Hae-Kyoung Kim

Ca2+-DEPENDENT AND -INDEPENDENT MECHANISMS OF ISCHAEMIA-EVOKED RELEASE OF [3H]-DOPAMINE FROM RAT STRIATAL SLICES

1. Ischaemia was induced by 5 min of deprivation of glucose and an additional 5 min of deprivation of glucose and oxygen from Mg2+‐free artificial cerebrospinal fluid in vitro.

Platelet-activating factor enhances tumour metastasis via the reactive oxygen species-dependent protein kinase casein kinase 2-mediated nuclear factor-κB activation

Platelet‐activating factor (PAF) promotes tumour metastasis via activation of the transcription factor nuclear factor‐κB (NF‐κB). We here investigated the role of the protein kinase CK2 (formerly Casein Kinase 2 or II) in PAF‐induced NF‐κB activation and tumour metastasis, given that PAF has been reported to increase CK2 activity, and that CK2 plays a key role in NF‐κB activation. PAF increased CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. CK2 inhibitors inhibited the PAF‐mediated NF‐κB activation and expression of NF‐κB‐dependent pro‐inflammatory cytokines and anti‐apoptotic factors. Pre‐treatment with the antioxidant N‐Acetyl‐L‐Cysteine (NAC) resulted in a significant inhibition in PAF‐induced enhancement of CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. H2O2 and known reactive oxygen species inducers, lipopolysaccharide (LPS) and tumour necrosis factor‐α (TNF‐α) enhanced CK2 activity, phosphorylation and protein expression, which was again inhibited by antioxidant. PAF, LPS and TNF‐α induced increased CK2 activity, phosphorylationand protein expression, which were inhibited by p38 inhibitor. PAF, LPS or TNF‐α increased pulmonary metastasis of B16F10, which was inhibited by antioxidants, CK2 inhibitor and p38 inhibitor. Our data suggest that (i) reactive oxygen species activate CK2 via p38, which, in turn, induces NF‐κB activation, and (ii) PAF, LPS and TNF‐α increase pulmonary tumour metastasis via the induction of the reactive oxygen species (ROS)/p38/CK2/NF‐κB pathway.

Glutamine suppresses DNFB-induced contact dermatitis by deactivating p38 mitogen-activated protein kinase via induction of MAPK phosphatase-1.

L-glutamine (Gln) is a nonessential amino acid that is the most abundant amino acid in plasma. Gln has been reported to have an anti-inflammatory activity that involves deactivation of mitogen-activated protein kinases (MAPKs) in a MAPK phosphatase (MKP)-1-dependent manner. This study investigated the role of Gln in the inhibition of DNFB-induced allergic contact dermatitis (CD) in the ears of mice, and specifically the involvement of Gln in p38 MAPK inhibition. Topical application of Gln or the p38 inhibitor, SB202190, suppressed DNFB-induced CD. Gln application inhibited DNFB-induced p38 phosphorylation. Western blot analysis revealed that Gln application resulted in early phosphorylation and protein induction of MKP-1. MKP-1 small interfering RNA (siRNA), but not control siRNA, abrogated Gln-mediated early phosphorylation, protein induction of MKP-1, deactivation of p38, and Gln-mediated suppression of CD. The extracellular signal-regulated kinase (ERK) inhibitor, U0126, blocked Gln-induced MKP-1 phosphorylation and protein induction, as well as Gln suppression of CD. These results suggest that Gln suppresses DNFB-induced CD via deactivation of p38 MAPK through the early induction of MKP-1, the negative regulator of p38, in an ERK-dependent manner.

Glutamine suppresses airway neutrophilia by blocking cytosolic phospholipase A(2) via an induction of MAPK phosphatase-1.

Neutrophils are inflammatory cells that may contribute in a crucial way to the pathophysiology of steroid-resistant severe asthma. We previously reported that the nonessential amino acid l-glutamine (Gln) suppressed the recruitment of neutrophils into the airway in a murine model of asthma. In this study, we investigated the mechanisms by which Gln exerts beneficial effects in airway neutrophilia. We used the model we previously developed, which is suitable for examining sequential early asthmatic events, including neutrophil infiltration. Gln suppressed airway neutrophilia in a CXC chemokine-independent way. Airway neutrophilia was associated with cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO) activities. p38 MAPK, the upstream pathway of cPLA2 and 5-LO, played a key role in inducing airway neutrophilia. Gln inhibited not only the phosphorylation of cPLA2 and p38 MAPK but also leukotriene B4 levels in the airways. Gln induced the early induction of MAPK phosphatase-1 (MKP-1) protein, a negative regulator of p38. MKP-1 small interfering RNA abrogated all the effects of Gln. Our results suggest that pathways involving p38/cPLA2/5-LO have a major role in airway neutrophilia. Gln suppresses airway neutrophilia via inhibiting p38 MAPK and its downstream pathways in an MKP-1–dependent way, which may provide a novel therapeutic strategy for pulmonary neutrophilic inflammatory diseases.

Biphasic Late Airway Hyperresponsiveness in a Murine Model of Asthma

Background: Nonspecific airway hyperresponsiveness (AHR) is one of the cardinal features of bronchial asthma. Early AHR is caused by chemical mediators released from pulmonary mast cells activated in an IgE-dependent way. However, the mechanism of late AHR remains unclear. Methods: Features of airway allergic inflammation were analyzed, including antigen-induced AHR, using a murine model of asthma. The model was suitable for examining the sequential early molecular events occurring after the initial airway exposure to antigen. Results: AHR increased at 10–12 h after airway challenge, followed by the second-phase response, which was larger and broader in resistance at 18–30 h. Pretreatment of sensitized animals with anti-tumor necrosis factor (TNF) before airway challenge or induction of allergic asthma in TNF–/– mice resulted in abrogation of the first-phase late AHR. Intratracheal instillation of TNF induced a single peak of AHR at 10 h. IgE and IgG immune complexes induced the development of the first-phase late AHR by TNF production. Pretreatment with cytosolic phospholipase inhibitor and 5-lipoxygenase inhibitors abolished the first-phase late AHR as well as the leukotriene B4 levels in the airway. CpG-oligodeoxynucleotide (ODN) pretreatment reduced airway levels of Th2 cytokines, eosinophil infiltration and second-phase late AHR. However, CpG-ODN did not reduce TNF levels or the magnitude of first-phase late AHR. Conclusion: Biphasic late AHR occurs in a murine model of asthma. First- and second-phase late AHR is caused by TNF and Th2 response, respectively.

Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A2 via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca2+]i) in MHS macrophage cells. Ionomycin increased [Ca2+]i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca2+→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca2+ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca2+→ ERK pathway, which plays a key role in suppression of inflammatory reactions.

IgG immune complex induces the recruitment of inflammatory cells into the airway and TNF-mediated late airway hyperresponsiveness via NF-κB activation in mice.

Background. Many of the inflammatory proteins that are expressed in asthmatic airways are regulated, at least partially, by nuclear factor (NF)-κB. Blockade of NF-κB activity has resulted in attenuation of the cardinal features of asthma. Thus, delineating the mechanisms involved in NF-κB activation in asthma might provide an interesting approach to improving the management of asthma. However, despite its importance, the mechanism for NF-κB activation in asthma has not yet been determined. Objective. To examine the role of IgE and IgG antibodies (Abs) in the activation of NF-κB in mouse lungs. Methods. To examine the effect of IgE, mice underwent intratracheal (i.t.) instillation of an IgE immune complex (IgE-IC) (anti-2,4-dinitrophenyl hapten (DNP) IgE + DNP-BSA or DNP-OVA) and anaphylactogenic anti-IgE (LO-ME-2). For IgG, mice underwent i.t. instillation with a complex of anti-chicken gamma globulin (CGG) IgG1 mAb + CGG. NF-κB activation was determined by gel shift assay. Small interfering RNA was used for blockade of p50 expression. The effect of tumor necrosis factor (TNF) blockade was determined using anti-TNF Ab. A previously established murine model of asthma was used to assess airway hyperresponsiveness (AHR). Results. A single i.t. instillation of either IgE-IC or LO-ME-2 failed to induce activation of NF-κB in the lungs. In contrast, single i.t. instillation of IgG-IC was capable of inducing NF-κB activation, as well as NF-κB-dependent proinflammatory molecules, such as TNF and CXC chemokines. Pretreatment of p50 small interfering RNA decreased bronchoalveolar lavage fluid levels of TNF and macrophage inflammatory protein-2 induced by IgG-IC instillation. Single i.t. instillation of IgG-IC caused the recruitment of neutrophils and macrophages into the airway and TNF-mediated late AHR, but failed to induce Th2 cell-mediated asthmatic phenotypes. Conclusion. IgG, but not IgE, is the major Ab that induces not only NF-κB activation and NF-κB-dependent proinflammatory molecules in the lungs but also subsequent recruitment of inflammatory cells into the airway and TNF-mediated late AHR.

Glutamine suppresses dinitrophenol fluorobenzene-induced allergic contact dermatitis and itching: inhibition of contact dermatitis by glutamine.

BACKGROUND Cytoplasmic phospholipase A(2) (cPLA(2)) is importantly implicated in a variety of inflammatory diseases by liberating arachidonic acid from phospholipids. The increased cPLA(2) activities as well as increased levels of cPLA(2) metabolites are associated with pathogenesis of many inflammatory skin disorders including atopic dermatitis. The non-essential amino acid l-glutamine (Gln) has been reported to have an anti-inflammatory activity. Regarding the molecular mechanism of Gln, we have recently shown that Gln effectively inhibits cPLA(2) phosphorylation and activity. OBJECTIVE To examine whether Gln could suppress allergic contact dermatitis (CD) induced on mouse ears by dinitrophenol fluorobenzene (DNFB). METHODS Mice were sensitized five times on their ears with a 0.15% solution of DNFB in a 3 day interval. To examine Gln effects, Gln solution (4% in saline) was applied three times a day onto both sides of DNFB-applied ears from the last day of DNFB application. The inflammatory reactions of ears were evaluated by measuring ear thickness and hematoxylin and eosin (H&E) staining. Mouse scratching behavior was objectively evaluated using a MicroAct apparatus. cPLA(2) phosphorylation and activity were analyzed using Western blotting and a cPLA(2) assay kit, respectively. RESULTS Topical application of Gln significantly attenuated inflammatory symptoms (ear thickness, histological inflammatory skin reactions) as well as itching. Gln inhibited cPLA(2) phosphorylation and enzymatic activity. Arachidonyl trifluoromethyl ketone (AACOCF(3)) inhibited cPLA(2) activity in DNFB-challenged ears and attenuated DNFB-induced ear inflammation and itching. CONCLUSION The results indicate that Gln suppresses DNFB-induced dermatitis and itching, at least in part, by inhibiting cPLA(2) activity.

Glutamine Prevents Late-Phase Anaphylaxis via MAPK Phosphatase 1-Dependent Cytosolic Phospholipase A2 Deactivation.

Background: Cytosolic phospholipase A2 (cPLA2) plays a key role in the development of late-phase anaphylaxis. L-Glutamine (Gln), a nonessential amino acid, has anti-inflammatory activity via inhibiting cPLA2. Methods: We used a penicillin-induced murine model of anaphylaxis, and late-phase anaphylaxis was quantified by measuring the increase in the hematocrit (Ht) value. Various inhibitors, small interfering RNA, and knockout mice were used in inhibition experiments. Phosphorylation and protein expression of cPLA2, ERK, and MAPK phosphatase 1 (MKP-1) were detected by Western blotting. Results: Leukotriene (LT) B4 was found to be another potent inducer of late-phase anaphylaxis besides the known mediator platelet-activating-factor (PAF). Gln efficiently prevented late-phase anaphylaxis when it was administered up to 3 h after challenge injection via inhibiting cPLA2. Inhibition studies indicated that p38 MAPK was the major upstream regulator of cPLA2. Gln dephosphorylated p38 and cPLA2 via up-regulating the negative regulator of p38 MAPK, i.e., MKP-1 protein. MKP-1 blockade abrogated all the effects of Gln. Conclusion: Of the cPLA2 metabolites, PAF and LTB4 play a key role in the development of late-phase anaphylaxis, and Gln prevents the reaction via MKP-1-dependent deactivation of cPLA2.

GPCR Kinase (GRK)-2 Is a Key Negative Regulator of Itch: l-Glutamine Attenuates Itch via a Rapid Induction of GRK2 in an ERK-Dependent Way

Many itch mediators activate GPCR and trigger itch via activation of GPCR-mediated signaling pathways. GPCRs are desensitized by GPCR kinases (GRKs). The aim of this study is to explore the role of GRKs in itch response and the link between GRKs and glutamine, an amino acid previously shown to be an itch reliever. Itch responses were evoked by histamine, chloroquine, and dinitrochlorobenzene-induced contact dermatitis (CD). Phosphorylation and protein expression were detected by immunofluorescent staining and Western blotting. GRK2 knockdown using small interfering RNA enhanced itch responses evoked by histamine, chloroquine, and dinitrochlorobenzene-induced CD, whereas GRK2 overexpression using GRK2-expressing adenovirus reduced the itch responses. Glutamine reduced all itch evoked by histamine, chloroquine, and dinitrochlorobenzene-induced CD. Glutamine-mediated inhibition of itch was abolished by GRK2 knockdown. Glutamine application resulted in a rapid and strong expression of GRK2 in not only dinitrochlorobenzene-induced CD (within 10 minutes) but also cultured rat dorsal root ganglion cells, F11 (within 1 minute). ERK inhibitor abrogates glutamine-mediated GRK2 expression and inhibition of itch in dinitrochlorobenzene-induced CD. Our data indicate that GRK2 is a key negative regulator of itch and that glutamine attenuates itch via a rapid induction of GRK2 in an ERK-dependent way.