HGF, EGF, TGF-α, FGF, SCF, and CTGF
Growth factors such as hepatocyte growth factor (HGF) and transforming growth factor-α (TGF-α) have limited stimulatory growth effect in vivo unless their target cells have been primed, e.g. by IL-6 (3). HGF is produced by mesenchymal cells of various tissues, including non-parenchymal liver cells, and induces pleiotropic effects ranging from stimulation of proliferation, motility (from which the alternative name “scatter factor”), morphogenesis, and in certain conditions survival of normal hepatocytes and extrahepatic epithelial cells to regulation of hematopoiesis, angiogenesis, fibrinolysis and coagulation (136, 137). HGF is secreted as a HGF precursor which is stored bound to the extracellular matrix and when needed cleaved to the active growth factor by proteases, including urokinase plasminogen activator (uPA) and Factor Xa (138, 139). The multiple functions of HGF are mediated by its binding to the membrane-spanning tyrosine kinase receptor c-Met, which is expressed in epithelial cells, including hepatocytes, biliary cells, and progenitor cells (137). By virtue of these properties, the HGF/c-Met system appears an important paracrine and endocrine modulator of mesenchymal-epithelial interactions required during development and repair/regeneration of a variety of tissues, including the liver (136, 137, 140–142). The plasma levels of HGF increase during liver regeneration in rodents and humans and are augmented in patients with chronic liver diseases (3, 136, 143). Ectopic expression of human HGF under the control of albumin regulatory sequences in mouse liver is able to accelerate the rate of liver repair and regeneration after partial hepatectomy (144). However, when administered intravenously or injected directly into the liver, HGF induces very little DNA synthesis unless the liver has been primed by one-third hepatectomy (145), thus categorizing HGF as a mitogenic growth factor without the capacity to “prime” the hepatocyte for replication.
Similarly, the full action of TGF-α and EGF on hepatocytes as growth factors also requires that the liver has been primed by one-third hepatectomy (145). TGF-α, a structurally and functionally EGF-related peptide, is synthesized by hepatocytes as transmembrane pro-TGF-α that in a juxtacrine manner can bind and activate the transmembrane tyrosine kinase receptor for EGF (EGFR) situated on adjacent cells. Pro-TGF-α undergoes extracellular proteolytic cleavage releasing mature soluble TGF-α and binds the EGFR mediating both autocrine and paracrine effects (146, 147). In quiescent liver, most TGF-α exists in a membrane-anchored form, but during liver regeneration secreted forms, including mature protein and partially cleaved variants, are newly synthesized and/or released from cell membranes and extracellular matrix. Among the cells in the body, hepatocytes contain the highest density of EGFR. Despite similar affinity for the EGFR and shared capability of activating the receptor tyrosine kinase activity, TGF-α exerts more potent mitogenic effects on hepatocytes than EGF (147). The expression of TGF-α is highly induced in the hepatocyte-mediated regeneration after 70% PHx, CCl4 or GalN (148, 149), and ectopic, constitutive expression of a TGF-α transgene causes pronounced hepatocyte proliferation and liver enlargement in young mice and an increased hepatocyte turnover in older transgenic mice as compared to control animals (150). However, the fact that a full mitogenic effect of TGF-α as well as EGF when administered intravenously or injected directly into the normal rodent liver can only be obtained in combination with one-third hepatectomy also categorizes these growth factors as mitogenic but without the capacity to “prime” the hepatocyte for replication (145).
Another growth factor that is transcriptionally upregulated during liver regeneration by mature cells is acidic fibroblast growth factor (aFGF) (151), which belongs to the large family of FGFs with high affinity for heparan sulfate proteoglycans of the extracellular matrix. These growth factors bind to and activate one or more members of the family of transmembrane tyrosine kinase FGF receptors (FGFRs), generating several cell type-specific and cell maturation-dependent effects, including proliferation, growth arrest, differentiation or apoptosis (152). In the normal adult rat liver only a low level of FGFR-2 in hepatocytes has been detected, which significantly increases after PHx, whereas the expression of FGFR-1 is only minimally induced (153).
Expression studies have indicated that TGF-α, HGF, and aFGF also play an important role in progenitor cell-mediated liver regeneration. Indeed, all three growth factors and their corresponding receptors are transcriptionally upregulated during the period of the active proliferation and differentiation of progenitor cells in the rat liver subjected to the 2-AAF/PHx protocol (73, 148, 151, 153, 154), and they appear to drive the early proliferation of the progenitor cell compartment (110, 154). Interestingly, the stellate cells, which proliferate concomitantly and in close contact with progenitor cells (41, 105), appear to be the main source of TGF-α (148), HGF (73), and aFGF (151), while the corresponding cognate receptors are strongly expressed in progenitor cells (73, 148, 153, 155), suggesting that the regulation of progenitor cell proliferation and differentiation by the three growth factors occurs primarily in a paracrine manner. In contrast to the predominant expression of FGFR-2 in proliferating hepatocytes after simple PHx, both FGFR-1 and -2 are highly induced during the transit amplification and differentiation of progenitor cells in the 2-AAF/PHx protocol. In particular, FGFR-1 appears mainly expressed in progenitor cells, while FGFR-2 is upregulated in both progenitor and stellate cells (153), suggesting a unique role for FGFR-1 signalling in progenitor cells. The intercellular interaction between stellate cells and progenitor cells is in part mediated by cell-specific membrane heparan sulfate proteoglycans, which are molecules also mediating the binding of growth factors to the receptors and the cellular connections with the extracellular matrix (reviewed in 30).
Urokinase-type plasminogen activator (uPA) is a component of the plasminogen activator/plasmin system, which plays important roles in liver remodelling and regeneration after partial hepatectomy (138, 156). The plasminogen activator/plasmin system has a direct role in migration of cells by cleaving components of the extracellular matrix, e.g. fibrinogen (an acute-phase protein). Furthermore, it has an indirect role in proliferation of cells by activation of growth factors, such as HGF, TGF-α, and TGF-β (138, 147, 157). In the 2-AAF/PHx protocol, expression of uPA, uPA receptor (uPAR), and plasminogen activator inhibitor type 1 (PAI-1) are localized to progenitor cells in ductular reactions with plasminogen activation taking place in the nearby surroundings of the progenitor cells (158). Infusion of HGF and/or EGF, and/or uPA into rats in which the proliferation of ductal and periductal cells is initiated by low dose of 2-AAF alone, has shown that each of these three proteins can expand the population of progenitor cells in vivo, and that this effect is synergistic when they are combined (110). However, at least in this study, HGF and EGF seem preferentially to influence different cell populations, since exposure to 2-AAF combined with infusion of HGF results in proliferation of similar numbers of biliary and stellate cells, whereas infusion of EGF and any combination hereof results in prevalent expansion of biliary cells. In addition to mitogenic effects, infusion of EGF or HGF leads to decreased numbers of cells undergoing apoptosis in response to 2-AAF (110). Collectively, these results indicate that, although 2-AAF acts as priming mitogenic stimulus for putative progenitor cells, the combination of growth factors and uPA is necessary for survival, motility, and expansion of these cells into the liver parenchyma.
The stem cell factor (SCF)/c-kit growth factor/receptor system has also been ascribed an important function during progenitor cell proliferation and differentiation. The growth factor/receptor system is expressed during the progenitor cell response in the 2-AAF/PHx protocol in rat where SCF and c-kit localize to progenitor cells in ductular structures (63, 64). Furthermore, when rats carrying a mutation in the c-kit receptor are subjected to the 2-AAF/PHx model, the overall progenitor cell response is suppressed, suggesting that SCF plays a role in the initial activation of the progenitor compartment (159).
Finally, connective tissue growth factor (CTGF) may play an important role in regeneration from both mature cells and progenitor cells in the adult rat liver. CTGF, a secretory protein and member of the ctgf/cyr61/nov (CCN) protein family, acts on many cell types regulating proliferation, apoptosis, differentiation, angiogenesis, migration, adhesion and extracellular matrix production (160). CTGF expression is increased during liver regeneration following PHx or GalN injury, but also increases in concert with the progenitor cell response in the 2-AAF/PHx protocol. Progenitor cell populations isolated from liver regenerating in response to the 2-AAF/PHx protocol and sorted out by their expression of Thy-1 contain CTGF mRNA, and inhibition of CTGF expression by the synthetic prostacyclin derivative, iloprost, are associated with a significant decrease in the number of proliferating progenitor cells and reduced expression of the progenitor cell marker AFP (161).