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ABSTRACT: Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A(1) also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-β1, without alterations in Col-I α1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-β1 further increased aggregated Col-I. These data indicate that Col-I and aggregated, insoluble procollagen I undergo intracellular degradation via autophagy. A cytoprotective role of autophagy is implicated in kidney injury, and we demonstrate that low-dose carbon monoxide, shown to exert cytoprotection against renal fibrosis, induces autophagy to suppress accumulation of Col-I induced by TGF-β1. We also show that TGF-β1 induces autophagy in MMC via TAK1-MKK3-p38 signaling pathway. The dual functions of TGF-β1, as both an inducer of Col-I synthesis and an inducer of autophagy and Col-I degradation, underscore the multifunctional nature of TGF-β1. Our findings suggest a novel role of autophagy as a cytoprotective mechanism to negatively regulate and prevent excess collagen accumulation in the kidney.
Journal of Biological Chemistry 02/2012; 287(15):11677-88. · 4.77 Impact Factor
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ABSTRACT: Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-β1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-β1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-β1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3(-/-)) mice. We show that TGF-β1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-β1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-β1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-β1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.
Journal of Biological Chemistry 09/2010; 285(48):37909-19. · 4.77 Impact Factor
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ABSTRACT: Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that signals through the interaction of type I (TbetaRI) and type II (TbetaRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-beta1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-beta1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TbetaRI. TGF-beta1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TbetaRI is crucial for its interaction with TAK1. Both TbetaRI-mediated TAK1 phosphorylation and TGF-beta1-induced TAK1 phosphorylation do not require kinase activity of TbetaRI. Moreover, TbetaRI-mediated TAK1 phosphorylation correlates with the degree of its association with TbetaRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-beta receptors, but TAB1 is indispensable for TGF-beta1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TbetaRI and TGF-beta1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-beta1-induced interaction of TbetaRI and TbetaRII triggers dissociation of TAK1 from TbetaRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TbetaRI.
Journal of Biological Chemistry 07/2009; 284(33):22285-96. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: TGF-β1 is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to
activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However,
the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated
by TGF-β1 in primary mouse mesangial cells. Under unstimulated condition, endogenous TAK1 is stably associated with TβRI.
TGF-β stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates
that the juxtamembrane region including GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1
phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1
phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not
interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2
are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in MMC. Taken together, our data indicate
that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated
through TAB1-mediated autophosphorylation, and not by the receptor kinase activity of TβRI.
Journal of Biological Chemistry 06/2009; · 4.77 Impact Factor
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ABSTRACT: The colchicine-derived CT20126 compound has recently been shown to exert an immune regulatory effect and prolong the survival of allograft skins. In this study, we explored the anti-inflammatory and anti-arthritic effects of CT20126 in vivo and in vitro as well as investigated its underlying action mechanism. CT20126 suppressed the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha, and interleukin-1beta as well as the production of nitric oxide and prostaglandin E(2) in lipopolysaccharide (LPS)-treated macrophages as well as LPS-administered mice. This drug also inhibited the production of nitric oxide, prostaglandin E(2), and the chemokines, RANTES, GROalpha, and ENA-78, in cytokine-stimulated human synoviocytes. CT20126 suppressed NF-kappaB activation and iNOS promoter activity, which correlated with its inhibitory effect on phosphorylation-dependent IkappaB kinase activation, IkappaB phosphorylation and degradation, and NF-kappaB nuclear translocation, in LPS-stimulated macrophages. This compound also inhibited LPS-induced NF-kappaB-inducing kinase (NIK) and Akt phosphorylation, which are upstream of NF-kappaB activation. Furthermore, CT20126 significantly decreased the incidence and severity of arthritis as well as inhibited the expression of inflammatory cytokines, chemokines, iNOS, and cyclooxygenase-2 in the paws of collagen-induced arthritic mice. These findings indicate that CT20126 exerts an anti-inflammatory effect through NF-kappaB-responsive inflammatory gene expression by inhibiting the NIK- and Akt-dependent canonical NF-kappaB pathway and can be used as a therapeutic agent for rheumatoid arthritis related to chronic inflammation.
Biochemical pharmacology 08/2008; 76(1):79-90. · 4.25 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) have been implicated in the regulation of NF-kappaB activation, which plays an important role in inflammation and cell survival. However, the molecular mechanisms of ROS in NF-kappaB activation remain poorly defined. We found that the non-provitamin A carotenoid, lutein, decreased intracellular H(2)O(2) accumulation by scavenging superoxide and H(2)O(2) and the NF-kappaB-regulated inflammatory genes, iNOS, TNF-alpha, IL-1beta, and cyclooxygenase-2, in lipopolysaccharide (LPS)-stimulated macrophages. Lutein inhibited LPS-induced NF-kappaB activation, which highly correlated with its inhibitory effect on LPS-induced IkappaB kinase (IKK) activation, IkappaB degradation, nuclear translocation of NF-kappaB, and binding of NF-kappaB to the kappaB motif of the iNOS promoter. This compound inhibited LPS- and H(2)O(2)-induced increases in phosphatidylinositol 3-kinase (PI3K) activity, PTEN inactivation, NF-kappaB-inducing kinase (NIK), and Akt phosphorylation, which are all upstream of IKK activation, but did not affect the interaction between Toll-like receptor 4 and MyD88 and the activation of mitogen-activated protein kinases. The NADPH oxidase inhibitor apocynin and gp91(phox) deletion reduced the LPS-induced NF-kappaB signaling pathway as lutein did. Moreover, lutein treatment and gp91(phox) deletion decreased the expressional levels of the inflammatory genes in vivo and protected mice from LPS-induced lethality. Our data suggest that H(2)O(2) modulates IKK-dependent NF-kappaB activation by promoting the redox-sensitive activation of the PI3K/PTEN/Akt and NIK/IKK pathways. These findings further provide new insights into the pathophysiological role of intracellular H(2)O(2) in the NF-kappaB signal pathway and inflammatory process.
Free Radical Biology and Medicine 07/2008; 45(6):885-96. · 5.42 Impact Factor
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ABSTRACT: Nitrosative stress, a nitric oxide (NO)-mediated nitrosylation of redox-sensitive thiols, has been linked to the regulation of signal transduction, gene expression, and cell growth and apoptosis and thus may be widely implicated in both physiological and pathological actions of NO. Protein S-nitrosylation has been observed to occur in vitro and in vivo in pathophysiological conditions. Apoptosis can be regulated by S-nitrosylation of the redox-sensitive cysteine residue in the active site of all caspase family proteases. Detection and measurement for the modification and inactivation of caspases by S-nitrosylation remain a new challenge because of the lability of the S-nitrosothiol moiety. This chapter describes approaches for assaying and identifying S-nitrosylated caspase enzymes in vitro and in vivo. These methods permit rapid and reproducible assays of S-nitrosylated caspases in biological and clinical specimens and should be useful for studies defining a pathophysiological role of NO in several apoptosis-associated human diseases.
Methods in Enzymology 02/2008; 441:317-27. · 2.04 Impact Factor
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ABSTRACT: We here investigated the functional effect of 4-O-methylgallic acid (4-OMGA), a major metabolite of gallic acid abundant in red wine, on vascular inflammation and its action mechanism. 4-OMGA inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-alpha (TNF-alpha), resulting in the suppression of leukocyte adhesion to HUVECs. In addition, 4-OMGA inhibited the promoter activities of ICAM-1 and VCAM-1 and the activity of nuclear factor-kappaB (NF-kappaB) without affecting cytosolic IkappaB kinase (IKK) activation, inhibitor of kappaB (IkappaB) phosphorylation and degradation, and nuclear translocation of NF-kappaB. This compound did not alter nitric oxide (NO) generation, but inhibited reactive oxygen species (ROS) production in TNF-alpha-stimulated HUVECs, suggesting that NO and ROS are not involved in 4-OMGA-mediated inhibition of NF-kappaB activity. Moreover, 4-OMGA directly blocked the binding activity of NF-kappaB to its consensus DNA oligonucleotide, when pre-incubated with the nuclear extract from TNF-alpha-stimulated HUVECs, but not with the oligonucleotide alone. This inhibition was completely abolished by the addition of dithiothreitol. 4-OMGA exhibits an anti-inflammatory property by interfering with the formation of the NF-kappaB-DNA complex in the nuclei through direct and redox-sensitive interactions and may play an important role in the prevention of inflammatory responses such as the atherosclerotic process.
European Journal of Pharmacology 01/2007; 551(1-3):143-51. · 2.52 Impact Factor
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ABSTRACT: 4-O-methylgallic acid (4-OMGA) is an in vivo major metabolite of gallic acid which is abundant in red wine, tea, legumes and fruit. We examined the in vitro and in vivo effects of 4-OMGA on the production and expression of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) as well as the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). 4-OMGA inhibited the expression and production of these inflammatory genes and mediators in RAW264.7 cells and primary macrophages stimulated with lipopolysaccharide (LPS). This compound also reduced the serum levels of these inflammatory mediators in endotoxemic mice. 4-OMGA inhibited iNOS promoter activity and NF-kappaB activation in LPS-treated RAW264.7 cells. 4-OMGA inhibited the LPS-mediated increase in reactive oxygen species production and exogenous H(2)O(2)-induced NF-kappaB activation. Moreover, this compound blocked IkappaBalpha phosphorylation and degradation and nuclear translocation of the cytosolic NF-kappaB p65 subunit, which highly correlated with its inhibitory effect on IkappaB kinase activity and inflammatory mediator production. These results suggest that 4-OMGA suppresses inflammation-associated gene expression by blocking NF-kappaB activation through the inhibition of redox-sensitive IkappaB kinase activity, suggesting that this compound may be beneficial for treating endotoxemia.
International Immunopharmacology 11/2006; 6(10):1597-608. · 2.38 Impact Factor
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ABSTRACT: beta-Carotene has shown antioxidant and anti-inflammatory activities; however, its molecular mechanism has not been clearly defined. We examined in vitro and in vivo regulatory function of beta-carotene on the production of nitric oxide (NO) and PGE(2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, TNF-alpha, and IL-1beta. beta-Carotene inhibited the expression and production of these inflammatory mediators in both LPS-stimulated RAW264.7 cells and primary macrophages in a dose-dependent fashion as well as in LPS-administrated mice. Furthermore, this compound suppressed NF-kappaB activation and iNOS promoter activity in RAW264.7 cells stimulated with LPS. beta-Carotene blocked nuclear translocation of NF-kappaB p65 subunit, which correlated with its inhibitory effect on IkappaBalpha phosphorylation and degradation. This compound directly blocked the intracellular accumulation of reactive oxygen species in RAW264.7 cells stimulated with LPS as both the NADPH oxidase inhibitor diphenylene iodonium and antioxidant pyrrolidine dithiocarbamate did. The inhibition of NADPH oxidase also inhibited NO production, iNOS expression, and iNOS promoter activity. These results suggest that beta-carotene possesses anti-inflammatory activity by functioning as a potential inhibitor for redox-based NF-kappaB activation, probably due to its antioxidant activity.
Experimental and Molecular Medicine 09/2005; 37(4):323-34. · 2.48 Impact Factor
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ABSTRACT: Development of nontoxic and biologically safe antiangiogenic agent has been highlighted as a promising way to treat angiogenesis related diseases including cancer. Herein, we isolated 4-O-methylgallic acid (4-OMGA) from the seed of Canavalia gladiata, a dietary legume, on the basis of the growth inhibitory activity for bovine aortic endothelial cells (BAECs). The compound potently inhibits endothelial cell invasion and tube formation stimulated with basic fibroblast growth factor (bFGF) at low micromolar concentrations where it shows no cytotoxicity to the cells. In addition, 4-OMGA inhibits vascular endothelial cell growth factor (VEGF) production under hypoxic condition and the production of reactive oxygen species (ROS) in the endothelial cells stimulated with VEGF. These results demonstrate that 4-OMGA is a compound having potential for an antiangiogenic agent.
Biochemical and Biophysical Research Communications 06/2005; 330(4):1268-74. · 2.48 Impact Factor
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ABSTRACT: Development of nontoxic and biologically safe antiangiogenic agent has been highlighted as a promising way to treat angiogenesis related diseases including cancer. Herein, we isolated 4-O-methylgallic acid (4-OMGA) from the seed of Canavalia gladiata, a dietary legume, on the basis of the growth inhibitory activity for bovine aortic endothelial cells (BAECs). The compound potently inhibits endothelial cell invasion and tube formation stimulated with basic fibroblast growth factor (bFGF) at low micromolar concentrations where it shows no cytotoxicity to the cells. In addition, 4-OMGA inhibits vascular endothelial cell growth factor (VEGF) production under hypoxic condition and the production of reactive oxygen species (ROS) in the endothelial cells stimulated with VEGF. These results demonstrate that 4-OMGA is a compound having potential for an antiangiogenic agent.
Biochemical and Biophysical Research Communications - BIOCHEM BIOPHYS RES COMMUN. 01/2005; 330(4):1268-1274.
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Hee-Jun Na,
Seon-Jin Lee,
Yun-Chul Kang,
Young-Lai Cho,
Woo-Dong Nam,
Peter K M Kim,
Kwon-Soo Ha,
Hun-Taeg Chung,
Hansoo Lee,
Young-Guen Kwon,
Jong Sung Koh,
Young-Myeong Kim
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ABSTRACT: Farnesylation of p21(ras) is an important step in the intracellular signaling pathway of growth factors, hormones, and immune stimulants. We synthesized a potent and selective farnesyltransferase inhibitor (LB42708) with IC(50) values of 0.8 nM in vitro and 8 nM in cultured cells against p21(ras) farnesylation and examined the effects of this inhibitor in the settings of inflammation and arthritis. LB42708 suppressed NF-kappaB activation and iNOS promoter activity by suppressing the I-kappaB kinase activity and I-kappaBalpha degradation. The inhibitor suppressed the expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta and the production of NO and PGE(2) in immune-activated macrophages and osteoblasts as well as LPS-administrated mice. Furthermore, in vivo administration of LB42708 significantly decreased the incidence and severity of arthritis as well as mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta in the paws of collagen-induced arthritic mice compared with controls. These observations indicate that the anti-inflammatory and antiarthritic effects of the farnesyltransferase inhibitor may be ascribed to the inhibition of I-kappaB kinase activity and subsequent suppression of NF-kappaB-dependent inflammatory gene expression through the suppression of p21(ras) farnesylation. Together, these findings reveal that the inhibitory effect of LB42708 on p21(ras)-dependent NF-kappaB activation may have potential therapeutic value for arthritis and other inflammatory diseases.
The Journal of Immunology 08/2004; 173(2):1276-83. · 5.79 Impact Factor
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ABSTRACT: IL-4 is emerging as a candidate cytokine for the treatment of inflammatory and autoimmune diseases. We have reported that IL-4 has anti-angiogenic activity and inhibits the growth of human umbilical vein endothelial cells (HUVEC) in response to vascular endothelial growth factor (VEGF) or fibroblast growth factor-2 (FGF-2). Cell cycle analysis of this effect revealed that IL-4 arrests the growth of FGF-2-stimulated HUVEC in G0 + G1 phases. The absence of subdiploid cells showed that it did not induce apoptosis. Growth arrest was dose-dependent, but the percentage of G0 + G1 phase cells never exceeded 85%. An immunoblot analysis demonstrated that expression of p53 and p21(Waf1) was increased and that of cyclin D1 and cyclin E decreased by IL-4. These results show that IL-4 inhibits endothelial cell growth by altering the expression of cell cycle regulatory molecules.
Molecules and Cells 09/2003; 16(1):92-6. · 2.18 Impact Factor
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Seon-Jin Lee,
Se-Kyung Bai,
Kwang-Soon Lee,
Seung Namkoong, Hee-Jun Na,
Kwon-Soo Ha,
Jeong-A Han,
Sung-Vin Yim,
Kwang Chang,
Young-Guen Kwon,
Sung Ki Lee,
Young-Myeong Kim
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ABSTRACT: Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.
Molecules and Cells 09/2003; 16(1):97-105. · 2.18 Impact Factor
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Jae-Hoon Choi,
Tae-Sook Jeong,
Dae-Yong Kim,
Young-Myeong Kim, Hee-Jun Na,
Ki-Hwan Nam,
Sae-Bom Lee,
Hyoung-Chin Kim,
Sei Ryang Oh,
Yang-Kyu Choi,
Song-Hae Bok,
Goo Taeg Oh
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ABSTRACT: Hematein, a natural compound, is a known anti-inflammatory and antiatherogenic agent in the rabbit model. The authors investigated the effects of this compound on atherogenesis and possible mechanisms of the actions in the hyperlipidemic mice. Low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed a high-cholesterol diet alone for 8 weeks developed the fatty streak lesion in the aortic sinus, whereas this lesion was significantly reduced by hematein treatment without a change in plasma lipid levels compared with control mice. Hematein treatment reduced plasma levels of lipid peroxide and superoxide generation in LPS-stimulated peritoneal macrophage. Hematein treatment inhibited NF-kappaB-DNA binding activity in peritoneal macrophages from Ldlr-/- mice and the activation of NF-kappaB in RAW264.7 macrophages. This compound suppressed plasma nitrite/nitrate levels in Ldlr-/- mice and NO production and iNOS expression in LPS+IFNgamma-stimulated peritoneal macrophages. Hematein treatment also suppressed the activity of iNOS promoters in RAW264.7 macrophages, and reduced the plasma levels of TNF-alpha and IL-1beta and the production of these cytokines in LPS+IFNgamma-stimulated peritoneal macrophages. These results suggest that hematein inhibits atherosclerotic lesion formation, possibly by reducing proinflammatory mediators through a decrease in reactive oxygen species generation and NF-kappaB activation.
Journal of Cardiovascular Pharmacology 09/2003; 42(2):287-95. · 2.29 Impact Factor
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Jae-Hoon Choi,
Tae-Sook Jeong,
Dae-Yong Kim,
Young-Myeong Kim, Hee-Jun Na,
Ki-Hwan Nam,
Sae-Bom Lee,
Hyoung-Chin Kim,
Sei Ryang Oh,
Yang-Kyu Choi,
Song-Hae Bok,
Goo Taeg Oh
[show abstract]
[hide abstract]
ABSTRACT: Hematein, a natural compound, is a known anti-inflammatory and antiatherogenic agent in the rabbit model. The authors investigated the effects of this compound on atherogenesis and possible mechanisms of the actions in the hyperlipidemic mice. Low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed a high-cholesterol diet alone for 8 weeks developed the fatty streak lesion in the aortic sinus, whereas this lesion was significantly reduced by hematein treatment without a change in plasma lipid levels compared with control mice. Hematein treatment reduced plasma levels of lipid peroxide and superoxide generation in LPS-stimulated peritoneal macrophage. Hematein treatment inhibited NF-κB-DNA binding activity in peritoneal macrophages from Ldlr-/- mice and the activation of NF-κB in RAW264.7 macrophages. This compound suppressed plasma nitrite/nitrate levels in Ldlr-/- mice and NO production and iNOS expression in LPS+IFNγ-stimulated peritoneal macrophages. Hematein treatment also suppressed the activity of iNOS promoters in RAW264.7 macrophages, and reduced the plasma levels of TNF-α and IL-1β and the production of these cytokines in LPS+IFNγ-stimulated peritoneal macrophages. These results suggest that hematein inhibits atherosclerotic lesion formation, possibly by reducing proinflammatory mediators through a decrease in reactive oxygen species generation and NF-κB activation.
Atherosclerosis is a complex and chronic inflammatory disease process involving elastic and muscular arteries. 1 The uncontrolled proliferation of the vascular myointimal cells, production of foam cells, and progressive vascular occlusion is thought to be a chronic inflammatory response of the vascular wall to increasing cellular oxidative stress. 2 Exposure of cells to oxidative stress induces a local inflammatory response and the release of cytokines and growth factors. Oxidative stress, such as oxidized low-density lipoprotein (LDL) and reactive oxygen species (ROS), has been reported to activate NF-κB and various NF-κB-dependent inflammatory molecules, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS), and interferon γ (IFN-γ). 3 Several studies have demonstrated that the reduction of ROS formation by some of antioxidants, including N-acetyl cysteine, α-lipoate, and α-tocopherol, inhibits the NF-κB activation and NF-κB-dependent gene expression. 4-6
The expression of iNOS, regulated mainly by NF-κB, has been found in atherosclerotic lesions and produces high amounts of NO, which contributes to the process of atherosclerosis. 7-9 On the other hand, a small quantity of NO produced by the endothelial NOS (eNOS) has been associated with prevention of endothelial cell dysfunction, an effect that may be beneficial for an atherosclerosis. 10 Recently, the deficiency of iNOS has been demonstrated to reduce the progression of atherosclerosis. 11 Therefore, large quantities of NO could combine with superoxide to form the strong oxidant peroxynitrite that may be involved in the pathogenesis of atherosclerosis. 12 Previously, several antioxidants have been reported to have an inhibitory action of iNOS expression and activity. 13,14
In our previous studies, hematein, a compound isolated from the dried heartwood of Caesalpinia Sappan L., reduced VCAM-1 expression by suppressing NF-κB activation in human umbilical vein endothelial cells. 15 In addition, dietary hematein inhibited the formation of fatty streak lesions in rabbits fed a high-cholesterol diet, possibly by reducing VCAM-1 and MCP-1 expression. 16 Therefore, we hypothesized that the anti-inflammatory and antiatherogenic action of hematein is mediated by its antioxidative action. However, it has not been investigated whether hematein has antioxidative action in vivo. In this study, we attempted to investigate the antioxidative action and antiatherogenic mechanism of hematein using Ldlr-/- mice and peritoneal macrophage.
Journal of Cardiovascular Pharmacology 07/2003; 42(2):287-295. · 2.29 Impact Factor
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Joon-Hong Park, Hee-Jun Na,
Young-Guen Kwon,
Kwon-Soo Ha,
Seon-Jin Lee,
Chun-Ki Kim,
Kwang-Soon Lee,
Toshie Yoneyama,
Kazuyuki Hatakeyama,
Peter K M Kim,
Timothy R Billiar,
Young-Myeong Kim
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ABSTRACT: Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.
Journal of Biological Chemistry 01/2003; 277(49):47073-9. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Nitrosative stress, a nitric oxide (NO)‐mediated nitrosylation of redox‐sensitive thiols, has been linked to the regulation of signal transduction, gene expression, and cell growth and apoptosis and thus may be widely implicated in both physiological and pathological actions of NO. Protein S‐nitrosylation has been observed to occur in vitro and in vivo in pathophysiological conditions. Apoptosis can be regulated by S‐nitrosylation of the redox‐sensitive cysteine residue in the active site of all caspase family proteases. Detection and measurement for the modification and inactivation of caspases by S‐nitrosylation remain a new challenge because of the lability of the S‐nitrosothiol moiety. This chapter describes approaches for assaying and identifying S‐nitrosylated caspase enzymes in vitro and in vivo. These methods permit rapid and reproducible assays of S‐nitrosylated caspases in biological and clinical specimens and should be useful for studies defining a pathophysiological role of NO in several apoptosis‐associated human diseases.
Methods in Enzymology.
