12-O-tetradecanoyl-phorbol-13-acetate-dependent up-regulation of dopaminergic gene expression requires Ras and neurofibromin in human IMR-32 neuroblastoma.

The dopaminergic transcriptional programme 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 signalling. To investigate possible interactions between these two pathways before they converge on Raf activation, we evaluated whether phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA)-dependent PKC activation required Ras for regulation of TH expression in IMR-32 cells. We found that long-term treatment with TPA, which induces down-regulation of PKC-alpha, led to induction of both protein and message levels of TH by autocrine factors. This was dependent on endogenous Ras, but independent of the transcription factor Nurr1. Moreover, this mechanism of action mimicked the effects of overexpression of the Ras-GAP domain of neurofibromin, GAP-related domain (GRD) I, which is part of the upstream mechanism for regulation of Ras activation and a PKC-alpha substrate. Overexpression of Ras also led to transcriptional and translational up-regulation of TH, independent of Nurr1 induction, as well as distinct phenotypic changes consistent with cell hypertrophy and increased secretory activity shown by induction of expression of vesicular monoamine transporter 2 and synaptosomal-associated protein-25. Most interestingly, overexpression of GRDI and down-regulation of the endogenous GRDII neurofibromin led to significant increases in Nurr1 message, possibly reflecting a transcriptional hierarchy during development. Taken together, these studies suggest that PKC-alpha, neurofibromin and Ras are essential in regulation of TH gene expression in IMR-32 cells.