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ABSTRACT: Immune regulating functions of lipid mediators are being recognized. Prostaglandins (PGs) are derived from phospholipid by cyclooxygenase-2 (COX-2) in inflammatory tissues. Based upon our previous data that imply an immune modulating activity of prostacyclin, we hypothesized that PGs promote the humoral immune responses in vivo. To test this hypothesis, we examined the effect of a prostacyclin analog beraprost and PGE2 on the antibody responses that were induced by immunizing mice with keyhole limpet hemocyanin (KLH). Beraprost was used due to the extremely unstable property of prostacyclin. Our results showed that beraprost indeed promoted the production of anti-KLH antibodies, which was dose-dependent and specific to beraprost because PGE2 did not modulate the antibody response compared with controls. The enhancing effect of beraprost was reproduced in the secondary responses, suggesting that memory B cell generation during the primary response was significantly affected by beraprost. Analysis of the isotypes of anti-KLH antibodies revealed that beraprost stimulated the production of IgA and IgG subisotypes but not IgM. However, germinal center B cell generation and the affinity of anti-KLH antibodies were not affected by beraprost administration. To confirm these results we immunized COX-2 knockout mice with KLH and analyzed whether the results with wild-type mice were reflected in the absence of PGs. The primary and secondary antibody responses were significantly impaired in the KO animals. The levels of anti-KLH IgG subisotypes and IgA were severely reduced in KO mice whereas those of IgM were comparable to controls. These results reveal an unrecognized function of PG in the humoral immune responses.
International immunopharmacology 03/2013; · 2.21 Impact Factor
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Yang Cheng,
Yue Wang,
Daisuke Ito,
Deok-Hoon Kong, Kwon-Soo Ha,
Jun-Hu Chen,
Feng Lu,
Jian Li,
Bo Wang,
Eizo Takashima,
Jetsumon Sattabongkot,
Takafumi Tsuboi,
Eun-Taek Han
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ABSTRACT: Merozoite surface protein 1 of Plasmodium vivax (PvMSP1), a glycosylphosphatidylinositol-anchored protein (GPI-AP), is a malaria vaccine candidate for P. vivax. The paralog of PvMSP1, named P. vivax merozoite surface protein 1 paralog (PvMSP1P; PlasmoDB PVX_099975), gene was recently identified and predicted as a GPI-AP. The similarities in genetic structural characteristics between PvMSP1 and PvMSP1P (e.g., size of open reading frames, two epidermal growth factor-like domains, and GPI-anchor motif in the C-terminus) led us to study this protein. In the present study, different regions of the PvMSP1P protein, demarcated based on the processed forms of PvMSP1, were expressed successfully as recombinant proteins [i,e., 83- (A, B, and C), 30, 38, 42, 33, and 19 fragments]. We studied the naturally acquired immune response against each fragment of recombinant PvMSP1P and the potential binding ability of each fragment to erythrocytes. The N-terminal (83A) and two C-terminal fragments (33 and 19) reacted strongly with sera from P. vivax-infected patients, with 50-68% sensitivity and 95-96% specificity, respectively. Due to colocalization of PvMSP1P with PvMSP1, we supposed that PvMSP1P plays a similar role as PvMSP1 during erythrocyte invasion. An in vitro cytoadherence assay showed that PvMSP1P, especially the C-terminal 19-kDa region, could bind to erythrocytes. We also found that human sera from populations naturally exposed to vivax malaria and antisera obtained by immunization using the recombinant molecule PvMSP1P-19 inhibited in vitro binding of human erythrocytes to PvMSP1P-19. These results provide further evidence that the PvMSP1P might be an essential parasite adhesion molecule in the P. vivax merozoite and is a potential vaccine candidate against P. vivax.
Infection and immunity 03/2013; · 4.21 Impact Factor
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Sookon Kim,
Chun-Ki Kim,
Kwang-Soon Lee,
Ji-Hee Kim,
Haejun Hwang,
Dooil Jeoung,
Jongseon Choe,
Moo-Ho Won,
Hansoo Lee, Kwon-Soo Ha,
Young-Geun Kwon,
Young-Myeong Kim
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ABSTRACT: ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Rubus coreanus has been used as a traditional herbal medicine for alleviation of inflammatory and vascular diseases in Asian countries. AIM OF THE STUDY: The anti-atherogenic effect of unripe Rubus coreanus fruit extract (URFE) and its underlying mechanism were analyzed in mice fed a high-fat diet (HFD) and in cell culture system. MATERIALS AND METHODS: Mouse were freely given HFD alone or supplemented with URFE for 14 weeks, followed by analysis of atherosclerotic lesions and serum lipid levels. For in vitro assay, macrophages were pretreated with URFE, followed by stimulation with lipopolysaccharide (LPS). Expression levels of inflammatory genes (TNF-α, IL-1β, and iNOS) and phase II genes (heme oxygenase-1, glutamate cysteine lygase, and peroxiredoxine-1) as well as intracellular reactive oxygen species (ROS) level and NF-κB activation pathway were analyzed in cultured macrophages as well as mouse sera and aortic tissues. RESULTS: URFE supplementation reduced HFD-induced atherosclerotic lesion formation which was correlated with decreased levels of lipids, lipid peroxides, and inflammatory mediators (TNF-α, IL-1β, and nitric oxide) in sera as well as suppression of inflammatory gene in aortic tissues. In addition, pre-treatment of macrophages with URFE also suppressed LPS-induced NF-κB activation, ROS production, and inflammatory and phase II gene expressions. Inhibition of phase II enzyme and protein activities attenuated the suppressive effects URFE on ROS production, NF-κB activation, and inflammatory gene expression. CONCLUSION: These results suggest that URFE attenuates atherosclerosis by improving blood lipid profile and inhibiting NF-κB activation via phase II antioxidant gene expression.
Journal of ethnopharmacology 01/2013; · 2.32 Impact Factor
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ABSTRACT: This study was designed to investigate the effects of the prenylated flavonoid kurarinone on TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis and its underlying mechanism. A low dose of kurarinone had no significant effect on apoptosis, but this compound markedly promoted tumor cell death through elevation of Bid cleavage, cytochrome c release and caspase activation in HeLa cells treated with TRAIL. Caspase inhibitors inhibited kurarinone-mediated cell death, which indicates that the cytotoxic effect of this compound is mediated by caspasedependent apoptosis. The cytotoxic effect of kurarinone was not associated with expression levels of Bcl-2 and IAP family proteins, such as Bcl-2, Bcl-xL, Bid, Bad, Bax, XIAP, cIAP-1 and cIAP-2. In addition, this compound did not regulate the deathinducing receptors DR4 and DR5. On the other hand, kurarinone significantly inhibited TRAIL-induced IKK activation, IκB degradation and nuclear translocation of NF-κB, as well as effectively suppressed cellular FLICE-inhibitory protein long form (cFLIPL) expression. The synergistic effects of kurarinone on TRAIL-induced apoptosis were mimicked when kurarinone was replaced by the NF-κB inhibitor withaferin A or following siRNA-mediated knockdown of cFLIPL. Moreover, cFLIP overexpression effectively antagonized kurarinone-mediated TRAIL sensitization. These data suggest that kurarinone sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-κB-dependent cFLIP expression, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.
Experimental and Molecular Medicine 08/2012; · 2.48 Impact Factor
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ABSTRACT: C-peptide is a bioactive peptide with a potentially protective role in diabetes complications; however, its molecular mechanism of protection against cardiovascular damage caused by hyperglycemia-induced apoptosis remains unclear. We investigated the protective mechanism of C-peptide against hyperglycemia-induced apoptosis using human umbilical vein endothelial cells and streptozotocin diabetic mice. High glucose (33 mmol/L) induced apoptotic cell death in endothelial cells via sequential elevation of intracellular Ca(2+) and reactive oxygen species (ROS) as well as subsequent activation of transglutaminase 2 (TG2). C-peptide (1 nmol/L) prevented endothelial cell death by inhibiting protein kinase C- and NADPH oxidase-dependent intracellular ROS generation and by abolishing high glucose-induced TG2 activation, without affecting intracellular Ca(2+) levels. Consistently, in the aorta of streptozotocin diabetic mice, hyperglycemia stimulated transamidating activity and endothelial cell apoptosis that was inhibited by C-peptide replacement therapy (35 pmol/min/kg) using osmotic pumps (control and diabetes, n = 8; diabetes + C-peptide, n = 7). In addition, C-peptide prevented hyperglycemia-induced activation of transamidation activity and apoptosis in the heart and renal cortex of streptozotocin diabetic mice. Thus, C-peptide protects endothelial cells from hyperglycemia-induced apoptotic cell death by inhibiting intracellular ROS-mediated activation of TG2. Furthermore, TG2 may be a promising avenue of therapeutic investigation to treat diabetic vasculopathies.
Diabetes 08/2012; · 8.29 Impact Factor
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ABSTRACT: Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.
Molecular & Cellular Proteomics 07/2012; · 7.40 Impact Factor
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ABSTRACT: Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.
Journal of Biological Chemistry 07/2012; 287(38):32027-39. · 4.77 Impact Factor
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ABSTRACT: Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).
The Analyst 07/2012; 137(16):3814-20. · 4.23 Impact Factor
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Ji-Hee Kim,
Yoon Kyung Choi,
Kwang-Soon Lee,
Dong-Hui Cho,
Yi-Yong Baek,
Dong-Keon Lee, Kwon-Soo Ha,
Jongseon Choe,
Moo-Ho Won,
Dooil Jeoung,
Hansoo Lee,
Young-Guen Kwon,
Young-Myeong Kim
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ABSTRACT: Keap1 is a cytoplasmic repressor of the transcription factor Nrf2, and its degradation induces Nrf2 activation, leading to upregulation of antioxidant phase II genes. We investigated the roles of phase II genes in vascular inflammation and septic injury using Keap1 siRNA and elucidated its underlying mechanism. Selective knockdown of Keap1 with siRNA promoted Nrf2-dependent expression of phase II genes in endothelial cells, such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), and peroxiredoxin-1 (Prx1), resulting in the elevation of cellular glutathione levels and suppression of tumor necrosis factor (TNF)-α-induced intracellular H(2)O(2) accumulation. Keap1 knockdown inhibited TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by suppressing NF-κB activation via inhibition of its upstream modulators, Akt, NIK, and IKK, resulting in the elevation of monocyte adhesion to endothelial cells. Importantly, these events were reversed by HO-1 and GCL inhibitors and Prx1-specific siRNA. Keap1 knockdown also inhibited endotoxin-induced expression of inducible nitric oxide synthase (iNOS) and TNF-α by upregulating HO-1, GCL, and Prx1 expression in macrophages. Moreover, in vivo Keap1 knockdown increased the expression of phase II genes and suppressed the expression of ICAM-1, VCAM-1, iNOS, and TNF-α in an endotoxemic mouse model, resulting in significant protection against liver and lung injuries and lethality. Our results indicate that Keap1 knockdown prevents NF-κB-mediated vascular inflammation and endotoxic shock by suppressing NF-κB-mediated inflammatory gene expression via upregulation of Nrf2-mediated antioxidant genes. Thus, siRNA targeting Keap1 may provide a new therapeutic approach for inflammation-associated vascular diseases and sepsis.
Free radical biology & medicine 04/2012; 53(3):629-40. · 5.42 Impact Factor
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ABSTRACT: We developed a new high-throughput approach to measure matrix metalloproteinase (MMP)-3 activity using Cy3-conjugated gelatin
arrays. We fabricated Cy3-conjugated gelatin arrays by immobilizing Cy3-conjugated gelatin onto well-type amine arrays. The
ratio of Cy3-NHS to gelatin was optimized to improve the efficiency of the arrays. MMP-3 activity was determined by the decrease
of fluorescence intensity due to MMP-3-mediated degradation of Cy3-conjugated gelatin. The gelatinolytic activity of MMP-3
increased in the presence of Brij-35, a non-ionic detergent, in a dose-dependent manner. Cy3-conjugated gelatin arrays were
successfully applied for the analyses of MMP-3 activity, which showed a dose-dependent degradation of the Cy3-conjugated substrate.
This chip-based activity assay using a Cy3-conjugated gelatin array is simple, rapid, cost-effective, and has a potential
for activity-based proteomic researches and screening of MMP-3 inhibitors.
KeywordsMatrix metalloproteinase-3-Well-type amine arrays-On-chip activity assay-Cy3-conjugated gelatin arrays-High-throughput
BioChip journal 04/2012; 4(3):210-216. · 0.86 Impact Factor
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ABSTRACT: We have developed an assay using peptide arrays based on phase transition from the glass substrate to the liquid for monitoring quantitative protease activity in real-time. Peptide arrays were fabricated using a bifunctional cross-linker, N-[γ-maleimidobutyryloxy] sulfosuccinimide ester, and a substrate peptide containing two functional groups, cysteine and tetramethyl-6-carboxyrhodamine (TAMRA) on the C- and N-terminus, respectively. The phase transition-based peptide arrays were characterized by analyzing the substrate peptide cleaved from the solid substrate by matrix metalloproteinase-3 (MMP-3). We successfully used this assay to determine the quantitative proteolytic activity of MMP-3 in a dose-dependent manner. In addition, parameters including Michaelis constant (K(m)), maximum rate of enzymatic reaction (V(max)), and half maximal inhibitory concentration (IC(50)) were determined by analyzing the concentrations of substrate peptide cleaved by MMP-3. Therefore, this new assay has potential for the quantitative analysis of enzyme kinetics of protease and informs research developments in drug discovery utilizing kinetic studies.
Biosensors & bioelectronics 04/2012; 36(1):147-53. · 5.43 Impact Factor
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ABSTRACT: Transglutaminase 2 (TG2) is a versatile protein that is implicated in significant biological processes, including cell death and degenerative diseases. A possible role of TG2 in the apoptotic death of cancer cells induced by photodynamic therapy (PDT) was suggested recently; however, the mechanism by which TG2 regulates apoptotic responses to PDT remains to be elucidated. In this study, we investigated the key signaling pathways stimulated during apoptotic cell death following PDT and whether inhibition of TG2 activation using pharmacological approaches and siRNAs affects the signaling pathways. PDT caused the release of both cytochrome c and apoptosis-inducing factor (AIF) by damaging mitochondria, which resulted in caspase-dependent and caspase-independent apoptotic cell death, respectively. Released AIF translocated to the nucleus and, synergistically with the caspase-dependent pathway, led to apoptotic cell death. Both the caspase cascade and the activation of AIF following PDT were mediated by TG2 activation. In addition, PDT-activated calpain was responsible for the sequential events of Bax translocation, the collapse of ΔΨ(m), caspase-3 activation, and AIF translocation, all of which were provoked by TG2 activation. Together, these results demonstrate that PDT with a chlorin-based photosensitizer targets TG2 by activating calpain-induced Bax translocation, which induces apoptotic cell death through both caspase-dependent and AIF-mediated pathways. Moreover, these results indicate that TG2 may be a possible therapeutic target for PDT treatment of cancer.
Journal of Biological Chemistry 03/2012; 287(18):14377-88. · 4.77 Impact Factor
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ABSTRACT: For minimizing systemic experimental variation in the analysis of antibody array data, we developed a novel median-centered/IgM-tagged-internal standard (TIS) assay normalization using median-centering and TIS assay-based determination of serum IgM concentrations. We evaluated five normalization methods by analyzing correlation coefficients and coefficients of variation for six serum proteins using human serum samples from normal controls (n=25) and patients with liver cirrhosis (n=25) or hepatocellular carcinoma (HCC; n=29). Median-centered normalization improved correlation coefficients, while IgM-based normalizations improved coefficients of variation. The TIS assay was more efficient, economical, and reproducible for determining IgM concentrations than enzyme-linked immunosorbent assay. Additionally, we normalized antibody array data for six serum proteins using the median-centered/IgM-TIS assay, and evaluated serum biomarkers through distribution analysis of normalized fluorescence intensities and receiver operating characteristic analyses for the diagnosis of liver cirrhosis and HCC. Apolipoprotein A-1 and a combination of alpha-fetoprotein and C-reactive protein were determined to be potential serological biomarkers for liver cirrhosis and HCC, respectively. Thus, median-centered/IgM-TIS assay normalization is a useful approach for analyzing antibody array data and evaluating serological biomarkers for the diagnosis of liver disease or cancers.
Analytica chimica acta 03/2012; 718:92-8. · 4.31 Impact Factor
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Jian Li,
Daisuke Ito,
Jun-Hu Chen,
Feng Lu,
Yang Cheng,
Bo Wang, Kwon-Soo Ha,
Jun Cao,
Motomi Torii,
Jetsumon Sattabongkot,
Takafumi Tsuboi,
Eun-Taek Han
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ABSTRACT: Pf12 in Plasmodium falciparum has been characterized as a merozoite surface protein and the Pf12 gene is actively transcribed during schizont stage. An orthologous gene, Pv12, has been identified in genome of P. vivax, but the protein product has not been characterized. The Pv12 is a 362 amino acid long polypeptide encoded by a single exon gene PVX_113775, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Pv12 contains two predicted six-cysteine (6-Cys) domains, which may be constrained by predicted disulfide bonds, and a transmembrane domain and a predicted GPI anchor attachment site in C-terminal region. The recombinant Pv12 protein is recognized by serum antibodies of patients naturally exposed to P. vivax and the native Pv12 protein from parasite extract is also recognized by immune mouse serum. The Pv12 is localized in rhoptry; an apical organelle of the merozoite, and the localization pattern of Pv12 is distinct from that of Pf12 in P. falciparum. The present study suggests that Pv12 is immunogenic in humans during parasite infection and it could play an important role in erythrocyte invasion.
Parasitology International 02/2012; 61(3):443-9. · 2.13 Impact Factor
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ABSTRACT: Nitric oxide synthase (NOS) isoforms are hemoenzymes that are only active as homodimers. We have examined the effect of the substrate-analogue inhibitors, N(G)-monomethyl-L-arginine (L-NMA), N(G)-nitro-L-arginine (L-NNA), N(G)-nitro-L-arginine methyl ester (L-NAME), N(5)-(1-iminoethyl)-L-ornithine (L-NIO), and N(6)-(1-iminoethyl)-L-lysine (L-NIL), the guanidine-containing inhibitor aminoguanidine (AG), and the amidine moiety-containing iNOS-specific inhibitor 1400W, on the formation of NOS dimer. Of these inhibitors, L-NMA effectively not only inhibited iNOS dimerization, but also destabilized its dimeric form in RAW264.7 cells stimulated with lipopolysaccharide plus interferon-γ, but not eNOS dimerization in endothelial cells. Importantly, this inhibition was highly correlated with NO production. These inhibitory effects were significantly reversed by addition of L-arginine. However, L-NNA, L-NAME, and AG in part or significantly increased dimerization of iNOS and eNOS in intact cells, and the other inhibitors assessed did not alter dimerization of iNOS and eNOS. These data taken together suggest that substituted groups of an arginine guanidino moiety play an important role in NOS dimerization as well as its catalytic activity. Our results indicate that l-NMA can inhibit iNOS-dependent NO production by preventing iNOS dimerization and destabilizing its dimeric form.
Biochemical and Biophysical Research Communications 02/2012; 418(1):49-55. · 2.48 Impact Factor
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ABSTRACT: Photodynamic therapy (PDT) is a promising therapeutic modality for cancer treatment; however, a more detailed understanding is needed to improve the clinical use of this therapy. PDT induces cancer cell death by apoptosis, necrosis, and autophagy, and these mechanisms can be concurrently occurred. PDT destroys cancer cells by inducing apoptosis through diverse signaling pathways coupled with Bcl-2 family members, caspases, and apopotosis-inducing factor. When the apoptotic pathway is unavailable, PDT can cause cancer cell death through induction of a necrotic or autophagic mechanism. Autophagy is occurred in a Bax-independent manner and can be stimulated in parallel with apoptosis. PDT directly destroys cancer cells by inducing either apoptotic or necrotic death. PDT also can induce autophagy as a death or a survival mechanism. These mechanisms are dependent on a variety of parameters including the nature of the photosensitizer, PDT dose, and cell genotype. Understanding the complex cross talk between these pathways may improve the effectiveness of PDT. Here, we discuss the interplay between these mechanisms based on recent evidence and suggest prospects with regard to advances in PDT.
International review of cell and molecular biology 01/2012; 295:139-74. · 4.48 Impact Factor
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Chun-Ki Kim,
Dong Hui Cho,
Kyu-Sun Lee,
Dong-Keon Lee,
Chan-Woong Park,
Wan Gi Kim,
Sang Jun Lee, Kwon-Soo Ha,
Oh Goo Taeg,
Young-Guen Kwon,
Young-Myeong Kim
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ABSTRACT: Ginseng berry possesses higher ginsenoside content than its root, which has been traditionally used in herbal medicine for many human diseases, including atherosclerosis. We here examined the antiatherogenic effects of the Korean ginseng berry extract (KGBE) and investigated its underlying mechanism of action in vitro and in vivo. Administration of KGBE decreased atherosclerotic lesions, which was inversely correlated with the expression levels of phase II genes to include heme oxygenase-1 (HO-1) and glutamine-cysteine ligase (GCL). Furthermore, KGBE administration suppressed NF-κB-mediated expression of atherogenic inflammatory genes (TNF-α, IL-1β, iNOS, COX-2, ICAM-1, and VCAM-1), without altering serum cholesterol levels, in ApoE(-/-) mice fed a high fat-diet. Treatment with KGBE increased phase II gene expression and suppressed lipopolysaccharide-induced reactive oxygen species production, NF-κB activation, and inflammatory gene expression in primary macrophages. Importantly, these cellular events were blocked by selective inhibitors of HO-1 and GCL. In addition, these inhibitors reversed the suppressive effect of KGBE on TNF-α-mediated induction of ICAM-1 and VCAM-1, resulting in decreased interaction between endothelial cells and monocytes. These results suggest that KGBE ameliorates atherosclerosis by inhibiting NF-κB-mediated expression of atherogenic genes via upregulation of phase II enzymes and thus has therapeutic or preventive potential for atherosclerosis.
Evidence-based Complementary and Alternative Medicine 01/2012; 2012:490301. · 4.77 Impact Factor
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Byung-Hee Chung,
Sookon Kim,
Jong-Dai Kim,
Jung Joon Lee,
Yi-Yong Baek,
Dooil Jeoung,
Hansoo Lee,
Jongseon Choe, Kwon-Soo Ha,
Moo-Ho Won,
Young-Guen Kwon,
Young-Myeong Kim
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ABSTRACT: Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS(-/-) mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor N(G)-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca(2+) levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca(2+) levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol- treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca(2+) chelator, calmodulin antagonist, and CaMKKβ siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca(2+)-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca(2+)/CaMKKβ-dependent eNOS phosphorylation and Ca(2+)-dependent eNOS dimerization.
Experimental and Molecular Medicine 12/2011; 44(3):191-201. · 2.48 Impact Factor
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ABSTRACT: Taurine, a non essential sulfur-containing amino acid, plays a critical role in cardiovascular functions. We here examined the effect of taurine on angiogenesis and its underlying signal pathway. Taurine treatment increased angiogenesis in vitro and in vivo, which was followed by activation of the phosphatidylinositol 3-kinase (PI3K)/Akt, MEK/ERK, and Src/FAK signaling pathways. Further, taurine promoted endothelial cell cycle progression to the S and G2/M phases by up-regulating the positive cell cycle proteins, particularly cyclins D1 and B, as well as down-regulating the negative cell cycle proteins, p53 and p21(WAF1/CIP1), resulting in Rb phosphorylation. This angiogenic event was inhibited by inhibitors of PI3K and MEK. In addition, a PI3K inhibitor blocked the activation of Akt and ERK, while Akt knockdown did not affect taurine-induced ERK activation, indicating that PI3K is an upstream mediator of both MEK and Akt. Taurine-induced endothelial cell migration was suppressed by Src inhibitor, but not by other inhibitors, suggesting that the increase in cell migration is regulated by Src-dependent pathway. Moreover, inhibition of cellular taurine uptake by β-alanine and taurine transporter knockdown promoted taurine-induced cell proliferation, ERK and Akt activation, and in vivo angiogenesis, suggesting that extracellular taurine induces angiogenesis. However, taurine did not induce vascular inflammation and permeability in vitro and in vivo. These data demonstrate that extracellular taurine promotes angiogenesis by Akt- and ERK-dependent cell cycle progression and Src/FAK-mediated cell migration without inducing vascular inflammation, indicating that it is potential use for the treatment of vascular dysfunction-associated human diseases.
European journal of pharmacology 11/2011; 674(2-3):188-99. · 2.59 Impact Factor
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ABSTRACT: We developed an on-chip activity assay system to simultaneously determine the transamidating activities of blood coagulation factor XIII (FXIII) and transglutaminase 2 (TG2) by use of fibrinogen arrays. FXIII and TG2 are transglutaminase family members that are involved in various physiological functions, including vascular pathophysiology, bone development, and cancer progression. However, investigation of their differential functions is limited by the lack of high-throughput and isozyme-specific activity assays. For the on-chip activity assay, we fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane surface of well-type arrays, and we determined transamidating activity by probing biotinylated fibrinogen with Cy3-conjugated streptavidin on arrays. We optimized assay conditions, such as buffer pH, concentrations of dithiothreitol and 5-(biotinamido)pentylamine, and incubation time, and we created equations to determine specific FXIII and TG2 activities in samples. We successfully applied this assay system to monitor changes in FXIII and TG2 activities in THP-1 monocytic cells differentiated with phorbol 12-myristate13-acetate and interleukin-4. This activity assay is sensitive and suitable for high-throughput determination of FXIII and TG2 activities and thus has a strong potential for investigating the differential functions of these isozymes in cell signaling and cardiovascular pathophysiology research.
Analytical Chemistry 09/2011; 83(22):8718-24. · 5.86 Impact Factor
