Research Article| Volume 30, ISSUE 2, P265-274, February 1999

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes


      Background/Aims: Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor.
      Methods: DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3.
      Results: Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees.
      Conclusions: Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Hepatology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Michalopoulos GK
        • DeFrances MC
        Liver regeneration.
        Science. 1997; 276: 60-66
        • Block GD
        • Locker J
        • Bowen WC
        • Petersen BE
        • Katyal S
        • Strom SC
        • Riley T
        • et al.
        Population expansion, clonal growth, and specific differentiation patterns in primary cultures of rat hepatocytes induced by HGF/SF, EGF and TGFα in a chemically defined (HGM) medium.
        J Cell Biol. 1996; 132: 1133-1149
        • Naldini L
        • Vigna E
        • Narsimhan RP
        • Guadino G
        • Zarnegar R
        • Michalopoulos GK
        • et al.
        Hepatocyte growth factor stimulates the tyrosine activity of the receptor encoded by the proto-oncogene c-MET.
        Oncogene. 1991; 6: 501-504
        • Bottaro DP
        • Rubin JF
        • Faletto DL
        • Chan AM-L
        • Kmiecik TE
        • Vande Woude GF
        • et al.
        Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.
        Science. 1991; 251: 802-804
        • Hunter T
        • Cooper JA
        Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells.
        Cell. 1981; 24: 741-752
        • Ullrich A
        • Schlessinger J
        Signal transduction by receptors with tyrosine kinase activity.
        Cell. 1990; 61: 203-212
        • Schlessinger J
        • Ullrich A
        Growth factor signaling by receptor tyrosine kinases.
        Neuron. 1992; 9: 383-391
        • Mars WM
        • Kim T-H
        • Stolz DB
        • Liu M-L
        • Michalopoulos GK
        Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.
        Cancer Res. 1996; 56: 2837-2843
        • Busso B
        • Nicodeme E
        • Chesne C
        • Guillouzo A
        • Belin D
        • Hyafil F
        Urokinase and type I plasminogen activator inhibitor production by normal hepatocytes: modulation by inflammatory agents.
        Hepatology. 1994; 20: 186-190
        • Zhong Z
        • Wen Z
        • Darnell JE
        STAT-3: a Stat family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukine-6.
        Science. 1994; 264: 95-98
        • Schindler C
        • Darnell Jr, JE
        Transcriptional response to polypeptide ligands: the JAK-STAT pathway.
        Ann Rev Biochem. 1995; 64: 621-651
        • Boccaccio C
        • Ando M
        • Tamagone L
        • Bardelli A
        • Michieli P
        • Battistini C
        • et al.
        Induction of epithelial tubules by growth factor HGF depends on the STAT pathway.
        Nature. 1998; 391: 285-288
        • Shima N
        • Tsuda E
        • Goto M
        • Yano K
        • Hayasaka H
        • Ueda M
        • et al.
        Hepatocyte growth factor and its variant with a deletion of five amino acids are distinguishable in their biological activity and tertiary structure.
        Biochem Biophys Res Commun. 1994; 200: 808-815
        • Strom SC
        • Dorko K
        • Thompson MT
        • Pisarov LA
        • Nüssler AK
        Large scale isolation and culture of human hepatocytes.
        in: Utilisation Therapeutics des Islets de Langerhans et des Hepatocytes. Les Editions INSERM, Paris1998
        • Kostrubsky VE
        • Lewis LD
        • Wood SG
        • Sinclair PR
        • Wrighton SA
        • Sinclair JF
        Effects of taxol on Cytochrom P450 3A and acetaminophen toxicity in cultured rat hepatocytes: comparison of dexamethasone.
        Toxicol Appl Pharmacol. 1997; 142: 79-86
        • Lowry O
        • Rosebrough N
        • Farr A
        • Randall R
        Protein measurement with Folin phenol reagent.
        J Biol Chem. 1951; 193: 265-275
        • Runge D
        • Runge DM
        • Bowen WC
        • Locker J
        • Michalopoulos GK
        Matrix induced re-differentiation of cultured rat hepatocytes and changes of CCAAT/enhancer binding proteins.
        Biol Chem. 1997; 378: 873-881
        • Laemmli UK
        Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
        Nature. 1970; 227: 680-685
        • Fausto N
        Growth factors in liver development, regeneration and carcinogenesis.
        Prog Growth Factor Res. 1991; 3: 219-234
        • Gómez-Lechón MJ
        • Guillén I
        • Pondosa X
        • Fabra R
        • Trullenque R
        • Nakamura T
        • et al.
        Cell cycle progression proteins (cyclins), oncogene expression, and signal transduction during the proliferative response of human hepatocytes to hepatocyte growth factor.
        Hepatology. 1996; 23: 1012-1019
        • Leffert HL
        • Koch KS
        • Lad PJ
        • Shapiro IP
        • Skelly H
        • de Humptinne B
        Hepatocyte regeneration, replication and differentiation.
        in: Arias IM Jakoby WB Popper H Schachter D Shafritz DA The Liver Biology and Pathobiology. Raven, New York1988: 833-850
        • Burger CH
        • Wick M
        • Brusselbach S
        • Muller R
        Differential induction of metabolic genes after mitogen stimulation and during normal cell cycle progression.
        J Cell Sci. 1994; 107: 241-252
        • Woodworth CD
        • Isom HC
        Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortilized by simian virus 40 and maintained in chemically defined medium.
        MCB. 1987; 7: 3740-3748
        • Kim KH
        • Han SB
        • Hyun BH
        • Ahn CJ
        • Cha YN
        • Jeong KS
        • et al.
        Functional human hepatocytes: isolation from small liver biopsy samples and primary cultivation with liver-specific functions.
        J Toxicol Sci. 1995; 20: 565-578
        • Schuetz JD
        • Schuetz EG
        • Thottassery JV
        • Guzelian PS
        • Strom S
        • Sun D
        Identification of a novel dexamethasone responsive enhancer in the human CYP3A5 gene and its activation in human in rat liver cells.
        Mol Pharm. 1996; 49: 63-72
        • Inoue C
        • Yamamoto H
        • Nakamura T
        • Ichihara A
        • Okamoto H
        Nicotinamide prolongs survival of primary cultured hepatocytes without involving loss of hepatocyte-specific function.
        J Biol Chem. 1989; 264: 4747-4750
        • Rosenberg MR
        • Strom SC
        • Michalopoulos GK
        Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes.
        In Vitro. 1982; 18: 775-782
        • Mitaka T
        • Sattler CA
        • Sattler GL
        • Sargent LM
        • Pitot HC
        Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor.
        Hepatology. 1991; 13: 21-30
        • Althaus FR
        • Lawrence SD
        • Sattler GL
        • Pitot HC
        The effect of nicotinamide on unscheduled DNA synthesis in cultured hepatocytes.
        Biochem Biophys Res Commun. 1980; 95: 1063-1070
        • Weidner KM
        • Cesare SD
        • Sachs M
        • Brinkman V
        • Behrens J
        • Birchmeier W
        Interaction between Gabl and the c-met receptor tyrosine kinase is responsible for epithelial morphogenesis.
        Nature. 1996; 384: 173-179
        • McWhinney CD
        • Hunt RA
        • Conrad KM
        • Dostal DE
        • Baker KM
        The type I angiotensin II receptor couples to Statl and STAT-3 activation through Jak2 kinase in neonatal rat cardiac myocytes.
        J Mol Cell Cardiol. 1997; 29: 2513-2524