Mitochondrial uncouplers inhibit hepatic stellate cell activation

BMC Gastroenterology

Leo A van Grunsven²  Etienne Sokal¹  Mustapha Najimi¹  Laurent Dollé²  Jan Best²  Eduardo L Guimarães² 

[1] Laboratory of Pediatric Hepatology and Cell Therapy, Université Catholique de Louvain (UCL), Brussel, Belgium;Department of Cell Biology, Liver Cell Biology Lab, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels, 1090, Belgium

关键词: Fibrosis;    Uncoupler;    Mitochondria;    Hepatic stellate cell;   

Others  :  1113039
DOI  :  10.1186/1471-230X-12-68

 received in 2012-01-06, accepted in 2012-04-23,  发布年份 2012

【 摘 要 】

Background

Mitochondrial dysfunction participates in the progression of several pathologies. Although there is increasing evidence for a mitochondrial role in liver disease, little is known about its contribution to hepatic stellate cell (HSC) activation. In this study we investigated the role of mitochondrial activity through mild uncoupling during in vitro activation of HSCs.

Methods

Cultured primary human and mouse HSCs were treated with the chemical uncouplers FCCP and Valinomycin. ATP levels were measured by luciferase assay and production of reactive oxygen species was determined using the fluorescent probe DCFH-DA. Possible cytotoxicity by uncoupler treatment was evaluated by caspase 3/7 activity and cytoplasmic protease leakage. Activation of HSCs and their response to the pro-fibrogenic cytokine TGF-β was evaluated by gene expression of activation markers and signal mediators using RT-qPCR. Proliferation was measured by incorporation of EdU and protein expression of α-smooth muscle actin was analyzed by immunocytochemistry and western blot.

Results

FCCP and Valinomycin treatment mildly decreased ATP and reactive oxygen species levels. Both uncouplers increased the expression of mitochondrial genes such as Tfam and COXIV while inducing morphological features of quiescent mouse HSCs and abrogating TGF-β signal transduction. Mild uncoupling reduced HSC proliferation and expression of pro-fibrogenic markers of mouse and human HSCs.

Conclusions

Mild mitochondrial uncoupling inhibits culture-induced HSC activation and their response to pro-fibrogenic cytokines like TGF-β. These results therefore suggest mitochondrial uncoupling of HSCs as a strategy to reduce progression of liver fibrosis.

【 授权许可】

   
2012 Guimaraes et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Friedman SL: Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver. Physiol Rev 2008, 88:125-172.
• [2]Atzori L, Poli G, Perra A: Hepatic stellate cell: A star cell in the liver. Int J Biochem Cell Biol 2009, 41:1639-1642.
• [3]Coleman WB, Cunningham CC: Effects of chronic ethanol consumption on the synthesis of polypeptides encoded by the hepatic mitochondrial genome. Biochim Biophys Acta - Bioenerg 1990, 1019:142-150.
• [4]Cahill A, Wang X, Hoek JB: Increased Oxidative Damage to Mitochondrial DNA Following Chronic Ethanol Consumption. Biochem Biophys Res Commun 1997, 235:286-290.
• [5]Mantena SK, King AL, Andringa KK, Eccleston HB, Bailey SM: Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases. Free Radic Biol Med 2008, 44:1259-1272.
• [6]Chavin KD, Yang S, Lin HZ, Chatham J, Chacko VP, Hoek JB, Walajtys-Rode E, Rashid A, Chen C-H, Huang C-C, et al.: Obesity Induces Expression of Uncoupling Protein-2 in Hepatocytes and Promotes Liver ATP Depletion. J Biol Chem 1999, 274:5692-5700.
• [7]Fujisawa K, Nishikawa T, Kukidome D, Imoto K, Yamashiro T, Motoshima H, Matsumura T, Araki E: TZDs reduce mitochondrial ROS production and enhance mitochondrial biogenesis. Biochem Biophys Res Commun 2009, 379:43-48.
• [8]Tejerina S, De Pauw A, Vankoningsloo S, Houbion A, Renard P, De Longueville F, Raes M, Arnould T: Mild mitochondrial uncoupling induces 3T3-L1 adipocyte de-differentiation by a PPAR{gamma}-independent mechanism, whereas TNF{alpha}-induced de-differentiation is PPAR{gamma} dependent. J Cell Sci 2009, 122:145-155.
• [9]Camille CCdS, Fernanda MC, Lívea FB, Marisa HGM, Alicia JK: Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance and longevity. Aging Cell 2008, 7:552-560.
• [10]Guimarães ELM, Empsen C, Geerts A, van Grunsven LA: Advanced glycation end products induce production of reactive oxygen species via the activation of NADPH oxidase in murine hepatic stellate cells. J Hepatol 2010, 52:389-397.
• [11]Najimi M, Khuu DN, Lysy PA, Jazouli N, Abarca J, Sempoux C, Sokal EM: Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes? Cell Transplant 2007, 16:717-728.
• [12]Mannaerts I, Nuytten NR, Rogiers V, Vanderkerken K, van Grunsven LA, Geerts A: Chronic administration of valproic acid inhibits activation of mouse hepatic stellate cells in vitro and in vivo. Hepatology 2010, 51:603-614.
• [13]Norifumi K, Shuichi S, Masayasu I, Tetsuo K: Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and kupffer cells. Hepatology 1998, 27:1265-1274.
• [14]Kong X, Wang R, Xue Y, Liu X, Zhang H, Chen Y, Fang F, Chang Y: Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis. PLoS One 2010, 5:e11707.
• [15]Ventura-Clapier R, Garnier A, Veksler V: Transcriptional control of mitochondrial biogenesis: the central role of PGC-1{alpha}. Cardiovasc Res 2008, 79:208-217.
• [16]Liying L, Pascale G, Jeanne Tran Van N, Boris J, Ariane M, Aamir H, Sophie L: Heme oxygenase-1 is an antifibrogenic protein in human hepatic myofibroblasts. Gastroenterology 2003, 125:460-469.
• [17]Serviddio G, Sastre J, Bellanti F, Viña J, Vendemiale G, Altomare E: Mitochondrial involvement in non-alcoholic steatohepatitis. Mol Aspects Med 2007, 29:22-35.
• [18]Siegmund V, Qian T, de Minicis S, Harvey-White J, Kunos G, Vinod KY, Hungund B, Schwabe RF: The endocannabinoid 2-arachidonoyl glycerol induces death of hepatic stellate cells via mitochondrial reactive oxygen species. FASEB J 2007, 21:2798-2806.
• [19]Mann J, Mann DA: Transcriptional regulation of hepatic stellate cells. Adv Drug Deliv Rev 2009, 61:497-512.
• [20]Butow RA, Avadhani NG: Mitochondrial Signaling: The Retrograde Response. Mol Cell 2004, 14:1-15.
• [21]Modrianský M, Gabrielová E: Uncouple my heart: the benefits of inefficiency. J Bioenerg Biomembr 2009, 41:133-136.
• [22]Rohas LM, St-Pierre J, Uldry M, Jager S, Handschin C, Spiegelman BM: A fundamental system of cellular energy homeostasis regulated by PGC-1α. Proc Natl Acad Sci 2007, 104:7933-7938.
• [23]Poli G: Pathogenesis of liver fibrosis: role of oxidative stress. Mol Aspects Med 2000, 21:49-98.
• [24]Greenwel P, Domínguez-Rosales J-A, Mavi G, Rivas-Estilla AM, Rojkind M: Hydrogen peroxide: A link between acetaldehyde-elicited α1(i) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells. Hepatology 2000, 31:109-116.
• [25]Han YH, Kim SW, Kim SH, Kim SZ, Park WH: 2,4-Dinitrophenol induces G1 phase arrest and apoptosis in human pulmonary adenocarcinoma Calu-6 cells. Toxicol in Vitro 2008, 22:659-670.
• [26]Hoehn KL, Salmon AB, Hohnen-Behrens C, Turner N, Hoy AJ, Maghzal GJ, Stocker R, Van Remmen H, Kraegen EW, Cooney GJ, et al.: Insulin resistance is a cellular antioxidant defense mechanism. Proc Natl Acad Sci 2009, 106:17787-17792.
• [27]Silic-Benussi M, Cannizzaro E, Venerando A, Cavallari I, Petronilli V, La Rocca N, Marin O, Chieco-Bianchi L, Di Lisa F, D'Agostino DM, et al.: Modulation of mitochondrial K+ permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1. Biochim Biophys Acta - Bioenerg 2009, 1787:947-954.
• [28]Tsukamoto H, She H, Hazra S, Cheng J, Wang J: Fat paradox of steatohepatitis. J Gastroenterol Hepatol 2008, 23:S104-S107.
• [29]Tsukamoto H, She H, Hazra S, Cheng J, Miyahara T: Anti-adipogenic regulation underlies hepatic stellate cell transdifferentiation. J Gastroenterol Hepatol 2006, 21:S102-S105.
• [30]Vila-Bedmar R, Lorenzo M, Fernandez-Veledo S: Adenosine 5'-Monophosphate-Activated Protein Kinase-Mammalian Target of Rapamycin Cross Talk Regulates Brown Adipocyte Differentiation. Endocrinology 2010, 151:980-992.
• [31]Caligiuri A, Bertolani C, Guerra CT, Aleffi S, Galastri S, Trappoliere M, Vizzutti F, Gelmini S, Laffi G, Pinzani M, et al.: Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells. Hepatology 2008, 47:668-676.
• [32]Kleuser B, Rieter H, Adam G: Selective Effects by Valinomycin on Cytotoxicity and Cell Cycle Arrest of Transformed versus Nontransformed Rodent Fibroblasts in Vitro. Cancer Res 1985, 45:3022-3028.
• [33]Hellerbrand C, Stefanovic B, Giordano F, Burchardt ER, Brenner DA: The role of TGF[beta]1 in initiating hepatic stellate cell activation in vivo. J Hepatol 1999, 30:77-87.
• [34]Chow JYC, Dong H, Quach KT, Van Nguyen PN, Chen K, Carethers JM: TGF-β mediates PTEN suppression and cell motility through calcium-dependent PKC-α activation in pancreatic cancer cells. Am J Physiol Gastrointest Liver Physiol 2008, 294:G899-G905.
• [35]Bernardi P: Mitochondrial Transport of Cations: Channels, Exchangers, and Permeability Transition. Physiol Rev 1999, 79:1127-1155.
• [36]Lim HW, Lim HY, Wong KP: Uncoupling of oxidative phosphorylation by curcumin: Implication of its cellular mechanism of action. Biochem Biophys Res Commun 2009, 389:187-192.
• [37]Xu J, Fu Y, Chen A: Activation of peroxisome proliferator-activated receptor-{gamma} contributes to the inhibitory effects of curcumin on rat hepatic stellate cell growth. Am J Physiol Gastrointest Liver Physiol 2003, 285:G20-G30.