Proteomic Effects of the Coagulation Proteinase Thrombin on LX-2 Hepatic Stellate Cells

Summary Background: The aim of this study was to characterize the effects of the coagulation proteinase thrombin on proteomic level in human hepatic stellate LX-2 cells. Methods: Proteomic analyses were performed using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). The protein profiles obtained from LX-2 cell lysates using strong anion exchanger Q10 ProteinChip arrays were statistically analyzed. Results: The peak intensities of 50 protein/peptide clusters were identified as being different between nonstimulated and LX-2 cells treated with thrombin for 6 h and 24 h, respectively. As the most significantly enhanced single signal in LX-2 cells stimulated with thrombin, a protein with a molecular mass of 13.560 kDa has been identified that corresponds exactly to calcium dependent phospholipase 2 (cPLA2). Thrombin-induced increase in the cPLA2 protein expression in LX-2 cells was confirmed by using the Western blotting technique. Conclusions: Together with the finding that thrombin induced phosphorylating activation of cPLA2 in LX-2 cells, our data point to an important function of the thrombin-mediated modulation of cytosolic phospholipase A2 in hepatic stellate cells.


Summary
Background: The aim of this study was to characterize the effects of the coagulation proteinase thrombin on proteomic level in human hepatic stellate LX-2 cells. Methods: Proteomic analyses were performed using surfaceenhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). The protein profiles obtained from LX-2 cell lysates using strong anion exchanger Q10 ProteinChip arrays were statistically analyzed. Results: The peak intensities of 50 protein/peptide clusters were identified as being different between nonstimulated and LX-2 cells treated with thrombin for 6 h and 24 h, respectively. As the most significantly enhanced single signal in LX-2 cells stimulated with thrombin, a protein with a molecular mass of 13.560 kDa has been identified that corresponds exactly to calcium dependent phospholipase 2 (cPLA2). Thrombin-induced increase in the cPLA2 protein expression in LX-2 cells was confirmed by using the Western blotting technique. Conclusions: Together with the finding that thrombin induced phosphorylating activation of cPLA2 in LX-2 cells, our data point to an important function of the thrombinmediated modulation of cytosolic phospholipase A2 in hepatic stellate cells.

Introduction
Beside its critical role in blood coagulation, the serine proteinase thrombin (EC 3.4.21.5) is known to evoke biological responses from a variety of cells, e.g. platelets, fibroblasts, vascular smooth muscle cells and monocytes (1)(2)(3), and may influence a number of cellular responses that play a role in subsequent proinflammatory and profibrotic processes in different organs including the liver (4).
Recent data suggest a role for thrombin in the activation of hepatic stellate cells (HSCs) that has been recognized as a central event in the development of liver fibrosis and finally cirrhosis and malignancy. More specifically, thrombin has been shown to stimulate matrix synthesis and the regulation of MCP1-production of cultured hepatic stellate cells (6)(7)(8). Hepatic stellate cells also express the proteinase activated receptors 1 and 4 (8), members of a novel subfamily of G-protein coupled receptors mediating the cellular effects of thrombin in different cell types.
Surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS) is a proteomic analysis (10) that has been demonstrated to be a very suitable approach to evaluate complex variations on protein level in tissue and cells (11,12).
In this study, we used SELDI-MS to evaluate the effect of thrombin on protein expression profiles in cells of the human stellate cell line LX-2 that has been characterized comprehensively as a suitable model for investigations on liver fibrosis (13). For that purpose, lysates from nonstimulated LX-2 cells and LX-2 cells stimulated with thrombin (1.0 NIH-U/mL) for 6 h and 24 h, respectively, were evaluated using strong anion exchanger Q10 ProteinChip arrays.

Reagents
Human alpha-thrombin (3085 NIH-Units/mg protein) was purchased from Haemochrom Diagnostica Supplies (Essen, Germany). All the other reagents were of the highest purity available.

Cell culture
LX-2 cells (gift from Prof. Scott Friedman, Mount Sinai School of Medicine, New York, USA) were routinely cultured in Dulbeccos modified Eagle's medium (DMEM) supplemented with 2% fetal calf serum at 37 °C in a humidified atmosphere of 5% CO 2 . The medium was changed every 2-3 days. For subculturing, treatment with trypsin/EDTA was used.
Proteins were analyzed on strong anion exchange arrays (Q10; BioRad) as described elsewhere (14). In brief, array spots were preincubated by a washing/loading buffer containing 100 mmol/L Tris-buffer, pH 8.5 with 0.02% Triton X-100 for Q10 arrays and 100 mmol/L Tris-buffer, pH 4.5 with 0.02% Triton X-100 for CM10 arrays followed by application of 2 mL of sample extract on ProteinChip Arrays, which were incubated at room temperature for 90 min in a humidity chamber. After washing three times with the same buffers and two final washing steps with water, 2 × 0.5 mL sinapinic acid (saturated solution in 0.5% TFA/50% acetonitrile) were applied. Mass analysis was performed in a Protein -Chip system (PCS 4000, Ciphergen Biosystems Inc, Fremont, CA) according to an automated data collection protocol. The instrument was externally calibrated with a ProteinChip OQ Kit (Biorad) for improved mass accuracy. Spectra were normalized with total ion current and cluster analyses of the detected signals and the determination of respective P-values were carried out with the CiphergenExpress Program (Version 3.0; Ciphergen Biosystems Inc, Fremont, CA). For P-value calculation, normalized spectra with signals in the range between 2.5 and 200 kDa exhibiting a signal-to-noise ratio (S/N) of at least 10 were selected and analyzed with the Mann-Whitney U test for nonparam etric data.
Protein was determined using the DC Protein Assay System from BioRad Laboratories according to the manufacturer's instructions.
Proteins from cell lysates were separated on a 12% SDS/PAGE and transferred to nitrocellulose membranes (BioRad). After blocking in 1% BSA/1% skimmed milk for 1 h, the nitrocellulose strips were incubated overnight with the respective first antibody. For the estimation of phosphorylating activation of cPLA2 phospho (Ser 505) cPLA2-antibody (Cell Signaling Technology, No. 2831) and for estimation of the cPLA2 protein expression, an antibody to total cPLA2 (Cell Signaling Technology, No. 2832) was used. Strips were washed two times with 0.05% (v/v) Tween 20 washing buffer, incubated for 45 min with the secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase) and washed again two times as described above. The immunoblots were stripped and reprobed with an antibody to total cPLA2 (cPLA2 activation experiments) or with an antibody to betaactin (Sigma-Aldrich, No. A 5441; cPLA2 protein expression experiments) to confirm equal protein loading. Secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase or anti-mouse IgG conjugated to horseradish peroxidase) was detected by using the chemiluminescence (ECL) Western blotting detection system (Amersham) and exposure to Kodak X-Omat films.
Immunoreactive bands for phosphorylated cPLA2, total cPLA2 and beta-actin were quantified using the image processing program Image J 1.43 (National Institutes of Health, Bethesda, Maryland, USA).

Results
Protein lysates from nonstimulated LX-2 cells and LX-2 cells stimulated with thrombin (1.0 NIH-Units/mL) for 6 h and 12 h, respectively, were applied to strong anion exchanger Q10 ProteinChip arrays and analysed individually by SELDI-MS on a PCS 4000 instrument to detect any differentially expressed proteins. In the low range of 2.5-20 kDa, up to 50 peaks were detected. After evaluation with Cipher -genExpress Program, a mass with a highly significant P-value was identified. This signal of 13.560 kDa was upregulated in thrombin-stimulated LX-2 cells discriminated significantly between nonstimulated LX-2 cells and cells stimulated with thrombin (1.0 NIH-Units/mL) for both 6 h (P=0.05) and 24 h (P=0.01). The measured intensities for nonstimulated and thrombin-treated LX-2 cells are depicted in Figure 1. By enquiring the ExPasy protein database (http://www.expasy.org) with a specified Mw range of 13560 Da +/-0.4 % and a pI range from 6 to 10, we found that the size of the protein with the mass of 13.560 kDa matches to the chain 21-138 of calciumdependent phospholipase A2 (UniProt: PA2G5_ HUMAN; P39877; Mw13591, pI 8.73).
Our data obtained with the use of proteinchip technology singled out a role for cPLA2 in thrombin's signalling activity in LX-2 hepatic stellate cells. To confirm these data, we investigated the effect of thrombin on cPLA2 protein expression by Western blotting. We found that stimulation of LX-2 cells with thrombin (1.0 NIH-U/mL) resulted in a significant increase of cPLA2 protein expression (Figure 2A). Since thrombin is known to activate cPLA2 in different cell types (15)(16)(17), we wished furthermore to evaluate thrombin-mediated activation of the enzyme in LX-2 cells, using a Western blot approach that monitors increases in phospho(P)-cPLA2 following the exposure of cells to thrombin. As demonstrated in Figure 2B, stimulation of LX-2 cells for 20 min caused an approximately 2-fold increase in P-cPLA2 immunoreactivity, relative to the signal observed in nonstimulated LX-2 cells.

Discussion
Liver fibrosis is the major complication of most chronic liver diseases, leading eventually to cirrhosis and finally to carcinoma. It is characterized by a massive deposition of extracellular matrix (ECM) components in the liver parenchyma. Synthesis of ECM components is performed by fibrogenic cells that derive from the activation of quiescent precursors such as hepatic stellate cells. Therefore, these cells play a key role in liver fibrosis and are also discussed as crucial cellular elements for the malignant process in the liver (5). In this study, we demonstrate an effect of the serine proteinase thrombin on the proteomic level in cells from the human hepatic stellate cell line LX-2 using SELDI ProteinChip technology. LX-2 cells have been extensively characterized and retain key features of cytokine signalling, neuronal gene expression, retinoid metabolism, and fibrogenesis, making them highly suitable for culture based studies of human hepatic fibrosis (13).
Proteomic analysis revealed that treatment of LX-2 cells with thrombin upregulated a single protein with a molecular mass corresponding to the chain 21-138 of calcium-dependent phospholipase A2. The effect of thrombin on the PLA2 protein expression was confirmed by Western blotting. Moreover, thrombin could be shown to induce phosphorylating activation of cPLA2 in LX-2 cells. Phospholipase A2 enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to release free arachidonic acid, which in turn is metabolized to prostaglandins by the cyclooxygenase pathway and to leukotrienes by the 5-lipoxygenase pathway (for review see e.g. 18). PLA2 is known to be involved in hepatic stellate cell activation (19) and is critically implicated in thrombininduced signalling in different cell types including platelets (20,21) and endothelial cells (22)(23)(24). In addition, from recently published data, a role for thrombin in liver fibrosis and hepatocellular carcinoma progression has been suggested (25,26).
Our study provides evidence for a link between thrombin and PLA2 in hepatic stellate cells. Thrombin's role in HSC cell activation during liver fibrosis and carcinogenesis has to be explored in further studies, including experiments on primary cultures of hepatic stellate cells and animal models.
Taken together, our data demonstrate that proteomic analysis is a suitable approach to evaluate effects of a single stimulus on protein level in hepatic stellate cells. With our results we support the concept of a function for the coagulation proteinase thrombin in hepatic stellate cell activation, and more generally, for coagulation enzymes in liver fibrosis.