transactivation in a dose-dependent manner. Further studies using c-Jun mutants revealed that the protein level of c-Jun, but not phosphorylation itself, was important for the suppression of Nur77 transactivation. Nur77 directly interacted with c-Jun in vivo, which blocked the DNA binding activity of Nur77. Together, these results suggest that ROS signaling-mediated c-Jun upregulation suppresses the expression of steroidogenic enzyme genes by inhibiting Nur77 transactivation, resulting in the reduction of testicular steroidogenesis. These findings may provide a mechanistic explanation for the age-related decline in testicular steroid hormone production.

Type I and II interferons enhance dendritic cell maturation and migration capacity by regulating CD38 and CD74 that have synergistic effects with TLR agonists

The major limitation for the maturation of dendritic cells (DCs) using Toll-like receptor (TLR) agonists is their decreased ability to migrate into lymph nodes compared with conventional DCs. CD38 can be used as a multifunctional marker to modulate migration, survival and Th1 responses of DCs. CD74 has been shown to negatively regulate DC migration. The goal of this study was to investigate the combinations of TLR agonists and interferons (IFNs) that most effectively regulate CD38 and CD74 expression on DCs. Synergistic TLR agonist stimulation in combination with IFN-α and IFN-γ was the best method for regulating CD38 and CD74 expression and inducing the highest secretion of IL-12p70. An in vitro migration assay showed that DCs treated with this combination had significantly enhanced migratory ability, similar to that observed in cells expressing CD38, CD74 and CCR7. The results of this study suggest that an alternative maturation protocol in which two TLR ligands are combined with type I and II IFNs generates potent DCs that have both a high migratory capacity and high IL-12p70 production.

Enhancement of antitumor effect using dendritic cells activated with natural killer cells in the presence of Toll-like receptor agonist

Dendritic cells (DCs) play a role in natural killer (NK) cell activation, while NK cells are also able to activate and mature DCs. Toll-like receptors (TLRs) on the surface of DCs and NK cells induce the maturation and activation of these cells when engaged with their cognate ligand. We investigated to generate potent DCs by maturation with NK cells in the presence of TLR agonist in vitro and tested the efficacy of these DC vaccinations in mouse colon cancer model. The optimal ratios of DCs versus NK cells were 1:1 to 1:2. Immature DCs were mature with NK cells in the presence of lipopolysaccharide, which is TLR4 agonist, and further addition of IL-2 induced phenotypically and functionally mature bone marrow-derived DCs. These potent DCs exhibited not only high expression of several costimulatory molecules and high production of IL-12p40 and IL-12p70, but also high allogeneic T cells stimulatory capacity, and the induction of the high activities to generate tumor-specific CTLs. Consistently, vaccination with these DCs efficiently inhibited CT-26 tumor growth in mouse colon cancer model when compared to other vaccination strategies. Interestingly, combination therapy of these DC-based vaccines and with low-dose cyclophosphamide showed dramatic inhibition effects of tumor growth. These results suggest that the DCs maturated with NK cells in the presence of TLR agonist are potent inducer of antitumor immune responses in mouse model and may provide a new source of DC-based vaccines for the development of immunotherapy against colon cancer.

Orphan Nuclear Receptor Nur77 Induces Zinc Finger Protein GIOT-1 Gene Expression, and GIOT-1 Acts as a Novel Corepressor of Orphan Nuclear Receptor SF-1 via Recruitment of HDAC2

Kruppel-associated box (KRAB) domain-containing proteins consist of potential transcriptional repression modules. Previously, gonadotropin-inducible ovarian transcription factor-1 (GIOT-1) was identified as a novel KRAB-containing zinc finger protein and shown to have transcriptional repression activity. Here, we demonstrate that orphan nuclear receptor Nur77 regulates GIOT-1 gene expression in testicular Leydig cell lines and that GIOT-1 acts as a novel corepressor of the orphan nuclear receptor steroidogenic factor 1 (SF-1). Mutation analysis of the GIOT-1 promoter and overexpression analysis of dominant-negative Nur77 revealed that luteinizing hormone activates GIOT-1 gene expression through Nur77. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Nur77 directly binds to the GIOT-1 promoter. GIOT-1 represses the SF-1 transactivation, and specific interaction between GIOT-1 and SF-1 was observed. We also demonstrate an interaction between GIOT-1 and histone deacetylase 2 (HDAC2). GIOT-1-mediated transrepression was recovered by down-regulation of HDAC2 expression with small interfering RNA of HDAC2. Knock down of the endogenous GIOT-1 results in significant enhancement of CYP17 expression in Leydig cells. In conclusion, this study of cross-talk between GIOT-1 and orphan nuclear receptors will provide new insights into the role of KRAB-containing zinc finger proteins in nuclear receptor action.

All-trans retinoic acid inhibits the differentiation, maturation, and function of human monocyte-derived dendritic cells

All-trans retinoic acid (ATRA) affects on the function of antigen presenting cells with somewhat controversies. We investigated the effects of ATRA on differentiation, maturation and function of human monocyte-derived dendritic cells (DCs). Low dose (10(-14)M) or high dose (10(-6)M) of ATRA was added either when monocytes were differentiated into immature DCs (imDCs) or mature DCs (mDCs) were induced. Apoptotic cell populations were dramatically increased in imDCs or mDCs with increasing concentration of ATRA. The productions of IL-12p40 and IL-12p70 were significantly suppressed in imDCs or mDCs induced by the addition of ATRA in the dose-dependent manner, whereas IL-10 was increased. DCs cultured with ATRA induced the differentiation of naïve T cells towards a helper T cell type 2 (Th2) response and expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cells. Allostimulatory capacity of DCs was suppressed with increasing concentration of ATRA. These findings suggest that ATRA inhibits the effects on the differentiation, maturation and function of human monocyte-derived DCs in vitro and also enhance the differentiation of naïve T cell toward the Th2 type.

Alpha-type 1-polarized dendritic cells loaded with apoptotic allogeneic myeloma cell line induce strong CTL responses against autologous myeloma cells

To induce a potent cytotoxic T lymphocyte (CTL) response, various tumor antigens should be loaded onto dendritic cells (DCs). In multiple myeloma (MM), it is difficult to obtain a sufficient number of autologous tumor cells as a source of tumor antigens in the clinical setting. We investigated the feasibility of immunotherapy in patients with MM, using myeloma-specific CTLs generated in vitro by alpha-type 1-polarized DCs (αDC1s) loaded with the ultraviolet B-irradiated allogeneic myeloma cell line, ARH77. αDC1s significantly increased the expression of several costimulatory molecules without differences in loading with tumor antigens. αDC1s showed a high production of interleukin-12 during maturation and after subsequent stimulation with CD40L but were not significantly affected by loading tumor antigens. Myeloma-specific CTLs against autologous myeloma cells from MM patients were induced by αDC1s pulsed with apoptotic ARH77 cells. Our data indicate that autologous DCs loaded with an allogeneic myeloma cell line can generate potent myeloma-specific CTL responses against autologous myeloma cells and might provide a practical method for cellular immunotherapy in patients with MM.

Modulation of Androgen Receptor Transactivation by the SWI3-Related Gene Product (SRG3) in Multiple Ways

ABSTRACT The SWI3-related gene product (SRG3), a component of the mouse SWI/SNF complex, has been suggested to have an alternative function. Here, we demonstrate that in the prostate transactivation of the androgen receptor (AR) is modulated by SRG3 in multiple ways. The expression of SRG3, which is developmentally regulated in the prostate, is induced by androgen through AR. SRG3 in turn enhances the transactivation of AR, providing a positive feedback regulatory loop. The SRG3 coactivation of AR transactivation is achieved through the recruitment of coactivator SRC-1, the protein level of which is upregulated by SRG3, providing another pathway of positive regulation. Interestingly, SRG3 coactivation of AR transactivation is fully functional in BRG1/BRM-deficient C33A cells and the AR/SRG3/SRC-1 complex formed in vivo contains neither BRG1 nor BRM protein, suggesting the possibility of an SRG3 function independent of the SWI/SNF complex. Importantly, the AR/SRG3/SRC-1 complex occupies androgen response elements on the endogenous SRG3 and PSA promoter in an androgen-dependent manner in mouse prostate and LNCaP cells, respectively, inducing gene expression. These results suggest that the multiple positive regulatory mechanisms of AR transactivation by SRG3 may be important for the rapid proliferation of prostate cells during prostate development and regeneration.

Successful cross-presentation of allogeneic myeloma cells by autologous alpha-type 1-polarized dendritic cells as an effective tumor antigen in myeloma patients with matched monoclonal immunoglobulins

AbstractsFor wide application of a dendritic cell (DC) vaccination in myeloma patients, easily available tumor antigens should be developed. We investigated the feasibility of cellular immunotherapy using autologous alpha-type 1-polarized dendritic cells (αDC1s) loaded with apoptotic allogeneic myeloma cells, which could generate myeloma-specific cytotoxic T lymphocytes (CTLs) against autologous myeloma cells in myeloma patients. Monocyte-derived DCs were matured by adding the αDC1-polarizing cocktail (TNFα/IL-1β/IFN-α/IFN-γ/poly-I:C) and loaded with apoptotic allogeneic CD138+ myeloma cells from other patients with matched monoclonal immunoglobulins as a tumor antigen. There were no differences in the phenotypic expression between αDC1s loaded with apoptotic autologous and allogeneic myeloma cells. Autologous αDC1s effectively took up apoptotic allogeneic myeloma cells from other patients with matched subtype. Myeloma-specific CTLs against autologous target cells were successfully induced by αDC1s loaded with allogeneic tumor antigen. The cross-presentation of apoptotic allogeneic myeloma cells to αDC1s could generate CTL responses between myeloma patients with individual matched monoclonal immunoglobulins. There was no difference in CTL responses between αDC1s loaded with autologous tumor antigen and allogeneic tumor antigen against targeting patients myeloma cells. Our data indicate that autologous DCs loaded with allogeneic myeloma cells with matched immunoglobulin can generate potent myeloma-specific CTL responses against autologous myeloma cells and can be a highly feasible and effective method for cellular immunotherapy in myeloma patients.