Kyeong Man Kim

Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.

Induction of human promyelocytic leukemia HL-60 cell differentiation into monocytes by silibinin: involvement of protein kinase C.

The effect of silibinin, an active component of Silybum marianum, on cellular differentiation was investigated in the human promyelocytic leukemia HL-60 cell culture system. Treatment of HL-60 cells with silibinin inhibited cellular proliferation and induced cellular differentiation in a dose-dependent manner. Cytofluorometric analysis and morphologic studies indicated that silibinin induced differentiation of HL-60 cells predominantly into monocytes. Importantly, strongly synergistic induction of differentiation into monocytes was observed when silibinin was combined with 5 nM 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], a well-known differentiation inducer of HL-60 cells into the monocytic lineage. Silibinin enhanced protein kinase C (PKC) activity and increased protein levels of both PKCalpha and PKCbeta in 1,25-(OH)(2)D(3)-treated HL-60 cells. PKC and extracellular signal-regulated kinase (ERK) inhibitors significantly inhibited HL-60 cell differentiation induced by silibinin alone or in combination with 1,25-(OH)(2)D(3), indicating that PKC and ERK may be involved in silibinin-induced HL-60 cell differentiation.

Agonist-Induced Endocytosis and Receptor Phosphorylation Mediate Resensitization of Dopamine D2 Receptors

The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.

Heterologous down-regulation of angiotensin type 1 receptors by purinergic P2Y2 receptor stimulation through S-nitrosylation of NF-κB

Cross-talk between G protein-coupled receptor (GPCR) signaling pathways serves to fine tune cellular responsiveness by neurohumoral factors. Accumulating evidence has implicated nitric oxide (NO)-based signaling downstream of GPCRs, but the molecular details are unknown. Here, we show that adenosine triphosphate (ATP) decreases angiotensin type 1 receptor (AT1R) density through NO-mediated S-nitrosylation of nuclear factor κB (NF-κB) in rat cardiac fibroblasts. Stimulation of purinergic P2Y2 receptor by ATP increased expression of inducible NO synthase (iNOS) through activation of nuclear factor of activated T cells, NFATc1 and NFATc3. The ATP-induced iNOS interacted with p65 subunit of NF-κB in the cytosol through flavin-binding domain, which was indispensable for the locally generated NO-mediated S-nitrosylation of p65 at Cys38. β-Arrestins anchored the formation of p65/IκBα/β-arrestins/iNOS quaternary complex. The S-nitrosylated p65 resulted in decreases in NF-κB transcriptional activity and AT1R density. In pressure-overloaded mouse hearts, ATP released from cardiomyocytes led to decrease in AT1R density through iNOS-mediated S-nitrosylation of p65. These results show a unique regulatory mechanism of heterologous regulation of GPCRs in which cysteine modification of transcriptional factor rather than protein phosphorylation plays essential roles.

Hydroquinone, a reactive metabolite of benzene, enhances interleukin-4 production in CD4+ T cells and increases immunoglobulin E levels in antigen-primed mice

Exposure to cigarette smoke is known to increase the risk of the development of allergic disease. The mechanism is not well understood. In this study, we determined the effect of hydroquinone (HQ), a major metabolite of benzene present in large quantities in cigarette tar, on interleukin‐4 (IL‐4) production by CD4+ T cells. HQ significantly enhanced IL‐4 production by keyhole limpet haemocyanin (KLH)‐primed CD4+ T cells in a dose‐dependent manner. The enhancing effect of HQ on IL‐4 production was maximal at a concentration of 50 µm. It increased the level of IL‐4 production approximately 10‐fold. HQ enhanced IL‐4 mRNA expression and also IL‐4 gene promoter activity, suggesting that the enhancing effect of HQ on IL‐4 production may occur at the transcriptional level. Furthermore, the injection of KLH‐primed mice with HQ resulted in a significant increase in the levels of IL‐4 and immunoglobulin E. These findings provide evidence that HQ, a major component of cigarette tar, may enhance allergic immune responses by inducing the production of IL‐4 in CD4+ T cells.

β-arrestin2 in infiltrated macrophages inhibits excessive inflammation after myocardial infarction.

Beta-arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. β-arrestin1 and β-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of β-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that β-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. β-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, β-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in β-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of β-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of β-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of β-arrestin2 KO mice is enhanced by MI, which is similar to that in β-arrestin2 KO mice. In contrast, the inflammation after MI in β-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that β-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the β-arrestin2 gene is knocked out. β-arrestin2 plays a protective role in MI-induced inflammation.

Anti-allergic actions of the leaves ofCastanea crenata and isolation of an active component responsible for the inhibition of mast cell degranulation

The anti-allergic actions of the leaves ofCastanea crenata, (Fagaceae) were studied. The water extract demonstrated potent anti-allergic actions inin vivo andin vitro experiments. The oral or intraperitoneal administration of the extract (100 or 200 mg/kg) caused a significant inhibition of the 48 hr-PCA (up to 90%) and the vascular permeability induced by histamine or serotonin in rats (about 80%). The anaphylactic release of β-hexosaminidase from RBL-2H3 cells was also significantly inhibited by the extract in a dose-dependent manner with an IC50 value of 230 μg/ml. The activity-guided fractionation of the extract, based on the, determination of inhibitory effect upon the release of β-hexosaminidase, led to the isolation of quercetin as an active principle responsible for the inhibition of degranulation.

Vaccination with an ovalbumin/interleukin-4 fusion DNA efficiently induces Th2 cell-mediated immune responses in an ovalbumin-specific manner

To more effectively drive immune responses toward antigen-specific T helper type 2 (Th2) cellmediated responses, we constructed a mammalian expression vector (pOVA/IL4) carrying a fused gene in which the ovalbumin (OVA) cDNA was covalently linked to murine interleukin-4 (IL-4) cDNA. A biologically active OVA/IL4 protein was expressed by the transfected COS cells with the pOVA/IL4 DNA, as demonstrated by Western blotting and cytokine bioassay. Intramuscular injection of BALB/c mice with the pOVA/IL4 DNA increased both the production of OVA-specific IL-4 by CD4+ T cells and the ratio of anti-OVA IgG1 to anti-OVA IgG2a isotypes, while the injection with the pOVA DNA alone, or with the mixture of the pOVA and plL4 DNA did no or little increase. Furthermore, the OVA-specific, Th2 cell-mediated immune responses were significantly enhanced by multiple injections with the pOVA/IL4 DNA. These studies indicate that the direct linkage of an OVA gene to an IL-4 gene in the expression plasmid confines the effects of IL-4 to the OVA-specific cells, efficiently driving the immune response toward OVA-specific., Th2 cell-mediated responses.

Luteolin mediates the antidepressant-like effects of Cirsium japonicum in mice, possibly through modulation of the GABAA receptor

Cirsium japonicum (CJ) has been shown to possess antidepressant-like properties. In the present study, we sought to identify which constituent of CJ might be responsible for its antidepressant effects and determine probable mechanism of action. The ethanol extract of CJ was administered to mice then behavioral changes were evaluated in the forced-swimming test (FST) and open-field test (OFT). In addition, its effects on norepinephrine (NE) reuptake and intracellular chloride (Cl−) flux were determined, in vitro. The effects of CJ’s major constituents (linarin, pectolinarin, chlorogenic acid, luteolin) were also evaluated. CJ showed antidepressant-like effect by significantly reducing immobile behavior of mice in the FST, without increasing locomotor activity in the OFT. CJ had no effect on monoamine (NE) uptake, but it significantly promoted Cl− ion influx in human neuroblastoma cells. This CJ-induced Cl− influx was significantly blocked by co-administration of the competitive GABAA receptor antagonist, bicuculline. Among the major constituents of the CJ extract, only luteolin produced similar antidepressant-like effect, in vivo, and Cl− ion influx, in vitro. Altogether, the present results suggest that the antidepressant-like effect of CJ was most probably induced by its constituent luteolin, mediated through potentiation of the GABAA receptor-Cl− ion channel complex.

Effects of dopaminergic drugs on the mast cell degranulation and nitric oxide generation in RAW 264.7 cells

Effects of dopaminergic drugs on the degranulation of mast cells (RBL-2H3 cells) and the nitric oxide production from macrophage cells (RAW 264.7) were studied. Among the dopaminergic agonists and antagonists tested, bromocriptine, 7-OH-DPAT, haloperidol, and clozapine showed potent inhibitions of mast cell degranualtion (IC50 value, 5 μM). However, these dopaminergic agents did not affect the tyrosine phosphorylations of the signaling components of the high affinity IgE receptor (FcεRI), such as Syk, PLCγ1, and PLCγ2.; This suggested that these signaling components were not involved in the inhibition of the mast cell degranulation by these compounds. On the other hand, dopamine, bromocriptine, 7-OH-DAPT, and haloperidol markedly inhibited the nitric oxide production from RAW 264.7 cells (IC50 values, 10–20 μM). Bromocriptine, a dopamine agonist that is routinely used for the treatment of Parkinsons disease, inhibited the expression of the inducible nitric oxide synthase at an early stage of the LPS-induced protein expression in a dose-dependent manner. The results suggested that these dopaminergic agents, when used for the treatment of dopamine receptors-related diseases, such as Schizophrenia or Parkinsons disease, might have additional beneficial effects.