Retinoic acid stimulates the protein kinase C pathway before activation of its β-nuclear receptor during human teratocarcinoma differentiation

We previously reported that protein kinase C (PKC) stimulation through phorbol ester (TPA) treatment enhances the effects of all-trans retinoic acid (RA) on immunophenotypic differentiation and RA nuclear receptor (RAR) activation in the multipotential human teratocarcinoma (TC) cell line NTera-2 clone D1 (abbreviated NT2 D1). This study extends prior work in NT2 D1 cells by demonstrating that PKC pathway activation is an early effect of RA treatment which regulates RAR transcriptional activity. RA activated the PKC pathway prior to induction of RAR-β expression at 6 h, which is an established early marker of RAR activation in NT2 D1 cells. RA caused a transient 1.3-fold increase in intracellular diacylglycerol (DG) at 2 min and a translocation of the gamma isozyme of PKC (PKC-γ) within 5 min. Transient co-transfection studies provided evidence that PKC pathway activation plays a role in the regulation of RAR-β expression. In these studies a constitutively active PKC-γ augmented the RA-mediated transactivation of a luciferase reporter containing the native RAR-β promoter which has a retinoic-acid-response element (RARE). These findings reveal that PKC pathway activation is an early step in RA-mediated human TC differentiation and that PKC-γ can potentiate the effects of RA on RAR transcriptional activation.