Nerve growth factor-mediated enzyme induction in primary cultures of bovine adrenal chromaffin cells: specificity and level of regulation

Abstract

Primary cultures of bovine adrenal chromaffin cells provide large quantities of a homogeneous population of target cells for nerve growth factor (NGF) and, thus, are a suitable system for studying the molecular mechanism of action of NGF. In this study, we have shown that NGF mediates the specific induction of the key enzymes in catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). Acetylcholinesterase (AChE), an enzyme which catalyzes the breakdown of acetylcholine, is also induced by NGF. We have compared NGF-mediated TH and AChE induction and have provided pharmacological evidence that TH induction involves a post-transcriptional, polyadenylation-dependent event (blockable by 9-beta-arabinofuranosyladenine but not by alpha- amanitin), whereas AChE induction requires transcription (blockable by alpha-amanitin). DBH and PNMT appear to be regulated via the same mechanism as TH. The time course of TH induction is such that NGF must be continuously present for at least the first 36 hr (during which time TH levels remain unchanged), and then the entire increase takes place during the subsequent 12 hr. In contrast, AChE induction proceeds linearly with time of NGF exposure. These data suggest that there may be multiple mechanisms by which NGF regulates enzyme induction. We have also compared the effects of cAMP with those of NGF. As compared to NGF, cAMP produces a different pattern of enzyme induction (in addition to TH, DBH, PNMT, and AChE, dopa decarboxylase (DDC) is also induced), it acts rapidly (a 12-hr exposure produces the full effect), and it acts only at the transcriptional level (its effects are blocked by alpha-amanitin). These data provide evidence that cAMP does not act as a second messenger for NGF with regard to enzyme induction.

Nerve growth factor regulates tyrosine hydroxylase gene transcription through a nucleoprotein complex that contains c-Fos.

“…Previous studies have demonstrated that TH activity is elevated following NGF treatment in various rat clonal pheochromocytoma cell lines (Goodman and Herschman 1978;Hatanaka 1981), primary adrenal chromaffin cell cultures (Acheson et al 1984), and rat superior cervical ganglia organ cultures (Rohrer et al 1978). Initial analysis of TH mRNA levels during NGF induction of PC12 cell differentiation revealed a slight increase (approximately twofold) in message levels by 4 hr of NGF treatment, which was followed by a decrease to initial basal levels in mRNA appearance (Leonard et al 1987).…”

“…Both in vivo and in vitro studies have demonstrated that TH enzyme activity can be modulated by various extracellular effector molecules that include stress (Thoenen 1970) agents that deplete catech0!amine stores (i.e., reserpine; Mueller et al 1969), glucocorticoids (Hanbauer et al 1975;Lucas and Thoenen 1977;Edgar and Thoenen 1978;Tank et al 1986), cAMP (Kumakara et al 1979;Acheson et al 1984;Tank et al 1986), insulin (Schubert et al 1980), epidermal growth factor (EGF; Goodman et al 1980), as well as NGF (Goodman and Herschman 1978;Rohrer et al 1978;Hatanaka 1981;Naujoks et al 1982;Acheson et al 1984). Several mechanisms have been proposed for regulating TH activity, including changes in the rate of enzyme synthesis (Hanbauer 1975;Goodman and Herschman 1978;Schubert et al 1980;Tank et al 1986), post-translational modifications of preexisting enzyme molecules (i.e., phosphorylation; Vulliet et al 1980;Campbell et al 1986), and other post-transcriptional events (Rohrer et al 1978;Acheson et al 1984). More recently, it has been demonstrated that the TH gene is transcriptionally regulated in response to EGF , glucocorticoids (Tank et al 1986;Harrington et al 1987;, and cAMP (Tank et al 1986;).…”

Multiple Protein Factors Interact with the cis-Regulatory Elements of the Proximal Promoter in a Cell-Specific Manner and Reg\ ulate Transcription of the Dopamine b-Hydroxylase Gene

“…Among several genes specifically expressed in noradrenergic neurons, dopamine ␤-hydroxylase (DBH; EC 1.14.17.1) is a hallmark protein, because noradrenaline is synthesized by this enzyme (Kirshner and Goodall, 1957;Friedman and Kaufman, 1965). Regulation of the DBH gene provides a challenging system for studying neuron-specific gene regulation in general, as well as cell type-specific gene expression in the brain, for the following reasons: (1) DBH is expressed restrictively in noradrenergic and adrenergic neurons and neurosecretary cells in the nervous system; (2) differential expression of DBH among catecholaminergic neurons underlies phenotypic subspecifications among catecholaminergic neurons; that is, whereas tyrosine hydroxylase (TH) is expressed both in dopaminergic and noradrenergic neurons, DBH is expressed only in noradrenergic neurons; and (3) expression of DBH is modulated in response to a variety of trans-synaptic signals, hormones, growth factors, and stress (Otten and Thoenen, 1976;Sabban et al, 1983;Acheson et al, 1984;Faucon Biguet et al, 1986;Lewis et al, 1987;Badoyannis et al, 1991;McMahon et al, 1992; K. T. Lamouroux et al, 1993;Wessel and Joh, 1993).…”

Nerve Growth Factor Regulates Adrenergic Expression

“…Although NGF and other neurotrophins are present in developing and mature adrenal chromaffin cells (Suter-Crazzolara et al, 1996), their ability to regulate adrenergic expression in these cells seems controversial. NGF has been shown to increase PNMT activity in bovine chromaffin cells (Acheson et al, 1984), but not rat chromaffin cells (Muller and Unsicker, 1986). It does not seem to induce PNMT expression in PC-12 cells derived from rat adrenal medullary pheochromocytomas (Unsworth et al, 1999).…”