Bok Yun Kang

Oxidized Low Density Lipoprotein Inhibits Interleukin-12 Production in Lipopolysaccharide-activated Mouse Macrophages via Direct Interactions between Peroxisome Proliferator-activated Receptor-γ and Nuclear Factor-κB

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a κB site within the IL-12 p40 promoter. In this study, we found that oxidized low density lipoprotein (oxLDL) inhibited this LPS-stimulated production of IL-12 in a dose-dependent manner while native LDL did not. OxLDL inhibited p40 promoter activation in monocytic RAW264.7 cells transiently transfected with p40 promoter/reporter constructs, and the repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor-κB (NF-κB) (p40-κB). Activation of macrophages by LPS in the presence of oxLDL resulted in markedly reduced binding to the κB site, as demonstrated by the electrophoretic mobility shift assays. In contrast, native LDL did not inhibit the IL-12 p40 promoter activation and NF-κB binding to the κB sites, suggesting that oxidative modification of LDL was crucial for the inhibition of NF-κB-mediated IL-12 production. 9-Hydroxyoctadecadienoic acid, a major oxidized lipid component of oxLDL, significantly inhibited IL-12 production in LPS-stimulated mouse macrophages and also suppressed NF-κB-mediated activation in IL-12 p40 promoter. The NF-κB components p50 and p65 directly bound peroxisome proliferator-activated receptor-γ (PPAR-γ) in vitro. In cotransfections of CV-1 and HeLa cells, PPAR-γ inhibited the NF-κB transactivation in an oxLDL-dependent manner. From these results, we propose that oxLDL-mediated suppression of the IL-12 production from LPS-activated mouse macrophages may, at least in part, involve both inhibition of the NF-κB-DNA interactions and physical interactions between NF-κB and PPAR-γ.

Retinoids Inhibit Interleukin-12 Production in Macrophages through Physical Associations of Retinoid X Receptor and NFκB

Lipopolysaccharide (LPS) increases the production of interleukin-12 (IL-12) from mouse macrophages via a κB site within the IL-12 p40 promoter. In this study, we found that retinoids inhibit this LPS-stimulated production of IL-12 in a dose-dependent manner. The NFκB components p50 and p65 bound retinoid X receptor (RXR) in a ligand-independent manner in vitro, and the interaction interfaces involved the p50 residues 1–245, the p65 residues 194–441, and the N-terminal A/B/C domains of RXR. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the κB site, which significantly decreased upon addition of retinoids, as demonstrated by the electrophoretic mobility shift assays. In cotransfections of CV-1 and HeLa cells, RXR also inhibited the NFκB transactivation in a ligand-dependent manner, whereas a mutant RXR lacking the AF2 transactivation domain, which serves as ligand-dependent binding sites for transcription integrators SRC-1 and p300, was without any effect. In addition, coexpression of increasing amounts of SRC-1 or p300 relieved the retinoid-mediated inhibition of the NFκB transactivation. From these results, we propose that retinoid-mediated suppression of the IL-12 production from LPS-activated macrophages may involve both inhibition of the NFκB-DNA interactions and competitive recruitment of transcription integrators between NFκB and RXR.

Inhibition of interleukin-12 and interferon-γ production in immune cells by tanshinones from Salvia miltiorrhiza

Pharmacological control of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) production may be a key therapeutic strategy for modulating immunological diseases dominated by Th1-derived cytokine responses. In this study, we investigated the effects of three different tanshinone pigments from Salvia miltiorrhiza (tanshinone I, dihydrotanshinone, and cryptotanshinone) on IL-12 production in mouse macrophages and on IFN-gamma production in lymph node cells. All tested tanshinones significantly inhibited IL-12 production in lipopolysaccharide (LPS)-activated macrophages and also IFN-gamma production in keyhole limpet hemocyanin (KLH)-primed lymph node cells in a dose-dependent manner. Dihydrotanshinone was more effective than tanshinone I or cryptotanshinone. Tanshinones significantly inhibited the expression of IL-12 p40 gene at the mRNA level. Furthermore, tanshinones potently inhibited the promoter activation of IL-12 p40 gene and nuclear factor (NF)-kappaB binding to the kappaB site, suggesting that tanshinones may negatively regulate IL-12 production at the transcription level. These results may explain some known biological activities of tanshinones including their anti-inflammatory effect, and suggest a possible use of tanshinones in the treatment of immunological diseases dominated by Th1-derived cytokine responses.

Curcumin inhibits Th1 cytokine profile in CD4+ T cells by suppressing interleukin‐12 production in macrophages

Interleukin‐12 (IL‐12) plays a central role in the immune system by driving the immune response towards T helper 1 (Th1) type responses which are characterized by high IFN‐γ and low IL‐4 production. In this study we investigated the effects of curcumin, a natural product of plants obtained from Curcuma longa (turmeric), on IL‐12 production by mouse splenic macrophages and the subsequent ability of these cells to regulate cytokine production by CD4+ T cells. Pretreatment with curcumin significantly inhibited IL‐12 production by macrophages stimulated with either lipopolysaccharide (LPS) or head‐killed Listeria monocytogenes (HKL). Curcumin‐pretreated macrophages reduced their ability to induce IFN‐γ and increased the ability to induce IL‐4 in Ag‐primed CD4+ T cells. Addition of recombinant IL‐12 to cultures of curcumin‐pretreated macrophages and CD4+ T cells restored IFN‐γ production in CD4+ T cells. The in vivo administration of curcumin resulted in the inhibition of IL‐12 production by macrophages stimulated in vitro with either LPS or HKL, leading to the inhibition of Th1 cytokine profile (decreased IFN‐γ and increased IL‐4 production) in CD4+ T cells. These findings suggest that curcumin may inhibit Th1 cytokine profile in CD4+ T cells by suppressing IL‐12 production in macrophages, and points to a possible therapeutic use of curcumin in the Th1‐mediated immune diseases.

Sirt2 interacts with 14-3-3 β/γ and down-regulates the activity of p53

Sirt2 is a mammalian member of the Sirtuin family of NAD(+) (nicotinamide adenine dinucleotide)-dependent protein deacetylases. Although Sir-2.1 (a Caenorhabditis elegans Sirt2 ortholog) has been reported to interact with PAR-5/FTT-2 (a C. elegans 14-3-3 homolog), the molecular significance of the interaction between Sirt2 and 14-3-3 proteins in mammalian cell is not understood. Here, we report that Sirt2 interacts with 14-3-3 beta and gamma among various 14-3-3 isoforms, and that this interaction is strengthened by AKT. Furthermore, Sirt2 deacetylates and down-regulates the transcriptional activity of p53, and 14-3-3 beta/gamma augment deacetylation and down-regulation of the p53 transcriptional activity by Sirt2 in an AKT-dependent manner. Treatment of cells with nicotinamide, an inhibitor of Sirtuins, relieves the inhibition of p53 by Sirt2 and 14-3-3 beta/gamma. Therefore, our results suggest that the interaction between Sirt2 and 14-3-3 beta/gamma is a novel mechanism for the negative regulation of p53 beside the well-characterized Mdm2-mediated repression.

Enhanced interleukin-4 production in CD4+ T cells and elevated immunoglobulin E levels in antigen-primed mice by bisphenol A and nonylphenol, endocrine disruptors: involvement of nuclear factor-AT and Ca2+

Bisphenol A (BPA) and p‐nonylphenol (NP) are representative endocrine disruptors (EDs) that may have adverse effects on human health. The influence of these compounds on allergic immune responses remains unclear. In this study, we have examined the effects of BPA and NP on production of interleukin‐4 (IL‐4), a pro‐inflammatory cytokine closely associated with allergic immune responses. Both BPA and NP significantly enhanced IL‐4 production in keyhole limpet haemocyanin (KLH)‐primed CD4+ T cells in a concentration‐dependent manner. Treatment with BPA or NP in vivo resulted in significant increase of IL‐4 production in CD4+ T cells and of antigen‐specific immunoglobulin E (IgE) levels in the sera of KLH‐primed mice. Furthermore, BPA and NP enhanced the activation of IL‐4 gene promoter in EL4 T cells transiently transfected with IL‐4 promoter/reporter constructs, and the enhancing effect mapped to a region in the IL‐4 promoter containing binding sites for nuclear factor (NF)‐AT. Activation of T lymphocytes by phorbol 12‐myristate 13‐acetate/ionomycin resulted in markedly enhanced binding activities to the NF‐AT site, which significantly increased upon addition of BPA or NP, as demonstrated by the electrophoretic mobility shift assay, indicating that the transcription factor NF‐AT was involved in the enhancing effect of BPA and NP on IL‐4 production. The enhancement of IL‐4 production by BPA or NP was significantly reduced by nitrendipine, a blocker of Ca2+ influx, and by FK506, a calcineurin inhibitor. FK506 inhibited the NF‐AT–DNA binding activity and IL‐4 gene promoter activity enhanced by BPA or NP. These results represent the first report describing possible enhancement of allergic response by EDs through increasing IL‐4 production in CD4+ T cells and antigen‐specific IgE levels in the sera via the stimulation of Ca2+/calcineurin‐dependent NF‐AT activation.

Inhibition of interleukin-12 production in lipopolysaccharide-activated macrophages by curcumin.

Pharmacological control of interleukin-12 production may be a key therapeutic strategy for modulating immunological diseases dominated by type-1 cytokine responses. In this study we investigated the effects of curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1, 6-heptadiene-3,5-dione) on the production of interleukin-12 from mouse macrophages stimulated with lipopolysaccharide. Curcumin potently inhibited the production of interleukin-12 in a dose-dependent manner. The effect of curcumin on interleukin-12 p40 promoter activation was analyzed by transfecting RAW264.7 monocytic cells with p40 promoter/reporter constructs. The repressive effect mapped to a region in the p40 promoter containing a binding site for nuclear factor kappaB (p40-kappaB). Furthermore, activation of macrophages by lipopolysaccharide resulted in markedly enhanced binding activity to the kappaB site, which significantly decreased upon addition of curcumin. These results suggest that curcumin-induced inhibition of interleukin-12 production in macrophages may explain some of the biological effects of curcumin including its anti-inflammatory activity.

Acetylation of Sirt2 by p300 attenuates its deacetylase activity.

Histone deacetylases (HDACs) are subdivided into three classes--HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.

Differential anti-inflammatory pathway by xanthohumol in IFN-γ and LPS-activated macrophages

Macrophages are the main cells responsible for the innate immunity, and their activation by lipopolysaccharide (LPS) from Gram-negative bacteria or interferon (IFN)-gamma from host immune cells is important for controlling infections. However, the overwhelming activation of macrophages can cause a severe inflammatory state. This study investigated the inhibitory mechanism of xanthohumol (XN) against the inflammatory effectors (IL-1beta, TNF-alpha, and iNOS) in activated RAW264.7 macrophages by using different stimuli such as LPS, IFN-gamma, or LPS plus IFN-gamma. XN is a major prenylated chalcone found in hops, which is used to add bitterness and flavor to beer. XN reduced the expression of the LPS receptor components such as TLR4 and MD2 resulting in the suppression of NF-kappaB activation in LPS-activated RAW264.7 cells. In the IFN-gamma stimulated RAW264.7 cells, the binding activity of STAT-1alpha and IRF-1 was inhibited by XN. This suggests that differential signaling pathways are used by XN for the inhibition of excess inflammatory mediators depending on the stimuli in macrophages.

Topoisomerase I inhibition and cytotoxicity of licochalcones A and E from Glycyrrhiza inflata.

Licochalcones A (1) and E (2), retrochalcones or reversely constructed chalcones, isolated from the roots ofGlycyrrhiza inflata, were evaluated for their cytotoxicities against four different human tumor cell lines; A549 (lung), SK-OV-3 (ovarian), SK-MEL-2 (melanoma) and HCT-15 (colon), using the sulforhodamine B (SRB) assay. The effects of these compounds toward the DNA topoisomerase l (topo l) inhibitory activity were also measured using the supercoiled DNA unwinding assay. All compounds showed moderate cytotoxicities against the four different human tumor cell lines and inhibited the topo l activity in dose-dependent manners. The inhibition of topo l by licochalcones A (1) and E (2) may explain the cytotoxicities of these compounds against the human tumor cell lines.