Calcium Mobilization through L-type Channels in Hepatic Stellate Cell Is Essential for TGF-b-mediated CTGF

( Jonghwa Kim ),( Sohee Kang ),( Soohyun Park ),( Ju-yeon Cho ),( Won Sohn ),( David A. Brenner ),( Yong-han Paik ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: In hepatic fibrogenesis hepatic stellate cell (HSC) is a major cell type responsible for producing a major profibrogenic cytokine TGF-b, and connective tissue growth factor (CTGF), a major fibrogenic mediator in several organs. The multi-functional nature of TGF-b signaling in hepatic fibrogenesis is still elusive. At the previous The Liver Week(2015) we reported that Pyk2 is essential for TGF-b-mediated, Smad-independent CTGF induction. Pyk2 is known to be calcium- sensitive, and TGF-b was reported to increase intracellular calcium level. Therefore, we investigated the relation between intracellular calcium levels and pro-fibrogenic TGF-b/Pyk2 axis in hepatic stellate cell. Methods: Immortalized human stellate cell line, LX-2, has been cultured. After TGF-b treatment, expression of CTGF and a-SMA were assessed with RT-PCR and western blot. Pharmacological inhibitor and siRNA-mediated knockdown were used to modulate the activities and expression levels of protein. Intracelllular calcium mobilization was measured with Fura-4/AM. Activation of Pyk2 was addressed in western blot using different phosphorylation site-specific antibodies. Results: CTGF expression was up-regulated within 1hr in TGF-b stimulated LX-2. This up-regulation was greatly suppressed by siRNA-mediated knockdown and pharmacological inhibitor of Pyk2. TGF-b treatment increased phosphorylation of Pyk2 on tyrosine 402, 579/580, and 881. Consistent with the previous reports, TGF-b increased intracellular calcium concentration in Fura-4-preloaded LX-2. CTGF induction by TGF-b was blocked in dose dependent manner by pre-treatment with BAPTA-AM, an intracellular calcium chelator, while A23187, a calcium ionopore, increased CTGF induction even in the absence of TGF-b, suggesting that increase of intracellular calcium level is enough to induce CTGF expression. In addition, A23187 increased phosphorylation of Pyk2, and CTGF induction by A23187 is also greatly reduced by siRNA of Pyk2. Pre-treament of LX-2 with Nifedipine (an L-type calcium channel blocker) suppressed CTGF induction by TGF-b in dose-dependent manner, while FPL64176 (L-type channel activator) increased CTGF expression without TGF-b. The CTGF up-regulations by TGF-b, A23187, and FPL64176 were all suppressed by siRNA-mediated knockdown and pharmacological inhibitors of Pyk2. Conclusions: In hepatic stellate cell, TGF-b increases the intracellular calcium level through L-type calcium channel, leading to downstream Pyk2 signaling for CTGF induction.

Review : NADPH oxidase mediated oxidative stress in hepatic fibrogenesis

( David A. Brenner ),( Yong Han Paik ) 대한간학회 2011 Clinical and Molecular Hepatology(대한간학회지) Vol.17 No.4

NADPH oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. ROS is involved as key secondary messengers in numerous signaling pathways, and NADPH oxidases complex has been increasingly recognized as key elements of intracellular signaling of hepatic fibrogenesis. In the liver, NADPH oxidase is functionally expressed both in the phagocytic form and in the non-phagocytic form. The non-phagocytic NADPH oxidase complex is structurally and functionally similar to the phagocytic NADPH, resulting in reduction of molecular oxygen to generate superoxide. There are six homologous NOX proteins in the non-phagocytic forms of NADPH oxidase. An emerging concept is that both phagocytic and nonphagocytic NADPH oxidase components in hepatic stellate cells (HSCs) mediate hepatic fibrosis, suggesting its potential role as a pharmacological target for anti-fibrotic therapy. The molecular components and signaling pathways of various NADPH oxidase homologues that are critical for the fibrotic activity in HSCs need to be more clearly identified. (Korean J Hepatol 2011;17:251-257)

Molecular mechanisms of hepatic fibrosis

( David A. Brenner ) 대한소화기학회 2002 대한소화기학회 추계학술대회 Vol.2002 No.-

The nicotinamide adenine dinucleotide phosphate oxidase (NOX) homologues NOX1 and NOX2/gp91^phox mediate hepatic fibrosis in mice

Paik, Yong‐,Han,Iwaisako, Keiko,Seki, Ekihiro,Inokuchi, Sayaka,Schnabl, Bernd,Ö,sterreicher, Christoph H.,Kisseleva, Tatiana,Brenner, David A. Wiley Subscription Services, Inc., A Wiley Company 2011 Hepatology Vol.53 No.5

<P><B>Abstract</B></P><P>Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a multicomponent enzyme that mediates electron transfer from nicotinamide adenine dinucleotide phosphate to molecular oxygen, which leads to the production of superoxide. NOX2/gp91<SUP>phox</SUP> is a catalytic subunit of NOX expressed in phagocytic cells. Several homologues of NOX2, including NOX1, have been identified in nonphagocytic cells. We investigated the contributory role of NOX1 and NOX2 in hepatic fibrosis. Hepatic fibrosis was induced in wild‐type (WT) mice, NOX1 knockout (NOX1KO) mice, and NOX2 knockout (NOX2KO) mice by way of either carbon tetrachloride (CCl<SUB>4</SUB>) injection or bile duct ligation (BDL). The functional contribution of NOX1 and NOX2 in endogenous liver cells, including hepatic stellate cells (HSCs), and bone marrow (BM)‐derived cells, including Kupffer cells (KCs), to hepatic reactive oxygen species (ROS) generation and hepatic fibrosis was assessed <I>in vitro</I> and <I>in vivo</I> using NOX1 or NOX2 BM chimeric mice. Hepatic NOX1 and NOX2 messenger RNA expression was increased in the two experimental mouse models of hepatic fibrosis. Whereas NOX1 was expressed in HSCs but not in KCs, NOX2 was expressed in both HSCs and KCs. Hepatic fibrosis and ROS generation were attenuated in both NOX1KO and NOX2KO mice after CCl<SUB>4</SUB> or BDL. Liver fibrosis in chimeric mice indicated that NOX1 mediates the profibrogenic effects in endogenous liver cells, whereas NOX2 mediates the profibrogenic effects in both endogenous liver cells and BM‐derived cells. Multiple NOX1 and NOX2 components were up‐regulated in activated HSCs. Both NOX1‐ and NOX2‐deficient HSCs had decreased ROS generation and failed to up‐regulate collagen α1(I) and transforming growth factor β in response to angiotensin II. <I>Conclusion:</I> Both NOX1 and NOX2 have an important role in hepatic fibrosis in endogenous liver cells, including HSCs, whereas NOX2 has a lesser role in BM‐derived cells. (H<SMALL>EPATOLOGY</SMALL> 2011;)</P>

Gliotoxin-mediated apoptosis of activated human hepatic stellate cells

Kweon, Young-Oh,Paik, Yong-Han,Schnabl, Bernd,Qian, Ting,Lemasters, John J.,Brenner, David A. 경북대학교 병원 2003 경북대학교병원의학연구소논문집 Vol.7 No.1

Background: Activated hepatic stellate cells(HSCs) play a central role in liver fibrogenesis, and apoptosis of activated HSCs might be essential to clear HSCs from injured liver. Gliotoxin induces apoptosis of activated human and rat HSCs by an unknown mechanism. Aim: This study investigated the role of reactive oxygen species(ROS) and membrane permeability transition(MPT) in gliotoxin-induced apoptosis of activated human HSCs. Methods: Primary and immortalized human HSCs were analyzed using confocal microscopy for ROS with dichlorodihdrofluorescence diacetate(DCFH-DA) fluorophore and for the mitochoudrial membrane potential(MMP) using tetramethylrhodamine methylester(TMRM). Results: Gliotoxin at higher concentrations(≥7.5μM) markedly increased ROS formation, and ROS production was also evident at concentrations of gliotoxin causing necrotic cell death(≥32.5μM). Gliotoxin rapidly(begins about 20min at 1.5μM and 10min at 7.5μM) disrupts MMP at a concentration as low as 300nM. MMP disruption was followed by cytochrome c release and caspase-3 activation. The MPT inhibitors, cyclosporine A(5μM) plus trifluoperazine(12.5μM), blocked depolarization of the mitochondrial membrane and release of cytochrome c, but did not block apoptosis in HSCs. Conclusions: Gliotoxin(0.3-7.5μM) induces apoptosis of activated human HSCs with induction of MPT, cytochrome c release and caspase-3 activation, whereas at higher doses(>325.μM), it induces necrosis. However, gliotoxin also activates a mitochondrial independent pathway.

LC, Acute : O-055 ; Protection from liver fibrosis by a PPAR agonist

( Keiko Iwaisako ),( Yong Han Paik ),( Michael Haimer ),( Kojiro Taura ),( Yuzo Kodama ),( Claude Sirlin ),( Elizabeth Yu ),( Ruth T Yu ),( Michael Downes ),( Ronald M Evans ),( David A Brenner ),( Be 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.1

Background: Peroxisome Proliferator-Activated Receptor delta (PPAR), a member of the nuclear receptor family, is emerging as a key metabolic regulator with pleiotropic actions on various tissues including fat, skeletal muscle and liver. The aim of our study was to assess the effect of a new and highly selective PPAR agonist KD3010 in experimental mouse models of liver fibrosis induced by carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Methods: Male adult C57/B6 mice were treated daily with vehicle or KD3010 by oral gavage. Liver fibrosis was induced by repeated intraperitoneal injections of CCl4 or BDL. For in vitro study, primary hepatocytes were isolated and incubated with/without KD3010. Results: PPAR agonist KD3010 ameliorates liver injury induced by CCl4 injections. Deposition of extracellular matrix proteins was lower in the KD3010 group as compared to the control group. The hepatoprotective and antifibrotic effect of KD3010 was confirmed in a model of cholestasis-induced liver injury and fibrosis using BDL for three weeks. Primary hepatocytes incubated with KD3010 were protected from serum starvation or CCl4-induced cell death, in part due to reduced reactive oxygen species (ROS) production. Conclusions: The PPAR agonist KD3010 has hepatoprotective and antifibrotic effects in animal models of liver fibrosis suggesting a new mechanistic and therapeutic approach in treating patients with chronic liver diseases.

Role of NADPH Oxidases in Liver Fibrosis

Paik, Yong-Han,Kim, Jonghwa,Aoyama, Tomonori,De Minicis, Samuele,Bataller, Ramon,Brenner, David A. Mary Ann Liebert 2014 ANTIOXIDANTS AND REDOX SIGNALING Vol.20 No.17

<P>Hepatic fibrosis is the common pathophysiologic process resulting from chronic liver injury, characterized by the accumulation of an excessive extracellular matrix. Multiple lines of evidence indicate that oxidative stress plays a pivotal role in the pathogenesis of liver fibrosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme complex that generates reactive oxygen species (ROS) in response to a wide range of stimuli. In addition to phagocytic NOX2, there are six nonphagocytic NOX proteins.</P>

Mitochondrial Permeability Transition (MPT) in not obligatory in Gliotoxin induced Apoptosis of Activated Human Hepatic Stellate Cells

권영오 ( Young Oh Kwon ),( Ting Quin ),( John J Lemaster ),( Bernd Schnabl ),( David A Brenner ) 대한소화기학회 2002 대한소화기학회 춘계학술대회 Vol.2002 No.-

<Background> Hepatic stellate cells (HSCs) play a central role in the development and resolution of liver fibrosis. Recently, apoptosis has been shown to be an important mechanism for terminating the proliferation of activated HSCs. Gliotoxin is a toxic e

대한간학회 제 8 차 춘계학술대회 초록집 : Lamivudine 치료에 의한 간섬유화의 호전과 치료전 조직 활동도

권영오,조창민,탁원영,김성국,최용환,(David A Brenner),(Michael Fried) 대한간학회 2002 Clinical and Molecular Hepatology(대한간학회지) Vol.8 No.3(S)

Body Weight Gain Is Exaggerated by Diet Type on ad Libitum Feeding

( Hyeok Choon Kwon ),( Seung Kyu Choi ),( Seung Woo Nam ),( David A. Brenner ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Obesity raises the risk of morbidity from metabolic syndrome including dyslipidemia, diabetes mellitus and fatty liver disease. Weight loss is associated with lowering the risk of metabolic syndrome. To lose body weight, reduced-calorie diet and increased physical activity are required. We report foz/foz (11- bp truncating deletion in exon 8 of Alms1 gene) mice which has disordered appetite regulation fed with western diet, high fat diet and normal chow ad liblitum showed exaggerated body weight gain according to diet pattern. Methods: 4~5 weeks old foz/foz mice and wild type (WT) littermates were fed with western diet, choline-deficient high fat diet(CD-HFD) and normal chow for 12 weeks, ad liblitum. Every week, food intake and body weight were measured and after 12 weeks, metabolic indices were assessed by evaluating body weight, liver weight, the level of fasting glucose and aminotransferase. Results: Total amount of food intake showed no significant difference among western diet, choline-deficient high fat diet and normal chow in spite of different flavor and taste. Average calorie intake was 35% higher in WD and CD-HFD compared with normal chow. However, body weight gain was 75% higher in WD and 49% higher in CD-HFD. AST was 10 times higher in WD and CD-HFD compared with normal chow. All three groups showed glucose intolerance. Conclusions: To reduce the risk of metabolic syndrome associated with obesity, reduced calorie intake and increased physical activity is mandatory. However, to keep reduced calorie intake is challeged while restriction of food intake only. This finding showed that western diet induced most exaggerated body weight gain and body weight gain is influenced more by diet pattern rather than calori itself. Finally, changing behavioral eating pattern is recommended to keep the reduced body weight and improve NASH.