The Ras/Erk Pathway Induces Primitive Endoderm but Prevents Parietal Endoderm Differentiation of F9 Embryonal Carcinoma Cells

Abstract

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear.We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTHinduced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK.In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

MST4, a new Ste20-related kinase that mediates cell growth and transformation via modulating ERK pathway

“…Likewise, constitutively active ERK was shown to be su cient to elicit cell transformation (Greulich et al, 1996;Huang et al, 1999;Plattner et al, 1999;Verheijen et al, 1999). These ®ndings are in good agreement with our results that MEK1/ERK activation by MST4 is the underlying cause of cellular transformation induced by MST4.…”

The phosphoinositide-3-kinase/Akt pathway mediates the transient increase in Nanog expression during differentiation of F9 cells

“…The phosphorylation of GSK 3β, a direct downstream target of Akt, almost paralleled that of Akt. Phosphorylated ERK levels were evidently pronounced at the late stage of differentiation, which is consistent with previous findings that activation of Ras/MEK/ERK signaling promotes endodermal differentiation of ES and EC cells (Verheijen et al, 1999;Yoshida-Koide et al, 2004). In contrast, levels of phosphorylated p38 started to reduce following RA treatment, demonstrating that the kinetics of p38 phosphorylation is different from that of Nanog expression during F9 cell differentiation.…”

Retinoic Acid and the Development of the Endoderm

“…PTHrP bound to its G-protein coupled PTH/PTHrP receptor (GPCR) stimulates adenylyl cyclase to increase cAMP levels [192]. These and other studies have shown that while RA induced differentiation is accompanied by an increase in Ras/ERK activity leading to PrE, this activity must be attenuated by increased PKA signaling in order for cells to develop into PE [189,193,194]. Evidence from in vivo expression and localization studies and from work with ES cells support this model [195][196][197][198].…”

“…Nevertheless, the host of profiling studies mentioned above documenting the ability of RA to regulate the expression of a plethora of genes in F9 cells has been complemented by several detailed studies on specific genes including Rex-1 [183], laminin B1 [184], Mct8 [185], Disabled-2 [186,187] and PTH/PTHrP [188], to name a few. In the case of the latter, and as described above, RA exposure followed by subsequent treatment of PTH or PTHrP is sufficient to induce F9 cells to form PE [189][190][191]. PTHrP bound to its G-protein coupled PTH/PTHrP receptor (GPCR) stimulates adenylyl cyclase to increase cAMP levels [192].…”