Twenty years ago, Sawada demonstrated that the hepatocyte proliferative response to EGF was markedly greater in young rats in comparison to old animals and suggested that aging impaired the responsiveness of the cells in old rats to growth factors [28
] (). These studies provided impetus to the controversy concerning the impact of aging on the hepatocyte proliferative response to growth factors. For some years, researchers have suspected that aging impairs specific growth-regulating molecules and/or their receptors, which, in turn, compromises the regenerative response. Despite Sawada's observation that old hepatocytes did not respond to EGF stimulation as well as did liver cells from young animals, they also reported that there were no age-related losses in either the number of hepatocellular EGF receptors or in their binding affinity. However, Marti, in our laboratory, demonstrated a 60% age-related decline in EGF binding to hepatocyte plasma membranes in rats [38
]. Interestingly, Ishigami et al. almost simultaneously reported the absence of any age-related change in hepatocyte EGF binding capacity, but did report a marked decline in EGF-induced DNA synthesis [39
]. It should be noted that Ishigami et al. used primary hepatocyte cultures, whereas Marti et al. used hepatocyte plasma membranes isolated from intact livers. The preparation of primary hepatocyte cultures involves the use of collagenase and other enzymes that cleave hepatocyte surface proteins nonspecifically, for example, EGF receptors, resulting in cells from both young and old donors expressing equivalently diminished numbers of receptors. The isolation of hepatocellular plasma membranes does not employ enzymes, and the inherent number of receptors and, assumedly, their affinity for their ligand(s) remain intact during this procedure. Interestingly, we observed an 80% age-related decline in the amount of radiolabeled EGF associated with rat hepatocyte nuclei [40
]. Furthermore, Ohtake et al. reported age-related losses of the hepatocyte high-affinity EGF receptor as well as in the level of receptor phosphorylation, a critical step in EGF activation [41
Figure 4 Effect of age on hepatocyte response to EGF and serum growth factors in young and old resting and posthepatectomy rats. Note that the posthepatectomy responses to both EGF (PHX) and EGF/serum were significantly greater in the young rats in comparison (more ...)
Several studies have reported diminished activation of a hepatocyte extracellular receptor kinase (ERK) in old rodents in comparison to young animals following partial hepatectomy [30
]. This decline leads to reduced EGF receptor phosphorylation and, subsequently, to decreased binding of the adapter protein, Shc, to the receptor, a critical event in the EGF-induced hepatocyte proliferation pathway (). Subsequent studies by Kamat and others focused on the molecular pathways that regulate hepatocyte proliferation [34
]. These investigators reported significant age-related declines in the expression of hepatocyte EGF receptor mRNA and protein, as well as in EGF receptor phosphorylation and the subsequent activation of ERK (Figures and ).
Figure 5 Effect of age on hepatic EGF receptor activation. Data derived from [30, 31].
Figure 6 Effect of age on liver EGF activation, phosphorylation, and subsequent activation of the extracellular receptor kinase (ERK). Data derived from .
Figure 7 Effect of age on the efficacy of growth hormone induced activation of the pathway resulting in hepatocyte proliferation. Data derived from .
Growth hormone (GH) is another mitogenic factor that has been implicated in hepatic regeneration. Krupczak-Hollis et al. reported that GH treatment of old, partially hepatectomized rats enhances hepatocyte proliferation in comparison to similarly aged, nontreated cohorts [42
]. Furthermore, the endogenous hepatocellular levels of GH and its receptor decline with age, whereas the level of cyclin D3
, which activates C/EBPα
(CCAAT/enhancer-binding proteins) phosphorylation, increases. This phosphorylation enhances C/EBPα
complexing with (a) a retinoblastoma gene product, (b) a chromosomal remodeling protein (Brm), and (c) a histone deacetylase to yield an inhibitor of a transcription factor required for hepatocyte proliferation, the Forkhead Box gene, FOXM1B.
The importance of transcription factors in the liver regeneration process has been illustrated in a series of studies by Wang and associates delineating the critical role played by the FOXM1B gene in hepatocyte proliferation [43
]. Using a mouse model deficient in FOXM1B, these investigators showed that adenovirus transfection with FOXM1B restored the liver regenerative capacity in mature animals to a level that exceeded that measured in young adult mice. Nontransfected FOXM1B-deficient mice did not exhibit enhanced hepatocyte proliferation.
In the resting liver, the hepatocellular levels of cyclin D3 and C/EBPα are high, thus inhibiting hepatocyte proliferation. In senescent animals, the cyclin D3 level remains high, activating the phosphorylation of C/EBPα and enhancing the formation of the larger proliferation inhibitory complex. However, following partial hepatectomy in young adult animals, the cyclin D3 level drops, as does the level of the inhibitor, C/EBPα, permitting the expression of essential transcription factors, for example, FOXM1B and cell cycle genes, and, ultimately, rapid hepatocyte proliferation ().
Proposed effect of aging on the molecular factors that regulates hepatocyte proliferation following partial hepatectomy.
Recently, Chen and colleagues identified a mechanism that regulates FOXM1B transcriptional activation and the liver regeneration process [45
]. These researchers showed that the farnesoid X receptor (FXR), a transcription factor that regulates a variety of metabolic pathways, is critical for liver regeneration since FXR-deficient mice exhibit a diminished regenerative capacity. In addition, FOXM1B was identified as a direct FXR target gene, and diminished FXR binding to FOXM1B may contribute to decreased hepatocyte regeneration in the elderly.