Effect of age and cognitive status on basal level AP-1 activity in rat hippocampus.

Activator protein-1 (AP-1) was examined at multiple levels (mRNA, DNA binding, composition) in hippocampus of young and aged rats that were behaviorally characterized for spatial memory. GFAP mRNA was measured as a gene product known to increase with aging and to be regulated by AP-1. The activity of Jun-amino terminal-kinase (JNK) was also assessed. Levels of c-jun and c-fos mRNAs were unchanged with aging or spatial learning ability. Abundance of GFAP mRNA was significantly increased in aged hippocampus but did not correlate with spatial learning. Total AP-1 binding activity was unaltered with age or cognitive ability. In hippocampus of young, aged unimpaired and aged impaired rats, AP-1 consists mainly of c-Jun, phosphorylated c-Jun (p-c-Jun), JunD, and smaller amounts of c-Fos. JNK is constitutively active in young and aged hippocampus. We conclude that the basal expression of c-fos and c-jun mRNA, overall AP-1 binding activity and AP-1 composition are not influenced by aging or cognitive ability.     

Activation of an intron enhancer within the keratin 18 gene by expression of c-fos and c-jun in undifferentiated F9 embryonal carcinoma cells.

The mouse forms of human keratins 18 and 8 (K18 and K8) are the first members of the large intermediate filament gene family to be expressed during embryogenesis. To identify potential regulatory elements of the human K18 gene, various recombinant constructions were expressed in cultured cells. An enhancer element was found in the first intron that functions on both the K18 and thymidine kinase promoters in differentiated cells. In F9 embryonal carcinoma cells, the level of expression was low in the presence or absence of the first intron. Cotransfection of F9 cells with K18 constructs that include the first intron and increasing amounts of an expression vector of c-jun results in a modest increase in the reporter gene expression. Cotransfection of the same construct with increasing amount of the mouse c-fos gene results in activation of the reporter gene by as much as 15-fold, with a near linear response to the amount of c-fos gene added. Site-specific mutagenesis of a putative AP-1 site within the intron abolishes trans-activation by c-fos in F9 cells. Furthermore, induction of c-fos in a derivative of F9 cells results in increased expression of the endogenous mouse form of K18. Cotransfection with c-jun or c-fos expression vectors had little effect on the expression of the K18 reporter construct in a parietal endodermal cell line already expressing the endogenous mouse gene. These results identify an enhancer within the first intron of K18 that may interact directly with c-jun and c-fos via a conserved AP-1-binding site. K18 expression in undifferentiated F9 cells may be limited by the low levels of c-jun and c-fos.     

Transforming growth factor-beta inhibits lipopolysaccharide-stimulated expression of inflammatory cytokines in mouse macrophages through downregulation of activation protein 1 and CD14 receptor expression

The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines. Several studies showed that transforming growth factor-beta1 (TGF-beta1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines. In this study, we investigated whether TGF-beta1 inhibition of LPS-induced expression of inflammatory cytokines in the septic shock results from downregulation of LPS-stimulated expression of CD14, an LPS receptor. TGF-beta1 markedly inhibited LPS stimulation of CD14 mRNA and protein levels in mouse macrophages. LPS-stimulated expression of CD14 was dramatically inhibited by addition of antisense, but not sense, c-fos and c-jun oligonucleotides. Since TGF-beta1 pretreatment inhibited LPS-stimulated expression of c-fos and c-jun genes and also the binding of nuclear proteins to the consensus sequence of the binding site for activation protein 1 (AP-1), a heterodimer of c-Fos and c-Jun, in the cells, TGF-beta1 inhibition of CD14 expression may be a consequence of downregulation of AP-1. LPS-stimulated expression of interleukin-1beta and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide. Also, TGF-beta1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages. In addition, TGF-beta1 inhibited expression of the two cytokines in several organs of mice receiving LPS. Thus, our results suggest that TGF-beta1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-beta1 inhibition of LPS-induced septic shock.     

AP-1 activity is negatively regulated by cannabinol through inhibition of its protein components, c-fos and c-jun

Regulation of the activator protein-1 (AP-1) complex is very intricate because it involves phosphorylation state, protein-protein, and protein-DNA interactions. In these studies, the regulation of AP-1 activity, with emphasis on c-fos and c-jun regulation, was investigated using cannabinol (CBN) in primary mouse splenocytes in vitro. Cannabinoid compounds exhibit immunosuppressive actions that are putatively mediated through Gi-protein coupled receptors that negatively regulate adenylate cyclase. However, recent studies suggest that cannabinoids modulate other signaling cascades. Indeed, we demonstrate that CBN inhibited binding to AP-1-containing sites from the interleukin-2 promoter. This inhibition of binding was, in part, due to decreased nuclear expression of c-fos and c-jun. We further determined that the effects of CBN were due to posttranslational modifications of these phosphoproteins and showed that CBN inhibited the activation of ERK MAP kinases. Thus, cannabinoid-induced immunosuppression involves disruption of the ERK signaling cascade.     

The effect of HIV-1 regulatory proteins on cellular genes: derepression of the IL-2 promoter by Tat

In HIV-infected individuals dysregulation of the immune system is characterized by severe disorders of the cytokine network. Increase secretion of IL-2, the major T cell growth and differentiation cytokine, may play a decisive role in sensitization of T cells for activation induced apoptosis and indirect death of activated T cells through augmented virus replication. We investigated the cause of enhanced IL-2 secretion and found that the HIV Tat induces this effect. We demonstrate that increased IL-2 secretion is due to Tat-enhanced IL-2 promoter activation. Tat derepresses and activates the distal AP-1 site (position -185 to -177) in the IL-2 promoter. In nonstimulated T cells a repressor complex containing NF-IL6, JunB, c-Fos and Fra-1 is formed on the AP-1(IL-2/d) site and represses IL-2 promoter activity. After T cell activation, a heterodimeric activator containing p65 and c-Jun binds to the AP-1(IL-2/d) site. HIV Tat enhances activation of NF-kappaB and consequently, activates the AP-1(IL-2/d) site. Our data provide evidence for a novel mechanism by which HIV Tat dysregulates IL-2 production and therefore may contribute to the HIV-1 infection in a way yet to be clarified.     

Interferon-gamma induces the expression of immediate early genes c-fos and c-jun in astrocytes.

The expression of the proto-oncogenes, c-fos and c-jun, in cultured mouse astrocytes and its induction by the potent astrocyte activator interferon-gamma (IFN-gamma), were examined by Northern blot and flow cytometry. Both proto-oncogenes were induced in a dose-dependent manner, peaking around 100 U/ml of IFN-gamma. The kinetics of expression is very transient for c-fos, reaching a maximum at 30 min and decreasing rapidly thereafter. The c-jun remained high throughout the stages analysed. Cycloheximide superinduced c-fos and c-jun induction by IFN-gamma, thus indicating that both act as immediate early genes. The products of c-fos and c-jun, proteins FOS and JUN, that act in conjunction forming the regulatory factor AP-1, were detected 1 hr after stimulation in virtually all cells, using flow cytometry. The induction in astrocytes of both proto-oncogenes could be the first stage of immunological activation of these central nervous system cells by immune interferon.     

Differential regulation of fos family genes in the ventrolateral and dorsomedial subdivisions of the rat suprachiasmatic nucleus

Extensive studies have established that light regulates c-fos gene expression in the suprachiasmatic nucleus, the site of an endogenous circadian clock, but relatively little is known about the expression of genes structurally related to c-fos, including fra-1, fra-2 and fosB. We analysed the photic and temporal regulation of these genes at the messenger RNA and immunoreactive protein levels in rat suprachiasmatic nucleus, and we found different expression patterns after photic stimulation and depending on location in the ventrolateral or dorsomedial subdivisions. In the ventrolateral suprachiasmatic nucleus, c-fos, fra-2 and fosB expression was stimulated after a subjective-night (but not subjective-day) light pulse. Expression of the fra-2 gene was prolonged following photic stimulation, with elevated messenger RNA and protein levels that appeared unchanged for at least a few hours beyond the c-fos peak. Unlike c-fos and fra-2, the fosB gene appeared to be expressed constitutively in the ventrolateral suprachiasmatic nucleus throughout the circadian cycle; immunohistochemical analysis suggested that delta FosB was the protein product accounting for this constitutive expression, while FosB was induced by the subjective-night light pulse. In the dorsomedial suprachiasmatic nucleus, c-fos and fra-2 expression exhibited an endogenous circadian rhythm, with higher levels during the early subjective day, although the relative abundance was much lower than that measured after light pulses in the ventrolateral suprachiasmatic nucleus. Double-label immunohistochemistry suggested that some of the dorsomedial cells responsible for the circadian expression of c-Fos also synthesized arginine vasopressin. No evidence of suprachiasmatic nucleus fra-1 expression was found.In summary, fos family genes exhibit differences in their specific expression patterns in the suprachiasmatic nucleus, including their photic and circadian regulation in separate cell populations in the ventrolateral and dorsomedial subdivisions. The data, in combination with our previous results [Takeuchi J. et al. (1993) Neuron 11, 825-836], suggest that activator protein-1 binding sites on ventrolateral suprachiasmatic nucleus target genes are constitutively occupied by DeltaFosB/JunD complexes, and that c-Fos, Fra-2, FosB and JunB compete for binding after photic stimulation. The differential regulation of fos family genes in the ventrolateral and dorsomedial suprachiasmatic nucleus suggests that their circadian function(s) and downstream target(s) are likely to be cell specific.