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

Abstract

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-a, 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-a 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-a, neurofibromin and Ras are essential in regulation of TH gene expression in IMR-32 cells.

Cell Type-Specific Upregulation of Parkin in Response to ER Stress

“…Anti-Parkin, anti-Glup, and anti-Hdj2 antibodies were described elsewhere (8,9,11). , anti-Hsp70 (K- 20), and anti-Hsc-70 (K- 19) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin (C4), anti-TCP1␣ (CTA-184), and anti-tyrosine hydroxylase (TH) (MAB318) antibodies were obtained from Roche Diagnostics (Mannheim, Germany), StressGen (San Diego, CA), and Chemicon (Billerica, MA), respectively.…”

Interplay Between Protein Kinase C Isoforms Alpha and Epsilon, Neurofibromin, and the Ras/MAPK Pathway in Neuroblastoma Differentiation

“…This phenotype is characterized by increased neuritic outgrowth and upregulation of neuron-specific genes, such as GAP-43, neuropeptide Y, and tyrosine hydroxylase (Pahlman et al 1981;Troller et al 2001;Olsson & Nanberg 2001), increases in noradrenaline content (Pahlman et al, 1984; www.intechopen.com Heikkila et al 1993), and development of membrane excitability (Jalonen & Akerman, 1988). Besides SH-SY5Y and the parental cell lines SK-N-SH and SK-N-SN, IMR32 cells also respond to TPA treatment with induction of neuronal genes that specify neurotransmitter phenotypes (Mangoura et al, 2006b). PKC activation may mediate significant crosstalk with the RARs, as TPA induces the upregulation of RAR expression in an nPKC/Ras/Rafdependent manner (Perez-Juste & Aranda, 1999).…”

“…PKC activation may mediate significant crosstalk with the RARs, as TPA induces the upregulation of RAR expression in an nPKC/Ras/Rafdependent manner (Perez-Juste & Aranda, 1999). Also in IMR32 cells, a synergistic transcriptional action of PKC and Ras with NF1 has been demonstrated in the induction of TH expression by TPA (Mangoura et al, 2006b). The prominent role of PKCs in TPA-induced SH-SY5Y differentiation has been established in many studies and the most important aspects have been reviewed by Larsson, Pahlman, and co-workers (Larsson, 2006;Edsjo et al, 2007), where they note that PKC is primarily responsible for TPA-induced differentiation, in particular for the induction of neuritic outgrowth.…”

“…Quantification suggests a 2-3 fold increase of neurofibromin's Ser2808 phosphorylation in differentiated cells ( lower panel). c) mRNA levels of PKC , PKC , and GAPDH were measured as described previously (Mangoura et al, 2006b). d) Abundance and phosphorylated forms of indicated proteins were assayed by Western blotting in total cell lysates; IP indicates that PKC was immunoprecipitated from cell lysates and asterisk a crossreactive protein in the immunoprecipitates.…”

“…Clinical trial data have also suggested that induced differentiation may be an alternative therapeutic approach, and retinoic acid analogues have been introduced in the clinical practice (Reynolds & Lie, 2000;Brodeur, 2003). The clinical observation of spontaneous differentiation into benign ganglioneuromas has provided the basis for studying neuronal differentiation of NB cells in culture (Reynolds & Lie, 2000;Edsjo et al, 2003;Hahn et al, 2008), and such studies have provided invaluable mechanistic insight into the fundamental mechanisms of neuronal differentiation and neurotransmitter phenotype acquisition (Mangoura et al, 2006b;Edsjo et al, 2007). The agents, mostly used to study NB cell differentiation in culture are phorbol esters, membrane permeable non-hydrolyzable cAMP analogues, and the clinically relevant vitamin A metabolite retinoic acid, alone or in combination with specific neurotrophic and growth factors (Table 1).…”