Ling-Chu Chang

Inhibition of nitric oxide production by the carbazole compound LCY-2-CHO via blockade of activator protein-1 and CCAAT/enhancer-binding protein activation in microglia

Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-stimulated murine microglial N9 and BV-2 cells and in LPS-stimulated N9 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-kappaB (NF-kappaB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor IL6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBPbeta, but not those of p65, p50, C/EBPdelta, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), MAPK-activated protein kinase-2 (MAPKAPK-2), STAT-1, CREB or c-Jun in LPS/IFNgamma-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBPbeta at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBPbeta expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP activation.

Examination of the signal transduction pathways leading to activation of extracellular signal-regulated kinase by formyl-methionyl-leucyl-phenylalanine in rat neutrophils

The signaling pathways leading to extracellular signal‐regulated kinase (ERK) activation in formyl‐methionyl‐leucyl‐phenylalanine (fMLP)‐stimulated rat neutrophils were examined. fMLP‐stimulated ERK activation based on immunoblot analysis with antibodies against the phosphorylation form of ERK was attenuated by the pretreatment of cells with pertussis toxin but not with a dual cyclo‐oxygenase/lipoxygenase inhibitor BW755C. Exposure of cells to the tyrosine kinase inhibitor genistein, phosphatidylinositol 3‐kinase (PI3K) inhibitors wortmannin and LY294002, or protein kinase C (PKC) inhibitors Gö6976, Gö6983, and GF109203X inhibited fMLP‐stimulated ERK phosphorylation in a concentration‐dependent manner. In addition, both the phospholipase C (PLC) inhibitor U73122 and the Ca2+ chelator BAPTA attenuated ERK activation. These results indicate that Gi/o protein, tyrosine kinase, PI3K, PKC, and PLC/Ca2+, but not arachidonate metabolites, act upstream of fMLP‐stimulated ERK activation.

Signal transduction pathways for activation of extracellular signal-regulated kinase by arachidonic acid in rat neutrophils.

Phosphorylation of extracellular signal‐regulated kinase (ERK) inresponse to arachidonic acid (AA) was rapid and transient, peaking at 1min and disappearing after 3 min, and it was accompanied by an increasein ERK activity in rat neutrophils. We examined the upstream regulationof AA‐stimulated ERK activation using one of the following signalingpathway inhibitors to pretreat rat cells: the ERK kinase inhibitorU0126 or PD98059, the Gi/o inhibitor pertussis toxin (PTX),the tyrosine kinase inhibitor genistein, the phosphatidylinositol3‐kinase (PI3K) inhibitor wortmannin or LY294002, the Ca2+chelator 1,2‐bis(O‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid, or the phospholipase C(PLC) inhibitor U73122. All of these inhibitors attenuated AA‐inducedERK activation. Activation of ERK was also effectively attenuated bythe cyclooxygenase and lipoxygenase inhibitor BW755C and by theleukotriene biosynthesis inhibitor MK886, but the cyclooxygenaseinhibitor indomethacin did not attenuate ERK activation. After exposingcells to three distinct protein kinase C (PKC) inhibitors, we foundthat Gö6976 significantly attenuated ERK phosphorylation butpotentiated ERK activity. Neither Gö6983 nor GF109203Xaffected AA‐induced responses. These data suggest that the lipoxygenasemetabolite(s) produced mediates AA‐stimulated ERK activation and thatthis effect is upstream regulated by PT‐sensitive G protein,nonreceptor tyrosine kinase, PI3K, and PLC/Ca2+ signalingpathways in rat neutrophils.

Inhibition of superoxide anion generation by CHS-111 via blockade of the p21-activated kinase, protein kinase B/Akt and protein kinase C signaling pathways in rat neutrophils

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111) inhibited superoxide anion (O(2)(-)) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect, and attenuated migration. CHS-111 had no effect on the arachidonic acid-induced NADPH oxidase activation or the GTPgammaS-stimulated Rac2 membrane translocation in cell-free systems, whereas it effectively attenuated the membrane recruitment of p40(phox), p47(phox) and p67(phox), phosphorylation of Ser residues in p47(phox), association between p47(phox) and p22(phox), and Rac activation in fMLP-stimulated neutrophils. Moreover, the phosphorylation and membrane recruitment of p21-activated kinase (PAK), PAK kinase activity and the interaction of PAK with p47(phox) were inhibited by CHS-111. CHS-111 effectively reduced Akt kinase activity and the association between Akt and p47(phox), moderately inhibited the membrane recruitment of Akt and phospho-PDK1, and slightly attenuated Akt (Thr308) phosphorylation, whereas it had no effect on Akt (Ser473) phosphorylation or p110gamma membrane translocation. The membrane recruitment of protein kinase C (PKC)-alpha, -betaI, -betaII, -delta and -zeta, PKC phosphorylation and PKC kinase activity was attenuated by CHS-111, whereas CHS-111 did not affect the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or downstream MAPK-activated protein kinase-2. Higher concentrations of CHS-111 were required to decrease fMLP-stimulated intracellular free Ca(2+) concentration ([Ca(2+)](i)) elevation in the presence but not in the absence of extracellular Ca(2+), and to reduce cellular cyclic AMP but slightly increase cyclic GMP levels. Taken together, these results suggest that CHS-111 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PAK, Akt and PKC signaling pathways.

Inhibition of plasma extravasation by abruquinone A, a natural isoflavanquinone isolated from Abrus precatorius

Polymyxin B-induced hind-paw edema was suppressed by abruquinone A, an isoflavanquinone isolated from Abrus precatorius, in normal as well in adrenalectomized mice. Unlike dexamethasone, abruquinone A did not increase the liver glycogen content in fasting adrenalectomized mice. The volume of exuded plasma was significantly reduced by abruquinone A in neurogenic inflammation, passive cutaneous anaphylactic reaction and compound 48/80-induced ear edema. Histamine-, serotonin-, bradykinin- and substance P-induced plasma extravasation in ear edema was also suppressed by abruquinone A. Abruquinone A, like isoproterenol, significantly reduced the bradykinin- and substance P-induced plasma extravasation in normal as well as in compound 48/80-pretreated mice. In addition, abruquinone A suppressed the bradykinin- and substance P-induced ear edema to a significantly greater extent than diphenhydramine/methysergide did. In the in vitro experiments, abruquinone A suppressed the compound 48/80-induced histamine and beta-glucuronidase released from isolated rat peritoneal mast cell preparations. These results suggest that the anti-inflammatory effect of abruquinone A is mediated partly via the suppression of the release of chemical mediators from mast cells and partly via the prevention of vascular permeability changes caused by mediators. The glucocorticoid activity and the release of glucocorticoid hormones from the adrenal gland are probably not involved.

Expression of adenylyl cyclase isoforms in neutrophils.

In the present study, we have identified the expression of adenylyl cyclase (AC) isoforms in rat neutrophils according to the mRNA analysis and the distinct mode of regulation of isoform activity. Agarose gel electrophoresis of reverse transcription-polymerase chain reaction (RT-PCR)-amplified products resulted in a single band of the expected size for each product with nucleotide sequences corresponding to AC1 to AC9. AC1 was abundant, while AC2, 6 and 9 were of moderate expression among the AC isoforms in neutrophils based on the quantitative real-time RT-PCR analysis. Exposure of neutrophils to Ca(2+) ionophore A23187, isoproterenol and forskolin stimulated cellular cyclic AMP accumulation. EDTA and the calmodulin (CaM) antagonist, trifluoperazine, prevented the A23187-induced response. Pretreatment with pertussis toxin (PTX) inhibited the alpha(2)-adrenergic agonist, UK14304-induced cellular cyclic AMP elevation. In addition, UK14304 augmented the cyclic AMP elevation when cells were stimulated by isoproterenol. Phorbol 12-myristate 13-acetate (PMA) attenuated the augmentation response of UK14304 and isoproterenol. Treatment of the membrane preparations from rat neutrophils with Ca(2+)/CaM, forskolin, isoproterenol, GTPgammaS or Gbetagamma all increased cyclic AMP production. The addition of protein kinase C (PKC) catalytic fragment and Gbetagamma augmented the Ca(2+)/CaM- and isoproterenol-stimulated AC activity, respectively. However, forskolin and the activated protein kinase A (PKA) attenuated the GTPgammaS- and isoproterenol-stimulated AC activity, respectively. KT5720, a PKA inhibitor, reversed the inhibition by PKA. Taken together, these data suggest the presence of four groups of AC isoforms in rat neutrophils.

Signaling mechanisms of inhibition of phospholipase D activation by CHS-111 in formyl peptide-stimulated neutrophils

A selective phospholipase D (PLD) inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) inhibited the O(2)(-) generation and cell migration but not degranulation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel benzyl indazole compound 2-benzyl-3-(4-hydroxymethylphenyl)indazole (CHS-111), which inhibited O(2)(-) generation and cell migration, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC(50) 3.9±1.2μM). CHS-111 inhibited the interaction of PLD1 with ADP-ribosylation factor (Arf) 6 and Ras homology (Rho) A, and reduced the membrane recruitment of RhoA in fMLP-stimulated cells but not in GTPγS-stimulated cell-free system. CHS-111 reduced the cellular levels of GTP-bound RhoA, membrane recruitment of Rho-associated protein kinase 1 and the downstream myosin light chain 2 phosphorylation, and attenuated the interaction between phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and Arf6, whereas it only slightly inhibited the guanine nucleotide exchange activity of human Dbs (DH/PH) protein and did not affect the arfaptin binding to Arf6. CHS-111 inhibited the interaction of RhoA with Vav, the membrane association and the phosphorylation of Vav. CHS-111 had no effect on the phosphorylation of Src family kinases (SFK) but attenuated the interaction of Vav with Lck, Hck, Fgr and Lyn. CHS-111 also inhibited the interaction of PLD1 with protein kinase C (PKC) α, βI and βII isoenzymes, and the phosphorylation of PLD1. These results indicate that inhibition of fMLP-stimulated PLD activity by CHS-111 is attributable to the blockade of RhoA activation via the interference with SFK-mediated Vav activation, attenuation of the interaction of Arf6 with PLD1 and PIP5K, and the activation of Ca(2+)-dependent PKC in rat neutrophils.

Discovery of 3-(4-bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide as a novel dual cyclooxygenase/5-lipoxygenase inhibitor that also inhibits tumor necrosis factor-α production

In the present study we have discovered compound 1, a benzo[1.3.2]dithiazolium ylide-based compound, as a new prototype dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX). Compound 1 was initially discovered as a COX-2 inhibitor, resulting indirectly from the COX-2 structure-based virtual screening that identified compound 2 as a virtual hit. Compounds 1 and 2 inhibited COX-1 and COX-2 in mouse macrophages with IC(50) in the range of 1.5-18.1microM. Both compounds 1 and 2 were also found to be potent inhibitors of human 5-LOX (IC(50)=1.22 and 0.47microM, respectively). Interestingly, compound 1 also had an inhibitory effect on tumor necrosis factor-alpha (TNF-alpha) production (IC(50)=0.44microM), which was not observed with compound 2. Docking studies suggested the (S)-enantiomer of 1 as the biologically active isomer that binds to COX-2. Being a cytokine-suppressive dual COX/5-LOX inhibitor, compound 1 may represent a useful lead structure for the development of advantageous new anti-inflammatory agents.

The influence of acetylshikonin, a natural naphthoquinone, on the production of leukotriene B4 and thromboxane A2 in rat neutrophils.

Both A23187 and formyl-Met-Leu-Phe (fMLP) induced the release of arachidonic acid and the production of thromboxane B(2) and leukotriene B(4) from rat neutrophils that were inhibited by acetylshikonin in a concentration-dependent manner. Acetylshikonin blocked exogenous arachidonic acid-induced leukotriene B(4) and thromboxane B(2) production in neutrophils and inhibited the enzymatic activity of ram seminal vesicles cyclooxygenase and human recombinant 5-lipoxygenase, whereas it had no effect on cytosolic phospholipase A(2) activity, in cell-free systems. 3-Morpholinosydnonimine- and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE)-mediated dihydrorhodamine 123 oxidation (to assess the lipid peroxide and peroxynitrite scavenging activity) was reduced by acetylshikonin. The membrane recruitment of cytosolic phospholipase A(2) was inhibited, but the phosphorylation of cytosolic phospholipase A(2) was enhanced, by acetylshikonin in the A23187-induced response. Acetylshikonin alone stimulated extracellular signal regulated kinase (ERK) phosphorylation and enhanced this response in cells stimulated with A23187 and fMLP. The phosphorylation of ERKs and cytosolic phospholipase A(2) was attenuated by U0126, a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Acetylshikonin facilitated both A23187- and fMLP-mediated translocation of 5-lipoxygenase to the membrane. Acetylshikonin attenuated both fMLP- and ionomycin-mediated [Ca(2+)](i) elevation. These results indicate that the inhibition of eicosanoid production by acetylshikonin is due to the attenuation of cytosolic phospholipase A(2) membrane recruitment via the decrease in [Ca(2+)](i) and to the blockade of cyclooxygenase and 5-lipoxygenase activity.

2-Benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils.

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated phospholipase D (PLD)-mediated products, phosphatidic acid (PA) and phosphatidylethanol (PEt) formation in rat neutrophils in a concentration-dependent manner with IC(50) values of 15.8+/-2.5 and 13.9+/-2.0 microM, respectively. The underlying cellular signaling mechanism of CCY1a inhibition was investigated. CCY1a inhibited the plateau phase but not the initial Ca(2+) spike of fMLP-stimulated Ca(2+) signal. CCY1a did not inhibit the [Ca(2+)](i) change in Ca(2+)-free medium in response to fMLP, but inhibited the [Ca(2+)](i) change by the subsequent addition of Ca(2+). In addition, CCY1a treatment attenuated the fMLP-induced protein tyrosine phosphorylation. The membrane translocation of ADP-ribosylation factor (ARF) and Rho A proteins in neutrophils stimulated with fMLP was attenuated by CCY1a in a concentration-dependent manner. In a cell-free system, neither the membrane association of ARF and Rho A caused by GTPgammaS nor the phorbol myristate acetate-stimulated membrane translocation of Rho A was suppressed significantly by CCY1a. These results indicate that the attenuation of protein tyrosine phosphorylation, blockade of Ca(2+) entry, and the suppression of ARF and Rho A membrane translocation are probably obligatory for the CCY1a inhibition of PLD activity in rat neutrophils in response to fMLP.