Fos regulates neuronal activity in the nucleus accumbens.

Repeated exposure to drugs of abuse induces a variety of persistent changes in the brain and the dopamine D1 receptor plays a major role in the process. To understand intracellular mechanisms contributing to cocaine-induced neuroadaptations, we previously examined the role of the immediate early gene Fos using a mouse in which Fos is disrupted primarily in D1 receptor-expressing neurons in the brain. We found that both dendritic remodeling of medium spiny neurons and behavioral sensitization induced by repeated exposure to cocaine are attenuated in the mutant mice. Moreover, the expression of genes encoding several transcription factors, neurotransmitter receptors and intracellular signaling molecules following repeated cocaine administration is altered in the mutant mice compared to that in wild-type mice. In the present study, we have investigated the role of Fos in regulating neuronal excitability at a cellular level and found that medium spiny nucleus accumbens neurons in the mutant mice exhibit increased excitability and attenuated inhibitory responses to stimulation of D1 receptors compared to those in wild-type mice. Our findings suggest that Fos functions in D1 receptor-bearing neurons to regulate neuronal activity which may contribute to the persistence of drug-induced changes.

Involvement of NF-kappaB and AP-1 activation in icariin promoted cardiac differentiation of mouse embryonic stem cells.

Icariin has been reported to facilitate the differentiation of mouse embryonic stem (ES) cells into cardiomyocytes; however, the mechanism on cardiomyogenic cell lineage differentiation has not been fully elucidated yet. In the present studies, an underlying signaling network including p38, extracellular signal-regulated kinase 1, 2 (ERK1, 2), nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) transcription factors c-jun and c-fos was assumed in icariin induced cardiomyogenesis. Icariin rapidly activated p38 and ERK1, 2 in embryoid bodies, treatment with p38 antagonist 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) or ERK1, 2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) significantly abolished icariin induced cardiac commitment, MEF2C gene expression and nuclear translocation, as well as cardiac-specific protein alpha-actinin expression, indicating that p38 and ERK1, 2 are specifically involved in icariin stimulated cardiomyogenic cell lineage differentiation of ES cells. Further, IkappaBalpha phosphorylation and NF-kappaB p65 translocation to the nucleus appeared rapidly when embryoid bodies exposed to icariin, and the expression of IkappaBalpha or NF-kappaB p65 in cytoplasm was decreased concomitantly. Moreover, icariin increased c-jun and c-fos mRNA and protein expression. Either SB203580 or U0126 displayed inhibitory effect on icariin induced NF-kappaB and AP-1 activation. It could be concluded that p38 and ERK1, 2 are activated in a coordinated manner, which in turn contribute to NF-kappaB and AP-1 activation in icariin induced cardiomyogenic cell lineage differentiation of mouse ES cells.

Centrally administered neuropeptide S activates orexin-containing neurons in the hypothalamus and stimulates feeding in rats.

Neuropeptide S (NPS) is a newly identified transmitter that modulates arousal and anxiety. To determine potential neuronal targets for NPS, we studied the pattern of neuronal activation as indicated by the expression of Fos. Centrally administered NPS increased Fos-like immunoreactivity (FLI) in the paraventricular, dorsomedial nuclei and lateral hypothalamic area (LHA) of the hypothalamus, the midline thalamic nuclei, and the amygdala, many parts of which are involved in the regulation of emotion, arousal, and feeding. In particular, we noted that Fos-immunoreactive (Fos-ir) levels were increased in orexin-expressing neurons in the LHA. We then studied whether an icv injection of NPS increased food intake. The injection of NPS (1 nmol) significantly stimulated feeding at 2 h in rats, but there was no difference in food intake at 4 h or 24 h. These results suggest that arousal and feeding induced by NPS in the central nervous system may be related to the activation of orexin-expressing neurons.

Effect of adrenalectomy on cholecystokinin-8-induced Fos-like immunoreactivity in myenteric neurons and the dorsal vagal complex in rats.

OBJECTIVE: To investigate the effect of adrenalectomy on cholecystokinin-8 (CCK-8)-induced Fos-like immunoreactivity (Fos-LI) in the myenteric neurons of the dorsal vagal complex (DVC) in rats. ANIMALS: 16 male Sprague Dawley rats. PROCEDURES: Rats were allocated to 1 of 2 groups and underwent adrenalectomy or a sham adrenalectomy procedure. Rats were challenged with a supraphysiologic dose of CCK-8 (40 microg/kg) or physiologic saline (0.9% NaCl) solution (0.5 mL) administered IP; after 90 minutes, rats were euthanized, and Fos-LI was quantified in the DVC (at the levels of the area postrema, nucleus tractus solitarii, and dorsal motor nucleus of the vagus) and the myenteric neurons of the duodenum and jejunum by use of a diaminobenzidine reaction enhanced with nickel. The Fos-LI-positive cells were counted by use of an automated system and manually in the DVC and intestinal samples, respectively. Counts of Fos-LI in the different hindbrain levels and myenteric neurons were compared between the adrenalectomy--and shamtreated groups and between the CCK-8- and saline solution-treated groups. RESULTS: After adrenalectomy, CCK-8-induced Fos-LI was attenuated only in the myenteric neurons of the duodenum. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the adrenal gland has a role in the activation of myenteric neurons by CCK-8 in rats.

Sensitized attentional performance and Fos-immunoreactive cholinergic neurons in the basal forebrain of amphetamine-pretreated rats.

BACKGROUND: The consequences of repeated exposure to psychostimulants have been hypothesized to model aspects of schizophrenia. This experiment assessed the consequences of the administration of an escalating dosing regimen of amphetamine (AMPH) on attentional performance. Fos-like immunoreactivity (Fos-IR) in selected regions of these rats' brains was examined to test the hypothesis that AMPH-sensitized attentional impairments are associated with increased recruitment of basal forebrain cholinergic neurons. METHODS: Rats were trained in a sustained attention task and then treated with saline or in accordance with an escalating dosing regimen of AMPH (1-10 mg/kg). Performance was assessed during the pretreatment and withdrawal periods and following the subsequent administration of AMPH "challenges" (.5, 1.0 mg/kg). Brain sections were double-immunostained to visualize Fos-IR and cholinergic neurons. RESULTS: Compared with the acute effects of AMPH, AMPH "challenges," administered over 2 months after the pretreatment was initiated, resulted in significant impairments in attentional performance. In AMPH-pretreated and -challenged animals, an increased number of Fos-IR neurons was observed in the basal forebrain. The majority of these neurons were cholinergic. CONCLUSIONS: The evidence supports the hypothesis that abnormally regulated cortical cholinergic inputs represent an integral component of neuronal models of the attentional dysfunctions of schizophrenia.

Dopamine manipulation alters immediate-early gene response of striatal parvalbumin interneurons to cortical stimulation.

Cortical projections provide the major excitatory inputs to the striatum. In addition to innervating medium spiny cells, these axons contact striatal interneurons that are parvalbumin-immunoreactive (PV-ir). PV-ir interneurons make synaptic connections with many medium spiny cells, and thus can modulate striatal output. The striatum also receives dopaminergic projections from the substantia nigra, but it has been challenging to study the impact of dopamine (DA) cell injury on corticostriatal activity in vivo due to limitations in the methods used to induce cortical activity. Using epidural application of the GABA(A) antagonist picrotoxin, which produces a topographically restricted region of striatal immediate-early gene expression, we have investigated the effect of DA cell injury or DA receptor antagonism on immediate-early gene (IEG) expression in striatal medium spiny cells and PV-ir interneurons. Epidural application of picrotoxin to the rat's M1 motor cortex induced Fos in ipsilateral dorsolateral striatum. Animals previously given 6-hydroxydopamine (6-OHDA) injections into the ascending DA pathways had greater total numbers of cortical stimulation-induced striatal Fos-ir cells but fewer Fos-ir/PV-ir cells, compared to sham-operates. In a separate experiment, rats given cortical stimulation and treated with the DA D2-class antagonist eticlopride (0.10 mg/kg) exhibited fewer Fos-ir/PV-ir cells than did vehicle-treated rats. Taken together, these results indicate that DA may importantly control striatal output via influences on PV-ir interneurons. Possible mechanisms for these influences are discussed.

Helicobacter pylori infection activates FOS and stress-response genes and alters expression of genes in gastric cancer-specific loci.

We studied human gene expression changes caused by Helicobacter pylori infection by using an in vitro model and 13k cDNA microarrays. A gastric cancer cell line was infected with H. pylori strain NCTC 11637. H. pylori infection was found to induce differential expression of genes in chromosomal locations known to contain frequent chromosomal aberrations and gene mutations specific to gastric cancer. Based on the results of time series experiments, the primary transcription target of the infection seemed to be FOS, the expression of which significantly increased after H. pylori infection. H. pylori infection also activated transcription of several stress-response genes. H. pylori infection may predispose the host cell to DNA damage in the chromosomal locations specific to gastric cancer by activating transcription and promoting histone removal from these sites, thus exposing its target DNA to mutations.

Early stages of memory formation in filial imprinting: Fos-like immunoreactivity and behavior in the domestic chick.

Early stages of memory formation in filial imprinting were studied in domestic chicks. Chicks trained for 15 min showed strong imprinting, demonstrated by a strong preference for their training stimulus, and the time course of this preference over 2 days after training was similar to that of chicks trained for 60 min. The chicks therefore learned characteristics of the training stimulus very early during training. The intermediate and medial hyperstriatum ventrale (IMHV) is a part of the chick forebrain that is crucial for imprinting. Previous experiments have shown a learning-specific increase in Fos-like immunoreactivity, used as a marker of neuronal activity, in the IMHV after training for 60 min. The time course of Fos expression in the IMHV was measured after training for 15 min and 60 min. The same pattern of expression was found for both training times, showing a peak 120 min after the start of training. The time course of expression was stimulus-dependent. Fos expression in the IMHV, but not the hippocampus, was significantly correlated with strength of imprinting. It is concluded that the learning-specific change in Fos expression in the IMHV is associated with very early components of memory formation.

c-Fos but not v-Fos protein induces programmed cell death of v-myb-transformed monoblasts.

c-Fos and v-Fos belong to a group of proteins forming the transcription factor AP-1 that is important for regulation of proliferation, differentiation and programmed cell death in multiple cell types. In this study, we examined the role of c-Fos and v-Fos proteins in v-myb-transformed BM2 monoblasts. We show that while the v-Fos protein prolongs the G0G1 phase of the BM2 cell cycle, c-Fos leaves the cell cycle unaffected and, rather, induces programmed cell death. The apoptosis-promoting activity of the c-Fos protein is markedly enhanced in cells cultivated under serum-free conditions. c-Fos-induced apoptosis of BM2 cells occurred in the presence of Bcl-2 and was not dependent on the transcription activation function of the c-Fos protein. No differentiation-promoting activity of the Fos proteins was observed. The effects of Fos proteins on BM2 cells differ from those induced by Jun proteins, suggesting differential roles of individual components of the AP-1 transcription factor in regulation of essential cellular processes.

2-Deoxy-D-glucose-induced cytotoxicity and radiosensitization in tumor cells is mediated via disruptions in thiol metabolism.

Exposure to ionizing radiation is believed to cause cell injury via the production of free radicals that are thought to induce oxidative damage. It has been proposed that exposure to agents that enhance oxidative stress-induced injury by disrupting thiol metabolism may sensitize cells to the cytotoxic effects of ionizing radiation. Recently, it has been shown that glucose deprivation selectively induces cell injury in transformed human cells via metabolic oxidative stress (J. Biol. Chem., 273: 5294-5299; Ann. N.Y. Acad. Sci., 899: 349-362), resulting in profound disruptions in thiol metabolism. Because 2-deoxy-D-glucose (2DG) is a potent inhibitor of glucose metabolism thought to mimic glucose deprivation in vivo, the hypothesis that exposure to 2DG might be capable of inducing radiosensitization in transformed cells via perturbations in thiol metabolism was tested. When HeLa cells were exposed to 2DG (4-10 mM) for 4-72 h, cell survival decreased (20-90%) in a dose- and time-dependent fashion. When HeLa cells were treated with 6 mM 2DG for 16 h before ionizing radiation exposure, radiosensitization was observed with a sensitizer enhancement ratio of 1.4 at 10% isosurvival. Treatment with 2DG was also found to cause decreases in intracellular total glutathione content (50%). Simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC; 30 mM) protected HeLa cells against the cytotoxicity and radiosensitizing effects of 2DG, without altering radiosensitivity in the absence of 2DG. Furthermore, treatment with NAC partially reversed the 2DG-induced decreases in total glutathione content, as well as augmented intracellular cysteine content. Finally, the cytotoxicity and radiosensitizing effects of 2DG were more pronounced in v-Fos-transformed versus nontransformed immortalized rat cells, and this radiosensitization was also inhibited by treatment with NAC. These results support the hypothesis that exposure to 2DG causes cytotoxicity and radiosensitization via a mechanism involving perturbations in thiol metabolism and allows for the speculation that these effects may be more pronounced in transformed versus normal cells.

v-FBR-fos oncogene fails to rescue mammalian cells from growth arrest but affects the responses of human fibroblasts to heparin.

The effects of v-fos oncogene on the proliferation of mammalian cells were studied using several approaches. Constitutive overexpression of v-FBR-fos in normal human fibroblasts (MRC-5) and of v-FBR-fos in human chondrocytes (HAC21) failed to immortalise them, extend their in vitro lifespan, increase their growth rates or induce cellular transformation. Further, v-FBR-fos did not render MRC-5 growth factor-independent or alter their responsivenness to serum, but it markedly suppressed their heparin-induced proliferation. A conditionally immortalized, temperature-sensitive rat embryo fibroblast cell line (tsa14) which undergoes growth arrest upon inactivation of a thermolabile SV40 large T antigen by a temperature shift producing a phenotype that mimmicks the senescent phenotype, was also used to study the effects of v-FBR-fos on cell proliferation. Whereas a wild-type SV40 large T antigen rescued tsa14 from a temperature-dependent growth arrest, v-FBR-fos failed to do so. Hence, v-FBR-fos was not sufficient to, at least, complement the tsa14 growth defect. There was no change in the expression of c-jun and junB, members of the AP-1 transcriptional complex in MRC-5v-fos cells. These data show that v-FBR-fos is not sufficient to rescue mammalian cells from senescence but it can affect the responses of human fibroblasts to heparin suggesting a role of fos in cell proliferation.

Requirements for repression of retinoid X receptor by the oncoprotein P75gag-v-erbA and the thyroid hormone receptors.

The oncogenic counterpart of thyroid hormone receptor-alpha (TRRalpha), denoted P75gag-v-erbA, has served as a paradigm for the ability of TRs to repress basal levels of transcription. We show here that the retinoid X receptor (RXR), when activated by its specific ligand SR11237, is repressed by both the normal TRalpha and the P75gag-v-erbA. The repression caused by the two proteins is distinct and dependent on both the cell type and the hormone-response element through which RXR acts. In HeLa cells only TR repressed efficiently through the palindromic 2xIR0 element, whereas the proteins were equally efficient in JEG cells. This demonstrates that proteins distinct in the two cell types mediate the repression. RXR-dependent induction via the natural response element of the cellular retinol-binding protein (CRBPII) gene was likewise (> or = 50%) repressed by TR, whereas P75gag-v-erbA did not repress during the same conditions. Furthermore, P75gag-v-erbA and its variants v-erbAtd359 (lacking repressing activity on TR) and v-erbAr12 (a highly active repressor of TR) efficiently repressed induction by a hybrid protein consisting of the DNA- binding domain of Gal4 and the ligand-binding region of RXR. The viral proteins did not, however, associate with RXR unless the two partners were allowed to heterodimerize upon binding to a specific response element, such as the 2xIR0 element or that of the CRBPII gene. In conclusion, we suggest that the efficient repression seen with the the 2xIR0 element is due to heterodimerization of TR or the viral oncoproteins with RXR and a concomitant inhibition of binding of the RXR-specific ligand that results in an inability of RXR to attract a cell type-specific cofactor. In addition, the data suggest that the interaction between RXR and P75gag-v-erbA on the CRBPII element is too weak to inhibit RXR from binding a ligand and therefore also to repress.

AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program.

Finkel-Biskis-Reilly osteosarcoma virus v-Fos inhibits adipogenesis and both the activity and expression of CCAAT/enhancer binding protein alpha, a key regulator of adipocyte differentiation.

Finkel-Biskis-Reilly (FBR) osteosarcoma virus v-Fos causes tumors of mesenchymal origin, including osteosarcomas, rhabdomyosarcomas, chondrosarcomas, and liposarcomas. Because the cell of origin in all these tumors is a pluripotent mesenchymal cell, the variety of tumors seen in mice which express FBR v-Fos implies that FBR v-Fos inhibits multiple differentiation pathways. To study the mechanism of FBR v-Fos' inhibition of mesenchymal differentiation, we utilized an in vitro model of adipocyte differentiation. We show by both morphological and biochemical means that FBR v-Fos inhibits adipocyte differentiation in vitro. This inhibition is due to FBR v-Fos' inhibition of the growth arrest characteristic of terminal differentiation and FBR v-Fos' inhibition of the expression and activity of a key regulator of this growth arrest, C/EBPalpha. The in vitro inhibition of adipogenesis by FBR v-Fos has in vivo significance as immunostaining of FBR v-Fos-induced tumors shows no CCAAT/enhancer binding protein (EBP)-alpha expression. These data implicate C/EBPalpha as a protein involved in the generation of liposarcomas.

Effect of decreased fte-1 gene expression on protein synthesis, cell growth, and transformation.

The fte-1 gene, previously cloned in our laboratory as a putative v-fos transformation effector gene (C.J. Kho and H. Zarbl, Proc. Natl. Acad. Sci. USA, 89: 2200-2204, 1992), has been shown to encode ribosomal protein S3a. Comparison of fte-1 expression in a variety of normal and transformed cells indicated that elevated expression of fte-1 mRNA was frequently associated with transformation of rodent and human cells. In an effort to understand how monoallelic disruption of fte-1 is able to block v-fos-induced cell transformation, we examined the pattern of fte-1 expression during cell cycle progression and determined its effects on protein synthesis and cell growth. In synchronously cultured human fibroblasts, fte-1 mRNA was found to accumulate in cells undergoing DNA synthesis, suggesting that its expression is correlated with S-phase progression. fte-1 does not function as a dominant oncogene because ectopic overexpression of fte-1 in normal Rat-1 fibroblasts failed to induce cell transformation. However, the expression of antisense fte-1 resulted in growth inhibition. Monoallelic disruption of the fte-1 gene in v-fos-transformed Rat-1 fibroblasts resulted not only in loss of the transformed phenotype but also in a decreased rate of protein synthesis due to decreased polysome formation. Taken together, these results indicate that the accumulation of ribosomal subunits and the rate of protein synthesis are important modulators of neoplastic transformation and cell growth.

The glucocorticoid receptor synergizes with Jun homodimers to activate AP-1-regulated promoters lacking GR binding sites.

Jun/Fos (AP-1) and steroid hormone receptors (SHR) are distinct families of transcription factors that convert extracellular signals into long-term genetic responses. Despite clear differences in their modes of activation and DNA binding specificities, a regulatory cross-talk between AP-1 and SHR such as the glucocorticoid receptor (GR), has been established. Here, we show that the hormone-activated GR negatively or positively modulates the expression of AP-1-dependent genes, depending on the subunits of the dimeric AP-1 complex. This type of regulation does not depend on the presence of a GR binding site in the promoter and is mediated through the DNA binding domain of Jun. Since individual subunits of AP-1 exhibit small differences in sequence specificity, specific subsets of AP-1-dependent genes may be regulated by steroid hormones in different directions.

Deregulation of genes encoding microfilament-associated proteins during Fos-induced morphological transformation.

The mechanism of Fos-induced transformation is still poorly understood. In the present study, we have asked whether genes whose products play a role in determining cell morphology might become deregulated in the course of Fos-induced transformation. A clear up-regulation in Fos-transformed rat fibroblasts was seen with ezrin, as well as tropomyosin (TM) -3 and -5B, while TM-1 was down-regulated. Significantly, the same genes were deregulated in a very similar, but hormone-inducible way in cells expressing a Fos-estrogen receptor fusion protein. In agreement with these results, Fos-expressing cells showed decreased levels of two TM isoforms of 36 and 38 kDa, and showed an impaired TM network. The significance of these observations is strengthened by the fact that the deregulation of TM expression has been shown to contribute to morphological transformation in other experimental systems. Deregulation of the TM and ezrin genes preceeds the induction of morphological transformation suggesting that this deregulation is not merely a consequence of transformation. On the other hand, deregulation follows the induction of direct Fos target genes. We therefore propose that a cascade of regulatory events is triggered by Fos oncoproteins which eventually leads to the deregulation of genes encoding cytoskeleton-associated proteins.

TGF alpha and v-fos cooperation in transgenic mouse epidermis induces aberrant keratinocyte differentiation and stable, autonomous papillomas.

To assess the synergistic effect of growth and transcription factor deregulation on carcinogenesis in vivo, mating experiments were performed between transgenic mice expressing human TGF alpha or v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos, HK1.TGF alpha and HK1.fos/alpha). While HK1.TGF alpha mice exhibited mild epidermal hyperplasia resulting in a wrinkled appearance, this hyperplasia was significantly increased in HK1.fos/alpha mice which also exhibited a novel opalescent and peeling skin phenotype. HK1.fos/alpha keratinocyte differentiation was considerably deregulated with cornified cells appearing in the granular layer, granular cells in the spinous layer and a sixfold increase in BrdU labeling over normal. In addition, hyperplastic HK1.fos/alpha epidermis exhibited aberrant loricrin, filaggrin and novel K13 expression associated with v-fos expression. Unlike adult HK1.TGF alpha controls, hyperplasia persisted in HK1.fos/alpha adults which also rapidly developed autonomous squamous cell papillomas. These results demonstrate that v-fos and TGF alpha over-expression can cooperate to reprogram keratinocyte differentiation and elicit the early stages of neoplasia. Moreover, TGF alpha over-expression appeared to play an early, initiating role in HK1.fos/alpha papilloma etiology, and a promotion role in the accelerated appearance of v-fos wound-associated preneoplastic phenotypes. However, the stable persistence of HK1.fos/alpha papillomas for up to 12 months, suggests that additional events are required for malignant conversion.

Elevated expression of the junB proto-oncogene is essential for v-fos induced transformation of Rat-1 cells.

We previously described the isolation of non-tumorigenic revertants from mutagenized populations of v-fos-transformed Rat-1 cells (Zarbl et al., 1987). In the present study we examined the possibility that the revertant phenotype resulted from mutations that altered the expression or activities of the c-jun or junB proto-oncogenes. The results demonstrated that levels of the c-jun mRNA and protein were unchanged in the revertants when compared to the transformed parental cells, and ectopic overexpression of c-jun failed to retransform the revertants. Although one mutant allele was detected in revertant EMS-1-19, overexpression of this mutant allele failed to inhibit v-fos induced cell transformation. Together these results indicated that the revertant phenotype did not result from altered expression or mutations in the c-jun gene. In contrast to the results obtained with c-jun, the levels of junB mRNA and protein were found to be reduced two- or threefold in revertant EMS-1-19. Ectopic overexpression of junB induced transformation of revertant EMS-1-19, but failed to transform Rat-1 cells. Moreover, about 10% of v-fos transformed cells transfected with vectors that express antisense junB mRNA acquired a non-transformed phenotype. Together these results indicate that expression of junB above a threshold level is essential for v-fos-induced transformation of Rat-1 fibroblasts.