Activation of the p53–p21Cip1 pathway is required for CDK2 activation and S-phase entry in primary rat hepatocytes

Abstract

p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative 'brake' to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21(Cip1) in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53(V143A)) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21(Cip1) was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21(Cip1) expression.

EGF Activates Autocrine TGFα to Induce Prolonged EGF Receptor Signaling and Hepatocyte Proliferation

“…The neutralizing antibody inhibited cyclin D induction and nuclear translocation of Cdk4 and Cdk2. This may be a consequence of Erk1/2 inhibition, leading to reduced cyclin D induction and Cdk4 activation, and possibly also of other effects on the intracellular trafficking of G1 cyclins [4,21]. Addition of the neutralizing antibody to TGFα altered sustained EGF receptor signaling even with EGF present in the medium.…”

“…Cultured hepatocytes were harvested and processed for Western immunoblot as previously described [20,21]. Briefly, cells were lysed and sonicated in a Tris-lysis buffer, (pH 7.4, with 60 mm Tris-HCl, 10% glycerol, 3% sodium dodecyl sulfate, 1 m EDTA, 0.2 mm AEBSF, 20 μm leupeptin, 200 units/mL aprotinin, 65 μm sodium orthovanadate and 10 mm β-glycerophosphate).…”

“…Immunofluorescence staining was performed as previously described [18,20,21]. Briefly, hepatocyte cultures were fixed in 70% ethanol or 4% PBS-buffered paraformaldehyde for 10 minutes.…”

“…Hepatocytes were isolated in a two-step in vitro version of the collagenase perfusion technique with modifications [19,20,21]. Cells were seeded at a density of 20 000 cells/cm2.…”

Neural Stem/Progenitor Cell Clones as Models for Neural Development and Transplantation

“…Upon activation, p53 can initiate a cell cycle arrest to allow DNA repair to take place, or signal cells to go on apoptosis if the DNA damage is unfixable. One mechanism of such function of p53 protein is that, upon activation, it can turn on the expression of p21, which forms complex with cyclindependent kinase 2 (CDK2) and inhibits its promoting activity in G1/S transition in the cell cycle (Wierod et al, 2008) (Fig. 2).…”