A role for both Ets and C/EBP transcription factors and mRNA stabilizationin the MAPK-dependent increase in p21(Cip1/WAF1/mda6) protein levels in primary hepatocytes

In primary hepatocytes and HepG2 hepatoma cells, prolonged activation of th e p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in DNA synthesis, mediated by increased expression of the cycl in-dependent kinase inhibitor protein p21 (Cip-1/WAF1/mda6) (p21) This stud y was performed to evaluate the contribution of transcriptional and post-tr anscriptional regulation in this response. prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and i ncrease, respectively, in DNA synthesis. Prolonged activation of the MAPK p athway in either wild-type or p21 antisense HepG2 cells also caused large d ecreases and increases, respectively, in DNA synthesis. MAPK signaling incr eased the phosphorylation of the transcription factors Ets2, C/EBP alpha an d C/EBP beta, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Fight hours a fter MAPK activation, loss of C/EBP beta or Ets2 function significantly red uced MAPK-stimulated transcription from the p21 promoter and abolished incr eased p21 protein expression. At this time, MAPK signaling increased both p 21 mRNA and p21 protein stabilities that were also demonstrated to be essen tial for a profound increase in p21 protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantl y reduced in cells without either C/EBP beta or Ets2 function; however, the se cells were now capable of exhibiting a partial increase in p21 protein e xpression, in contrast, loss of C/EBP alpha function modestly reduced MAPK- stimulated transcription from the p21 promoter but strongly inhibited the a bility of prolonged MAPK activation to increase protein levels of p21 This data suggested that prolonged enhancement of p21 protein levels may be unde r 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 promo ter activity via multiple transcription factors, which alone were insuffici ent for a robust prolonged increase in p21 protein levels in primary hepato cytes, and that to increase p21. protein levels also required enhanced stab ilization of p21 mRNA and p21 protein. Collectively, these data suggest tha t loss of transcription factor and mRNA/protein stabilization functions cor relates with an inability of MAPK signaling to cause growth arrest versus p roliferation in primary hepatocytes.