Met provides essential signals for liver regeneration

Abstract

The Met gene was discovered through its oncogenic potential when mutated (1). The protooncogene encodes a receptor tyrosine kinase that binds hepatocyte growth factor/scatter factor (HGF/SF) as its specific ligand (2). HGF/SF was first characterized as a factor that induces hepatocyte proliferation (HGF) and as a motility factor (SF) that dissociates and increases the motility of epithelial cells (3–6). Met mainly uses the Gab1 scaffolding protein in its initial step of signal transduction (7). Well characterized biochemical responses downstream of Met signaling are the activation of the Ras/Erk/mitogen-activated protein kinase (MAPK), PI3K/Akt, Rac/Pak, and Crk/Rap1 pathways (ref. 8; see also ref. 9 for a recent review). In cell culture, activation of Met results in increased cell proliferation and survival, as well as complex motogenic and morphogenic responses. Activation of the Ras/Erk/MAPK response is of particular importance for Met-elicited proliferation (10).

Liver regeneration that follows loss or damage of hepatic tissue is a well known response to liver injury and has been studied extensively on a molecular level (11–13). A commonly used experimental model for the analysis of liver regeneration is partial hepatectomy in which two-thirds of the rodent liver is removed, leaving the remaining lobes intact. Remaining hepatic tissue then grows to compensate for the loss, and regeneration is proportional to the amount of removed tissue. Thus, liver mass becomes reconstituted within days. Various experiments have indicated that signal exchange between the body and hepatic tissue regulates liver size, i.e., an optimal liver-to-body weight ratio that is required for correct metabolic function.

Hepatocytes are mostly quiescent highly differentiated cells that retain the ability to replicate and possess an enormous proliferation potential in vivo. Under normal conditions, hepatocytes have minimal replicative activity and a long half-life. After partial hepatectomy, hepatocytes synchronously exit G₀, reenter the cell cycle, and undergo one to two rounds of replication before returning to quiescence (11–13). Cell cycle entry of hepatocytes during regeneration occurs in two steps. During the initial “priming” phase, hepatocytes leave G₀ and enter G₁. This step is reversible and starts with the activation of immediate early genes (encoding transcription factors like c-fos and c-jun), followed by the expression of other genes that encode cell cycle regulators such as the G₁ cyclin, cyclin D (14–16). In a second step, hepatocytes leave G₁ and enter S phase, which is accompanied by phosphorylation of the retinoblastoma protein (pRb) and by up-regulated expression of a number of genes including cyclin E, cyclin A, and DNA polymerase (14, 17, 18). When the liver has reattained its original size, replication ceases, and the hepatocytes enter G₀ (refs. 19 and 20; for reviews, see also refs. 21–23).

Experimental evidence indicates that signals provided by growth factors are important during liver regeneration. HGF/SF, epidermal growth factor (EGF), or transforming growth factor α are potent mitogens for cultured hepatocytes. Transgenic overexpression of HGF/SF or HB-EGF in mice results in enhanced regeneration and/or increased liver size (24, 25). Levels of HGF/SF protein increase rapidly after partial hepatectomy, a fact that led to the discovery of the factor (4–6). Furthermore, signals elicited by cytokines including IL-6 and tumor necrosis factor (TNF) also play important roles during liver regeneration. Shortly after partial hepatectomy, increased levels of IL-6 and TNF-α appear in the blood, and hepatocytes respond by the activation of transcription factors like NF-κB, STAT3, and AP1 (14, 26). Experiments performed with mice mutant for the IL-6, gp130, or the TNF-α receptor 1 genes demonstrated the importance of cytokine signaling during liver regeneration (14, 26, 27). Activation of NF-κB, STAT3, and AP1 is impaired in such mutants, and the expression of immediate early and delayed genes occurs later and is reduced. However, despite a delayed S-phase entry, liver regeneration occurs in such mutant mice (27). Moreover, the analysis of genes encoding cell cycle proteins has demonstrated their role in liver regeneration. P21Cip1/Waf1 inhibits cell cycle-dependent kinases (cdk) 2 and 4 during cell cycle progression and mutation of the gene encoding P21CIP1/WAF1 results in accelerated progression through G₁ during liver regeneration (28). Similarly, c-jun or STAT3 are activated during the early stages of liver regeneration, and mutation of c-jun or STAT3 impairs the regeneration process (16, 29).

Signals elicited through binding of HGF/SF to Met are known to be of particular importance for liver development, and both HGF/SF and Met are expressed at high levels in the developing liver. Met ^{embryonic stem cells cannot contribute to the adult liver, and furthermore,} HGF/SF ^or Met ^{embryos display reduced liver size and liver-to-body weight ratio (}30–32). Proliferation and survival of hepatocytes are impaired in the mutants, but hepatocyte differentiation is normal. A defect in placental development causes SF/HGF ^or Met ^{embryos to die during the second phase of gestation. This lethality had precluded the genetic analysis of} Met or HGF/SF function in the adult liver. Conditional mutagenesis in mice, for instance the use of cre-loxP technology (33), now allows such genetic analyses. We report here the generation of a “floxed” Met allele and its use in the analysis of Met function during liver regeneration. We find a severe impairment of liver regeneration in the conditional Met mutant mice. Analysis of cell cycle progression in the mutant livers indicates that both exit of hepatocytes from G₀ and replication are impaired. Furthermore, high levels of phospho-pRb and proliferating cell nuclear antigen (PCNA) are found in the livers after partial hepatectomy of conditional Met mutant mice at a time point when replication has ceased. Our data thus demonstrate that inhibition of cell cycle progression overrides the signals for S-phase entry and hinders complete liver regeneration in Met mutants.

Acknowledgments

Notes

Abbreviations: HGF/SF, hepatocyte growth factor/scatter factor; cdk, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; pIpC, poly(deoxyinosinic/deoxycytidylic) acid; pRb, retinoblastoma protein.