NF1 mRNA ISOFORM EXPRESSION IN PC12 CELLS: MODULATION BY EXTRINSIC FACTORS

Linda J. Metheny and Gary R. Skuse

SUMMARY: The rat NF1 gene expresses several transcript isoforms which differ by the alternative splicing of exons 23a and 23b in the region encoding the GAP-related domain. The significance of this alternative splicing event is unclear and the factors which influence isoform expression are largely unknown. Here we show that a variety of factors can modulate the expression of these isoforms in PC12 cells. Nerve growth factor and dexamethasone lead to an increase in the type I isoform concurrent with a decrease in cellular proliferation. The upregulation by dexamethasone occurs in an RNA synthesis-dependent manner. Cycloheximide treatment leads to the detection of an additional species identified as the murine type III transcript. These results suggest that the NF1 alternative splicing event can respond to environmental cues. These changes in the type of NF1 transcript expressed may be important in the normal physiological regulation of neurofibromin andmodulate its role in cellular differentiation and development.

INTRODUCTION

The product of the neurofibromatosis type I (NF1) gene is a member of the class of negative growth regulatory proteins known as tumor suppressors. The NF1 encoded protein, neurofibromin, contains a region with homology to the GTPase activating protein (GAP) of mammals and to the inhibitor of RAS proteins (IRA1 and IRA2) of yeast. Ras is involved with theintracellular transduction of signals from receptors of growth and differentiation stimulatory factors. While bound to GDP, ras is inactive in its role as a signal transducer but is active when bound to GTP. Neurofibromin exerts a tumor suppressor function by facilitating the conversion of ras-GTP to ras-GDP thereby inhibiting ras-mediated signal transduction. In view of this it is likely that neurofibromin plays a role in the regulation of cell cycle progression in normal cells as well as in NF1-associated neoplasms.

Neurofibromatosis type I is a common genetic disease which predisposes to neoplasia as well as a number of other clinical features. The human NF1 gene is large, encompassing more than 300kb on chromosome 17, and is composed of at least 59 exons. Several alternatively-spliced variants have been identified. One of the alternatively-spliced exons, exon 23a, encodes 63 bases and lies within the GAP related domain (GRD). Exclusion of exon 23a results in a transcript termed type I while inclusion of 23a results in the NF1 type II transcript. Each of these transcript isoforms differs not only structurally but also in its respective GAP activity. An additional splice variant, termed type III, has previously been identified in murine species and includes an exon (denoted 23b) between 23a and 24; inclusion of this 41 base exon causes a frameshift which leads to a premature termination codon. This form is seen in various tissues but is highest in abundance in adrenal tissue. Type IV arises when exon 23b is included in the type I form. Expression of these isoforms appears to be tightly controlled in a tissue- and developmental stage-specific manner. This is particularly true in nervous system tissues, thus suggesting specific roles for these variants in development. Not surprisingly, homozygous transgenic mice created by targeted disruption of the NF1 gene (which
die in utero by 14.5 days gestation) demonstrate overproliferation of sympathetic ganglia and heterozygotes are predisposed to the formation of pheochromocytomas and other tumors at advanced age.

In order to investigate the role of the NF1 type I and II transcripts in the cellular response to extrinsic factors we measured the levels of each transcript after treatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) and other effectorswhich are known to alter gene expression in these cells. We observed changes in the ratio of type I to type II transcript. Here we report that both nerve growth factor and dexamethasone treatment result in upregulation of the type I transcript. That upregulation is dependent on de novo RNA synthesis and correlates with a decrease in cellular proliferation. Further, we have determined that the type III transcript is detected following cycloheximide treatment.