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.
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1. Glenn K Carney D Fenton J Cunningham D. Thrombin active-site regions required for fibroblast receptor binding and initiation of cell division. J Biol Chem 2004; 255: 6609-16.
2. Bar-Shavit R Mudd M Wilner G Mann K Fenton JWII. Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science 1893; 220: 728-31.
3. Berndt M Philipps D. Platelet membrane proteins: composition and receptor function. In: Gordon J editor. Platelets in biology and pathology. Amsterdam/North Holland 1981: 43-7.
4. Duplantier JG Dubuisson L Senant N Freyburger G Laurendeau I Herbert JM et al. A role for thrombin in liver fibrosis. Gut 2004; 53: 1682-7.
5. Kocabayoglu P Friedman SL. Cellular basis of hepatic fibrosis and its role in inflammation and cancer. Front Biosci (Schol Ed) 2013; 5: 217-30K.
6. Mallat A Gallois C Tao J et al. Platelet-derived growth factor-BB and thrombin generate positive and negative signals for human hepatic stellate cell proliferation. Role of a prostaglandin/cyclic AMP pathway and cross-talk with endothelin receptors. J Biol Chem 1998; 273: 27300-5.
7. Gaça MD Zhou X Benyon RC. Regulation of hepatic stellate cell proliferation and collagen synthesis by proteinase-activated receptors. J Hepatol 2002; 36: 362-9.
8. Fiorucci S Antonelli E Distrutti E Severino B Fiorentina R Baldoni M et al. PAR1 antagonism protects against experimental liver fibrosis. Role of proteinase receptors in stellate cell activation. Hepatology 2004; 39: 365-75.
9. Ramachandran R Noorbakhsh F Defea K Hollenberg MD. Targeting proteinase-activated receptors: therapeutic potential and challenges. Nat Rev Drug Discov 2012; 11: 69-86.
10. Tang N Tornatore P Weinberger SR. Current developments in SELDI affinity technology. Mass Spectrom Rev 2004; 23: 34-44.
11. Clarke W Zhang Z Chan DW. The application of clinical proteomics to cancer and other diseases. Clin Chem Lab Med 2003; 41: 1562-70.
12. Whelan LC Power KA McDowell DT Kennedy J Gallagher WM. Applications of SELDI-MS technology in oncology. J Cell Mol Med 2008; 12(5A): 1535-47.
13. Xu L Hui AY Albanis E Arthur MJ O'Byrne SM Blaner WS Mukherjee P Friedman SL Eng FJ. Human hepatic stellate cell lines LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 2005; 54: 142-51.
14. Melle C Bogumil R Ernst G Schimmel B et al. De tection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling. Proteomics 2006; 6: 2600-8.
15. Kramer RM Roberts EF Manetta JV Sportsman JR Jakubowski JA. Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) in human platelets. J Lipid Mediat 1993; 6: 209-16.
16. Puri RN. Phospholipase A2: its role in ADP- and thrombin-induced platelet activation mechanisms. Int J Biochem Cell Biol 1998; 30: 1107-22.
17. Gluck N Schwob O Krimsky M Yedgar S. Activation of cytosolic phospholipase A2 and fatty acid transacylase is essential but not sufficient for thrombin-induced smooth muscle cell proliferation. Am J Physiol Cell Physiol 2008; 294: C1597-603.
18. Leslie CC. Properties and regulation of cytosolic phospholipase A2. J Biol Chem 1997; 272: 16709-12.
19. Cardoso CC Paviani ER Cruz LA Guma FC Borojevic R Guaragna RM. Effect of pentoxifylline on arachidonic acid metabolism neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell. Mol Cell Biochem 2003; 254: 37-46.
20. Akiba S Sato T Fujii T. Evidence for an increase in the association of cytosolic phospholipase A2 with the cytoskeleton of stimulated rabbit platelets. J Biochem 1993; 113: 4-6.
21. Akiba S Kawauchi T Sato T. Acceleration of Ca2+ ionophore-induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets. Eur J Biochem 1999; 259: 643-50.
22. Garcia JG. Molecular mechanisms of thrombin-induced human and bovine endothelial cell activation. J Lab Clin Med 1992; 120: 513-19.
23. Sato Y Kataoka H Asada Y Marutsuka K Kamikubo Y Koono M Sumiyoshi A. Overexpression of tissue factor pathway inhibitor in aortic smooth muscle cells inhibits cell migration induced by tissue factor/factor VIIa complex. Thromb Res 1999; 94: 401-6.
24. Shah B Shah G. Antifibrotic effect of heparin on liver fibrosis model in rats. World J Gastrointest Pharmacol Ther 2012; 3: 86-92.
25. Kaufmann R Rahn S Pollrich K Hertel J Dittmar Y Hommann M et al. Thrombin-mediated hepatocellular carcinoma cell migration: cooperative action via proteinase-activated receptors 1 and 4. J Cell Physiol 2007; 211: 699-707.