PKCδ alternatively spliced isoforms modulate cellular apoptosis in retinoic acid-induced differentiation of human NT2 cells and mouse embryonic stem cells

NT2 cells are a human teratocarcinoma cell line that, upon treatment with retinoic acid (RA), begin differentiating into a neuronal phenotype. The transformation of undifferentiated NT2 cells into hNT neurons presents an opportunity to investigate the mechanisms involved in neurogenesis because a key component is cell apoptosis, which is essential for building neural networks. Protein kinase C8 (PKC8) plays an important role as a mediator of cellular apoptosis in response to various stimuli. PKC8 (81) is proteolytically cleaved at its hinge region (V3) by caspase 3 and the catalytic fragment is sufficient to induce apoptosis in various cell types. Mouse PKC delta 11 is rendered caspase resistant due to an insertion of 78 bp within the caspase recognition site in its V3 domain. No functional role has been attributed to these alternatively spliced variants of PKC delta. We sought to find a correlation between the onset of apoptosis, neurogenesis, and the expression of PKC delta isoforms. Our results indicate that RA regulates the expression of PKC8 alternative splicing variants in NT2 cells. Further, overexpression of PKC delta I promotes apoptosis while PKC delta II overexpression shields the cells from apoptosis. This is the first report to attribute physiological function to PKC delta I and -delta 11 isoforms. Next we demonstrated that mouse embryonic stem cells differentiate in vitro into dopaminergic neurons upon stimulation with RA and ciliary neurotrophic factor. These cells showed a simultaneous increase in tyrosine hydroxylase and PKC delta 11 expression. We suggest that the molecular mechanisms regulating differentiation and apoptosis could be understood by alternative expression of PKC delta isoforms.