Activator Protein-1 Binding Activity Research Articles

Light-Induced Variations in AP-1 Binding Activity and Composition in the Rat Suprachiasmatic Nucleus

Expression of immediate early genes, including fos-like and jun-like genes, in the suprachiasmatic nucleus is believed to be part of the mechanism for photic entrainment of circadian rhythms to the environmental light/dark cycle. However, the effects of a light stimulus on activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus remain unclear. The photic regulation of AP-1 DNA-binding activity and composition in the rat suprachiasmatic nucleus was evaluated by using an electrophoretic mobility shift assay. A light pulse given during subjective night induced an increase in AP-1 binding activity when either nuclear or whole-cell extracts from suprachiasmatic nuclei were used. Under constant dark conditions, proteins that are predominant components of AP-1 complexes are Fra-2 and Jun-D. Under light stimulation, c-Fos and Jun-B consistently increased, as expected, but this was also the case for Fra-2, Jun-D, and c-Jun, although to a lesser extent. An immunocytochemical study of the Fra-2 expression pattern demonstrated the presence of the protein in the ventrolateral as well as in the dorsomedial subdivisions of the suprachiasmatic nucleus. Light regulation of Fra-2 immunoreactivity, however, appeared to be restricted to the ventrolateral subdivision. It is concluded that light may be acting both by increasing constitutive AP-1 complexes and by inducing the expression of specific complexes.

Herpes Simplex Virus Type 1 Infection Stimulates p38/c-Jun N-terminal Mitogen-activated Protein Kinase Pathways and Activates Transcription Factor AP-1

Cells respond to environmental stress and proinflammatory cytokines by stimulating the Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase cascades. Infection of eukaryotic cells with herpes simplex virus type 1 (HSV-1) resulted in stimulation of both JNK/SAPK and p38 mitogen-activated protein kinase after 3 h of infection, and activation reached a maximum of 4-fold by 9 h post-infection. By using a series of mutant viruses, we showed that the virion transactivator protein VP16 stimulates p38/JNK, whereas no immediate-early, early, or late viral expressed gene is involved. We identified the stress-activated protein kinase kinase 1 as an upstream activator of p38/JNK, and we demonstrated that activation of AP-1 binding proceeded p38/JNK stimulation. During infection, the activated AP-1 consisted mainly of JunB and JunD with a simultaneous decrease in the cellular levels of Jun protein. We suggest that activation of the stress pathways by HSV-1 infection either represents a cascade triggered by the virus to facilitate the lytic cycle or a defense mechanism of the host cell against virus invasion.

Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells.

The expression, DNA binding, and transactivating activity of activator protein 1 (AP-1) was examined in a series of multidrug resistant (MDR) MCF-7 human breast cancer cells that have increasing levels of MDR1 gene expression. We observed an increase in the amount of both c-jun and c-fos mRNA in cells with 12-, 65-, or 200-fold higher resistance to adriamycin when compared to drug-sensitive MCF-7 wild type (WT) cells. Electrophoretic mobility shift assays (EMSA) demonstrated an increase in the DNA binding activity of an AP-1 complex in nuclear extracts from MDR MCF-7 cells when compared to extracts from WT cells. We observed a proportional increase in luciferase expression from a reporter vector containing consensus AP-1 binding sites in transiently transfected MDR cells when compared to WT cells, indicating that AP-1 mediated gene expression is increased in drug-resistant MCF-7 cells. Since the MDR1 promoter contains a putative AP-1 binding site, we used EMSA to examine AP-1 binding activity to an oligonucleotide probe that contained the relevant MDR1 promoter sequences (-123 to -108). Nuclear extracts from resistant MCF-7 cells displayed an increased level of DNA binding of Jun/Jun dimers to the probe, indicating that AP-1 was capable of binding to this promoter site. A luciferase reporter construct containing triplicate copies of the MDR1 promoter sequence was expressed at higher levels in transiently transfected MDR cells when compared to expression in WT cells. Co-transfection of WT cells with a c-jun expression vector and either of the AP-1 luciferase constructs demonstrated that c-jun could activate gene expression from both the consensus and the MDR1 AP-1 sites in a dose dependent manner. In addition, RT-PCR and western blot analysis showed that levels of MDR1 mRNA and Pgp were increased in c-jun transfected WT cells. Taken together, these data indicate that increased AP-1 activity may be an important mediator of MDR by regulating the expression of MDR1.

Enhanced AP-1 binding in brain induced by D1 and D2 agonists in methamphetamine-sensitized rats.

The activator protein-1 (AP-1) binding activities induced by a separate challenge with SKF38393 and quinpirole after 1 weeks' abstinence from chronic methamphetamine (4 mg/kg/day, 14 days) were increased significantly in the striatum, nucleus accumbens and cingulate cortex compared with the saline-treated controls. Quinpirole-, but not SKF38393-induced AP-1 binding activities were still significantly higher after a 4-week abstinence period in the chronic methamphetamine group than in the chronic saline control group. Downward sniffing, which occurred following a quinpirole-challenge, was significantly intensified after both a 1 and 4 weeks' abstinence from chronic methamphetamine. These results indicate that chronic administration of methamphetamine induces alterations of the interactions of dopamine D1 and D2 receptors which are reflected as enhanced AP-1 binding activities.

Transcriptional Activation of the Ovine Follicle-Stimulating Hormone β-Subunit Gene by Gonadotropin-Releasing Hormone: Involvement of Two Activating Protein-1-Binding Sites and Protein Kinase C**This work was supported by the North Carolina State University Agricultural Research Service, NICHD Grant 34863, and the Mellon Foundation.

FSH is an alpha/beta heterodimeric glycoprotein, the formation of which is regulated primarily by expression of its beta-subunit. Recent studies on transcriptional regulation of the ovine FSH beta-subunit gene (oFSHbeta) have defined two functional activating protein-1 (AP-1) enhancers in the proximal promoter (located at -120 and -83 bp) that are probably physiologically important for FSHbeta expression. As GnRH is a major regulator of FSHbeta expression and is also known to stimulate the synthesis of Jun and Fos family members (AP-1), we investigated the possibility that oFSHbeta transcription may be regulated by GnRH through AP-1. Here we report the use of an in vitro cell system involving transient transfection of GnRH receptors (GnRHR) into HeLa cells to define regulatory elements involved in GnRH-mediated induction of oFSHbeta. This system was used to show that expression of luciferase constructs containing either the -4741/+759 region of the oFSHbeta gene (-4741oFSHbeta-Luc) or the -846/+44 region of the human alpha gene (alpha-Luc; a positive control) was stimulated 3.1 +/- 0.3- and 7.7 +/- 1.9-fold, respectively, by 100 nM GnRH. Another luciferase expression plasmid containing the Rous sarcoma virus promoter (a negative control) showed no response to GnRH. Similar results with these constructs were obtained in COS-7 cells. Studies with progressive 5'-deletion constructs and site-specific mutations demonstrated that this stimulation was dependent on each AP-1 site in the proximal promoter of oFSHbeta. Gel shift assays demonstrated the ability of GnRHR in HeLa cells to increase AP-1 binding activity. Responses in the HeLa cell system were dependent on GnRH (ED50 = 0.5 nM) and GnRHR, which was identified by photoaffinity labeling. In addition, GnRHR-expressing HeLa cells exhibited a normal GnRH-dependent mobilization of intracellular calcium. Finally, as protein kinase C (PKC) is a known target of GnRH action in gonadotropes, the role of PKC in transcriptional regulation of oFSHbeta and alpha-subunit genes by GnRH in HeLa cells was investigated. Although 12-O-tetradecanoyl 13-acetate induction of alpha-Luc and -215oFSHbeta-Luc could be completely blocked in a dose-dependent manner by the specific PKC inhibitor bisindolylmaleimide I, only 57-65% of the GnRH-mediated stimulation of these promoters was blocked, demonstrating the involvement of PKC as well as other signaling systems in GnRH induction. These data define a molecular action of GnRH on oFSHbeta gene transcription that involves two proximal AP-1 enhancer elements and PKC activation. Furthermore, these studies establish the usefulness of HeLa and COS-7 cells to investigate specific aspects of GnRH action on gonadotropin subunit gene expression, as similar signaling pathways and transcription factors that are activated by GnRH in gonadotropes (such as PKC, mitogen-activated protein kinase, Ca2+, and AP-1) exist in these cells.

Effects of prednisolone on glomerular signal transduction cascades in experimental glomerulonephritis.

In vitro data support that activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) regulate the gene expression of numerous growth factors and cytokines involved in the development of glomerulonephritis (GN). However, the in vivo activation and role of these transcription factors are poorly understood. This study examines whether these transcription factors are activated in antithymocyte serum (ATS)-induced GN in vivo and whether prednisolone suppresses activation of them. As assessed by gel mobility shift assay, glomerular DNA binding activity of AP-1 containing both c-Jun and c-Fos and NF-kappaB composed of P-50 and P-65 subunits was significantly increased after ATS injection. Furthermore, as estimated by in-gel kinase assay, glomerular activity of extracellular signal-regulated kinases (ERK) and c-jun NH2-terminal kinases (JNK), which are mitogen-activated protein kinases (MAPK) known to activate AP-1 and NF-kappaB in vitro, was significantly increased after ATS injection, preceding the increase in AP-1 activity. Prednisolone treatment significantly prevented the increase in urinary protein and albumin excretion and glomerular cell proliferation in ATS-induced GN, indicating the beneficial effects of prednisolone on this GN. Prednisolone significantly suppressed the increased glomerular ERK and JNK activities and AP-1 binding activity, but not glomerular NF-kappa binding activity. This study provides the first evidence of the marked increase in glomerular MAPK activities, and AP-1 and NF-kappa binding activities in ATS-induced GN. The beneficial effect of prednisolone on this GN may be partially mediated by the suppression of MAPK, followed by the suppression of AP-1.

Activation of glomerular mitogen-activated protein kinases in angiotensin II-mediated hypertension.

The in vivo signal transduction pathway, responsible for hypertension-induced glomerular injury, remains to be clarified. In this study, the effect of angiotensin II (Ang II)-induced hypertension was examined on glomerular mitogen activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK), and on glomerular transcription factors activator protein-1 (AP-1) and Sp 1. MAPK activities were determined by in-gel kinase assay. DNA binding activity of AP-1 and Sp 1 was determined by gel mobility shift assay. Continuous infusion of Ang II (1000 ng/kg per min, intravenously) to conscious rats rapidly increased BP, followed by the rapid and transient activation of glomerular p42 and p44 ERK and p46 and p55 JNK with the peak at 15 to 180 min. Glomerular AP-1 binding activity was increased 2.6-fold (P < 0.01) at 24 h after the start of Ang II infusion. Supershift analysis showed that the activated AP-1 complexes contained c-Fos and c-Jun proteins. On the other hand, glomerular Sp 1 DNA binding activity was not changed throughout 7 d of Ang II infusion. These results provided the first in vivo evidence that Ang II-induced hypertension causes the activation of glomerular ERK and JNK, leading to the activation of AP-1. Thus, ERK and JNK signaling cascades, via the activation of AP-1, may be implicated in the development of hypertension-induced glomerular injury.

Peripheral Axotomy Induces Long-Term c-Jun Amino-Terminal Kinase-1 Activation and Activator Protein-1 Binding Activity by c-Jun and junD in Adult Rat Dorsal Root GangliaIn Vivo

One of the earliest documented molecular events after sciatic nerve injury in adult rats is the rapid, long-term upregulation of the immediate early gene transcription factor c-Jun mRNA and protein in lumbar dorsal root ganglion (DRG) neurons, suggesting that c-Jun may regulate genes that are important both in the early post-injury period and during later peripheral axonal regeneration. However, neither the mechanism through which c-Jun protein is increased nor the level of its post-injury transcriptional activity in axotomized DRGs has been characterized. To determine whether transcriptional activation of c-Jun occurs in response to nerve injury in vivo and is associated with axonal regeneration, we have assayed axotomized adult rat DRGs for evidence of jun kinase activation, c-Jun phosphorylation, and activator protein-1 (AP-1) binding. We report that sciatic nerve transection resulted in chronic activation of c-Jun amino-terminal kinase-1 (JNK) in L4/L5 DRGs concomitant with c-Jun amino-terminal phosphorylation in neurons, and lasting AP-1 binding activity, with both c-Jun and JunD participating in DNA binding complexes. The timing of JNK activation was dependent on the distance of the axotomy site from the DRGs, suggesting the requirement for a retrograde transport-mediated signal. AP-1 binding and c-Jun protein returned to basal levels in DRGs as peripheral regeneration was completed but remained elevated in the case of chronic sprouting, indicating that c-Jun may regulate target genes that are involved in axonal outgrowth.

Cytokine-mediated differential induction of hepatic activator protein-1 genes

Background: Tumor necrosis factor–α (TNF-α) and interleukin-6 (IL-6) increase the synthesis of hepatic acute-phase proteins; these effects appear mediated by activation of transcription factors. The purpose of this study was to determine the effects of TNF-α and IL-6 on expression of the jun family of activator protein-1 (AP-1) transcription factors with the human hepatoma cell line HepG2, a well-characterized model of the hepatic acute-phase response. Methods: HepG2 cells, treated with either TNF-α (100 ng/ml) or IL-6 (10 ng/ml), were extracted for RNA and protein (total and nuclear) and analyzed. Results: TNF-α increased c-jun and junD mRNA and c-Jun and JunD protein levels, as well as AP-1 binding activity. IL-6 increased c-jun mRNA, c-Jun protein, and AP-1 binding activity but did not affect either junD or junB expression. Conclusions: TNF-α and IL-6 induce a differential pattern of AP-1 expression in HepG2 cells; TNF-α increases both c-Jun and JunD, whereas IL-6 stimulates only c-Jun. Neither TNF-α nor IL-6 stimulates JunB. Multiple cytokines, released during stress, may act in concert to stimulate the AP-1 proteins, which ultimately culminate in the downstream synthesis of a variety of acute-phase proteins. (Surgery 1998;123:191-8.)

Cytokine-mediated differential induction of hepatic activator protein-1 genes

<h2>Abstract</h2> <b>Background:</b> Tumor necrosis factor–α (TNF-α) and interleukin-6 (IL-6) increase the synthesis of hepatic acute-phase proteins; these effects appear mediated by activation of transcription factors. The purpose of this study was to determine the effects of TNF-α and IL-6 on expression of the jun family of activator protein-1 (AP-1) transcription factors with the human hepatoma cell line HepG2, a well-characterized model of the hepatic acute-phase response. <b>Methods:</b> HepG2 cells, treated with either TNF-α (100 ng/ml) or IL-6 (10 ng/ml), were extracted for RNA and protein (total and nuclear) and analyzed. <b>Results:</b> TNF-α increased c-jun and junD mRNA and c-Jun and JunD protein levels, as well as AP-1 binding activity. IL-6 increased c-jun mRNA, c-Jun protein, and AP-1 binding activity but did not affect either junD or junB expression. <b>Conclusions:</b> TNF-α and IL-6 induce a differential pattern of AP-1 expression in HepG2 cells; TNF-α increases both c-Jun and JunD, whereas IL-6 stimulates only c-Jun. Neither TNF-α nor IL-6 stimulates JunB. Multiple cytokines, released during stress, may act in concert to stimulate the AP-1 proteins, which ultimately culminate in the downstream synthesis of a variety of acute-phase proteins. (Surgery 1998;123:191-8.)

Bcl-2 enhancement of malignant transformation in mouse epidermal JB6 cells.

Increased bcl-2 expression is a common feature of many types of human malignancies, which implies that bcl-2 plays an important role in tumorigenesis. To better understand the molecular mechanisms of bcl-2-induced oncogenesis, we examined the effects of bcl-2 expression on transformation of mouse epidermal JB6 cells induced by the tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA). Promotion-sensitive JB6 clone41 cells were transfected with the bcl-2-containing expression vector pD5-neo/bcl-2, and the soft agar growth of bcl-2-transfected cells and control cells were compared. bcl-2 overexpression in JB6 clone41 cells caused a TPA-induced soft-agar growth fivefold greater than the growth of nontransfected or vector-transfected (neo control) cells. bcl-2 expression in the absence of TPA did not lead to colony formation in soft agar. Because the level of the transcription factor activator protein 1 (AP-1) has been shown to be critical for the responsiveness of JB6 cells to TPA-induced transformation, we compared c-jun and c-fos expression as well as the AP-1-binding activity and the AP-1-mediated transactivation of the reporter construct TRE-CAT between bcl-2-expressing cells and control cells. When compared with control cells, bcl-2-transfected cells expressed significantly more c-fos but not c-jun after TPA treatment. Furthermore, the levels of AP-1 and AP-1-induced transactivation of TRE-CAT were greater in bcl-2-transfected cells than in control cells after TPA treatment. These results showed that bcl-2 cooperates with a tumor promoter such as TPA in the induction of malignant transformation in mouse epidermal cells and that bcl-2 enhances soft-agar growth by stimulating signaling pathways that led to increased AP-1 expression.

Interleukin-7 Upregulates the Interleukin-2–Gene Expression in Activated Human T Lymphocytes at the Transcriptional Level by Enhancing the DNA Binding Activities of Both Nuclear Factor of Activated T Cells and Activator Protein-1

In the present report, we studied the role of the stromal-derived cytokine interleukin-7 (IL-7) in the IL-2–gene regulation in activated T lymphocytes. Production of IL-2 requires the formation of transcription factors involved in the IL-2 –gene regulation. T-cell receptor (TCR)/CD3 engagement results in the activation of nuclear factor of activated T cells (NFAT), activator protein-1 (AP-1), and nuclear factor κB (NFκB), whereas the CD28 responsive complex (CD28RC) is activated in response to the CD28 signal. Costimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with IL-7 induces a fivefold enhanced IL-2–mRNA accumulation and a 2.5-fold enhanced protein secretion. The IL-2–gene transcription rate is increased 3.4-fold, indicating that the effect of IL-7 is in part mediated at the transcriptional level. The molecular mechanisms underlying the IL-7 effect involve the upregulation of the DNA binding activity of NFAT (60%) and AP-1 (120%), without affecting the activities of NFκB and CD28RC, which was confirmed by transfection assays. We also show that the IL-7–induced enhancement of the AP-1–DNA binding activity is not cyclosporin A-sensitive. Since AP-1 is part of the NFAT complex, we conclude that the IL-7–signaling pathway is involved in the activation of the fos and jun proteins of which AP-1 consists.

Interleukin-7 Upregulates the Interleukin-2–Gene Expression in Activated Human T Lymphocytes at the Transcriptional Level by Enhancing the DNA Binding Activities of Both Nuclear Factor of Activated T Cells and Activator Protein-1

AbstractIn the present report, we studied the role of the stromal-derived cytokine interleukin-7 (IL-7) in the IL-2–gene regulation in activated T lymphocytes. Production of IL-2 requires the formation of transcription factors involved in the IL-2 –gene regulation. T-cell receptor (TCR)/CD3 engagement results in the activation of nuclear factor of activated T cells (NFAT), activator protein-1 (AP-1), and nuclear factor κB (NFκB), whereas the CD28 responsive complex (CD28RC) is activated in response to the CD28 signal. Costimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with IL-7 induces a fivefold enhanced IL-2–mRNA accumulation and a 2.5-fold enhanced protein secretion. The IL-2–gene transcription rate is increased 3.4-fold, indicating that the effect of IL-7 is in part mediated at the transcriptional level. The molecular mechanisms underlying the IL-7 effect involve the upregulation of the DNA binding activity of NFAT (60%) and AP-1 (120%), without affecting the activities of NFκB and CD28RC, which was confirmed by transfection assays. We also show that the IL-7–induced enhancement of the AP-1–DNA binding activity is not cyclosporin A-sensitive. Since AP-1 is part of the NFAT complex, we conclude that the IL-7–signaling pathway is involved in the activation of the fos and jun proteins of which AP-1 consists.

The engagement of CD4 surface antigen in the HEL haemopoietic cell line up-regulates the transforming growth factor-beta1 (TGF-beta1) promoter activity.

In order to investigate the effect of CD4 engagement on the transforming growth factor beta1 (TGF-beta1) promoter activity in haemopoietic progenitors, HEL cells were transiently transfected with a plasmid vector containing -453/+11 nucleotides of the TGF-beta1 promoter fused with the bacterial chloramphenicol acetyltransferase (CAT) gene and then treated with various agonists. Both cross-linked CD4mAb and envelope gp120 were able to significantly up-regulate CAT activity with respect to the levels of activation observed in HEL cells treated with cross-linked CD8 mAb or p24. By using deletion mutants of the TGFbeta1 promoter, we found that the minimal DNA sequence still responsive to cross-linked CD4 mAb or gp120 was located between nucleotides -453/-323 of the TGF-beta1 promoter, which contains two activating protein 1 (AP1) binding sites. In electromobility shift assays (EMSA) we could demonstrate that CD4 engagement of HEL cells induced a significant increase of AP1 binding activity at the nuclear level. Furthermore, the steady-state mRNA of endogenous TGF-beta1 showed a small but reproducible increase when HEL cells were treated with cross-linked CD4mAb or gp120. Altogether, these findings suggest that the engagement of CD4 in HEL cells modulates TGF-beta1 expression, acting predominantly at the transcriptional level.

Characterization of NMDA- and AMPA-induced enhancement of AP-1 DNA binding activity in rat cerebellar granule cells

Effects of the glutamate receptor agonists, N-methyl- d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), on the activator protein-1 (AP-1) DNA binding activity were studied in primary cultures of rat cerebellar granule cells. Application of NMDA as well as of AMPA produced a concentration-dependent enhancement of AP-1 binding. Further examination revealed that only a brief exposure (10 min) to NMDA or AMPA was required for the initiation of a significant, four- to sixfold enhancement of AP-1 DNA binding activity. Blockade of the desensitization of AMPA receptors by cyclothiazide further reduced the exposure time needed to activate the AP-1 complex. The time needed to achieve a maximal increase of AP-1 binding activity varied depending on the glutamate receptor agonist used. NMDA gave maximal AP-1 stimulation after 60 min exposure, whereas stimulation with AMPA alone reached a maximum after 240 min exposure. When AMPA was applied together with cyclothiazide the maximal enhancement of AP-1 binding was reached much faster, within 120 min. Supershift analysis with specific antibodies against the members of Fos and Jun protein families (c-Fos, Fos B, c-Jun, Jun B, Jun D) revealed that the NMDA-induced AP-1 complex was composed predominantly of Jun D and c-Fos. The composition of the AP-1 complex activated by AMPA alone was similar to that produced by NMDA, but with an additional contribution of Fos B. In contrast, application of AMPA plus cyclothiazide induced an AP-1 transcription with contribution of Jun D, c-Fos, Fos B, c-Jun and Jun B proteins. These findings indicate that glutamate is able to enhance AP-1 DNA binding activity in cerebellar granule cells through both NMDA and AMPA glutamate receptors.

Effect of acute and chronic administration of methamphetamine on activator protein-1 binding activities in the rat brain regions.

The activator protein-1 (AP-1) binding activities in the three brain regions (striatum, nucleus accumbens, cingulate cortex) increased after a single methamphetamine (METH, 4 mg/kg) injection and reached maximum levels after 180 min. Pretreatment with SCH 23390 (0.5 mg/kg), a selective dopamine D1 receptor antagonist, significantly inhibited the enhanced AP-1 binding activities induced by acute METH (4 mg/kg) administration. In chronic experiments, rats were pretreated with METH (4 mg/kg) or saline for 14 days. The AP-1 binding activities after a 1-week abstinence from chronic administration of MAP increased significantly in all the brain regions compared with those of the saline-treated controls, whereas after a 4-week abstinence, the AP-1 binding activity decreased significantly in the cingulate cortex, but not striatum or nucleus accumbens, compared with the saline-treated control group. A METH challenge after a 4-week abstinence period induced significantly more intense stereotypy, but lower AP-1 binding activities in all the brain regions of rats treated with repeated METH than repeated saline injections. The super-shift assay revealed that after a 1- or 4-week abstinence, there was no significant difference between the Fos-related antigens (Fras) contents of the saline- and METH-treated groups in any brain region examined, and that the Jun family protein levels of the METH-treated group increased significantly in the striatum and nucleus accumbens after a 1-, but not 4-, week abstinence. These results suggest that chronic METH administration leads to delayed decay of the induced AP-1 binding activities and Jun component levels after abstinence for up to 1 week, but results in no change in or decreases these activities and attenuates METH challenge-induced AP-1 binding activities after abstinence for 4 weeks.

Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation.

Developmental studies of oncogene expression implicate the Fos and Jun family of transcription factors in the regulation of bone growth and differentiation. Promoters of many developmentally regulated genes, including osteocalcin, a marker of osteoblast differentiation, contain AP-1 sites that bind Fos/Jun dimers. Here, we demonstrate that the selective expression of fos- and jun-related genes is functionally related to the stage of osteoblast growth and differentiation in vitro. During osteoblast proliferation, nuclear protein levels of all seven activating protein-1 (AP-1) members are maximal. Subsequently, during the period of extracellular matrix maturation, levels decline. In fully differentiated osteoblasts, Fra-2 and (to a lesser extent) Jun D are the principal AP-1 members detectable by Western blot analysis. AP-1 complex composition and binding activity also exhibit developmental changes. All Fos and Jun family members are involved in AP-1 complex formation in proliferating cells, whereas Fra-2 and Jun D predominate in AP-1 complexes in differentiated osteoblasts. Overexpression of Fos and Jun family members in ROS 17/2.8 cells markedly affects the expression of an osteocalcin promoter-chloramphenicol acetyltransferase construct. Coexpression of only one AP-1 pair, Fra-2 and Jun D, stimulated reporter expression, whereas coexpression of other AP-1 pairs down-regulated expression (i.e. c-jun and any Fos family member) or had no effect (i.e. Fra-1 and Jun B). Promoter deletion analyses indicate that these effects are site specific. Consequential effects of Fra-2 on osteoblast differentiation are further demonstrated by antisense studies in which osteoblast differentiation and the development of a bone tissue-like organization were suppressed. Consistent with recent findings suggesting that AP-1 complex composition can selectively regulate gene transcription, our findings demonstrate that differential expression of Fos and Jun family members could play a role in the developmental regulation of bone-specific gene expression and, as a result, may be functionally significant for osteoblast differentiation.

Activator protein-1 binding activities in discrete regions of rat brain after acute and chronic administration of methamphetamine.

The activator protein-1 (AP-1) binding activities increased in three brain regions (striatum, nucleus accumbens, and cingulate cortex) after a single methamphetamine (METH) injection to rats. Pretreatment with SCH 23390, but not (-)-eticlopride, significantly inhibited the enhanced AP-1 binding activities induced by acute METH administration. The magnitude of enhancement of AP-1 binding activities 3 h after the last dose of chronic METH administration (4 mg/kg once daily for 14 days) was significantly attenuated as compared with those 3 h after a single METH administration. The AP-1 binding activities after a 1-, but not 4-, week abstinence from chronic administration of METH were still significantly higher than those of the saline-treated controls. A METH challenge after a 4-week abstinence period induced significantly lower AP-1 binding activities in rats chronically injected with METH than in rats chronically injected with saline. The supershift assay revealed that the levels of Jun family protein, but not Fos-related antigen, increased significantly in the striatum and nucleus accumbens of chronically METH-treated rats after a 1-week abstinence. These results suggest that chronic METH administration leads to delayed decay of the induced AP-1 binding activities and Jun component levels after abstinence for up to 1 week but results in no change in or decreases these activities and attenuates METH challenge-induced AP-1 binding activities after abstinence for 4 weeks.

Seizure activity results in a rapid induction of nuclear factor-kappa B in adult but not juvenile rat limbic structures.

Previous studies have indicated that increased formation of oxygen free radicals is likely to participate in the cascade of events leading to neuronal damage following kainic acid (KA)-induced seizure activity. As reactive oxygen species are involved in signal transduction pathways leading to nuclear factor-kappaB (NF-kappaB) activation, we examined the effects of KA treatment on the activation of NF-kappaB in adult and juvenile rat brain. For comparison, changes in two other transcription factors, activator protein-1 (AP-1) and Sp1, were also determined. In adult rat piriform cortex and hippocampus, significant induction of NF-kappaB was observed at 4 h after KA injection, and the maximal increase was reached at 8-16 h posttreatment. NF-kappaB binding activities returned to control levels by 5 days after injection. NF-kappaB binding activities were slightly decreased in adult rat cerebellum at 8 and 16 h after KA treatment. In the juvenile rat, no significant changes in NF-kappaB binding activity were observed in piriform cortex, hippocampus, and cerebellum after KA injection. Changes in AP-1 binding activity were qualitatively similar to those observed with NF-kappaB in adult but not juvenile rat brain, as AP-1 was significantly induced in juvenile piriform cortex and hippocampus following KA injection. On the other hand, little or no changes in Sp1 activity were detected in adult and juvenile rat brain. Our results provide further evidence that oxidative stress participates in neuronal damage resulting from KA-induced seizure activity.

Activating protein-1 cooperates with phorbol ester activation signals to increase HIV-1 expression.

To determine whether Jun and Fos, components of the activating protein-1 (AP-1) transcription factor, transactivate HIV-1 proviral expression. The effects of phorbol myristate acetate (PMA) and Jun or Fos transcription factors on HIV-1 expression were investigated using a provirus clone and long terminal repeat (LTR)-reporter gene constructs. The influence of PMA stimulation on AP-1 binding activity was determined with antibodies in gel mobility shift assays. Activation of HIV-1 provirus and transcription of HIV-1 LTR sequences in response to cotransfection of Jun or Fos expression plasmids into a permissive colon cancer cell line, SW480, were assessed by p24 core antigen capture and reporter gene assays, respectively. The effect of protein kinase C activation was evaluated by comparing cells grown in the presence or absence of PMA (20 ng/ml). Cotransfection of HIV-1 provirus and expression plasmids for c-Jun or JunB into SW480 cells resulted in increased p24 core antigen and this response was markedly increased following PMA stimulation of cells. c-Fos or JunD alone did not increase p24 production but markedly increased p24 production in PMA-stimulated cells. PMA increased c-Fos and JunD binding activity on an AP-1 binding site within the U5 region of the LTR, as shown in gel mobility shift assays. Functional analysis of this site by transient transfections demonstrated it was required to mediate c-Fos and JunD transactivation of the HIV-1 LTR. Specific Jun and Fos transcription factors can transactivate the HIV-1 provirus and this response is markedly increased in cooperation with cellular activation signals elicited by PMA. Taken together, the data indicate that AP-1 binding sites downstream of the transcriptional start site in the HIV-1 LTR are capable of binding c-Fos and JunD and may contribute to transactivation of HIV-1 provirus.