Induction of p53-dependent p21 limits proliferative activity of rat hepatocytes in the presence of hepatocyte growth factor.

BACKGROUND: Hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, enhances hepatocyte function without stimulating proliferation, depending on the physiological conditions. p53, a transcription factor, suppresses the cell proliferation by expressing p21(WAF1/CIP1) in various tissues. AIM: To investigate the mechanism through which the hepatocytes maintain mitotically quiescent even in the presence of HGF. METHODS: We studied the relationship between p53 and p21 expression and the effect of p53-p21 axis on hepatocyte proliferation in primary cultured rat hepatocytes stimulated by HGF. Hepatic p21 levels are determined serially after partial hepatectomy or sham operation in rats. RESULTS: DNA synthesis was markedly increased by HGF addition in rat hepatocytes cultured at low density but not at high density. Cellular p53 levels increased in the hepatocytes cultured at both the densities. p21 levels were increased and correlated with cellular p53 levels in hepatocytes cultured at high density but not at low density. When the activity of p53 was suppressed by a chemical inhibitor for p53, cellular p21 levels were reduced, and DNA synthesis was increased. Similarly, p21 antisense oligonucleotide increased the DNA synthesis. In rats after partial hepatectomy, transient elevation of hepatic p21 levels was observed. In contrast, in sham-operated rats, hepatic p21 levels were increased on sustained time scales. CONCLUSION: p53-related induction of p21 may suppress hepatocyte proliferation in the presence of HGF in the setting that mitogenic activity of HGF is not elicitable

Cellular p53 levels in hepatocytes treated with HGF.

<p>Rat hepatocytes were cultured in WE containing 10% FCS and various concentration of HGF for 18 hours. Closed bars denote hepatocytes cultured at high density. Open bars denote hepatocytes cultured at low density. Data are mean ± SEM of four dishes. *p<0.01 compared with the values in the absence of HGF.</p

Cellular p53 and p21 levels in hepatocytes treated with HGF.

<p>Rat hepatocytes were cultured in WE containing 10% FCS and various concentrations of HGF for 18 hours. Open circles denote hepatocytes cultured in the absence of HGF. Dotted open circles denote hepatocytes cultured with 2.5 ng/mL HGF. Dotted closed circles denote hepatocytes cultured with 5 ng/mL HGF. Closed circles denote hepatocytes cultured with 10 ng/mL HGF. (A) Hepatocytes cultured at high density. (B) Hepatocytes cultured at low density.</p

Changes in DNA synthesis of hepatocytes after HGF treatment.

<p>Rat hepatocytes were cultured in WE containing 10% FCS, 10 ng/mL HGF and 0.1 mmol/L BrdU, and were harvested serially. Closed circles denote hepatocytes cultured at high density. Open circles denote hepatocytes cultured at low density. Data are mean ± SEM of eight dishes. *p<0.01 compared with the values cultured for 0 hours.</p

The effect of pifithrin-α on DNA synthesis of hepatocytes in the presence of HGF.

<p>Rat hepatocytes were cultured at high density in the same medium as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078346#pone-0078346-g006" target="_blank">Figure 6</a>, except that the medium contained 1 mmol/L BrdU, for 24 hours. An open bar denotes hepatocytes cultured in the absence of HGF. Closed bars denote hepatocytes cultured with pifithrin-α in the presence of 10 ng/mL HGF. A dotted bar denotes hepatocytes cultured with DMSO in the presence of 10 ng/mL HGF. Data are mean ± SEM of eight dishes. *p<0.01 compared with the values treated only with HGF or values treated with HGF and DMSO.</p

Cellular p21 levels in hepatocytes treated with HGF.

<p>Rat hepatocytes were cultured in WE containing 10% FCS and various concentration of HGF for 18 hours. Closed bars denote hepatocytes cultured at high density. Open bars denote hepatocytes cultured at low density. Data are mean + SEM of four dishes. *p<0.05 compared with the values in the absence of HGF.</p

Serial changes in p21 protein levels of hepatocytes treated with HGF.

<p>Rat hepatocytes were cultured at high density in WE containing 10% FCS and 10 ng/mL HGF, and were harvested serially. Closed circles denote hepatocytes cultured at high density. Open circles denote hepatocytes cultured at low density. Data are mean ± SEM of four dishes. *p<0.01 compared with the values cultured for 0 hours.</p

Changes in hepatic p21 levels after two thirds partial hepatectomy in rats.

<p>Data are mean ± SEM of four rats. Open and closed circles indicate the hepatic p21 levels in partially hepatectomized rats and in sham-operated rats, respectively. *p<0.05 compared with the values at 0 hour, and #p<0.05 compared with the values of partially hepatectomized rats.</p

The effect of p21 antisense on DNA synthesis of hepatocytes in the presence of HGF.

<p>Rat hepatocytes were cultured at high density in WE containing 10% FCS, 10 ng/mL HGF, 1 mmol/L BrdU, along with various concentrations of either p21 antisense or nonsense oligonucleotide for 24 hours. An open bar denotes hepatocytes cultured in the absence of HGF. Closed bars denote hepatocytes cultured with p21 antisense oligonucleotide in the presence of 10 ng/mL HGF. A dotted bar denotes hepatocytes cultured with nonsense oligonucleotide in the presence of 10 ng/mL HGF. Data are mean ± SEM of eight dishes. *p<0.05 compared with the values treated only with HGF, and #p<0.01 compared with the values treated with HGF and nonsense oligonucleotide.</p