Activator Protein-1 DNA Binding Activity Research Articles

Induction of gene expression via activator protein-1 in the ascorbate protection against UV-induced damage

UV irradiation is a major insult to the skin. We have shown previously that exogenous vitamin C (ascorbate) accumulates in HaCaT keratinocytes, thus conferring the ability to prevent radical formation and cell death elicited by UV-B. Here, we have investigated the potential mechanisms accounting for the cytoprotective effects exerted by this antioxidant. Using a cDNA microarray hybridization, we identified several genes whose expression was up-regulated by ascorbate. We focused on the fra-1 gene, a member of the Fos family of transcription factors that down-regulates activator protein-1 (AP-1) target genes. Both in HaCaT and in normal human epidermal keratinocytes, we found Fra-1 mRNA induction as early as 2h after ascorbate loading. Electrophoretic mobility-shift assay and antibody supershift analysis revealed that ascorbate modulates AP-1 DNA-binding activity and that Fra-1 is in AP-1 complexes in treated cells. Furthermore, transient-transfection studies, using an AP-1 reporter construct, showed that ascorbate was able to inhibit both basal and UV-B-induced AP-1-dependent transcription. Ascorbate also modulates UV-B-induced AP-1 activity by preventing the phosphorylation and activation of the upstream c-Jun N-terminal kinase (JNK), thus inhibiting phosphorylation of the endogenous c-Jun protein. These data suggest that ascorbate mediates cellular responses aimed at counteracting UV-mediated cell damage and cell death by interfering at multiple levels with the activity of the JNK/AP-1 pathway and modulating the expression of AP-1-regulated genes.

Phorbol ester responsiveness of the glutathione S-transferase P1 gene promoter involves an inducible c-jun binding in human K562 leukemia cells

Overexpression of the glutathione S-transferase P1 (GSTP1) gene is related to drug resistance in human cancer cells. However, the mechanisms of the transcriptional activation of this gene remain unclear. In this study, we examined the molecular mechanisms underlying phorbol ester mediated gene regulation using human K562 leukemia cells as a model. Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor site was crucial for 12- O-tetradecanoyl phorbol 13-acetate (TPA)-mediated GSTP1 gene transcription. Electrophoretic mobility shift assays and transient transfection analysis demonstrated that both DNA binding and transactivation activities of AP-1 were induced by TPA. By supershift analysis, we identified transcription factors c-jun and fra-1 as well as NF-E2p45 as components of the induced binding complex. These results show for the first time that the phorbol ester TPA is involved in the molecular mechanism(s) mediating the activation of the GSTP1 promoter in a human leukemia model.

High cholesterol diet down regulates the activity of activator protein-1 but not nuclear factor-kappa B in rabbit brain

Cardiovascular risk factors and alterations in cholesterol metabolism are implicated in the pathogenesis of Alzheimer's dementia (AD). The hypercholesterolemic rabbit model of atheroslerosis and AD was utilized in this study to examine oxidative stress related changes in the brain. The high cholesterol diet induced dramatic increases in plasma and liver cholesterol concentrations, but brain cholesterol levels remained constant. Similar effects have been found regarding lipid oxidation products. The amounts of conjugated dienes, trienes and thiobarbituric acid reactive substances (TBARS) significantly increased in the plasma of cholesterol treated animals while the brain cortex showed no signs of increased lipid peroxidation. The oxidative damage sensitive nuclear transcription factor kappa B (NF-κB) and activator protein-1 (AP-1) diverged in their responses. Accordingly, the AP-1 DNA binding activity decreased by more than 50% in brain nuclear protein extracts while the NF-κB binding activity remained unaltered by the hypercholesterol diet. These results indicate that despite the relative resistance of the central nervous system to dietary manipulation of its lipid composition and lipid peroxidation products, chronic dietary intake of cholesterol can alter the function of certain proteins involved in regulation of gene expression in the brain.

Influence of proteasome and redox state on heat shock-induced activation of stress kinases, AP-1 and HSF

We studied the pattern of activation of stress kinases and of transcription factors activator protein-1 (AP-1) and heat shock factor (HSF) in FAO cells by combining two treatments, i.e. heating (42°C for 1 h) and proteasome inhibition, each known to cause cellular heat shock response. The co-treatment heat shock (HS) and proteasome inhibitor (a peptidyl aldehyde or lactacystin) showed cumulative effects on the intensity and duration of activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) at the end of the HS period and during recovery. Similarly, the thiol-reducing agents N-(2-mercaptoethyl)-1,3-diaminopropane and dithiothreitol strongly activated both JNK and p38 MAPK in cells undergoing HS. AP-1 DNA binding activity in response to proteasome inhibitors was so strong that it shadowed the stimulatory effect of HS in the combined treatment, but lactacystin, which is the most potent and specific proteasome inhibitor, decreased the binding late during recovery from HS. Thiol-reducing agents prevented AP-1 DNA binding induced by HS. The combined HS/proteasome inhibitors or HS/thiol-reducing agents treatments cooperatively activated HSF DNA binding. Expression of collagenase I and hsp 70 mRNAs reflects the different behavior of AP-1 and HSF transcription factors in cells exposed to HS and proteasome inhibition. The data seem to indicate that JNK and p38 MAPK activations are not necessarily coupled to DNA binding of AP-1, which can be either increased or inhibited when these kinases are activated. AP-1 and HSF show opposite patterns of response to HS in the presence of proteasome inhibitors or reducing agents.

Repeated administration of CGP 46381, a γ-aminobutyric acid B antagonist, and ethosuximide suppresses seizure-associated cyclic adenosine 3′5′ monophosphate response element- and activator protein-1 DNA-binding activities in lethargic ( lh/ lh) mice

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic ( lh/ lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control ( +/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.

Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo.

Most breast cancers, even those that are initially responsive to tamoxifen, ultimately become resistant. The molecular basis for this resistance, which in some patients is thought to involve stimulation of tumor growth by tamoxifen, is unclear. Tamoxifen induces cellular oxidative stress, and because changes in cell redox state can activate signaling pathways leading to the activation of activating protein-1 (AP-1), we investigated whether tamoxifen-resistant growth in vivo is associated with oxidative stress and/or activation of AP-1 in a xenograft model system where resistance is caused by tamoxifen-stimulated growth. Control estrogen-treated, tamoxifen-sensitive, and tamoxifen-resistant MCF-7 xenograft tumors were assessed for oxidative stress by measuring levels of antioxidant enzyme (e.g., superoxide dismutase [SOD], glutathione S-transferase [GST], and hexose monophosphate shunt [HMS]) activity, glutathione, and lipid peroxidation. AP-1 protein levels, phosphorylated c-jun levels, and phosphorylated Jun NH(2)-terminal kinase (JNK) levels were examined by western blot analyses, and AP-1 DNA-binding and transcriptional activities were assessed by electrophoretic mobility shift assays and a reporter gene system. All statistical tests are two-sided. Compared with control estrogen-treated tumors, tamoxifen resistant tumors had statistically significantly increased SOD (more than threefold; P=.004) and GST (twofold; P=.004) activity and statistically significantly reduced glutathione levels (greater than twofold; P<.001) and HMS activity (10-fold; P<.001). Lipid peroxides were not significantly different between control and tamoxifen-resistant tumors. We observed no differences in AP-1 protein components or DNA-binding activity. However, AP-1-dependent transcription (P=.04) and phosphorylated c-Jun and JNK levels (P<.001) were statistically significantly increased in the tamoxifen-resistant tumors. Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth.

The mechanism of taurine chloramine inhibition of cytokine (interleukin-6, interleukin-8) production by rheumatoid arthritis fibroblast-like synoviocytes.

Taurine chloramine (Tau-Cl) has been shown to inhibit the production of proinflammatory cytokines (interleukin-6 [IL-6] and IL-8) by fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients. The present study was conducted to elucidate the mechanism of inhibitory action exerted by Tau-Cl. The effects of Tau-Cl on 1) the transcription of genes coding for IL-6 and IL-8, and 2) the activity of nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors, which are crucial for the transcription of these cytokine genes, were investigated in FLS isolated from the synovial tissue of RA patients. FLS were cultured in vitro for 3-6 passages and stimulated with recombinant human IL-1beta (1 ng/ml) in the presence of either Tau or Tau-Cl, which were added simultaneously with the stimulus at concentrations of 250 microM or 500 microM. The relative expression of IL-6 and IL-8 messenger RNA (mRNA) was evaluated after 4 hours of stimulation, using competitive reverse transcriptase-polymerase chain reaction. The DNA binding activity of NF-kappaB and AP-1 was examined 30 minutes and 2 hours after cell stimulation, respectively, using electromobility gel shift assay. IL-1beta triggered a significant rise in the activity of transcription factors NF-kappaB and AP-1, followed by an elevation of cytokine IL-6 and IL-8 mRNA expression. Tau-Cl, but not Tau, reduced IL-1beta-triggered cytokine mRNA expression, exerting stronger inhibitory activity on the levels of IL-6 than on those of IL-8. Importantly, Tau-Cl also diminished the activity of NF-kappaB and, to a lesser extent, that of AP-1 transcription factor. Neither IL-1beta nor Tau-Cl affected the activity of octamer transcription factor 1. Tau-Cl inhibition of IL-6 and IL-8 synthesis in FLS from RA patients results from the ability of this compound to diminish the activity of the major transcriptional regulators (NF-kappaB and AP-1), which subsequently reduces the transcription of these cytokine genes.

The mechanism of taurine chloramine inhibition of cytokine (interleukin-6, interleukin-8) production by rheumatoid arthritis fibroblast-like synoviocytes

Taurine chloramine (Tau-Cl) has been shown to inhibit the production of proinflammatory cytokines (interleukin-6 [IL-6] and IL-8) by fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients. The present study was conducted to elucidate the mechanism of inhibitory action exerted by Tau-Cl.The effects of Tau-Cl on 1) the transcription of genes coding for IL-6 and IL-8, and 2) the activity of nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors, which are crucial for the transcription of these cytokine genes, were investigated in FLS isolated from the synovial tissue of RA patients. FLS were cultured in vitro for 3-6 passages and stimulated with recombinant human IL-1beta (1 ng/ml) in the presence of either Tau or Tau-Cl, which were added simultaneously with the stimulus at concentrations of 250 microM or 500 microM. The relative expression of IL-6 and IL-8 messenger RNA (mRNA) was evaluated after 4 hours of stimulation, using competitive reverse transcriptase-polymerase chain reaction. The DNA binding activity of NF-kappaB and AP-1 was examined 30 minutes and 2 hours after cell stimulation, respectively, using electromobility gel shift assay.IL-1beta triggered a significant rise in the activity of transcription factors NF-kappaB and AP-1, followed by an elevation of cytokine IL-6 and IL-8 mRNA expression. Tau-Cl, but not Tau, reduced IL-1beta-triggered cytokine mRNA expression, exerting stronger inhibitory activity on the levels of IL-6 than on those of IL-8. Importantly, Tau-Cl also diminished the activity of NF-kappaB and, to a lesser extent, that of AP-1 transcription factor. Neither IL-1beta nor Tau-Cl affected the activity of octamer transcription factor 1.Tau-Cl inhibition of IL-6 and IL-8 synthesis in FLS from RA patients results from the ability of this compound to diminish the activity of the major transcriptional regulators (NF-kappaB and AP-1), which subsequently reduces the transcription of these cytokine genes.

Src-Dependent Erk1/2-Mediated Signaling Pathways and Associated Ang Ii-Stimulated Growth Responses Are Altered in Vascular Smooth Muscle Cells from Arteries of Hypertensive Patients.

P154 Molecular mechanisms underlying vascular remodeling in human hypertension are unclear. We investigated c-Src and ERK1/2 activity, proto-oncogene expression, activating protein 1 (AP-1) DNA-binding activity, and DNA and protein synthesis in Ang II-stimulated vascular smooth muscle cells (VSMC) derived from small peripheral arteries from normotensive subjects (NT) (n=5) and age-matched untreated hypertensive patients (HT) (n=10). The media and media:lumen ratio were greater (p&lt;0.05) in arteries from HT than NT, indicating vascular remodeling. Ang II-dose-dependently increased synthesis of DNA and protein, with enhanced effects (p&lt;0.01) in VSMCs from HT. PD98059, a selective inhibitor of the ERK1/2 pathway, attenuated (p&lt;0.01) Ang II-stimulated growth. PD98059 effects were greater in HT than NT. In NT, Ang II transiently increased ERK1/2 phosphorylation. In HT, Ang II-stimulated ERK1/2 activation was significantly augmented (p&lt;0.05 vs NT) and sustained. PP2, a selective Src inhibitor, reduced ERK1/2 activity and normalized responses in HT. Ang II-induced c-Src phosphorylation was 2-3 fold greater in HT than NT. In HT, kinase activation was followed by overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity. PD98059 attenuated these responses. In HT cells transfected with c-fos antisense oligodeoxynucleotide (ODN), Ang II-stimulated DNA synthesis was reduced compared with sense ODN. These novel findings suggest that vascular remodeling is associated with augmented Ang II-stimulated VSMC growth, which is mediated via hyperactivation of c-Src-regulated, ERK1/2-dependent pathways leading to overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity. Our data define a signal transduction pathway in VSMCs that could contribute to structural changes and vascular remodeling in essential hypertension.

Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA.

The hormone bombesin (BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) act through specific GRP receptors (GRP-R) to affect multiple cellular functions in the gastrointestinal tract; the intracellular signaling pathways leading to these effects are not clearly defined. Previously, we demonstrated that the human gastric cancer SIIA possesses GRP-R and that BBS stimulates activator protein-1 (AP-1) gene expression. The purpose of our present study was to determine the signaling pathways leading to AP-1 induction in SIIA cells. A rapid induction of c-jun and jun-B gene expression was noted after BBS treatment; this effect was blocked by specific GRP-R antagonists, indicating that BBS is acting through the GRP-R. The signaling pathways leading to increased AP-1 gene expression were delineated using phorbol 12-myristate 13-acetate (PMA), which stimulates protein kinase C (PKC)-dependent pathways, by forskolin (FSK), which stimulates protein kinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSK stimulated jun-B expression but produced only minimal increases of c-jun mRNA and AP-1 binding activity. Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKC inhibitors) inhibited the induction of c-jun and jun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor) exhibited only minimal effects. Pretreatment with tyrphostin-25, a protein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediated induction of c-jun and jun-B, but the effect was not as pronounced as with H-7. Collectively, our results demonstrate that BBS acts through its receptor to produce a rapid induction of both c-jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction of AP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimal involvement of PKA.

Activated Raf inhibits myogenesis through a mechanism independent of activator protein 1-mediated myoblast transformation.

Skeletal myogenesis is acutely affected by growth factors and subsequent activation of their respective intracellular signaling cascades. Components of the mitogenic Ras/Raf/mitogen-activated protein kinase (MAPK) signaling module are potent inhibitors of myoblast differentiation. However, the means by which these kinases prevent myocyte formation and activation of the muscle gene program is unknown. Activator protein 1 (AP-1) is a transcriptional regulator the actions of which are up-regulated by signaling events, including elevated MAPK. Because activated Raf inhibits avian myogenesis in a MAPK-dependent fashion, we investigated the role of AP-1 as a mediator of the Raf-imposed block to myogenesis. Avian myoblasts overexpressing activated Raf contain elevated levels of AP-1 DNA binding and transcriptional activity. Introduction of an AP-1 dominant inhibitory protein (AFOS) into Raf-expressing myoblasts prevented acquisition of a transformed morphology. Interestingly, these cells remained differentiation-defective. Myogenic cells cotransduced with RCAS(A)-Raf BXB and RCAS(B)-AFOS remained mononuclear and myosin-negative and did not activate significantly muscle-specific reporter genes. These results argue that Raf inhibits muscle differentiation independent of AP-1-mediated cell transformation. Our results provide evidence for AP-1 as a critical component of the transforming capacity of activated Raf and evidence that AP-1 is not involved in the myogenic inhibitory effects of the kinase.

Gene, stimulus and cell-type specific regulation of activator protein-1 in mesangial cells by lipopolysaccharide and cytokines

Activator protein-1 (AP-1) plays an important role in the regulation of gene expression in mesangial cells (MC) during the pathogenesis of glomerular inflammatory disease. The precise regulation of the AP-1 family by agents that are known to activate MC is, however, poorly understood. The action of platelet-derived growth factor (PDGF) and, for the first time, lipopolysaccharide (LPS), interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) on AP-1 gene expression in MC was therefore studied. Whilst the expression of JunD was not affected by any of the mediators, the mRNA levels of c-fos and JunB were induced by LPS, IL-6, IFN-γ, PDGF and TNF-α, and that of c-jun by LPS, IFN-γ, PDGF and TNF-α. Electrophoretic mobility shift assays showed a time-dependent increase in AP-1 DNA binding activity with JunB representing the major mediator-inducible member involved in DNA–protein interactions. However, stimulus-specific changes in the kinetics and magnitude of AP-1 mRNA expression and DNA binding activity were identified and, additionally, the results showed the potential existence of cell-type-specific mechanisms in the regulation of the AP-1 family. These studies provide novel insights into the mediator-specific modulation of AP-1-regulated gene expression and the activation of MC in renal diseases.

CYTOKINE-MEDIATED DIFFERENTIAL REGULATION OF MACROPHAGE ACTIVATOR PROTEIN-1 GENES

The regulation of macrophage activator protein-1 (AP-1) gene expression by LPS and cytokines is of potentially crucial importance in the pathogenesis of several diseases. The action of LPS and four cytokines on AP-1 gene expression in the murine macrophage J774.2 cell line was, therefore, studied. Exposure of the cells to IL-6 produced no changes in the mRNA levels of all AP-1 members studied. In contrast, the expression of JunB, c-jun and c-fos, but not JunD, was increased by LPS, TNF-α, IFN-γ and IL-1, albeit with different kinetics and magnitude of induction. Electrophoretic mobility shift assays showed a close correlation between the expression of the AP-1 genes and the functional AP-1 DNA binding activity and, additionally, demonstrated the participation of heterodimeric interactions between the different members. These studies provide insights into the potential mechanisms that may be involved in the mediator-specific modulation of AP-1 regulated macrophage gene expression.

Chlamydia pneumoniae activates nuclear factor kappaB and activator protein 1 in human vascular smooth muscle and induces cellular proliferation.

Observational data strongly suggest an association between Chlamydia pneumoniae and atherosclerotic cardiovascular disease. However, few studies have mechanistically linked C. pneumoniae to vascular remodeling. The purpose of the present study was to examine the mechanistic relationship between C. pneumoniae and human vascular smooth muscle cell (VSMC) physiology. We sought to determine the influence of human VSMC infection by C. pneumoniae on (1) VSMC proliferation and (2) activation of the proinflammatory and proliferative transcription factors nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). C. pneumoniae was grown and isolated from Hep 2 cells. Human aortic VSMCs were inoculated with C. pneumoniae in the presence and absence of the azalide antibiotic azithromycin. Cell proliferation was assayed by direct cell counting 48 h following infection. Two hours following infection, nuclear extracts were isolated, and activation of both NF-kappaB and AP-1 was assessed by electrophoretic mobility shift assay. Compared with control, C. pneumoniae infection stimulated VSMC proliferation (P < 0.05) and induced both NF-kappaB and AP-1 DNA binding activity. These effects were eliminated by concurrent treatment with azithromycin. VSMC infection with C. pneumoniae activates proliferative intracellular signals and stimulates cell growth. These data implicate C. pneumoniae as a pathogenic mediator and a potential therapeutic target in the prevention of atherosclerotic disease.

Bile Acid-induced Activation of Activator Protein-1 Requires Both Extracellular Signal-regulated Kinase and Protein Kinase C Signaling

Elevated concentrations of fecal bile aids are known to promote colon cancer and increasing evidence suggests that alterations in cellular signaling and gene expression may play an important role in this process. In this study, we examined the molecular mechanisms underlying bile acid-mediated gene regulation using GADD153 as our model gene. Promoter deletion analyses revealed that the activator protein-1 (AP-1) transcription factor was crucial for deoxycholic acid (DCA)-mediated GADD153 gene transcription. Electrophoretic mobility shift assays and transient transfection analyses demonstrated that both DNA binding and transactivation activities of AP-1 were induced by DCA in a dose-dependent manner. The AP-1 complex induced by DCA consisted of JunD, Fra-1, and c-Fos. Examination of the signaling pathways stimulated by DCA showed that extracellular signal-regulated kinases (ERKs) were required for AP-1 activation. Inhibition of ERK by the mitogen-activated protein kinase/ERK kinase inhibitor PD 98059 or by expression of a dominant negative mutant ERK suppressed AP-1 activation. Notably, the PKC inhibitor, calphostin C, also abolished DCA-induced AP-1 activation but did not affect DCA-mediated ERK activation, suggesting that ERK and PKC function in separate signaling pathways that cooperatively mediate DCA-induced AP-1 activation. Hence, bile acid-stimulated signaling appears to converge on the AP-1 protooncogene.

The mood-stabilizing agent valproate inhibits the activity of glycogen synthase kinase-3.

Valproic acid (VPA) is a potent broad-spectrum anti-epileptic with demonstrated efficacy in the treatment of bipolar affective disorder. It has previously been demonstrated that both VPA and lithium increase activator protein-1 (AP-1) DNA binding activity, but the mechanisms underlying these effects have not been elucidated. However, it is known that phosphorylation of c-jun by glycogen synthase kinase (GSK)-3beta inhibits AP-1 DNA binding activity, and lithium has recently been demonstrated to inhibit GSK-3beta. These results suggest that lithium may increase AP-1 DNA binding activity by inhibiting GSK-3beta. In the present study, we sought to determine if VPA, like lithium, regulates GSK-3. We have found that VPA concentration-dependently inhibits both GSK-3alpha and -3beta, with significant effects observed at concentrations of VPA similar to those attained clinically. Incubation of intact human neuroblastoma SH-SY5Y cells with VPA results in an increase in the subsequent in vitro recombinant GSK-3beta-mediated 32P incorporation into two putative GSK-3 substrates (approximately 85 and 200 kDa), compatible with inhibition of endogenous GSK-3beta by VPA. Consistent with GSK-3beta inhibition, incubation of SH-SY5Y cells with VPA results in a significant time-dependent increase in both cytosolic and nuclear beta-catenin levels. GSK-3beta plays a critical role in the CNS by regulating various cytoskeletal processes as well as long-term nuclear events and is a common target for both lithium and VPA; inhibition of GSK-3beta in the CNS may thus underlie some of the long-term therapeutic effects of mood-stabilizing agents.

2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced changes in activities of nuclear protein kinases and phosphatases affecting DNA binding activity of c-Myc and AP-1 in the livers of guinea pigs

To study the effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on nuclear protein phosphorylation activities, male guinea pigs were treated in vivo with a single 1 μg/kg i.p. injection of TCDD, and the state of protein kinases and phosphatases in the nuclei of the hepatocytes was examined after 1, 10, and 40 days. TCDD was found to cause a rise in nuclear protein tyrosine kinase on day 1, and to a lesser extent on day 10, but this effect diminished almost completely on day 40. TCDD also caused a reduction in nuclear casein kinase II (CKII) activity at all time points. To study the biochemical events taking place at the early stage of the action of TCDD, a short-term in vitro model system was established using explant liver tissues maintained in tissue culture medium. It was found that TCDD caused a rapid reduction of the activity of nuclear CKII with an accompanying increase in the cytosol. Such changes in protein phosphorylation activities were also accompanied by an increase in the DNA binding activity of activator protein 1 (AP-1). The effect of TCDD on nuclear proteins binding to the c-Myc response element DNA was, on the other hand, biphasic: an initial increase of protein binding to the c-Myc response element was followed by suppression. To test the hypothesis that some of the above changes were caused by TCDD-induced changes in protein kinase activity, nuclear proteins isolated from hepatocytes of in vivo treated guinea pigs were incubated with exogenously added Mg 2+ and ATP under cell-free conditions. The results showed that this in vitro phosphorylation treatment exacerbated this tendency of increased AP-1 and decreased c-Myc binding to their respective response element DNAs, indicating that kinases and phosphatases present in the isolated nuclear protein preparation were active and capable of modifying protein binding to DNA. Such effects of Mg 2+ and ATP on AP-1 were blocked by heparin, indicating that CKII plays an important role in transducing the signal of TCDD into the nucleus.

The lipid peroxidation product 4-hydroxy-2,3-nonenal increases AP-1-binding activity through caspase activation in neurons.

The transcription factor activator protein-1 (AP-1) is activated in response to physiological activity in neuronal circuits and in response to neuronal injury associated with various acute and chronic neurodegenerative conditions. The membrane lipid peroxidation product 4-hydroxy-2,3-nonenal (HNE) is increasingly implicated in the disruption of neuronal calcium homeostasis that occurs in various paradigms of neuronal excitotoxicity and apoptosis. The possible mechanistic links between lipid peroxidation and alterations in gene transcription during neuronal apoptosis have not previously been examined. We now report that exposure of cultured rat cortical neurons to an apoptotic concentration of HNE results in a large increase in AP-1 DNA-binding activity. The protein synthesis inhibitor cycloheximide blocked the induction of AP-1, consistent with a requirement for induction of expression of AP-1 family members. The broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde blocked HNE-induced increases in AP-1 DNA-binding activity, demonstrating a requirement for caspase activation in the activation of AP-1. HNE induced phosphorylation of c-Jun N-terminal kinase (JNK), which was prevented by caspase inhibitors, indicating that HNE was acting at or upstream of JNK phosphorylation. The intracellular calcium chelator BAPTA-acetoxymethyl ester completely prevented stimulation of AP-1 DNA-binding by HNE, indicating a requirement for calcium. Moreover, agents that suppress mitochondrial calcium uptake (ruthenium red) and membrane permeability transition (cyclosporin A) attenuated AP-1 activation by HNE, suggesting a contribution of mitochondrial alterations to AP-1 activation. Collectively, our data suggest a scenario in which HNE disrupts neuronal calcium homeostasis and perturbs mitochondrial function, resulting in caspase activation. Activated caspases, in turn, induce activation of JNK, resulting in stimulation of AP-1 DNA-binding protein production. This transcriptional pathway induced by HNE may modulate the cell death process.

Assaying NF-kappa B and AP-1 DNA-binding and transcriptional activity.

Nuclear factor-KB (NF-KB) and activator protein 1 (AP-1) are well-characterized ubiquitously expressed transcription factors that play important roles in the response to cellular stress situations. In unstimulated resting cells, NF-KB and AP-1 are inactive. These transcription factors are induced by a great variety of stimuli and conditions that represent internal or external stress situations for the cells. These include pathological stimuli, such as viruses, bacteria, oxidative stress, hypoxia, and inflammatory mediators as well as internal cellular stress (e.g., endoplasmic reticulum overload) (, , , , , , ).

Cadmium-mediated oxidative stress in alveolar epithelial cells induces the expression of gamma-glutamylcysteine synthetase catalytic subunit and glutathione S-transferase alpha and pi isoforms: potential role of activator protein-1.

Exposure of rat alveolar epithelial cells to 10 micromol/L CdCl2 causes time-dependent increases in steady-state mRNA levels of the gamma-glutamylcysteine synthetase catalytic (heavy) subunit (gamma-GCS) and of glutathione S-transferase isoforms (GST-alpha and GST-pi). The expression of gamma-GCS was significantly increased as early as 2 h after addition of cadmium. Maximal induction of gamma-GCS mRNA (approximately 4-fold), at 8 h, was subsequently followed by increases in gamma-GCS activity/protein and glutathione (GSH) levels. Maximal elevations in GST-pi (approximately 2-fold) and GST-alpha (approximately 10-fold) transcripts, at 8 and 24 h, respectively, were also accompanied by enhanced GST activity. Cadmium-induced oxidative stress, assessed by alterations in GSH homeostasis and an accelerated rate of intracellular oxidant production, could constitute early events in the signal transduction pathway mediating these responses. The dimeric transcription factor, activator protein-1 (AP-1), may also play a regulatory role in this process. This association is suggested by transcriptional activation of the immediate-early response genes, c-fos and c-jun, within 15 min after exposure to cadmium and by the enhancement of AP-1 DNA binding activity, involving a c-Jun protein complex, which is maximally induced (approximately 4-fold) by 2 h. These molecular changes likely function together to protect alveolar epithelial cells against cadmium toxicity.