The Role Of RKIP In Rat Liver Fibrogenesis

Chronic damage results in a progressive accumulation of scarring proteins (hepatic fibrosis) that, with increasing severity, alters tissue structure and function, leading to cirrhosis and liver failure. Hepatic fibrosis is the important link that affects the chronic liver disease. Therefore, prevention and treatment of hepatic fibrosis is the focus of chronic liver disease. HBV and HCV type hepatitis are the most mainly chronic liver diseases in our country. Understanding the complex intercellular interactions regulating liver fibrogenesis is of increasing importance in view of predicted increases in chronic liver disease and the current paucity of effective therapies.Physiologically, hepatic stellate cells (HSCs) play a pivotal role in vitamin A metabolism and in maintaining the liver's architecture by producing components of extracellular matrix and regulating sinusoidal blood flow by contractility. During chronic liver injury, HSCs undergo phenotypical transformation (i.e. activation) to actively proliferate retinoid-deficient cells that express theα-smooth muscle actin (α-SMA) known as activated HSCs. Activated HSCs play an essential role in the pathogenesis of liver fibrosis and cirrhosis, the development of portal hypertension, and in the progression of liver cancer. Activated HSCs have the ability to proliferate, to synthesize a large variety of ECM molecules, to secrete cytokines and growth factors, and to migrate and contract. This activation process of HSCs is reproducible in vitro.The extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway has been shown to be activated in liver fibrosis and cirrhosis and is involved in cell growth, differentiation, and migration. Many different growth factor receptors, including PDGF receptor and EGF receptor, activate the ERK/MAPK signaling pathway through small G-protein Ras, which consequently binds Raf-1 kinase and thereby recruits Raf-1 to the inner surface of the cell membrane. After this event, Raf-1 phosphorylates MEK, which in turn phosphorylates and activates ERK. Phosphorylated ERK translocates into the nucleus and regulates gene expression via interactions with various transcription factors and subsequently, results in fibrosis related protein expression.Yeung et al, who identified the Raf kinase inhibitor protein (RKIP) as a protein that directly interacts with the kinase domain of Raf-1, first demonstrated that RKIP acts as an inhibitor of the ERK/MAPK signaling pathway. RKIP is a widely expressed and highly conserved cytosolic protein that does not share any significant homology with other kinase inhibitors. In its non-phosphorylated form, RKIP negatively regulates the Raf-1/MEK/ERK1,2 pathway by interfering with the activity of Raf-1, disrupting the Raf/MEK interaction, and preventing the activation of MEK and downstream components. In its phosphorylated state, RKIP dissociates from Raf-1 and combines with GRK-2, a negative regulator of G-protein coupled receptors (GPCRs). Available data indicate that the phosphorylation of RKIP by PKC stimulates both the Raf-1/MEK/ERK1,2 and GPCR pathways. In recent years, RKIP has been identified as a member of a novel class of molecules that suppress metastasis, with evidences from prostate cancer, malignant melanomas, breast cancer, insulinomas, colorectal cancer and hepatocellular carcinoma. Such activity appears to be opposite to that in MDCK epithelial cells, in which RKIP overexpression converts the cells to fibroblast-like morphology and promotes migration. Locostatin, a non-antibacterial oxazolidinone derivative, was discovered to abrogate RKIP's ability to bind and inhibit Raf-1 kinase by disrupting a protein-protein interaction. Locostatin also inhibits epithelial cell sheet migration. Another study suggests that RKIP plays important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion and negatively controlling cell-cell adhesion.Although the importance of RKIP in MDCK epithelial cells and metastatic cells of tumors has been well documented, there is no report so far on the role of RKIP in HSC behavior and hepatic fibrosis. The present study investigates the expression of RKIP in liver fibrogenesis and the involvement of RKIP in HSC proliferation and migration. The experiments contained three parts as below:Part 1: The dynamic expressions of RKIP, p-RKIP, ERK and p-ERK in liver of the bile duct ligated ratsObjective:To explore the dynamic expressions of RKIP, p-RKIP, p-ERK and ERK in liver of the bile duct ligated rats.Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL). Livers in model group were harvested at fixed timepoints: 1wk, 2wk, 3wk and 4wk after operation. Livers in sham operation group were harvested at 4wk after operation. Histopathological changes were evaluated by hematoxylin and eosin staining and by Masson's trichrome method. RKIP protein expression and phosphorylatin of RKIP and ERK in the livers were determined by Western blot, while the distribution of RKIP in the livers was assessed immunohistochemistically.Results:①Hematoxylin and eosin staining of liver established the bile duct ligated rats.②The location of RKIP in rat liver by immunohistochemistry assay: RKIP was located in the cytoplasm and plasma membrane, and expressed in hepatocytes, bile duct epitheliums, vascular endothelial cells and sinusoidal endothelial cells of the normal rat liver. With the development of hepatic fibrosis, the positive cells of RKIP decreased. RKIP expression was decreased in the myofibroblast of portal ducts and fiber septa, but was increased in the plasma membrane of hepatocytes. The positive areas of RKIP in the rat livers in model groups at week 1 to 4 were (87.1±1.4) %, (77.2±2.2) %, (60.9±2.3) % and (48.2±2.2) %. RKIP expression in model groups at week 2 to 4 was all lower than that in control group ((89.2±1.3) %, P<0.05).③Western blot analysis: RKIP expression in model groups at week 3 to 4 was decreased compared with the control group ((105.7±4.9) %, P<0.05). RKIP expression in model groups at week 1 to 4 was (104.0±4.2) %, (103.1±3.5) %, (54.5±2.8) % and (41.0±1.8) %. ERK expression was not changed, with (66.8±2.3) % in control group, and (70.4±2.3) %, (69.3±2.0) %, (68.1±1.4) %, (67.4±2.4) % in model groups at week 1 to 4. With the development of hepatic fibrosis, phosphorylatin of RKIP and ERK in the liver was increased. The expression of p-RKIP in control group was (12.4±1.9) %, with (43.6±2.2) %, (45.0±2.6) %, (83.9±2.9) % and (89.7±3.5) % in model groups at week 1 to 4. The expression of p-RKIP in control group was (25.8±3.2) %, with (95.5±3.8) %, (132.1±2.7) %, (277.7±4.8) % and (332.9±2.4) % in model groups at week 1 to 4.Conclusions: With the development of liver fibrosis, phosphorylatin of ERK in the livers was increased and Raf-1/MEK/ERK1,2 signaling pathway was activated in liver of the bile duct ligated rats. The activation of Raf-1/MEK/ERK1,2 signaling pathway was correlated with decreased RKIP expression and increased phosphorylated RKIP in the livers of bile duct ligated rats.Part 2: The RKIP expressions during rat HSC activation in vitro Objective: To isolate rat HSCs, and investigate the RKIP expressions during HSC activation.Methods: The viability of all cells was verified by phase contrast microscopy as well as the ability to exclude Trypan Blue. To evaluate the purity of the cultures, HSCs were tested by immunofluorescence. With the use ofα-SMA immunoreactivity and Western Blot as an activation parameter, HSCs were tested during culture in vitro. RKIP protein expression and phosphorylatin of RKIP and ERK in the HSCs were determined by Western blot, while the RKIP gene expression was assessed by real time RT-PCR.Results:①Primary rat HSCs were isolated successfully by sequential digestion of the liver with Pronase and collagenase, followed by single step density gradient centrifugation with Nycodenz.②Cell viability was greater than 92% as determined by Trypan Blue exclusion. HSC purity, as assessed by phase-contrast microscopy and vitamin A autofluorescence immediately after plating, was greater than 95%, with a yield ranging from 1.5×107 to 2.0×107 HSC/rat.③Primary HSCs show blue under ultraviolet light (328nm). The primary HSC is in rich of retinoid which squeezes the nuclear to one side. After 8 days culture, rat HSC is activated. Activated HSC loses retinoid and becomes myofibroblast-like cell.④Rat HSCs were checked byα-SMA staining with monoclonal antibody at day 1 (quiescent,α-SMA negative cells) and day 8 (activated,α-SMA positive cells). Western blots also showed that the activated HSCs wereα-SMA positive.⑤By real time RT-PCR analysis, RKIP mRNA expression was not changed during rat HSC culture in vitro. RKIP protein expression was significantly decreased in activated HSCs compared with those in quiescent status (RKIP/β-actin, 0.377±0.009, vs 0.926±0.008, P<0.01).⑥RKIP, Raf, and ERK phosphorylations were significantly increased (pRKIP/RKIP, 1.721±0.081 vs 1.087±0.021, P<0.01; pRaf/Raf, 0.216±0.019 vs 0.053±0.009, P<0.01; pERK/ERK, 0.922±0.014 vs 0.011±0.002, P<0.01). These results indicate that RKIP downregulation and RKIP phosphorylation upregulation activated the ERK/MAPK pathway.Conclusions: Primary rat HSCs were isolated successfully by sequential digestion of the liver with Pronase and collagenase, followed by single step density gradient centrifugation with Nycodenz. RKIP protein expression is downregulated and high phosphorylation of RKIP is followed by activated Raf/MEK/ERK pathway after activation of rat HSCs, suggesting that this protein is a possible factor in regulation of HSC cell proliferation and migration.Part 3: The role of RKIP in HSC proliferation and migrationObjective: To investigate the role of RKIP in HSC proliferation, apoptosis, and migration.Methods: Rat HSC cell line (HSC-T6) was used in this study, and cultured in HEPES-buffered DMEM. For transfection, pCMV5-HA-RKIP or empty vector plasmid (control) was added to HSC-T6 when 60%-70% confluence using Lipofectamine 2000 reagent. 48 hours later, the medium was renewed. After 36 hours of transient transfection, HSC-T6 cells in 6-well dishes were treated with 0.1% DMSO carrier solvent or with 50μM locostatin for 12 hours. The medium was renewed, and cells were rinsed with PBS thrice and extracted for SDS-PAGE in lysis buffer. Western blot analysis was undertaken by using the following primary antibodies: RKIP, pRKIP, Raf-1, pRaf-1, ERK1/2, pERK1/2 andβ-actin. HSC proliferation and apoptosis were evaluated with 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. To cell migration assay, G418 was added for the selection of stable clones. The method for Transwell cell migration assay was used. For wound closure assay, HSC-T6 cells were grown to confluence in DMEM supplemented with 10% FCS and then scratch wounded with a sterile plastic micropipette tip. At 0, 12, and 24 hours, photographs were taken at the same position of the wound.Results:①HSC-T6 cells transfected with RKIP plasmids overexpressed RKIP (RKIP/β-actin, 0.673±0.016, vs 0.227±0.025, P<0.01), and the phosphorylation of Raf-1 and ERK were significantly decreased in these cells (pRaf/Raf, 0.027±0.006 vs 0.853±0.022, P<0.01; pERK/ERK, 0.293±0.012 vs 1.027±0.060, P<0.01). Locostatin significantly decreased RKIP expression in transfected cells (RKIP/β-actin, 0.338±0.016, vs 0.673±0.016, P<0.01). Locostatin also increased the phosphorylation of RKIP, Raf-1, and ERK (pRKIP/RKIP, 1.090±0.128 vs 0.332±0.024, P<0.01; pRaf/Raf, 0.216±0.015 vs 0.027±0.006, P<0.01; pERK/ERK, 0.790±0.028 vs 0.293±0.012, P<0.01).②The proliferation of HSCs was significantly inhibited in cells overexpressing RKIP compared with those in the empty vector group (0.981±0.020 vs 0.860±0.022, P<0.01), while RKIP overexpression did not significantly affect HSC apoptosis.③RKIP overexpression significantly increased HSC-T6 cell migration rates compared to the control (161.00±9.17 vs 124.00±6.00, P<0.01), and locostatin abrogated this effect (43.00±7.94 vs 161.00±9.17, P<0.01), and HSC-T6 cell treated with locostatin (10.00±3.61 vs 161.00±9.17, P<0.01). To further confirm the effect of RKIP on cell migration, RKIP-overexpressing HSC-T6 cells were grown to confluence and then scrape wounded. Within 24 hours, RKIP-overexpressing cells grew to confluence again while cells transfected with the empty vector grew only to 73.2%. Locostatin significantly delayed wound closure of HSC-T6 cells as they grew only to 3.3%. Wound closure assay showed that overexpression of RKIP promoted wound closure while locostatin inhibited wound closure.Conclusions: RKIP inhibits Raf-1/MEK/ERK1,2 signaling and this inhibition impedes HSCs proliferation. RKIP promotes HSCs migration and wound closure.