Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun.

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STIMULUS-TRANSCRIPTION COUPLING IN THE NERVOUS SYSTEM: INVOLVEMENT OF THE INDUCIBLE PROTO-ONCOGENES

fos ANDjun James

I.

l 2 Morgan and Tom Curran

2 Departments of INeuroscience and Molecular Oncology and Virology, Roche Institute of M olecular Biology, Roche Research Center, Nutley, New Jersey 07 1 10 KEY WORDS:

oncogenes, gene regulation, stimulus-response coupling, plasticity, fos andjun

INTRODUCTION One of the major challenges confronting modern biologists is to delineate the molecular mechanisms by which cells adapt their phenotype in response to environmental signals. Although much has been learned about the rapid and short-lived changes that occur following stimulation, relatively little is known about the processes that convert ephemeral second messenger mediated events into long-term cellular phenotypic alterations. This ques tion is of particular relevance to the neurobiologist, who must explain how brief stimulation of neurons can lead to changes in function that can persist for the lifetime of the animal. Although there are no simple answers, recently neurobiologists have been aided in their quest by concepts and reagents borrowed from the field of oncogene research. In particular, the proto-oncogene Jos has provided a novel avenue for research and a useful marker with which the effects of pharmacological, electrical, and physiologicaf stimuli may be traced in the nervous system. Here we summarize the origin and function ofJos and the set of inducible genes to 421 o 1 47-D06Xj 9 1j030 I-D421 $02.00

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MORGAN & CURRAN

which it belongs, and we review the literature concerning its advent in the neurosciences.

THE ORIGIN OF ONCOGENES Oncogenes were first described as the genetic information responsible for the induction of tumors by RNA viruses of the class

Retroviridae.

In 1989

the Nobel Prize was awarded to M ichael J. Bishop and Harold Varmus


for their discovery (Stehelin et al 1976) of the cellular origin of retroviral oncogenes (for a summary of this work see Curran 1 989). Thus, the transforming genes of retroviruses have been usurped from the cellular genome. During this process, they are often mutated and their expression is deregulated. Cellular transformation occurs when these genes are ex pressed inappropriately, outside of natural constraints (for a detailed re view of the origin, structure, and function of oncogenes see Reddy et aI 1 988). The term "oncogene" is now more all-encompassing; it includes several transforming genes isolated directly from human tumors as well as the viral genes. Furthermore, there are many derivative terms, such as "anti oncogene" or "recessive oncogene." The normal ccllular genes from which retroviral oncogenes were derived are most correctly referred to as "proto oncogenes," meaning that they represent the progenitors of oncogenes (Bishop 1 985). Over the last five to ten years, the study of oncogenes has revealed that as a class, proto-oncogenes function in several aspects of signal transduction processes. That is, they function in the transmission of information between and within cells. They may do so as extracellular polypeptide messengers, cell-surface receptors, protein kinases, G-proteins, and nuclear transcription factors. This generality is not necessarily surprising, as cancer can be viewed as a breakdown in cellular communication processes. The cancer cell either fails to respond, or responds inappropriately, to environ mental signals, and this failure results in uncontrolled cell growth. The surprising finding was that the same signaling molecules that mediate growth regulation are involved in many other processes, including cellular differentiation and neuronal physiology. When viewed simply as messenger molecules, however, it is clear that proto-oncogenes can be adapted to many purposes in a variety of cell types. The los and jun proto-oncogenes discussed here may be regarded as encoding general transcription factors (Fos and Jun, respectively) that are induced by environmental signals. In each case they function in coupling short-term signals received at the cell surface to longer-term alterations in cellular phenotype by regulating expression of selected target genes. The specificity of the cellular response lies within the host cell, i.e. different target genes may be selected for

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regulation by Fos and lun in mitogenically stimulated fibroblasts than in glutamate-treated neurons.

Induction of c-fos Expression The termfos was first used to describe the oncogene encoded by the Finkel Biskis-linkins murine osteogenic sarcoma virus (FBl-MSV) (Curran & Teich 1982a). Like many acutely oncogenic retroviruses, FBl-MSV was derived by recombination of retroviral sequences with cellular genetic infor


mation. The normal cellular sequences from which the viral oncogene (v fo s) was derived is referred to as the fos proto-oncogene or c-fos (Curran et al 1983). Although mutations in fos were introduced during the gen eration of v-fos (Van Beveren et aI 1983), these are not absolutely necessary for the transforming function. Deregulated expression of the normal c-fos protein also causes cellular transformation (Miller et al 1984, Lee et al 1988). At first it was difficult to reconcile this observation. How could a normal protein cause cancer? It is now clear, however, that in most cell types the level of Fos is highly regulated and cells are usually exposed to elevated levels of Fos expression for only a brief period (for review see Curran 1988). Thus, cellular transformation by Fos results from con tinuous or deregulated expression. The exact molecular details responsible for transformation are not yet known. It is presumed that there are target genes in the transformed cell that are regulated by Fos that lead to a form of autocrine stimulation of cell growth. In the majority of cell types, the basal level of c-fos mRNA and protein expression are relatively low, although there are some exceptional cir cumstances in which cells maintain relatively high levels of expression (for reviews see Curran 1988, Cohen & Curran 1989). In thcsc ccll types, extracellular signals are required constantly to maintain elevated levels of expression. One of the features of c-fos that suggested it might function in signaling processes was the discovery that its expression could be induced transiently to very high levels by serum and polypeptide growth factors (Greenberg & Ziff 1984, Kruijer et al 1985, Muller et al 1984). Although several investigators interpreted these data to suggest that c-fos was exclus ively involved in regulation of the cell cycle, it was already clear from early on that induction was not necessarily linked to cell division (Bravo et al 1985) but was rather an immediate consequence of receptor stimulation (for review see Curran et al 1985). It is now established that many types of stimuli, some associated with the process of differentiation and some linked to neuronal excitation, elicit a very similar transient induction of c-fos mRNA and protein (see below). The time-course of the induction process is identical in most circumstances. Transcriptional activation occurs within 5 minutes and continues for 15-20 minutes (Greenberg &

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MORGAN & CURRAN

Ziff 1 984, Greenberg et al 1 985). mRNA accumulates and reaches peak values at 30-45 minutes post-stimulation (Muller et al 1 984); thereafter it declines with a relatively short half-life of

�

1 2 minutes. Synthesis of

c:fos protein follows mRNA expression and it is turned over with a half-life of about

2 hours (Muller et al 1 984, Curran et al 1 984). The induction

of c-fos transcription occurs in the presence of protein synthesis inhibitors, thus suggesting that the proteins required for expression are present in unstimulated cells and that they are activated by post-translational modi


fication. This feature led to the classification of fos and other rapidly induced genes as cellular immediate-early genes by analogy to the im mediate-early genes of viruses (Lau & Nathans 1 987, Curran & Morgan 1 987). It was suggested from studies on the effects of protein synthesis inhibitors that

c-fos transcription

was repressed by a labile factor whose

expression was restricted by cycloheximide (Greenberg et aI 1 986a). Recent data indicate that Fos itself, as well as several Fos-related proteins, may be involved in autorepression (see below). Several regulatory elements located in the 5' untranslated region of

c-fos have been

demonstrated to play a role in controlling the induction of

expression. The first regulatory element to be defined operationally was the serum response element (SRE) (Treisman 1 985, Gilman et al 1 986, Prywes & Roeder 1 987). This element has also been referred to as. the dyad symmetry element (DSE); however, because of precedence (Treisman 1985) and because the element is not completely symmetrical, we use the term SRE here. A more detailed review of the SRE and associated proteins is presented by Treisman ( 1 990). The nucleotide sequence defined as the SRE is located at - 3 10; it functions as an inducible enhancer element and binds a 67 kDa nuclear protein termed the serum response factor (SRF) (Treisman 1 986, 1 987, Gilman et al 1 986, Prywes & Roeder 1 987, Green berg et al 1 987). SRF is expressed in most cell types, and .it has been purified by affinity chromatography with SRE oligonucleotides (Treisman 1 987, Ryan et a1 1 989, Prywes & Roeder 1 987, Schroter et al 1 987). It is a 508-amino-acid nuclear phosphoprotein that has a notable lack of generic sequence motifs (Norman et al 1 988). The core sequence of the S RE is of the form CCAjT6GG, which is very similar to the regulatory element present in the cardiac actin gene known as the CArG box (Minty & Kedes 1 986). This core sequence is present in several different immediate-early genes, in which it functions as a serum-inducible regulatory element. Despite its name, the S RE does not only function in mediating trans criptional responses to serum stimulation. As well as contributing to basal transcription levels in unstimulated cells (Mohun et al 1 987, Konig et al 1 989), it functions in the mediation of responses to many different types of cell stimuli. It responds to protein kinase C-dependent and -independent

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signals but not to protein kinase A signals (Gilman 1 988). It may be involved in the cellular responses to insulin (Stumpo & Blackshear 1 986), epidermal growth factor (Fisch et al 1 987), and nerve growth factor (Vis vader et al 1988). The mechanism of action of SRF is not known; indeed two other proteins have also been shown to interact directly or indirectly with the SRE (Shaw et a1 1 989, Ryan et aI 1 989). The roles of each of these proteins in mediating responses to specific cell-surface stimuli are not clear . Although several second messenger signals activate c-fos transcription


via the SRE, another regulatory element at - 60 also functions as a basal and inducible regulatory element (Gilman et al 1 986, Fisch et al 1 987, Sheng et a1 1 988, Berkowitz et al 1 989). This site is similar to the cAMP responsive elements (CRE) that are present in several genes (Comb et al 1 986, Montminy et al 1 986). Although it can function as a CRE in c-fos (Berkowitz et al 1 989), this element also acts as a calcium responsive element (CaRE) in mediating inducibility of c-fos by depolarizing stimuli in pel2 cells (Sheng et al 1 990). Expression of c-fos in PC1 2 cells can be induced by voltage-gated calcium influxes (Morgan & Curran 1 986), neurotransmitters (Greenberg et al 1 986b), and barium ions (Curran & Morgan 1 986), all of which appear to act through this CRE/CaRE sequence . Stimulation by these agents correlates with a rapid phos phorylation of a cAMP-responsive element binding protein (CREB), although there is little effect on cAMP levels (Sheng et al 1 990). Thus, there is a complex interaction among several second messenger signals and the 5' region of c-fos. The regulatory elements probably do not act in isolation but are more likely to synergize in a combinatorial fashion in the mediation of nuclear responses to environmental signals. Although most of the studies on c-fos regulation have concentrated on alterations in the rate of initiation, some evidence suggests that there is also regulation of transcriptional elongation (Fort et al 1 987). This would provide an alternative method of altering levels of c-fos expression. The molecular basis of this regulation and its relative contribution to the control of c-fos expression are not yet known.

The

c-fos

Protein

The other major feature of c-fos that suggested it might fulfill a regulatory role in the cell is that it encodes a nuclear protein (Curran et aI 1 984). This observation was the first implication that Fos might be involved in the regulation of gene expression. This impression was reinforced by the dem onstration that Fos was associated with chromatin and could bind to DNA cellulose in vitro (Sambucetti & Curran 1 986, Renz et al 1 987). Furthermore, co-transfection studies indicated that Fos can function as an activator of gene expression (Setoyama et al 1 986).

426 The

MORGAN & CURRAN

c-los gene encodes a 62 kDa nuclear protein that undergoes exten

sive post-translational modification (Curran et aI 1984). Most of the modi fication corresponds to serine and threonine phosphorylation (Curran et al 1 984, Barber & Verma 1 987). At least in vitro the cAMP-dependent kinase can modify Fos (Curran et al 1 987). Fos participates in a nuclear protein complex with a cellular protein previously termed p39 (Curran et al 1 985). As discussed below, this protein has now been identified as Jun, the product of thejun proto-oncogene (Rauscher et aI 1 988a). This protein


was routinely detected in immunoprecipitates of both

v-fos and c-fos from

non-denatured cellular extracts (Curran & Teich 1 982b, Curran et a1 1984, 1 985). Indeed, immunoprecipitates from serum-stimulated cells with anti Fos antibodies are rather complex when analyzed on high-resolution, two dimensional gels . In addition to Fos and p39, antibodies directed against

c-fos amino acids 1 27-1 52 precipitate a set of Fos-related antigens (Fra) from stimulated cells as well as several other Fos-associated proteins (Fap) (Franza et al 1 987). These proteins are nuclear, and they are induced by many of the agents and conditions previously shown to increase Fos expression (Miiller et a1 1 984b, Franza et a1 1 987, Cohen & Curran 1 988).

Several Fos-related genes have now been cloned and sequenced (Cohen &

Curran 1 988, Cohen et a1 1 989, Zerial et a1 1 989, Matsui et al 1 990, Nishina et al 1 990). They share several regions of homology with Fos, including the DNA-binding domain and leucine zipper region (see below). Like

c-los, c-jun is a member of a multigene family that includes jun-B (Ryder et al 1 988) and jun-D (Ryder et al 1 989). Furthermore, all members of the

jun family form complexes with the protein encoded by the fos family (Nakabeppu et al 1 988, Cohen et al 1 989, Zerial et aI 1 989). The demonstration that Fos functions directly as a transcriptional regu lator in cooperation with Jun through so-called activator-protein- l (AP-l) (Lee et al 1 988) regulatory elements was the cumulation of several independent studies that led to the conclusion that Fos and Jun interact as a heterodimeric protein complex with the AP- l binding site (for a more detailed review of these investigations see Curran & Franza 1 988). The initial observation was a report of a sequence similarity between the pre dicted amino acid sequence of the v-jun oncogene and the DNA-binding domain of t he yeast transcription factor GCN4 (Vogt et al 1 987). The namejun was given to the oncogene carried by avian sarcoma virus ASV 1 7 because ju-nana is the Japanese translation o f 1 7 (Maki e t a l 1 987). The

v-jun product is expressed as a fusion protein containing an N-terminal portion of the retroviral gag gene (Maki et al 1 987). Indeed, this region of Jun was found to be functionally interchangeable with the DNA-binding domain of GCN4 (Struhl 1 988). The core DNA sequence (TGACTCA) recognized by GCN4 is closely related to the mammalian regulatory

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427

element known as the AP- l binding site (Hill et al 1986, Lee et al 1 987). The AP- l site was first identified in the enhancer regions of SV40 and the human metallothionein IIA (hMTIIA) gene and has subsequently been identified in the control regions of several viral and cellular genes that are stimulated by phorbol esters (Curran & Franza 1 988). These findings prompted an examination of the relationship between J un and AP- l . This led to the identification of Jun among the proteins associated with AP-l DNA-binding activity (Bohmann et aI 1 9 8 7). The connection between Fos


and transcription factor AP- I was made by a completely separate route. The inhibition of a gel-shift activity by anti-Fos antibodies suggested that Fos, or a Fos-related protein, contributed to the DNA-binding activity associated with the control region (FSE2) of a gene,

ap2,

expressed in

adipocytes (Distel et al 1 987). The recognition site for the Fos immuno reactive DNA-binding activity was closely related to the AP-l consensus sequence (Rauscher et al 1 988b, Franza et al 1 9 8 8) . Indeed, affinity puri fication analysis revealed the presence of several Fos- and Jun-related proteins (and some unrelated proteins) in preparations of AP- l DNA binding activity (Franza et al 1 988). Thus, Fos, Jun, and several related proteins were shown to interact with a similar nucleotide sequence motif, the AP- l binding site. This complexity was resolved to some extent by the identification of p39, the Fos-associated protein, as Jun (Rauscher et al 1 988a). Much has been learned since these reports, and it is now clear that

fos andjun are members of inducible gene families whose protein products form an array of homodimeric and heterodimeric complexes that function as transcriptional regulators by interacting with DNA sequences related to the AP- l and CRE motifs.

Fos and Jun Are Cooperative Transcription Factors Analysis of the DNA-binding and dimerization functions of Fos and Jun were simplified by the proposal of the leucine zipper structure (Landschulz et al 1988). The leucine zipper, first identified in the C/EBP transcription factor, was suggested to mediate dimerization by an interaction of leucine side chains, spaced seven amino acids apart, on adj acent

fJ.

helices. Muta

genesis studies pointed to a central role of the leucine zipper in the formation of Fos-Jun heterodimers (Bos et a1 1989, Gentz et al 1 989, Kouzarides & Ziff 1 988, Ransone et al 1 989, Schuermann et al 1 989, Turner & Tjian 1 989). Studies on synthetic zipper peptides (O'Shea et aI 1 989) and com parison of the complementary effects of mutations in Fos and lun (Gentz et al 1 989) revealed that the zipper most likely represents a parallel associ ation of a helices somewhat like the coiled-coil structure. This parallel association results in j uxtaposition of regions of each protein rich in basic amino acids, both of which contact DNA (Risse et al 1 9 89, Abate et al

428

MORGAN & CURRAN

1 990a). Dimerization is necessary for DNA-binding activity, and a basic region from each protein is required. There is a high degree of specificity among zipper-containing proteins . Although lun and lun-related proteins form both heterodimeric and homodimeric complexes, Fos and Fos-related proteins only form stable heterodimers with members of the lun family (Cohen et al 1 989, Naka beppu et aI 1988). The presence of Fos in a Fos-lun heterodimer increases the affinity of the complex for DNA by increasing the stability of the


protein-DNA interaction (Rauscher et al 1 9 88c). The specificities of these many protein heterodimers for AP- l sites is similar, though not necessarily identical. lun is also capable of interacting with one of the CRE-binding proteins that has a leucine zipper. This protein was identified by direct screening of a Agtil library with biotinylated lun (Macgregor et aI 1 990); however, it had previously been cloned by oligonucleotide-affinity screen ing and described as the CRE-binding protein, CRE-BP I (Maekawa et al 1 989). This protein has a leucine zipper and forms heterodimers with lun that bind with high affinity to CRE but not to AP- l sites (Macgregor et al 1 990), although it shares only a limited sequence similarity with c-jun. Thus, Jun may be regarded as a protein with relatively broad DNA binding specificity that in the presence of Fos or Fos-related proteins has a greater specificity for AP- l sites (TGACTCA) and in the presence of CRE-BP I has greater specificity for CRE sites (TGAGCTCA). These findings question the rather strict definitions of regulatory elements as AP-I or CRE sites and suggest that there is a complex cross talk among signal transduction pathways within the nucleus. Co-transfection experiments indicate that c-fos and c-jun act syner gistically in the transcriptional activation of genes containing AP-l binding sites, although the results of these experiments are somewhat variable (Chui et al 1988, Sassone-Corsi et al 1988a, Sonnenberg et al 1 989c). It is possible that additional, as yet unidentified, components contribute to transcriptional activation. Indeed, the DNA-binding activities of Fos and lun are stimulated dramatically by a cellular factor in vitro (Abate et al 1990a,b). Distinct activation domains have been identified in each protein, an acidic region in the case of Fos and an N-terminal region of lun, that stimulate transcription cooperatively in vitro (Abate et al I 990c). The role of Fos- and lun-related proteins in gene regulation is less well charac terized, although lunB has been reported to inhibit the function of lun (Schutte et al 1 989). In addition to its role in gene activation, Fos has also been shown to act as a negative regulator of its own expression. This repressor function may be required for the rapid decline in immediate-early gene expression that follows induction as the shut-off process requires ongoing protein

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429

synthesis. Thus, it was reasonable to suppose that one or more of the protein products of cellular immediate-early genes were involved. Indeed, at least in some circumstances, there is a refractory period for reinduction of c-fos expression lasting for several hours after the primary stimulus that is consistent with the period of expression of cellular immediate-early pro teins (Morgan et aI 1 987). The results of several experiments have suggested that Fos itself is a negative regulator of its own promoter (Wilson & Treisman 1988, Sassone-Corsi et al 1988b, Konig et al 1989). There has


been some confusion in the literature, however, as to whether this effect is mediated by the SRE (Konig et al 1 989, Subramanian et al 1 989) or a sequence that is related to the AP- l -binding site located next to the SRE (Sassone-Corsi 1 9 88b, Wilson & Treisman 1 988). Recent experiments de monstrate conclusively that the SRE in c-fos and several other immediate early genes is the target for Fos-mediated repression (Lucibello et a1 1989, Guis et al 1990). Controversy remains, however, over whether Jun con tributes to down regulation and whether the leucine zipper and basic regions of Fos play a role (Konig et al 1 989, Lucibello et al 1 989, Guis et al 1 990). Mutagenesis analysis has suggested that repression by Fos is mediated by an indirect effect of its C-terminus on the SRE (Guis et al 1990). This C-terminal region is conserved in the chicken (Fujiwara et al 1987, M olders et al 1 9 8 7) and

Xenopus c-fos

genes (Mohun et al 1989,

Kindy & Verma 1 990), and it is present in several Fos-related genes (Cohen

& Curran 1988, Zerial et al 1 989, Nishina et al 1 990, M atsui et al 1 990). Indeed, at least one of the Fos-related genes, Fra- l , can also cause repression of S RE-mediated transcription (Guis et al 1990). This would explain why the refractory period for reinduction of c-fos expression lasts for several hours after Fos has disappeared (Morgan et aI 1987), as several Fos-related proteins are expressed throughout this period (Sonnenberg et al 1988a,b). Thus, repression of immediate-early gene transcription may result from complex interactions among several immediate-early proteins and the SRE. The exact mechanism responsible for shut-off is unclear, but it could involve the interaction of Fos-related proteins with modifying enzymes, such as protein kinases and phosphatases, that in turn regulate the function of SRF. The cellular immediate-early response is an integrated process involving direct and indirect communication among several inducible transcription factors. Once triggered, a series of induction events takes place that gives rise to elevated levels of AP- l DNA binding activity that persist in stimu lated cells for at least 8 hours (Sonnenberg et al 1989a). These events are rather stereotypic; overlapping inductions of Fos-related proteins occur in a variety of situations. Although the exact role of these inducible tran scription factors is not known, it is likely that their function is tailored

430

MORGAN & CURRAN

to the specific requirements of distinct differentiated cell types. However, although the gene targets may be different and the long-term adaptive process to which they are coupled may be unrelated, the underlying molec ular events are likely to be the same in depolarized neurons as they are in serum-stimulated fibroblasts. The next sections deal with some specific situations associated with

the cellular immediate-early response in

neuronal cells. In reading these sections, the protein-protein and protein DNA interactions that govern the function of thefos andjun gene families


should be kept in mind.

Regulation of Immediate-Early Response in Cells Derived from the Nervous Sys tem EXPRESSION OF c-fos AND c-jun

PC 1 2 pheochromocytoma cells represent

a particularly useful model for investigating the biochemical and molecular genetic events that accompany neuronal differentiation (see Greene & Tischler 1982). One attractive aspect of this cell line is that when treated with nerve growth factor (NGF), it elaborates an extensive neuritic out growth and assumes the phenotype of a sympathetic neuron (Greene

&

Tischler

1 982).

The

process

of NGF-induced differentiation is

accompanied by alterations in gene expression, thereby raising the question of how the extracellular growth factor is coupled to the intracellular transcriptional machinery. This consideration led several groups to inves tigate the effects of NGF on cellular immediate-early gene expression by analogy to the effects of mitogenic polypeptide growth factors on fibro blasts. Indeed, NGF elicited a rapid and transient induction of c�fos in PC 1 2 cells (Curran & M organ 1 985, Greenberg et al 1 985, Kruijer et al 1985, Kujubu et a1 1 987, Milbrandt 1986). From the historical perspective it was these studies that paved the way for future investigations of the expression of c-fos and other cellular immediate-early genes in the nervous system. In addition to NGF, a diverse array of agents have been shown to induce c-fos expression in PC 1 2 cells, including FGF (Greenberg et al 1 9 8 5), EGF (Greenberg et al 1 985), IL-6 (Satoh et al 1988), nicotinic agonists (Greenberg et al 1 986b), depolarizing stimuli (Greenberg et al 1985, Morgan & Curran 1986, Kruijer et al 1 9 8 5), barium (Curran & Morgan 1 986), and cyclic AMP analogues (Greenberg et al 1 985, Kruijer et al 1 985). Although most of the initial studies followed the expression of c-fos alone in PC 12 cells, immunoprecipitation analyses revealed that the levels of p39 also increased following administration of NGF (Curran & Morgan 1 985, 1 986). With the recognition that p39 was the product of c-jun, subsequent studies then established that c-jun transcripts also exhibited a transient rise following NGF treatment (Wu et al 1 989). Thus, both c-jun

fOS

AND

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43 1

IN NEURONAL CELLS

and c-fos become transcriptionally activated shortly after application of NGF to PCI2 cells. In the related human cell line, SYSY, platelet-acti vating factor elicits a transient transcriptional activation of both c-fos and

c�iun (Squinto et al 1 990). OTHER IMMEDIATE-EARLY GENES

These results led others to determine

whether additional immediate-early genes are induced by NGF in P C 1 2 cells. Milbrandt and co-workers have investigated two rapidly inducible


genes in PC 1 2 cells that have been termed NGFI-A and NGFI-B (Mil brandt 1 987, 1 988). Both of these genes encode nuclear proteins that, like Fos and Jun, have the properties of transcriptional regulatory molecules. NGFI-A encodes a zinc-finger protein that is related to the

kruppel gene

of Drosophila (Milbrandt 1 9 8 7 , Changelian et aI 1 9 89). The identical gene has been described independently as zif268 (Christy et al 1988),

e

g r- 1

(Sukhatme et aI 1 988), Krox-24 (Lemaire e t al 1 988), and TIS-8 (Kujubu et al 1 987, Lim et al 1 987). In these studies, the gene was also found to be inducible by phorbol esters and several growth factors, including NGF. It might also be noted that another inducible gene related to egr- 1 , termed

egr-2 (Josephs et al 1 988) or Krox-20 (Chavrier et al 1 988), has been observed in the developing mammalian hind brain. A second NGF inducible gene, NGFI-B, encodes a protein belonging to the steroid recep tor superfamily (Milbrandt 1 988, Watson & Milbrandt 1 989). Like NGFI A, this gene has been independently identified and given the alternative designations nur77 (Hazel et al 1988), N 1 0 (Ryseck et al 1989), and TIS1 (Kujubu et al 1 987, Lim et aI 1 987). Other genes that are rapidly induced by NGF encode proteins that may not be involved in transcription regu lation. For example, the gene PC4 (Tirone & Shooter 1 989) appears to be a cytokine related to one of the interferons. Again this gene has been independently identified as TIS7 (Lim et al 1 987). Unfortunately, to date no serious attempt has been made to unify the nomenclature of these many genes. Other immediate-early genes have been identified (Almendral et a1 1 988, Cochran et al 1 98 3 , Lau & Nathans 1985, 1 987, Lim et al 1 987, Varnum et aI 1 989), and several of these are known to be induced by NGF in PC l 2 cells (Kujubu et al 1987). These are not discussed here, however, since their structure and function is either unknown or not yet shown to be relevant to studies of the nervous system.

L

CHARACTERISTICS OF THE IMMEDIATE-EAR Y RESPONSE

The discovery of

multiple inducible genes has raised a number of questions that have attracted some attention in recent years. Several general points have arisen. First, is the repertoire of immediate-early genes that can bc induced limited by cellular phenotype? Second, for a given cell type, do all stimulants

432

MORGAN & CURRAN

produce a stereo typic induction of the same gene set? Third, what second messenger systems mediate the induction of the cascade? Fourth, are the regulatory sequences present in the various immediate-early genes conserved? Fifth, are the target genes for proteins such as Fos and Jun the same in all cell types? It would be premature to draw too many conclusions from the available data regarding the phenotypic limitations on the expression of cellular immediate-early genes; however, the majority of immediate-early genes


exhibit a rather stereo typic induction in most cell types. For example, when the stimulus-transcription coupling cascade is activated either in cultured cells or in vivo,

c-fos, NGFI-A/egr- l , and junB seem to be

invariably induced (Curran 1 988; R. Molinar-Rode, T. Curran and J. I. Morgan, unpublished observations). This would suggest that the products of these genes play a role in stimulus-response coupling that is common to most cell types. In contrast, the immediate-early genes,jra-l (Cohen & Curran 1 988) and TIS 10 (Arenander et aI 1 989b), exhibit a phenotypically restricted pattern of expression. Thus, TIS I O is induced in astrocytes and fibroblasts but not in PC 12 cells; whereas fra- l is induced in fibroblasts and PC 1 2 cells but not in the CNS following seizure. As further genes are isolated and characterized and their expression studied in different cell types, more will be learned concerning the phenotypic restriction of im mediate-early gene induction. A related aspect of this question that has attracted experimental atten tion is whether the pattern of immediate-early gene expression is dependent upon the nature of the stimulus. The most exhaustive study to address this question in PC l 2 cells (Bartel et al 1 989) found that NGF and EGF were potent inducers of c-fos,

c-jun, and egr- l , whereas depolarization induced

c-fos but had only a weak effect upon the expression of c-jun and egr- l . In contrast, depolarization was a much more potent inducer of NGF I -B than either of the two growth factors. In cultured astrocytes, NGFI-B (TIS 1 ) also shows such differential regulation, since it i s induced by phorbol ester but not EGF or FGF (Arenander et al 1989c). In contrast, TIS8 (NGFI Ajegr- l) and c-fos are strongly induced by all three agents (Arenander et aI 1989c). The secondary rat astrocyte cultures studied in this case possess acetylcholine receptors that are coupled to phosphatidylinositol turnover. Activation of these receptors with carbachol leads to a maximal induction of TIS 1 (NGF I - B), TIS7, and TIS8 (NGFI-A/egr-l) but only a relatively weak induction of TIS I I and c-fos (Arenander et aI 1 9 89a). The latter two genes are maximally stimulated only when lithium is present in the medium. Selective agonists of various adrenergic receptors also exhibit specificity to the types of immediate-early genes that are induced in these astrocytes

los

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jun

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(Arenandcr et al 1989a). Pure f1-adrenergic agonists induce all of the immediate-early genes investigated (except TIS7 which is not induced) with kinetics indistinguishable from EGF and phorbol ester. a-Adrenergic agonists induce all the immediate-early genes studied, including TIS7, but with more transient kinetics than the beta-agonists. Treatment with mixed a- and j3-agonists induced all of the genes investigated and greatly pro tracted the time over which transcripts were detected in the astrocytes. Therefore, it appears that the efficiency of coupling of second messenger


systems to the responsive elements in the various immediate-early genes is not equal. It is the coupling efficiency that may then underlie the differential effects of the various inducers on c-fos and related inducible genes and account for phenotypic differences in the sensitivity of particular imme diate-early genes to induction via certain pathways. It could, for example, account for the fact that phorbol esters are weak inducers of c-fos in rat PC I 2 cells whereas they are potent inducers in the related human line, SY5Y (Morgan & Curran 1 989a). SECOND

MESSENGER

INDUCTION

PATHWAYS

MEDIATING

IMMEDIATE-EARLY

GENE

Several studies have attempted to probe directly the second

messenger pathways involved in

c-Ios

activation . In the PC 1 2 cell, at

least three distinct second-messenger systems can activate c-fos, namely those involving diacylglycerol-protein kinase C (Greenberg et al 1 986b), cyclic AMP (Greenberg et al 1 9 8 5), and calcium-calmodulin (Morgan & Curran 1 986). In addition, other intracellular signaling pathways are coupled to c-fos expression, since ligands that do not exclusively utilize the classical second messengers (notably NGF, EGF, FGF, and IL-6) can induce los expression (Greenberg et al 1 985, Morgan & Curran 1 989a, Curran & Morgan 1985, Kruijer et a1 1 985, Satoh et al 1 988). In cultured glia, multiple second messenger pathways also appear to converge on c los induction (Arenander et al 1 989a, Condorelli et al 1989, Mochetti et al 1 9 89). In PC12 cells, a critical intracellular determinant of c-Ios induction is the free calcium concentration . As noted above, depolarization of P C 1 2 cells b y elevation of the extracellular potassium concentration results i n a n induction of c-Ios (Morgan & Curran 1 986, Greenberg e t al 1 986b). This same result obtains when the cells are depolarized with veratridine, an alkaloid that holds sodium channels in their open state (Morgan & Curran 1 986). Unlike NGF, both veratridine and elevated potassium require calcium to be present in the medium for an induction of los to occur (Morgan & Curran 1986), thus suggesting that the voltage-depen dent gating of calcium ions elicits the induction of this gene. Two experi-

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MORGAN & CURRAN

ments have demonstrated this point. First, antagonists of the L-type vol tage-dependent calcium channel (such as the dihydropyridine, nisoldipine) block the induction of fos by depolarizing stimuli (Morgan & Curran 1986). Second, agonists of this channel (such as the dihydropyridine, BAY K8644) directly activate c-fos in a calcium-dependent manner (Morgan & Curran 1986). In addition, we noted that all calmodulin antagonists block fos induction by depolarizing agents (Morgan & Curran 1986). The foregoing data suggested that the elevated calcium stimulates fos


transcription by activating processes that involved calmodulin. The molec ular details of this portion of the signal transduction cascade are unclear. Though it would seem reasonable to propose that calmodulin would act via a calmodulin-dependent protein kinase, this has been difficult to establish experimentally. More is known, however, of the regulatory elements in c-fos that are responsive to the calcium-dependent inducers in PCl2 cells. Furthermore, an analysis of the convergence of second messenger path ways upon these elements points toward the biochemical steps that link calcium to the induction of c-fos. As introduced above, Sheng et al ( 1988) have established that the CaRE is required for inducibility by calcium dependent stimuli but not agents such as EGF, FGF, NGF, or phorbol esters; the latter inducers require the SRE. The CaRE is coincident with sequences necessary for c-fos induction by cyclic AMP, however. There fore, it has been suggested that calcium acts via calmodulin and calmodulin kinase to induce c-fos transcription via the CaRE. Since proteins that bind to CREs have been characterized, the link between cyclic AMP and alterations in transcription rates are better understood. It still cannot be discounted, however, that proteins associated with the CaRE are substrates for both the cyclic AMP and calmodulin pathways. A similar multiplicity of second messenger signaling to the c-fos promoter has been observed in other cell lines. For example in SY5Y, platelet-activating factor, a lipid mediator generated in the nervous system, induces fos via sequences that encompass the CaRE (Squinto et al 1990). As well as the discrimination between signaling pathways converging upon the transcriptional activation of c-fos, evidence has been provided for differential post-translational modification ofFos in PC 12 cells (Curran

& Morgan 1986). Fos undergoes less post-translational processing in PC 1 2 cells stimulated with calcium-dependent inducers than with calcium independent ones such as NGF (Curran & Morgan 1986). This deficit in modification can be rescued within 5 minutes by addition of NGF to PC 12 cells previously stimulated with calcium-dependent inducers such as barium or elevated potassium (F. J. Rauscher III, T. Curran, and J. I. Morgan, unpublished observations). Since the majority of Fos was localized in the nucleus during these experiments, this further suggests that

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extracellular inducers must be coupled to intracellular modifying enzymes that are in the nucleus.

The Expression of Immediate-Early Genes in the Nervous System For the studies of immediate-early gene expression in cultured cells and cell lines to be meaningful, it is necessary to demonstrate that these genes are regulated in vivo. A large number of studies have now established


that

c-fos, and several other immediate-early genes,

are induced within the

central nervous system under diverse circumstances (Table 1). In terms of delineating the molecular and biochemical details of the immediate-early response in the nervous system, the most informative investigations have involved various seizure paradigms. Although these pharmacological models are not particularly relevant to normal neural function, they have provided the basic molecular underpinning for the interpretation of more neurophysiologically relevant experiments that rely heavily upon im munohistochemical techniques.

Dynamics of Expression of c-fos , c-jun and Related Genes Following Pentylenetetrazole Seizures Administration of pentylenetetrazole (PTZ) to rodents results in the rapid onset of seizures and convulsions that persist for approximately 30 minutes. We have used this model to obtain brain tissue that has experi enced massive, relatively synchronous, stimulation. Within minutes ofPTZ seizures, there is an increase in the levels of mRNA encoded by c-fos (Morgan et al 1987, Saffen et al 1988, Sonnenberg et al 1 989a,b), c-jun, and jun B (Saffen et al 1 988, Sonnenberg et al 1 989a,c). In addition, the mRNA encoded by other immediate-early genes, such as egr- l (Sukhatme et al 1 988, Sonnenberg et al 1 989c) and NGFI- B (Watson & Milbrandt 1989), also increases dramatically. Although most of these genes show transient expression, as they do in cell lines, c-jun appears to have a somewhat more protracted time-course than either

c-fos

or jun B (Son

nenberg et al 1 989a,c). In addition to the various mRNA species detected by Northern transfer and hybridization, immunoblot analysis has revealed the presence of large amounts of several Fos-related proteins (Fra) in the brains ofPTZ-treated rats and mice (Sonnenberg et al 1989a,b). The levels of Fra increase dramatically following seizure, although the time-course of their accumu lation is slower than Fos (Sonnenberg et al 1 989a, M organ & Curran 1 989b), a situation similar to the delayed appearance of Fra- l in P C l 2 cells following NGF treatment (Cohen & Curran 1988). Indeed, the various

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MORGAN & CURRAN

Table I system

Situations involving the induction of c-fos in the mammalian nervous

Stimulus paradigm Pentylenetetrazol (Metrazole)


Kainic acid

Electrical stimulation

Surgical lesions and nerve transections N-methyl-D-aspartic acid

Picrotoxin Vitamin B6 antagonist-induced seizures Cortical devascularization Cerebral ischaemia D, -dopamine agonists p-Adrenergic agonists Morphine Opiate withdrawal Nociceptive and peripheral stimulation

Heat stress Adrenalectomy Light stimulation

Intracortical NGF

References Morgan et al 1987 Saffen et al 1988 Sonnenberg et al1989a--c Le Gal La Salle 1988 Sonnenberg et al 1 989b Popovici et al 1988 Dragunow & Robertson 1987b Douglas et al 1988 Daval et al 1989 Sonnenberg et al1989b Hunt et al 1 987 Sagar et al 1 988 Sharp et al 1 989b Winston et a1 1 990 Wisden et al 1 990 Shin et a1 1 990 White & Ga11 1987 Sharp et a1 1 989a,c Dragunow & Robertson 1988 Sonnenberg et al 1989b Kaczmarek et al 1988 Cole et al 1989 Sonnenberg et al 1989b Mizuno et a1 1989 Herrera & Robertson 1 989 Jorgenson et a1 1989 Onodera et a1 1989 Robertson et a11989a,b Gubits et a1 1989 Chang et al 1 988 Hayward et al 1990 Bullit 1989 Draisci & I adarola 1989 Menetrey et al 1 989 Presley et al 1990 Wisden et al 1 990 Dragunow et al 1 989b Jacobson et al 1990 Aronin et al1990 Sagar & Sharp, 1 990 Rea 1989 Rusak et al 1 990 Sharp et al 1 989a

los

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Fra appear and disappear sequentially, such that there is a temporal staggering of their expression in the nervous system (Sonnenberg et al 1 9 89a, Morgan & Curran 1989b). The significance of these waves of Fos related gene products in the brain is unknown, although we discuss some possibilities below. The induction of multiple Fos-like and lun-like proteins following PTZ seizure would imply that transcription factor AP- I levels also increase. This has been tested indirectly by gel-shift analysis in which a double


stranded DNA oligonucleotide containing the AP-l recognition sequence was used. Nuclear extracts from normal mouse brain possess binding activity to the AP-l DNA sequence; however, PTZ seizures result in a five to ten-fold increase of AP- l DNA-binding capacity (Sonnenberg et al 1 989a,b). In addition, antibodies directed against Fos (but that crossreact with the Fra) completely abolish both the basal and stimulated AP-l binding activity (Sonnenberg et al 1 989a). The enhanced DNA-binding activity persists for up to eight hours in the brain of PTZ-treated mice and is always abolished by anti-Fos antibodies even when there is no longer any Fos in the sample (Sonnenberg et aI 1 989a). This would imply that the Fra can also participate in AP- l DNA-binding complexes. Furthermore, it demonstrates that there are dynamic alterations in the molecular com position of AP- l with time after seizure (Morgan & Curran 1 989b). These results demonstrate that relatively brief periods of stimulation (a matter of seconds in some cases) lead to changes in the level of a known transcription factor that persist for at least eight hours in some cir cumstances. In addition, there is a dynamic alteration in the molecular composition of the AP- l transcription factor, which could affect its bio logical potency or target sequence specificity. A limitation in pursuing these possibilities has been the lack of known target genes for AP- l in the nervous system. Recently, it has been shown that transcription of the proenkephalin gene may be influenced by AP- l in transient transfection assays (Sonnenberg et al 1 989c). Furthermore, proenkephalin expression is known to be stimulated by PTZ seizures within one hour in granule cells of the dentate gyrus (Sonnenberg et al 1989c), as it is in other seizure models (White & Gall 1 987). The granule cells of the dentate gyrus are among the first neurons in the CNS to express

c�ros, c�iun,

and

junB

following administration of PTZ (Morgan et al 1 987, Saffen et al 1 988, Sonnenberg et al 1 989c). In addition, the proenkephalin gene contains a regulatory sequence that adheres to the AP-I consensus and binds Fos Jun heterodimers in vitro (Sonnenberg et al 1 989c). Interestingly, Fos JunB heterodimers do not bind efficiently to the AP- l -like sequence in proenkephalin and neither do they transactivate the proenkephalin pro moter in transient transfection assays (Sonnenberg et aI 1 989c). This is the

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first indication that the composition of the AP- l complex may affect binding specificity and, thereby, biological activity.

Localization of Fos in the Nervous System Fos-like immunoreactivity has been mapped in brains of both normal mice and those subjected to PTZ seizures (Morgan et a1 1987, Mugnaini et al 1989). These studies demonstrate that Fos immunostaining can be detected in the nuclei of neurons, but not glia, in both normal and PTZ-treated


mice . This is not because glia cannot be induced for fos expression, since they accumulate massive Fos-like immunoreactivity (FLI) following heat stress (Dragunow et a1 1989b) and brain lesions (Dragunow & Robertson 1 988). Although there is a great increase in FLI in the PTZ-treated brain, in both control and treated mice there is a highly characteristic neuro anatomical distribution of staining. In normal mouse brain, Fos staining mainly represents the distribution of a 35 kDa Fos-related antigen (Son nenberg et al 1 989a,b, Morgan & Curran 1 989b). This FLI is pre dominantly found in granule cells of the dentate gyrus, some pyramidal neurons within the hippocampus, neurons in the piriform and anterior cingulate cortices, the anterior olfactory nucleus, the bed nucleus of the

stria terminalis, the amygdala, and sporadically in the cerebral cortices (Morgan et al 1987). Following PTZ, the intensity of staining of cells in all these locations increases dramatically. The primary sites of induction are the piriform cortex and the dentate gyrus, as well as a number of nuclei within the amygdala (anterior nucleus), thalamus (habenular nucleus), and hypothalamus (paraventricular nucleus). A little later, many regions of the CNS become Fos-immunoreactive, including several layers of the cerebral cortex (2/3 and 5/6), the entire pyramidal neuron population of the hip pocampus, granule cells and periglomerular cells of the olfactory bulb, caudate-putamen, striatum, several regions in the limbic system, further nuclei within the thalamus and hypothalamus, the locus coeruleus, and a few brain stem and spinal cord nuclei. Large areas of the CNS, however, are not induced for FLI; such areas include both colliculi, the lateral geniculate nuclei, central gray, substantia nigra, cerebellum, and most of the brainstem and spinal cord (Morgan et al 1987, M ugnaini et al 1 989). These results demonstrate a number of relevant features of the biology and biochemistry of Fos. First, unstimulated neurons contain FLI, indi cating that this class of gene is expressed under normal physiological conditions. Second,

c-fos is rapidly induced in

neurons within a relatively

small number of brain regions but subsequently many more areas are recruited for Fos expression. Third, those areas of the CNS, such as dentate

fos

AND

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IN NEURONAL CELLS

439

gyrus and piriform cortex, that exhibit an immediate induction of c-fos are precisely those that have high basal levels of FLI. Fourth, since FLI increases between 2 and 4 hours post-seizure (Morgan et al 1987) but Fos is declining during this same period (Sonnenberg et al 1 989a), the immunohistochemical pictures are actually composites of the distribution of Fos and several Fos-related proteins. Indeed, at later times (subsequent to 4 hours), the contribution of Fos is minimal and the pictures are largely those of Fra-3 5 K and Fra-46 K. Since most of the figures depicting Fos


immunostaining in the CNS are taken at longer times after stimulation, they should be correctly defined as representing FLI rather than Fos or

fos protein, unless absolutely monospecific antibodies were employed. One extension of this analysis has been the use of immunoelectron microscopy to determine the subnuc1ear localization of FLI in neurons following PTZ seizures (Mugnaini et al 1989). Whereas fibroblasts and many cultured cell lines exhibit little subnuclear structure, neurons have a highly characteristic subnuclear morphology. In granule cells of the den tate gyrus, it can be shown that FLI is excluded from the characteristic perinucleolar and perinuclear heterochromatin . Rather, FLI appears as granular deposits located throughout the euchromatic regions of the cell nucleus (Mugnaini et aI 1989). Thus, FLI is associated with the component of the genome that contains actively transcribed genes. FOS IMMUNOSTAINING AS AN

ACTIVITY MAPPING TECHNIQUE

A further

application of the immunohistochemical approach has been the use of Fos staining as an indicator of neuronal activity, as suggested in some of the early studies (Hunt et al 1 987, Morgan et al 1987, Sagar et al 1988). The attraction here is that Fos immunostaining might provide a method to map the pattern of postsynaptic stimulation within the intact nervous system with single-cell resolution. Several investigations employing elec trical stimulation to the rat motor/sensory cortex have demonstrated that there is a coincidence between FLI and C 4C)-2-deoxyglucose uptake (Sagar et al 1988, Sharp et al 1989b), thus suggesting a tight correlation between the expression of c-fos and ongoing neuronal activity; however, the picture is more complicated. For example,

fos induction is evident even when

deoxyglucose uptake is unchanged or reduced, such as in the para ventricular nucleus following water deprivation (Sagar et al 1 988) or the CAl layer of hippocampus following ischemic damage (Jorgenson et al 1989). More subtle difficulties are also apparent and are best exemplified by the elegant studies of c-fos expression in the lumbar spinal cord following peripheral stimulation. Noxious stimulation of cutaneous sensory afferents

440

MORGAN & CURRAN

induces FLI in neurons within the superficial layers (laminae I and II) of the dorsal horn of the spinal cord, as first established by Hunt and col leagues (Hunt et al 1 987, Williams et al 1 989, Wisden et aI 1 990). The FLI positive cells appear to be interneurons and projection neurons that express preproenkephalin and preprodynorphin (Draisci & Iadarola 1 989). Sub sequent studies have shown that the pattern of Fos labeling is dependent both upon the route and mode of stimulation (Hunt et al 1 987, Bullitt 1 989, Draisci & Iadarola 1 989, Menetrey et al 1 989, Presley et al 1 990).


Furthermore, analgesics and anesthetics have been found to influence the levels and distribution of FLI. For example, systemic morphine, an analgesic, suppresses, but does not completely block, FLI induction by these noxious stimulants (Presley et al 1 990). Hunt et al ( 1 987), remarked that many more neurons must be involved in the response to peripheral stimulation than were positive for Fos. Menetrey et al ( 1 989) subsequently found that the number of FLI-positive neurons was markedly increased if no anesthetics were employed. It should be noted that anticonvul sants and sedatives such as benzodiazepines and barbiturates, which have been used in the hyperalgesia studies, are known to block

fos

induction

in the e N S (Morgan et a1 1 987). Taken together, these results point to a series of concerns regarding the strategy of equating Fos maps with those of neuronal activity,

sensu stricto. These

problems, both conceptual

and practical, have been reviewed recently (Morgan & Curran 1 9 89b, Dragunow & Faull 1 989). Though caution should be exerted in overinterpreting the presence or absence of FLI in a particular situation, it is still likely that maps of Fos expression will prove extremely useful. This expectation is engendered by several studies that established FLI increases in patterns and sites not anticipated from current knowledge. This has been particularly true of studies involving the administration of opiates or their withdrawal (Chang et al 1 988, Hayward et al 1 990), or the injection of NGF into the cerebral cortex (Sharp et al 1 989a). Other excellent examples are the induction of FLI by light in the retina (Sagar & Sharp 1 990) and suprachiasmatic nucleus (Rea 1 989, Rusak et al 1 990, Aronin et al 1 990). The latter studies raise the exciting possibility that there may be an association between the expression of c

fos

-

and the resetting of the circadian pacemaker by the

photoperiod. Although the induction of c-fos or FLI may not be an absolute reflection of a true increase in neuronal activity, it probably does represent a monitor of intracellular second-messenger levels. Thus it provides another window through which to view neurophysiology, and in conjunction with other methods, such as 2-deoxy-glucose uptake, can lead to completely new insights into information processing in the nervous system.

fos

AND

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IN NEURONAL CELLS

44 1

Neurophysiological Correlates of Immediate-Early Gene Induction KINDLING

Several studies have attempted to draw a closer correlation

between the induction of c-fos by convulsant agents such as PTZ (see Table 1 ) and the epileptic state in humans. In particular, kindling in rats has been investigated as a rodent model of epilepsy in humans. The kindled state is obtained by repeated subconvulsant electrical stimulation to vari


ous brain regions for approximately 1 to 2 weeks. Subsequently the animal will experience convulsions with the same challenge current and will often spontaneously go into convulsions without exogenous stimulation. The kindled state will normally persist for the lifetime of the animal. The electrical stimulus used to establish kindling will induce c-fos mRNA (Shin et al 1 990) and FLI (Dragunow & Robertson 1 987a,b) in the dentate gyrus and hippocampus. Thus, it could provide a link between the stimulus and the kindled condition. The dynamics (time-course and magnitude of response) of c-fos induction by the kindling stimulus is identical, however, in both kindled and naive animals (Shin et aI 1 990). Furthermore, there is no alteration in the basal level of c-fos expression in the brains of kindled rats (Shin et al 1 990). Thus, the product of c-fos is unlikely to be the biochemical basis for the kindled state. Whether Fos is essential for estab lishing kindling remains unclear; however, several interesting corollary findings emanated from these studies. First, it was found that after discharges, which are essential for establishing kindling, induce c-fos (Shin et al 1 990). Second, when recording from the contralateral hippocampus, it was noted that there was a threshold of afterdischarge number for c-fos induction . Thus, there appears to be an all-or-nothing relationship between excitation and c-fos expression, with little gradation of the c-fos response. A similar relationship has been observed for PTZ-induced seizures (Mor gan et al 1 987). In this sense, the immediate-early genes are apparently triggered by stimuli that exceed a threshold value. This threshold probably varies from one cell population to another. Therefore, although higher levels of stimulation may recruit more cells to express c-fos, the gene seems to be either off or fully on. LONG-TERM POTENTIATION

Another model that has drawn attention with

regards to c-fos expression is that of long-term potentiation (LTP) in the hippocampus (Cole et al 1 989, Douglas et al 1 988, Wisden et al 1 990). Regarded as a model for learning, LTP, like kindling, involves the delivery of electrical stimuli to the hippocampal formation, which results in a subsequent potentiation

of output

response

to

further challenges.

Although the stimuli that elicit LTP will also induce c-fos, the two may be dissociated (Cole et al 1 989, Douglas et al 1988, Wisden et al

442

MORGAN & CURRAN

1 990). These authors conclude that a high-frequency discharge of granule cells in the dentate gyrus induces c-fos expression (Douglas et al 1 988, Dragunow et al 1 9 89a) but that lower levels of excitation will evoke LTP but not induction of c-fos. Recently, Cole et al ( 1 989) and Wisden et al ( 1 990) have suggested that although c-fos expression is not strongly correlated with the establishment of LTP, the induction of another imme diate-early gene, NGFI-A, cannot be dissociated from this process. Like most of the studies involving c-fos, these interpretations rely upon temporal


and/or spatial correlations, since the precise function of the immediate early genes is still unknown. In addition, they may be complicated by inter animal variation and stress responses. Unfortunately, the key experiment, namely genetically eliminating c-fos, has not yet been performed. Thus, the precise role (if any) of the various immediate-early genes in processes such as L TP still awaits absolute confirmation. RESPONSE TO CORTlCIAL LESIONS

Besides various forms of seizure, lesions

to the CNS as well as nerve transection also increase c-fos mRNA and FLI with interesting spatial and temporal characteristics (Dragunow & Robertson 1 988, Herrera & Robertson 1 989, Sharp et al 1 989a,c). Typically, CNS lesions induce c-fos in the entire ipsilateral cortex (Dragunow &

Robertson 1 988, Herrera & Robertson 1 989, Sharp et al 1 989a). A situ

ation also observed with intracerebral injections of NGF (Sharp et al 1 989a). Soon after lesioning, FLI is produced predominantly in cells having the appearance and position of neurons. Subsequently, the neuronal FLI disappears but there ensues a protracted expression of FLI in what are identified as glia or some other class of non-neuronal cell located in and around the site of the lesion (Dragunow & Robertson 1 988). This result would suggest that the latter cells represent some form of reactive glia, perhaps stimulated by modulators released at the site of the wound. NEUROHORMONAL AND PHARMACOLOGICAL CORRELATES OF

c-fos

INDUCTION

Attempts have been made to examine the expression of c-fos and FLI in the nervous system following other forms of stimulation. It h ad been noted that water deprivation could lead to an induction of FLI in neurons within the paraventricular nucleus (Sagar et al 1 988), a region of the hypothalamus that is involved in osmoregulation. Interestingly, adrenal ectomy will elicit an induction of FLI in CRF-containing neurons within the hypothalamus (Jacobson et al 1 990). This result would suggest a physiological link among a neuroendocrine axis, the hypothalamic pituitary-adrenal pathway, and gene transcription; however, it has been recognized that nonspecific stressors can induce c-fos in the hypothalamus (Gubits et al 1 989). Thus, there must remain a caveat as to the specificity

fos

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443

of the correlation between physiological challenges and c-fos expression in the hypothalamus. A potential use of F os staining is to define the cellular targets of neuroac tive substances. As mentioned above, morphine can induce c-fos mRNA and FLT in neurons within the caudate-putamen (Chang et aI 1 988). Opiate withdrawal can also evoke an induction of c-fos in the locus coeruleus as well as other brain regions (Hayward et al 1 990). In rats bearing unilateral lesions in the substantia nigra, D I -dopamine receptor agonists such as CY


208-243 induce FLI in the striatum (Robertson et aI 1 989a,b). In contrast, the D2-dopamine agonist, LY 1 7 1 555, does not induce FLT although, like the D I -dopamine agonists, it evokes contraversive rotation in the lesioned animals. Amphetamines elicit ipsiversive rotation in these animals but also lead to an induction of FLl in the striatum (Robertson et aI 1 986b). Thus, rotational behavior in animals bearing nigral lesions can be separated from c-fos induction. In addition to dopamine, other catecholamines, such as adrenergic receptor agonists, can also elicit an induction of c-fos in the nervous system (Gubits et al 1 989). Indeed, these receptors may underlie some of the stress-mediated inductions of c-fas observed predominantly in the rat (Gubits et al 1 989). Recent research has been aimed at identifying the receptor systems that predominantly underlie the induction of c-fos in the mammalian eNS. Glutamate receptors appear to provide one key mechanism for inducing c-fas and several of the immediate-early genes. In cultured granule neurons from the cerebellum, glutamate, acting specifically at the N-methyl-D aspartate (NMDA) form of the glutamate receptor can induce FLI and c%��s mRNA (Szekely et al 1 987, 1 989). This result complements in vivo studies showing that NMDA-type glutamate receptors mediate c%��s induction in the hippocampus (Kaczmarek et al 1 988, Cole et al 1 989, Sonnenberg et al 1 989b) and other brain areas (Sonnenberg et al 1 989b). Indeed, a major component of PTZ-induced c%��s expression in the entire nervous system can be attributed to the activation of NMDA type glu tamate receptors (Sonnenberg et aI 1 989b). The induction of other imme diate-early genes by PTZ is probably also mediated via NMDA receptors to some degree (Saffen et al 1 988, Cole et al 1 989, Sonnenberg ct al 1 989a-c). In addition to the NMDA-type receptor, the occupation of the kainic acid form of the glutamate receptor is also associated with an induction of c%��s and FLI (Popovici et al 1 988, Le Gal La Salle 1 988, Sonnenberg et al 1 989b). One surprising finding in kainic acid-induced seizures is that c-fos mRNA is induced to very high levels with a protracted time-course of expression compared to all other forms of stimulation (Sonnenberg et al 1 989b. Surprisingly, Fos does not show a comparable increase in expression, but rather shows a low level of induction that is

444

MORGAN & CURRAN

also apparent over a protracted period of time (Sonnenberg et al 1 989b). This would suggest that in kainic acid seizures, either Fos is degraded rapidly or there is a failure to translate the abundant c-fos mRNA. Both explanations may have significance for our understanding of the behavior of the immediate-early gene products in such circumstances.

c-fos and c-jun Expression in the Developing Nervous System


Relatively few studies have addressed the question of c-fos expression in the developing nervous system. Ruppert & Wille ( 1 987), using Northern blot analysis, established that there was no significant endogenous regu lation of c-fos mRNA during postnatal development of the cerebellum; however, they did establish that c-fos could be induced if the cerebellum was dissociated by trituration and that the degree of this induction was age-related (Ruppert & Wille 1 987). A more recent in situ hybridization study by Caubert ( 1 989) points to the expression of c-fos mRNA in distinct brain regions during development. During the earlier stages of neuro embryogenesis, around embryonic day 1 2, there is a general high level of c-fos transcripts throughout the nervous system. During subsequent development, high-level expression of c-fos mRNA was confined to more limited regions of the spinal cord, forebrain, cerebellum, and retina (Cau bert 1 989). In an equivalent study (Wilkinson et al 1 989), c-jun and jun B transcripts were found to have differential patterns of expression in the developing mouse nervous system. In early periods of neurogenesis, jun transcripts were observed in proliferating neuroepithelial cells but not in post-mitotic progeny. A further point of interest was that jun expression appeared to be associated with motoneurons. In contrast,junB expression was most evident in non-neuronal elements, where it was not co-localized withfos transcripts (Wilkinson et aI 1 989). With the availability of probes to other members of the fos and jun families, it may now be possible to determine whether patterns of co-expression, and thereby functional interaction, may exist. Literature Cited Abate, C., Rauscher, F. J. III, Gentz, R., Curran, T. 1990a. Expression and puri fication of the leucine zipper and the DNA-bin ding domains of Fos and Jun: both Fos and Jun directly contact DNA. Proc. Natl. Acad. Sci. USA 87: 1 032-36

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fos H. R. 1 989a. Induction of c-fos and TIS genes in cultured rat astrocytes by neuro transmitters. J. Neurosci. Res. 24: 1 0714

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Transcriptional activation of c-fos and c-jun protooncogenes by serum growth factors in osteoblast-like MC3T3-E1 cells.

Estrogen regulates expression of the jun family of protooncogenes in the uterus.

The regulation and function of c-fos and other immediate early genes in the nervous system.

Mechanism of specificity in the Fos-Jun oncoprotein heterodimer.

[Involvement of the central nervous system in disseminated tuberculosis].

[Involvement of the central nervous system in cysticercosis].

Fos and Jun: oncogenic transcription factors.

Learning disabilities and central nervous system involvement.

Central nervous system involvement in the eosinophilia-myalgia syndrome.

Nuclear proto-oncogenes fos and jun.

Temporal and spatial expression of a fos-lacZ transgene in the developing nervous system.

Central nervous system involvement in infantile botulism.

Peripheral nervous system involvement in experimental tuberculosis.

Central nervous system involvement in sarcoidosis.

Central nervous system involvement in relapsing polychondritis.

Nervous system involvement in systemic lupus erythematosus.

Potential involvement of the extracranial venous system in central nervous system disorders and aging.

Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly.

fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system.

Central- and autonomic nervous system coupling in schizophrenia.

The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.

Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus.

Differential expression of early response genes, c-jun, c-fos, and jun B, in A5 cells.