RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats

et al. (2013) RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats. PLoS ONE 8(2): e55860. doi:10.1371/journal.pone.0055860 RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats Zheng Luo 0 Huimin Liu 0 Xiaomeng Sun 0 Rong Guo 0 Ruibing Cui 0 Xiangxing Ma 0 Ming Yan 0 Partha Mukhopadhyay, National Institutes of Health, United States of America 0 1 Department of Geriatric Gastroenterology, Qilu Hospital of Shandong University , Jinan, Shandong , China , 2 Key Laboratory of Cardiovascular Remodeling, Qilu Hospital of Shandong University , Jinan, Shandong , China , 3 Department of Hepatology and Gastroenterology of Yantai Yuhuangding Hospital of Qingdao University Medical College , Yantai, Shandong , China , 4 Department of Radiology, Qilu Hospital of Shandong University , Jinan, Shandong , China Discoidin domain receptor 2 (DDR2) is involved in fibrotic disease. However, the exact pathogenic implications of the receptor in early alcoholic liver disease are still controversial. We constructed plasmid vectors encoding short-hairpin RNA against DDR2 to investigate its role in alcoholic liver disease in an immortalized rat hepatic stellate cell line, HSC-T6, and in rats by MTT, RT-PCR and western blot analyses; immunohistochemistry and electron microscopy. Alcohol-induced upregulation of DDR2 was associated with the expression of matrix metalloproteinase 2, the transforming growth factor b1 signaling pathway and tissue inhibitor of metalloproteinase 1; collagen deposition; and extracellular matrix remodeling. Inhibition of DDR2 decreased HSC-T6 cell proliferation and liver injury in rats with 10-week-induced alcoholic liver disease. DDR2 may have an important role in the pathogenesis of early-stage alcoholic liver disease. Silencing DDR2 may be effective in preventing early-stage alcoholic liver disease. - Alcoholic liver disease (ALD) is a major cause of morbidity and mortality. It begins with the fatty liver and proceeds to liver inflammation, necrosis, progressive fibrosis and hepatocellular carcinoma. The stage before fibrosis can be cured if discovered in time and treated properly [1]. Histopathologic features of ALD include sinusoidal capillarization, deposition of collagen, and ballooning degeneration of hepatocytes [2]. Sinusoidal capillarization is characterized by loss of differentiation of liver sinusoidal endothelial cells and the formation of basement membrane in the Disse space. It impedes the metabolic exchange of nutrients and waste and precedes the onset of hepatic fibrosis. The formation of basement membrane results from increased sinusoidal deposition of laminin and collagen produced by activated hepatic stellate cells (HSCs) [3]. Ballooning degeneration of hepatocytes is a form of cell swelling, enlargement, rounding and reticulated cytoplasm [4]. The biochemical indexes of serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), especially their ratio suggest the degree of liver injury, and the ratio of AST/ALT indicates the degree of ALD [5]. Ethanol could increase liver weight, and cause losing appetite, nausea, at last body weight decreased. So the ratio of liver to body weight in ALD was increased [6,7,8]. HSCs, located in the perisinusoidal spaces, a small area between the sinusoids and hepatocytes, are the major effectors of ALD. During the early stages of hepatic injury, the normally quiescent, vitamin-A-storing HSCs transform into actively proliferating, collagen-producing myofibroblast-like cells [9]. Activated HSCs demonstrate a plastic and metabolitically active phenotype that is proliferative and fibrogenic, during resolution of liver injury, activated HSCs have been demonstrated to undergo apoptosis [10]. Acetaldehyde, a metabolite of alcohol, is associated with the activation and perpetuation of HSCs by increasing collagen I content [11,12]. HSCs participate in extracellular matrix (ECM) remodelling by producing transforming growth factor beta b1 (TGF-b1), tissue inhibitor of metalloproteinase 1 (TIMP-1), asmooth muscle actin (a-SMA), collagen and matrix metalloproteinases (MMPs, especially MMP2), which degrades the normal subendothelial ECM [13,14]. TGF-b1 has an important role as a profibrogenic factor in chronic liver disease, triggering the expression of TIMP-1, a key effector of fibrogenesis [15,16]. ECM is a dynamic regulator of cell function, whose degradation hastens its replacement by fibril-forming collagen and the formation of basement membrane, then further HSC proliferation and MMP2 production occur in a positive feedback loop [17]. The interaction of collagen and HSCs may be mediated by tyrosine kinase receptor-like discoidin domain receptor 2 (DDR2) [18]. DDR2 is expressed in some tumor cells and some fibrotic diseases of the heart, lung, kidney, cartilage, skin and liver [19,20,21]. DDR2 expression induced in liver fibrosis models was detected in activated HSCs but not hepatocytes or Kupffer cells [22,23,24]. DDR2 is characterized by 3 distinct regions: an extracellular domain for collagen binding, a transmembrane region and an intracellular kinase domain [25]. Binding of collagen(s) to the extracellular domain could induce tyrosine phosphorylation of the kinase domain [26]. Prolonged activation of the DDR2 kinase domain results in upregulation or activation of MMP2 linked to the control and neo-synthesis of ECM molecules. MMP2-mediated proliferation and invasion of HSCs can aggravate ALD [18,27,28]. However, DDR2, HSCs and macrophages may combine to attenuate chronic hepatic fibrosis [29,30]. So the exact role of DDR2 in ALD remains unknown. Targeted posttranscriptional gene silencing by RNA interference (RNAi) can inhibit gene expression in vitro and in vivo. Transduction of naked plasmid with short hairpin RNA (shRNA) is a safe and effective method of gene delivery. Rapid intravenous injection of physiological buffer in a large volume can achieve effective localization mainly in the liver [31]. In this study, we used shRNA of DDR2 to investigate its role in HSC activation, proliferation, necrosis and apoptosis, sinusoidal capillarization and collagen deposition in vitro and in vivo in rats with early stage ALD. Materials and Methods Cell Culture and Transfection HSC-T6 cells, an immortalized rat HSC cell line [32] with an activated HSC phenotype, were obtained from the Institute of Basic Medical Sciences of Qilu Hospital (originally from Liver Disease Research Center of San Francisco General Hospital, CA, USA). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, New Zealand, USA) under a humidified atmosphere of 5% CO2 at 37uC in 60-mm flasks. When cells reached 90% confluence, they were transfected with the plasmid PGPU6/GFP/Neo-shRNA targeting DDR2 (p.DDR2.shRNA; 8.0 mg with 10 ml Lipofectamine 2000 [Invitrogen, Shanghai] in each flask). The medium was changed after 6 (...truncated)