Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Abstract

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3-to 10-fold and three decrease 2-to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.Nerve growth factor (NGF) plays a major role in the growth, differentiation, and survival of sympathetic and sensory neurons (22,41). Recent studies have demonstrated the presence of NGF and its receptor in the central nervous system and have shown that certain neurons of the forebrain respond to NGF (35,51). One in vitro model system used to study the effects and mechanism of action of NGF is the rat PC12 pheochromocytoma cell line (27,28). In the presence of NGF, PC12 cells differentiate from replicating, chromaffinlike cells to nonreplicating, sympathetic-neuron-like cells. One of the more dramatic aspects of PC12 cell differentiation is the attainment of a neuronal phenotype by the extension of long, branching neurites. Within 1 week of NGF treatment, approximately 90% of the cells have neurites. NGF-treated PC12 cells also develop the ability to generate fast, Na+-dependent action potentials (13) and undergo alterations in the levels of a numDer of specific proteins, many of which change in an RNA synthesis-dependent manner (see, for example, reference 28 for review). Burstein and Greene (6) demonstrated that both RNA synthesis-dependent and -independent events are necessary for neurite o...

Intron 1 Is Required for Cell Type-Specific, But Not Injury-Responsive, Peripherin Gene Expression

“…Several potential regulatory sequences, including NGF-inducible and IL-6-binding elements, have been identified within the 5Ј flanking sequence of peripherin via in vitro studies (Thompson et al, 1992;Lecomte et al, 1998). NGF activator domains lie both distal and proximal to the TATA box (Thompson et al, 1992;Desmarais and Royal, 1996), and NGF is believed to be a primary activator of the peripherin gene (Leonard et al, 1987), but this has not been verified in vivo. Significantly, functional recovery after nerve regeneration is an NGF-independent process (Diamond et al, 1992).…”

K vitamins, PTP antagonism, and cell growth arrest

“…In NGF-treated PC12 cells, activation of ERK pathway leads to the induction of a class of immediate-early response genes (Segal and Greenberg, 1996). Many of these immediate-early response genes encode transcription factors, the products of which may contribute to the differentiation of NGF-treated PC-12 cells (Leonard et al, 1987). Among them, the c-fos proto-oncogene has been extensively studied.…”

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

“…Previous studies in our laboratory (Leonard et al 1987) utilizing differential screening of cDNA libraries from PC12 cells have identified several genes regulated by NGF. These studies suggest that NGF-induced differentiation is accompanied by the activation of various classes of genes whose onset of transcription occurs at different times following NGF stimulation.…”

“…This class includes the tyrosine hydroxylase (TH) gene (Leonard et al 1987). A third class includes the peripherin gene, which encodes a neuron-specific intermediate filament protein (Portier et al 1984;Leonard et al 1987Leonard et al , 1988Parysek et al 1988;Thompson and Ziff 1989). Peripherin is representative of a class of "late" genes whose transcription is induced -18 hr post-NGF, a time coinciding with the onset of neurite extension.…”

“…A second class of genes, the delayed early genes, is transiently activated within 1-2 hr of NGF stimulation and includes genes whose products may be important for early events during the transition from the chromaffin to the neuronal cell state. This class includes the tyrosine hydroxylase (TH) gene (Leonard et al 1987). A third class includes the peripherin gene, which encodes a neuron-specific intermediate filament protein (Portier et al 1984;Leonard et al 1987Leonard et al , 1988Parysek et al 1988;Thompson and Ziff 1989).…”