Pharmaceutical Research

, 26:1012 | Cite as

Characterization of the Enhancing Effect of Protamine on the Proliferative Activity of Hepatocyte Growth Factor in Rat Hepatocytes

  • Ke-Xin Liu
  • Yukio Kato
  • Kunio Matsumoto
  • Toshikazu Nakamura
  • Taiichi Kaku
  • Yuichi SugiyamaEmail author
Research Paper



The aim of the present study was to characterize the mechanism of the stimulatory effect of protamine on HGF activity.


The enhancing effects of protamine on the proliferative activity of HGF were investigated in vivo, in primary cultured rat hepatocytes, and in perfused rat liver.


In α-naphthylisothiocyanate-intoxicated rats, pretreatment with protamine increased HGF-induced autophosphorylation of the HGF receptor in liver. The maximum enhancing effect of protamine on HGF-induced DNA synthesis of hepatocytes required a 10 min-pretreatment period both in vivo and in vitro, and the stimulatory effect of protamine was not observed when it was administered simultaneously with HGF. Preperfusion of the liver with protamine for 10 min decreased the non-saturable portion of hepatic clearance for 125I-HGF, which is mainly mediated by cell-surface heparan-sulfate proteoglycan (HSPG). Inhibition of HGF binding to heparin by protamine was confirmed using heparin-coated sepharose. This inhibition also required 10 min of pretreatment, for protamine to bind heparin.


The enhancing effect of protamine on the mitogenic activity of HGF on hepatocytes requires pretreatment with protamine for a short period presumably required for its binding to cell-surface heparin, implying possible regulation of c-met autophosphorylation by HSPG.


clearance heparan-sulfate proteoglycan (HSPG) heparin hepatocyte growth factor (HGF) protamine 


  1. 1.
    T. Nakamura, T. Nishizawa, M. Hagiya, T. Seki, M. Shimonishi, A. Sugimura, K. Tashiro, and S. Shimizu. Molecular cloning and expression of human hepatocyte growth factor. Nature. 342:440–443 (1989). doi: 10.1038/342440a0.PubMedCrossRefGoogle Scholar
  2. 2.
    T. Nakamura, H. Teramoto, A. Icto, and A. Ichihara. Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures. Proc. Natl. Acad. Sci. U. S. A. 83:6489–6493 (1986). doi: 10.1073/pnas.83.17.6489.PubMedCrossRefGoogle Scholar
  3. 3.
    Y. Ishiki, H. Ohnishi, Y. Muto, K. Matsumoto, and T. Nakamura. Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and for potent anti-hepatitis action in vivo. Hepatology. 16:1227–1235 (1992).PubMedGoogle Scholar
  4. 4.
    Y. Kato, K. X. Liu, T. Nakamura, and Y. Sugiyama. Heparin–hepatocyte growth factor complex with low plasma clearance and retained hepatocyte proliferating activity. Hepatology. 20:417–424 (1994).PubMedCrossRefGoogle Scholar
  5. 5.
    J. K. Spix, E. Y. Chay, E. R. Block, and J. K. Klarlund. Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor. Exp. Cell Res. 313:3319–3325 (2007). doi: 10.1016/j.yexcr.2007.06.006.PubMedCrossRefGoogle Scholar
  6. 6.
    Y. Matsumoto, T. Motoki, S. Kubota, M. Takigawa, H. Tsubouchi, and E. Gohda. Inhibition of tumor–stromal interaction through HGF/Met signaling by valproic acid. Biochem. Biophys. Res. Commun. 366:110–116 (2008). doi: 10.1016/j.bbrc.2007.11.089.PubMedCrossRefGoogle Scholar
  7. 7.
    A. Catizone, G. Ricci, J. D. Bravo, and M. Galdieri. Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development. J. Endocrinol. 189:137–146 (2006). doi: 10.1677/joe.1.06528.PubMedCrossRefGoogle Scholar
  8. 8.
    E. Cacci, M. Salani, S. Anastasi, I. Perroteau, G. Poiana, S. Biagioni, and G. Augusti-Tocco. Hepatocyte growth factor stimulates cell motility in cultures of the striatal progenitor cells ST14A. J. Neurosci. Res. 74:760–768 (2003). doi: 10.1002/jnr.10799.PubMedCrossRefGoogle Scholar
  9. 9.
    M. Johansson, G. Mattsson, A. Andersson, L. Jansson, and P. O. Carlsson. Islet endothelial cells and pancreatic beta-cell proliferation: studies in vitro and during pregnancy in adult rats. Endocrinology. 147:2315–2324 (2006). doi: 10.1210/en.2005-0997.PubMedCrossRefGoogle Scholar
  10. 10.
    K. X. Liu, Y. Kato, T. Terasaki, T. Nakamura, and Y. Sugiyama. Change in hepatic handling of hepatocyte growth factor during liver regeneration in rats. Am. J. Physiol. 269:G745–G753 (1995).PubMedGoogle Scholar
  11. 11.
    K. X. Liu, Y. Kato, M. Yamazaki, O. Higuchi, T. Nakamura, and Y. Sugiyama. Decrease in the hepatic uptake clearance of hepatocyte growth factor (HGF) in CCl4-intoxicated rats. Hepatology. 17:651–660 (1993). doi: 10.1002/hep.1840170420.PubMedCrossRefGoogle Scholar
  12. 12.
    S. Hagiwara, T. Otsuka, Y. Yamazaki, T. Kosone, N. Sohara, T. Ichikawa, K. Sato, S. Kakizaki, H. Takagi, and M. Mori. Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride-induced chronic liver injury. Liver Int. 28:126–131 (2007).PubMedGoogle Scholar
  13. 13.
    Y. H. Lee, Y. J. Suzuki, A. J. Griffin, and R. M. Day. Hepatocyte growth factor regulates cyclooxygenase-2 expression via beta-catenin, Akt, and p42/p44 MAPK in human bronchial epithelial cells. Am. J. Physiol. 294:L778–L786 (2008).Google Scholar
  14. 14.
    E. Vigna, L. Naldini, L. Tamagnone, P. Longati, A. Bardelli, F. Maina, C. Ponzetto, and P. M. Comoglio. Hepatocyte growth factor and its receptor, the tyrosine kinase encoded by the c-MET proto-oncogene. Cell Mol. Biol. 40:597–604 (1994).PubMedGoogle Scholar
  15. 15.
    C. C. Lee, and K. M. Yamada. Alternatively spliced juxtamembrane domain of a tyrosine kinase receptor is a multifunctional regulatory site. Deletion alters cellular tyrosine phosphorylation pattern and facilitates binding of phosphatidylinositol-3-OH kinase to the hepatocyte growth factor receptor. J. Biol. Chem. 270:507–510 (1995). doi: 10.1074/jbc.270.2.507.PubMedCrossRefGoogle Scholar
  16. 16.
    A. Bardelli, C. Ponzetto, and P. M. Comoglio. Identification of functional domains in the hepatocyte growth factor and its receptor by molecular engineering. J. Biotechnol. 37:109–122 (1994). doi: 10.1016/0168-1656(94)90002-7.PubMedCrossRefGoogle Scholar
  17. 17.
    K. X. Liu, Y. Kato, M. Narukawa, D. C. Kim, M. Hanano, O. Higuchi, T. Nakamura, and Y. Sugiyama. The importance of the liver in the plasma clearance of hepatocyte growth factor in rats. Am. J. Physiol. 263:G642–G649 (1992).PubMedGoogle Scholar
  18. 18.
    K. X. Liu, Y. Kato, T. Terasaki, S. Aoki, K. Okumura, T. Nakamura, and Y. Sugiyama. Contribution of parenchymal and non-parenchymal liver cell to the clearance of hepatocyte growth factor from the circulation in rats. Pharm. Res. 12:737–1740 (1995).Google Scholar
  19. 19.
    K. X. Liu, Y. Kato, T. I. Kaku, K. Okumura, T. Nakamura, and Y. Sugiyama. Protamine enhances the proliferative activity of hepatocyte growth factor. Am. J. Physiol. 274:G21–G28 (1998).PubMedGoogle Scholar
  20. 20.
    K. X. Liu, Y. Kato, T. I. Kaku, T. Nakamura, and Y. Sugiyama. Existence of two nonlinear elimination mechanisms for hepatocyte growth factor in rats. Am. J. Physiol. 273(5 Pt 1):E891–E897 (1997).PubMedGoogle Scholar
  21. 21.
    L. E. Kemp, B. Mulloy, and E. Gherardi. Signalling by HGF/SF and Met: the role of heparin sulphate co-receptors. Biochem. Soc. Trans. 34(Pt 3):414–417 (2006). doi: 10.1042/BST0340414.PubMedGoogle Scholar
  22. 22.
    K. Matsumoto, and T. Nakamura. NK4 gene therapy targeting HGF-Met and angiogenesis. Front. Biosci. 13:1943–1951 (2008). doi: 10.2741/2813.PubMedCrossRefGoogle Scholar
  23. 23.
    Y. L. Yin, H. L. Chen, H. M. Kuo, and S. P. He. NK3 and NK4 of HGF enhance filamin production via STAT pathway, but not NK1 and NK2 in human breast cancer cells. Acta Pharmacol. Sin. 29:728–735 (2008). doi: 10.1111/j.1745-7254.2008.00799.x.PubMedCrossRefGoogle Scholar
  24. 24.
    A. Krishnan, K. Viker, H. Rietema, M. Telgenkamp, B. Knudsen, and M. Charlton. Prolonged engraftment of human hepatocytes in mice transgenic for the deleted form of human hepatocyte growth factor. Hepatol. Res. 37:854–862 (2007). doi: 10.1111/j.1872-034X.2007.00139.x.PubMedCrossRefGoogle Scholar
  25. 25.
    V. C. Yang, Y. Y. Fu, C. C. Teng, S. C. Ma, and J. N. Shanberge. A method for the quantitation of protamine in plasma. Thromb. Res. 74:427–434 (1994). doi: 10.1016/0049-3848(94)90158-9.PubMedCrossRefGoogle Scholar
  26. 26.
    S. Miyauchi, Y. Sawada, T. Iga, M. Hanano, and Y. Sugiyama. Comparison of the hepatic uptake clearances of fifteen drugs with a wide range of membrane permeabilities in isolated rat hepatocytes and perfused rat livers. Pharm. Res. 10:434–440 (1993). doi: 10.1023/A:1018952709120.PubMedCrossRefGoogle Scholar
  27. 27.
    M. Kato, Y. Kato, T. Nakamura, and Y. Sugiyama. Efficient extraction by the liver governs overall elimination of hepatocyte growth factor in rats. J. Pharmacol. Exp. Ther. 290:373–379 (1999).PubMedGoogle Scholar
  28. 28.
    K. Komamura, J. Miyazaki, E. Imai, K. Matsumoto, T. Nakamura, and M. Hori. Hepatocyte growth factor gene therapy for hypertension. Methods Mol. Biol. 423:393–404 (2008). doi: 10.1007/978-1-59745-194-9_31.PubMedCrossRefGoogle Scholar
  29. 29.
    R. Gong, A. Rifai, Y. Ge, S. Chen, and L. D. Dworkin. Hepatocyte growth factor suppresses proinflammatory NFkappaB activation through GSK3beta inactivation in renal tubular epithelial cells. J. Biol. Chem. 283:7401–7410 (2008). doi: 10.1074/jbc.M710396200.PubMedCrossRefGoogle Scholar
  30. 30.
    T. H. Cheng, W. T. Lee, J. S. Jeng, C. M. Wu, G. C. Liu, M. Y. Chiang, and Y. M. Wang. Synthesis and characterization of a novel paramagnetic macromolecular complex [Gd(TTDASQ-protamine)]. Dalton Trans. 21:5149–5155 (2006). doi: 10.1039/b604783a.CrossRefGoogle Scholar
  31. 31.
    E. Moreno, J. C. Meneu, J. Calvo, B. Perez, A. G. Sesma, A. Manrique, I. Vegh, A. M. Aragon, M. Grau, A. Gimeno, C. Jimenez, R. Gomez, A. Moreno, M. Abradelo, I. Garcia, and A. de la Calle. Modulation of hepatocyte growth factor plasma levels in relation to the dose of exogenous heparin administered: an experimental study in rats. Transplant. Proc. 37:3943–3947 (2005). doi: 10.1016/j.transproceed.2005.10.089.PubMedCrossRefGoogle Scholar
  32. 32.
    D. C. West, C. G. Rees, L. Duchesne, S. J. Patey, C. J. Terry, J. E. Turnbull, M. Delehedde, C. W. Heegaard, F. Allain, C. Vanpouille, D. Ron, and D. G. Fernig. Interactions of multiple heparin binding growth factors with neuropilin-1 and potentiation of the activity of fibroblast growth factor-2. J. Biol. Chem. 280:13457–13464 (2005). doi: 10.1074/jbc.M410924200.PubMedCrossRefGoogle Scholar
  33. 33.
    D. Naka, T. Ishii, T. Shimomura, T. Hishida, and H. Hara. Heparin modulates the receptor-binding and mitogenic activity of hepatocyte growth factor on hepatocytes. Exp Cell Res. 209:317–324 (1993). doi: 10.1006/excr.1993.1316.PubMedCrossRefGoogle Scholar
  34. 34.
    R. H. Schwall, L. Y. Chang, P. J. Godowski, D. W. Kahn, K. J. Hillan, K. D. Bauer, and T. F. Zioncheck. Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2. J .Cell Biol. 133:709–718 (1996). doi: 10.1083/jcb.133.3.709.PubMedCrossRefGoogle Scholar
  35. 35.
    M. Machide, K. Kamitori, and S. Kohsaka. Hepatocyte growth factor-induced differential activation of phospholipase Cgamma1 and phosphatidylinositol 3-kinase is regulated by tyrosine phosphatase, SHP-1 in astrocytes. J. Biol. Chem. 275:31392–31398 (2000). doi: 10.1074/jbc.M002817200.PubMedCrossRefGoogle Scholar
  36. 36.
    M. Hecht, M. Papoutsi, H. D. Tran, J. Wilting, and L. Schweigerer. Hepatocyte growth factor/c-Met signaling promotes the progression of experimental human neuroblastomas. Cancer Res. 64:6109–6118 (2004). doi: 10.1158/0008-5472.CAN-04-1014.PubMedCrossRefGoogle Scholar
  37. 37.
    V. Crljen, S. Volinia, and H. Banfic. Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes. Biochem. J. 365(Pt 3):791–799 (2002).PubMedGoogle Scholar
  38. 38.
    Y. Okano, K. Mizuno, S. Osada, T. Nakamura, and Y. Nozawa. Tyrosine phosphorylation of phospholipase C gamma in c-met/HGF receptor-stimulated hepatocytes: comparison with HepG2 hepatocarcinoma cells. Biochem. Biophys. Res. Commun. 190:842–842 (1993). doi: 10.1006/bbrc.1993.1125.PubMedCrossRefGoogle Scholar
  39. 39.
    J. A. Price, J. Caldwell, and N. J. Hewitt. The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes. Cryobiology. 53:182–193 (2006). doi: 10.1016/j.cryobiol.2006.05.008.PubMedCrossRefGoogle Scholar
  40. 40.
    V. B. Lokeshwar, S. S. Huang, and J. S. Huang. Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number. J. Biol. Chem. 264:19318–19326 (1989).PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Ke-Xin Liu
    • 1
    • 3
    • 4
  • Yukio Kato
    • 1
    • 6
  • Kunio Matsumoto
    • 2
    • 5
  • Toshikazu Nakamura
    • 2
  • Taiichi Kaku
    • 4
  • Yuichi Sugiyama
    • 1
    Email author
  1. 1.Graduate School of Pharmaceutical SciencesUniversity of TokyoBunkyo-kuJapan
  2. 2.Biomedical Research CenterOsaka University School of MedicineSuitaJapan
  3. 3.College of PharmacyDalian Medical UniversityDalianChina
  4. 4.Japan Bioproducts Industry Co. Ltd.Shibuya-kuJapan
  5. 5.Cancer Research InstituteKanazawa UniversityKanazawaJapan
  6. 6.Institute of Medical, Pharmaceutical and Health SciencesKanazawa UniversityKanazawaJapan

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