In primary hepatocytes and HepG2
hepatoma cells, prolonged activation of the p42/44
mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in
DNA synthesis, mediated by increased expression of the
cyclin-dependent kinase inhibitor
protein p21 (Cip-1/WAF1/mda6) (p21). This study was performed to evaluate the contribution of transcriptional and post-transcriptional regulation in this response. Prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and increase, respectively, in
DNA synthesis. Prolonged activation of the MAPK pathway in either wild-type or p21 antisense HepG2 cells also caused large decreases and increases, respectively, in
DNA synthesis. MAPK signaling increased the phosphorylation of the
transcription factors Ets2,
C/EBPalpha, and
C/EBPbeta, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Eight hours after MAPK activation, loss of
C/EBPbeta or Ets2 function significantly reduced MAPK-stimulated transcription from the p21 promoter and abolished increased p21
protein expression. At this time, MAPK signaling increased both p21
mRNA and p21 protein stabilities that were also demonstrated to be essential for a profound increase in p21
protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantly reduced in cells without either
C/EBPbeta or Ets2 function; however, these cells were now capable of exhibiting a partial increase in p21
protein expression. In contrast, loss of
C/EBPalpha function modestly reduced MAPK-stimulated transcription from the p21 promoter but strongly inhibited the ability of prolonged MAPK activation to increase
protein levels of p21. This data suggested that prolonged enhancement of p21
protein levels may be under posttranscriptional control. In agreement with this hypothesis, prolonged MAPK signaling further increased p21 mRNA stability at 36 h, compared with the 8-h time point. Our data argue that MAPK signaling increased p21 promoter activity via multiple
transcription factors, which alone were insufficient for a robust prolonged increase in p21
protein levels in primary hepatocytes, and that to increase p21
protein levels also required enhanced stabilization of p21
mRNA and p21
protein. Collectively, these data suggest that loss of
transcription factor and
mRNA/
protein stabilization functions correlates with an inability of MAPK signaling to cause growth arrest versus proliferation in primary hepatocytes.