Disassociation of MAPK activation and c-Fos expression in F9 embryonic carcinoma cells following retinoic acid-induced endoderm differentiation

Retinoic, acid induces cell differentiation and suppresses cell growth in a wide spectrum of cell lines, and down-regulation of activator protein-1 ac tivity by retinoic acid contributes to these effects. In embryonic stem cel l-like F9 teratocarcinoma cells, which are widely used to study retinoic ac id actions on gene regulation and early embryonic differentiation, retinoic acid treatment for 4 days resulted in suppression of cell growth and diffe rentiation into primitive and then visceral endoderm-like cells, accompanie d by a suppression of serum-induced c-Fos expression. The ALAPK (ERK) pathw ay was involved in mitogenic signaling in F9 cells stimulated with serum. S urprisingly, although c-Fos expression was reduced, the MAPK activity was n ot decreased by retinoic acid treatment. We found that retinoic acid treatm ent inhibited the phosphorylation of Elk-1, a target of activated MAPK requ ired for c-Fos transcription. In F9 cells, the MAPK/MEK inhibitor PD98059 s uppressed Elk-1 phosphorylation and c-Fos expression, indicating that MAPK activity is required for Elk-1 phosphorylation/activation. Phosphoprotein p hosphatase 2B (calcineurin), the major phosphatase for activated Elk-1, is not the target in the disassociation of MAPK activation and c-Fos expressio n since its inhibition by cyclosporin A or activation by ionomycin had no s ignificant effects on serum-stimulated c-Fos expression and Elk-1 phosphory lation. Thus, we conclude that retinoic acid treatment to induce F9 cell di fferentiation uncouples Ras/MAPK activation from c-Fos expression by reduct ion of Elk-1 phosphorylation through a mechanism not involving the activati on of phosphoprotein phosphatase 2B.