PKC and Ras differentially regulate gene expression of the dopaminergic marker TH and noradrenergic marker DBH

Abstract

We have successfully established for the first time in the literature cell lines from human fetal cells derived from the amniotic fluid (AF) and have documented that these cells may progress and stably express many of the same genes which define progenitor dopaminergic neurons. AF cells (AFCs) may thus provide an excellent model for studying the development of dopaminergic neurons. The dopaminergic and noradrenergic transcriptional program is highly regulated during development and in the adult, in response to activation of membrane receptor signalling cascades. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, is known to be regulated by receptors that act through protein kinase C (PKC) or Ras signaling. Therefore, we pharmacologically or genetically manipulated each signalling molecule by downregulating PKC with long term (24 h) exposure of cells to the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), by overexpressing Ras with transfection, or by downregulating activated Ras by overxepressing the GRDI domain of the neurofibromin protein, a potent Ras GAP. We found that treatment with TPA increased transcription of both TH and Nurr1 (a transcriptional 'hub' for the acquisition of a dopaminergic phenotype) by over 80%, whereas GRDI blocked almost all TH expression. Moreover, while Ras overexpression had no effect on these two genes, it induced the de novo expression of the noradrenergic phenotype marker DBH. Expression of VMAT2 increased with all molecular manipulations, while expression of several neuronal markers, namely Tau, b-tubulin, and syntaxin, was not affected by any condition. Interestingly, treatment of AFCs with cytochalasin, which disrupts microfilaments, caused a 50% decrease in Nurr1 transcript compared to control, a 4-fold increase in VMAT2 message, and a 40% decrease in Ptx3 message. Taken together, these studies suggest that PKC and Ras have important yet differential roles in regulating gene expression of the dopaminergic marker TH and noradrenergic marker DBH during neuronal progression. ©ΦAPMAKON-TÚTTOς.