Abstract
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.
Keywords
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References
- Liver regeneration.Science. 1997; 276: 60-66
- 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
- Hepatocyte growth factor stimulates the tyrosine activity of the receptor encoded by the proto-oncogene c-MET.Oncogene. 1991; 6: 501-504
- Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.Science. 1991; 251: 802-804
- Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells.Cell. 1981; 24: 741-752
- Signal transduction by receptors with tyrosine kinase activity.Cell. 1990; 61: 203-212
- Growth factor signaling by receptor tyrosine kinases.Neuron. 1992; 9: 383-391
- Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.Cancer Res. 1996; 56: 2837-2843
- Urokinase and type I plasminogen activator inhibitor production by normal hepatocytes: modulation by inflammatory agents.Hepatology. 1994; 20: 186-190
- STAT-3: a Stat family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukine-6.Science. 1994; 264: 95-98
- Transcriptional response to polypeptide ligands: the JAK-STAT pathway.Ann Rev Biochem. 1995; 64: 621-651
- Induction of epithelial tubules by growth factor HGF depends on the STAT pathway.Nature. 1998; 391: 285-288
- 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
- Large scale isolation and culture of human hepatocytes.in: Utilisation Therapeutics des Islets de Langerhans et des Hepatocytes. Les Editions INSERM, Paris1998
- Effects of taxol on Cytochrom P450 3A and acetaminophen toxicity in cultured rat hepatocytes: comparison of dexamethasone.Toxicol Appl Pharmacol. 1997; 142: 79-86
- Protein measurement with Folin phenol reagent.J Biol Chem. 1951; 193: 265-275
- Matrix induced re-differentiation of cultured rat hepatocytes and changes of CCAAT/enhancer binding proteins.Biol Chem. 1997; 378: 873-881
- Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685
- Growth factors in liver development, regeneration and carcinogenesis.Prog Growth Factor Res. 1991; 3: 219-234
- 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
- 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
- Differential induction of metabolic genes after mitogen stimulation and during normal cell cycle progression.J Cell Sci. 1994; 107: 241-252
- 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
- Functional human hepatocytes: isolation from small liver biopsy samples and primary cultivation with liver-specific functions.J Toxicol Sci. 1995; 20: 565-578
- 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
- Nicotinamide prolongs survival of primary cultured hepatocytes without involving loss of hepatocyte-specific function.J Biol Chem. 1989; 264: 4747-4750
- Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes.In Vitro. 1982; 18: 775-782
- Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor.Hepatology. 1991; 13: 21-30
- The effect of nicotinamide on unscheduled DNA synthesis in cultured hepatocytes.Biochem Biophys Res Commun. 1980; 95: 1063-1070
- Interaction between Gabl and the c-met receptor tyrosine kinase is responsible for epithelial morphogenesis.Nature. 1996; 384: 173-179
- 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
Article info
Publication history
Accepted:
September 17,
1998
Received in revised form:
September 4,
1998
Received:
May 4,
1998
Identification
Copyright
© 1999 Published by Elsevier Inc.
