We report the results of an extensive kinetic analysis of the effects of ACTH, cAMP derivatives (dibutyryl cAMP and 8-bromo-cAMP) and phorbol ester (phorbol-12-myristate-13-acetate) on the expression of fos and jun gene family members at the mRNA (Northern hybridization) and protein levels (immunoprecipitation and indirect immunofluorescence) in the mouse Y-1 adrenocortical cell line. FOS and JUN proteins are induced by ACTH independently of cell cycle stage. c-Fos, fos-B, fra-1, fra-2, c-jun, and jun-B genes are induced by ACTH, the kinetic profiles for mRNAs and respective protein products being similar, except for a 1-h protein delay. Jun-D mRNA is an exception, being constitutively expressed. However, JUN D protein is induced by ACTH. phorbol-12-myristate-13-acetate closely mimics these inductive effects of ACTH. On the other hand, cAMP derivatives are not effective in inducing the fos and jun genes, except for fra-2 mRNA, JUN D protein, and to some extent JUN B protein. Clearly, ACTH is endowed with the versatile capability of modulating fos and jun gene expression, suggesting that AP-1 transcription factors play a role in ACTH mechanisms of action. ACTH receptors are likely to activate signaling routes other than the classical cAMP/protein kinase A in order to induce FOS and JUN proteins.
We previously reported that ACTH, but not dibutyryl cAMP, rapidly induces the c-fos proto-oncogene in Y-1 adrenocortical cells. Here we show that PMA induces c-fos with similar kinetics when compared with ACTH (0.5-1 h peak) but reaches only 60% of the maximal ACTH induction and dcAMP is a weak c-fos inducer (15% of ACTH). However, combination of PMA and dcAMP has a synergistic effect leading to maximal c-fos induction. c-fos expression may play a role in the RNA synthesis-dependent corticosteroidogenesis response and/or growth regulation by ACTH. We also show that, in contrast to dcAMP, PMA is a poor steroidogenesis stimulator (15 to 17% of maximum ACTH-stimulated level), its activity being completely dependent on RNA synthesis. Combination of dcAMP and PMA yields an additive steroidogenesis stimulation, an effect that is also dependent on RNA synthesis. Although no strict correlation was found between c-fos induction and early steroidogenesis stimulation, particularly with respect to cAMP derivatives, the results suggest that a PKC pathway is likely to cooperate with the classical cAMP-PKA pathway in adrenal cells' RNA-dependent steroidogenesis.
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