Activator Protein-1 Site Research Articles

α-Melanocyte-Stimulating Hormone Counteracts the Suppressive Effect of UVB on Nrf2 and Nrf-Dependent Gene Expression in Human Skin

Human skin is constantly exposed to UV light, the most ubiquitous environmental stressor. Here, we investigated the expression and regulation of Nrf1-3, transcription factors crucially involved in protection against oxidative stress in human skin cells in vitro, ex vivo, and in situ. In particular, we examined whether alpha-MSH, a UV-induced peptide, is capable of modulating Nrf2 and Nrf-dependent gene expression. Nrf1, -2, and -3 were found to be expressed in various cutaneous cell types in vitro. Surprisingly, UVB irradiation at physiological doses (10 mJ/cm(2)) reduced Nrf2 and Nrf-dependent gene expression in normal keratinocytes and melanocytes in vitro as well as ex vivo in skin organ cultures. alpha-MSH alone significantly increased Nrf2 as well as Nrf-dependent heme oxygenase-1, gamma-glutamylcysteine-synthetase, and glutathione-S-transferase Pi gene expression in both keratinocytes and melanocytes. This effect of alpha-MSH occurred at physiological doses and was due to transcriptional induction, mimicked by the artificial cAMP inducer forskolin, and blocked by protein kinase A pathway inhibition. In silico promoter analysis of Nrf2 further identified several putative binding sites for activator protein 1 and cAMP response element-binding protein, transcription factors typically activated by alpha-MSH. Importantly, alpha-MSH prevented or even overcompensated the UVB-induced suppression of Nrf2 and Nrf-dependent genes not only in normal keratinocytes and melanocytes in vitro but also in skin organ cultures. These findings, for the first time, show regulation of Nrf2 and Nrf-dependent genes by alpha-MSH. Our data also highlight a novel facet in the cytoprotective and antioxidative effector mechanisms of alpha-MSH and perhaps of related melanocortin peptides.

Cooperation between GLI and JUN enhances transcription of JUN and selected GLI target genes

Sustained Hedgehog (HH) signaling is implicated in basal cell carcinoma of the skin and other types of cancer. Here we show that GLI1 and GLI2, the main transcriptional activators of the HH pathway, directly regulate expression of the activator protein 1 (AP-1) family member JUN, a transcription factor controlling keratinocyte proliferation and skin homeostasis. Activation of the JUN promoter by GLI is dependent on a GLI-binding site and the AP-1 sites known to be involved in self-activation of JUN. Transcription of JUN is greatly enhanced in the presence of GLI and requires activated JUN protein. GLI2act is a more potent activator than GLI1 in these experiments and physical interaction with phosphorylated JUN was only detected for GLI2act. The synergistic effect of GLI and JUN extends to the activation of further GLI target genes as shown by shRNA-mediated knockdown of JUN in human keratinocytes. Some of these cooperatively activated genes are involved in cell-cycle progression, which is consistent with a significant reduction of the proliferative potential of GLI in the absence of JUN. These results suggest a novel connection between HH/GLI pathway activity and JUN, which may contribute to the oncogenic activity of HH/GLI signaling in skin.

JunD and HIF‐1α mediate transcriptional activation of angiotensinogen by TGF‐β1 in human lung fibroblasts

Earlier work showed that TGF-beta1 potently increases angiotensinogen (AGT) gene mRNA in primary human lung fibroblasts. Here the mechanism of TGF-beta1-induced AGT expression was studied in the IMR90 human lung fibroblast cell line. The increase in AGT mRNA induced by TGF-beta1 was completely blocked by actinomycin-D. TGF-beta1 increased the activity of a full-length human AGT promoter-luciferase reporter (AGT-LUC) but did not alter AGT mRNA half-life. Serial deletion analyses revealed that 67% of TGF-beta-inducible AGT-LUC activity resides in a small domain of the AGT core promoter; this domain contains binding sites for hypoxia-inducible factor (HIF)-1 and activation protein-1 (AP-1) transcription factors. TGF-beta1 increased HIF-1alpha protein abundance and the activity of a hypoxia-responsive element reporter; overexpression of HIF-1 increased basal AGT-LUC activity. Both oligonucleotide pulldown and chromatin immunoprecipitation assays revealed increased binding of JunD and HIF-1alpha to the AGT core promoter in response to TGF-beta1. TGF-beta1-inducible AGT-LUC was reduced by an AP-1 dominant negative or by mutation of the AP-1 site. Knockdown of either JunD or HIF-1alpha individually by siRNA partially reduced AGT-LUC. In contrast, simultaneous knockdown of both JunD and HIF-1alpha completely eliminated TGF-beta1-inducible AGT-LUC activity. These data suggest that TGF-beta1 up-regulates AGT transcription in human lung fibroblasts through a mechanism that requires both JunD and HIF-1alpha binding to the AGT core promoter. They also suggest a molecular mechanism linking hypoxia signaling and fibrogenic stimuli in the lungs.

Ligand-selective transactivation and transrepression via the glucocorticoid receptor: Role of cofactor interaction

The mechanisms that determine ligand-selective transcriptional responses by the glucocorticoid receptor (GR) are not fully understood. Using a wide panel of GR ligands, we investigated the relationships between the potency and maximal response for transactivation via a glucocorticoid response element (GRE) and transrepression via both nuclear factor small ka, CyrillicB (NFsmall ka, CyrillicB) and activator protein-1 (AP-1) sites, relative binding affinity for the GR, as well as interaction with both coactivators and corepressors. The results showed ligand-selective differences in potency and efficacy for each promoter, as well as for a particular ligand between the three promoters. Ligand potency correlated with relative affinity for the GR for agonists and partial agonists in transactivation but not for transrepression. Maximal response was unrelated to relative affinity of ligand for GR for both transactivation and transrepression. A good and significant correlation between full length coactivator binding in two-hybrid assays and efficacy as well as potency of different receptor-steroid complexes for both transactivation and transrepression supports a major role for coactivator recruitment in determination of ligand-selective transcriptional activity. Furthermore, ligand-selective GR binding to GRIP-1, as determined by both two-hybrid and DNA pull down assays, correlated positively with ligand-selective efficacy for transactivation of both a synthetic GRE reporter with expressed GR as well as of an endogenous gene via endogenous GR. The receptor interacting domain of the corepressor SMRT exhibited strong interaction with both agonists and partial agonists, similar to the results for coactivators, suggesting a possible role for SMRT in activation of transcription. However, there was no correlation between ligand affinity for the GR and cofactor interaction. These results provide strong quantitative biochemical support for a model in which GR-mediated ligand-selective differential interaction with GRIP-1, SRC-1A, NCoR and SMRT is a major determinant of ligand-selective and promoter-specific differences in potency and efficacy, for both transactivation and transrepression.

Regulation of Interleukin-8 Gene at a Distinct Site of Its Promoter by CCAAT Enhancer-binding Protein Homologous Protein in Prostaglandin E2-treated Human T Cells

For a long period of time, the transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) has been thought to inhibit transcriptional activity for its ability to interact with CCAAT enhancer-binding protein family factors, thus preventing their binding to DNA. We have previously shown that in human T lymphocytes the CHOP phosphorylation induced by prostaglandin E(2) (PGE(2))-increased interleukin-8 (IL-8) gene expression. Given the CHOP positive role in the regulation of transcription, here we have investigated the molecular mechanism(s) by which CHOP increases IL-8 gene activity under PGE(2) stimulus. Transfection experiments with mutants showed both that the CHOP transactivation domain is essential for IL-8 transcription and that the IL-8/activator protein 1 (AP-1) promoter mutated in NF-kappaB and NF-IL-6, but not in the AP-1 site, harbors essential CHOP-responsive elements. CHOP silencing confirmed its role in the IL-8 transcriptional regulation and protein production, whereas c-Jun small interfering RNA experiments showed that the PGE(2)-induced activation of IL-8 promoter is mainly c-Jun-independent. Moreover, PGE(2) induced CHOP-DNA complexes only when the entire IL-8/AP-1 promoter or the wild type sequences encompassing the AP-1 upstream region were employed. Mutations introduced in these sequences prevented the DNA-CHOP complex formation. The IL-8/AP-1 mutant promoter lacking the sequence immediately upstream the AP-1 site is PGE(2)-unresponsive. Finally, chromatin immunoprecipitation data confirmed in vivo that PGE(2) induces CHOP binding to the IL-8 promoter. Taken together, our results suggest that the increased expression of CHOP in response to PGE(2) exerts a positive transcriptional regulation of the IL-8 promoter mediated by direct binding to a novel consensus site.

Induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective Nrf2 activator 3H‐1,2‐dithiole‐3‐thione

Peroxiredoxins are an important family of cysteine-based antioxidant enzymes that exert a neuroprotective effect in several models of neurodegeneration. However, under oxidative stress they are vulnerable to inactivation through hyperoxidation of their active site cysteine residues. We show that in cortical neurons, the chemopreventive inducer 3H-1,2-dithiole-3-thione (D3T), that activates the transcription factor Nuclear factor erythroid 2-related factor (Nrf2), inhibits the formation of inactivated, hyperoxidized peroxiredoxins following oxidative trauma, and protects neurons against oxidative stress. In both neurons and glia, Nrf2 expression and treatment with chemopreventive Nrf2 activators, including D3T and sulforaphane, up-regulates sulfiredoxin, an enzyme responsible for reducing hyperoxidized peroxiredoxins. Induction of sulfiredoxin expression is mediated by Nrf2, acting via a cis-acting antioxidant response element (ARE) in its promoter. The ARE element in Srxn1 contains an embedded activator protein-1 (AP-1) site which directs induction of Srxn1 by synaptic activity. Thus, raising Nrf2 activity in neurons prevents peroxiredoxin hyperoxidation and induces a new member of the ARE-gene family, whose enzymatic function of reducing hyperoxidized peroxiredoxins may contribute to the neuroprotective effects of Nrf2 activators.

Regulation of Bovine Tumor Necrosis Factor-α-Induced Protein 6 in Ovarian Follicles during the Ovulatory Process and Promoter Activation in Granulosa Cells

To study the regulation of bovine TNFalpha-induced protein 6 (TNFAIP6) prior to ovulation, preovulatory follicles obtained after the treatment with human chorionic gonadotropin (hCG) were used. RT-PCR analyses showed that levels of TNFAIP6 mRNA were low before hCG but significantly increased after hCG treatment in follicles. Further analyses and immunohistochemistry indicated that this increase in transcript and protein levels occurred in theca and granulosa cells. To investigate molecular mechanisms involved in TNFAIP6 transactivation, the activity of bovine TNFAIP6 promoter was studied in granulosa cell cultures. Mutant studies identified the minimal region conferring full-length promoter activity, in which activator protein-1 (AP1) and cAMP response element (CRE) elements were required for promoter activity. Overexpression of dominant-negative AP1 and activating transcription factor/cAMP response element-binding protein (CREB) inhibited forskolin-inducible promoter activity. DNA binding assays demonstrated the importance of AP1 and CRE for activity and identified JunD, FosB, Fra2, CREB1, and CREB2 as being part of the AP1 complex, and FosB, Fra2, and CREB1 for the CRE complex. Chromatin immunoprecipitation assays confirmed binding of these proteins with endogenous TNFAIP6 promoter. Treatment with forskolin, prostaglandin E2, and catalytic subunit protein kinase (cPKA) stimulated, but H89, PKA inhibitor peptide, and indomethacin inhibited, TNFAIP6 promoter activity and gene expression in granulosa cells. Collectively, this study is the first to describe that the ovulatory process in cows is associated with a gonadotropin-dependent induction of TNFAIP6 in ovarian follicles and provide the molecular basis through which AP1 and CRE sites and PKA activation played important roles in the regulation of TNFAIP6 in granulosa cells.

Activator Protein-1 and Smad Proteins Synergistically Regulate Human Follicle-Stimulating Hormone β-Promoter Activity

GnRH1 stimulates the synthesis and secretion of FSH and LH from the anterior pituitary gland. The molecular mechanisms through which GnRH1 produces these effects in humans have not been determined. Here, we examined transcriptional regulation of the human FSHbeta (FSHB) subunit using reporter assays in immortalized murine gonadotrope cells. GnRH1 dose and time dependently stimulated FSHB promoter activity, with peak stimulation occurring at 8 h. GnRH1 rapidly stimulated various MAPK cascades, though the ERK1/2 and p38 pathways appeared to be most critical for FSHB induction. Indeed, constitutively active forms of both Raf1 kinase and MAP2K6 (MKK6) were sufficient to stimulate reporter activity. GnRH1 stimulated activator protein-1 (AP-1) (FosB, c-fos, JunB, and cJun) synthesis and complex formation, the latter of which bound to a conserved cis-element within -120 bp of the transcription start site. A second, lower affinity, site was mapped more proximally. Mutations of both cis-elements diminished GnRH1-stimulated promoter activity, though disruption of the higher affinity site had a more dramatic effect. A dominant-negative Fos protein dose dependently inhibited GnRH1-stimulated FSHB transcription, confirming a role for endogenous AP-1 proteins. MAPK kinase 1 (MEK1) and p38 inhibitors significantly attenuated GnRH1-stimulated c-fos, FosB, and JunB synthesis, suggesting a mechanism whereby the ERK1/2 and p38 signaling pathways regulate FSHB transcription. Activins and inhibins potently regulate FSH synthesis in rodents, but their roles in FSH regulation in humans are less clear. Activin A, though weak on its own, synergized with GnRH1 to stimulate human FSHB promoter activity. In contrast, activin A partially inhibited GnRH1-stimulated LHbeta subunit (LHB) transcription. The GnRH1 and activin A signaling pathways appear to converge at the level of the high-affinity AP-1 site. Fos and Jun proteins synergistically regulate reporter activity through this element, and their effects are potentiated by coexpression of either Smad2 or Smad3, effectors in the activin signaling cascade. In summary, GnRH1 and activin A synergistically regulate human FSHB subunit transcription. The combined actions of AP-1 and Smad proteins acting through a conserved AP-1 element provide a candidate mechanism for this effect. The ability of activins to potentiate selectively the effects of GnRH1 on FSHB expression suggests a model for preferential increases in FSH secretion at the luteal-follicular transition of the menstrual cycle.

The AP-1 site is essential for the promoter activity of NOX1/NADPH oxidase, a vascular superoxide-producing enzyme: Possible involvement of the ERK1/2-JunB pathway

NADPH oxidase is a major source of the superoxide produced in cardiovascular tissues. The expression of NOX1, a catalytic subunit of NADPH oxidase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F 2α, and platelet-derived growth factor (PDGF). It was reported previously that the inducible expression of NOX1 is governed by the activating transcription factor-1 (ATF-1)-myocyte enhancer factor 2B (MEF2B) cascade downstream of phosphoinositide 3 (PI3) kinase. It was also reported that extracellular signal-regulated kinase (ERK) 1/2 is involved in the expression of NOX1. To further clarify the factors involved in NOX1 induction downstream of ERK1/2, the promoter region of the NOX1 gene was analyzed. A consensus activator protein-1 (AP-1) site was found at −98/−92 in the 5′-flanking region of the rat NOX1 gene. The introduction of mutations at this site abolished PGF 2α-induced transcriptional activation in a luciferase assay. Electrophoresis mobility shift assays demonstrated that PGF 2α and PDGF augmented the binding of JunB to this sequence. PD98059, an inhibitor of MAPK/ERK kinase, suppressed the expression of JunB induced by PGF 2α or PDGF. These results suggest that the ERK1/2-JunB pathway is a key regulator of the inducible expression of the NOX1 gene in vascular smooth muscle cells.

Transforming Growth Factor-β–Mediated Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Expression and Apoptosis in Hepatoma Cells Requires Functional Cooperation between Smad Proteins and Activator Protein-1

Transforming growth factor-beta (TGF-beta) has been shown to induce apoptotic cell death in normal and transformed hepatocytes. We recently identified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as an important mediator of TGF-beta-induced apoptosis in hepatoma cells. In this study, we have further explored the mechanism by which TGF-beta up-regulates TRAIL expression. The 5'-flanking region of the TRAIL gene was isolated and characterized. Deletion mutants of the 5'-untranslated region of the TRAIL gene revealed a region comprising nucleotides -1950 to -1100 responsible for TRAIL induction following treatment with TGF-beta. Within this region, we have identified an activator protein-1 (AP-1) site indispensable for TGF-beta-mediated induction of TRAIL. Activation of this AP-1 site is mediated by a JunD.FosB heterodimer. Expression of DNSmad4, DNJunD, or DNFosB significantly impairs TGF-beta-mediated activation of the TRAIL promoter. Furthermore, with tRNA interference targeting Smad4, junD, FosB, we could abolish TRAIL expression and, subsequently, TGF-beta-induced TRAIL-mediated apoptosis in hepatoma cells. Our results reveal a new AP-1 site within the TRAIL promoter functionally involved in TGF-beta-induced TRAIL expression and apoptosis in hepatomas and thus provide evidence for the underlying mechanism by which TGF-beta might regulate cell death in liver cancer.

Luteolin reduces IL-6 production in microglia by inhibiting JNK phosphorylation and activation of AP-1

Luteolin, a flavonoid found in high concentrations in celery and green pepper, has been shown to reduce production of proinflammatory mediators in LPS-stimulated macrophages, fibroblasts, and intestinal epithelial cells. Because excessive production of proinflammatory cytokines by activated brain microglia can cause behavioral pathology and neurodegeneration, we sought to determine whether luteolin also regulates microglial cell production of a prototypic inflammatory cytokine, IL-6. Pretreatment of primary murine microlgia and BV-2 microglial cells with luteolin inhibited LPS-stimulated IL-6 production at both the mRNA and protein levels. To determine how luteolin inhibited IL-6 production in microglia, EMSAs were performed to establish the effects of luteolin on LPS-induced binding of transcription factors to the NF-kappaB and activator protein-1 (AP-1) sites on the IL-6 promoter. Whereas luteolin had no effect on the LPS-induced increase in NF-kappaB DNA binding activity, it markedly reduced AP-1 transcription factor binding activity. Consistent with this finding, luteolin did not inhibit LPS-induced degradation of IkappaB-alpha but inhibited JNK phosphorylation. To determine whether luteolin might have similar effects in vivo, mice were provided drinking water supplemented with luteolin for 21 days and then they were injected i.p. with LPS. Luteolin consumption reduced LPS-induced IL-6 in plasma 4 h after injection. Furthermore, luteolin decreased the induction of IL-6 mRNA by LPS in hippocampus but not in the cortex or cerebellum. Taken together, these data suggest luteolin inhibits LPS-induced IL-6 production in the brain by inhibiting the JNK signaling pathway and activation of AP-1 in microglia. Thus, luteolin may be useful for mitigating neuroinflammation.

Epidermal Growth Factor Induces G Protein-Coupled Receptor 30 Expression in Estrogen Receptor-Negative Breast Cancer Cells

Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.

T-Cell Receptor/CD28-Mediated Activation of Human T Lymphocytes Induces Expression of Functional μ-Opioid Receptors

Opiates function as immunomodulators, partly by their effects on T cells. Opioids act via mu-, delta-, and kappa-opioid receptors, among which the mu-type is of particular interest, because morphine-like opioids preferentially bind to it. Here we report that mu-opioid receptor mRNA was induced after CD3/28-mediated activation of primary human T lymphocytes and Jurkat T cells, neither of which expresses the gene constitutively. Moreover, a reporter gene construct containing 2624 base pairs of the mu-opioid receptor promoter was transactivated by CD3/28 stimulation. Transcriptional induction of the mu-opioid receptor gene was mediated by activator protein-1 (AP-1), nuclear factor-kappaB, and nuclear factor of activated T cells (NFAT). NFAT was found to bind to three sequences of the mu-opioid receptor promoter, located at nucleotides -1064, -785, and -486. Although the -486 element is in close proximity to a putative AP-1 site, there was no evidence for a combined AP-1/NFAT site. Furthermore, we demonstrated that the induction of interleukin-2 mRNA and protein in activated T cells was inhibited by morphine in cells, in which mu-opioid receptors had been induced by CD3/28 monoclonal antibodies (mAbs), and that this effect was blocked by the mu-opioid receptor-specific antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2). CD3/28 mAb-induced interleukin-2 transcription was also inhibited by the opioids fentanyl and loperamide. This indicates that the induced mu-opioid receptor mRNA is translated into functional receptor protein. Furthermore, a mu-opioid receptor-enhanced green fluorescent protein-fusion protein was localized in membranes of Jurkat cells and internalized in response to [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin but not morphine. In conclusion, these data emphasize the role of opioids in the modulation of T lymphocyte signaling.

Reactive oxygen species mediate oxidized low-density lipoprotein-induced endothelin-1 gene expression via extracellular signal-regulated kinase in vascular endothelial cells

Oxidized low-density lipoprotein (oxLDL) promotes expression and secretion of endothelin-1 (ET-1), however, the precise mechanism involved is unclear. This study was designed to identify the regulatory mechanism of oxLDL-induced ET-1 expression in endothelial cells. ET-1 mRNA expression, secretion and promoter activity were evaluated by reverse transcriptase-PCR (RT-PCR), enzyme immunometric and luciferase assays, respectively. oxLDL (35 microg/ml) significantly enhanced reactive oxygen species (ROS), mRNA expression, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), all of which were nullified by the antioxidant N-acetyl cysteine (NAC). oxLDL stimulated the extracellular signal-regulated kinase (ERK) phosphorylation in HUVECs, which was blocked by NAC and the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD98059. NAC and PD98059 stopped oxLDL-elicited increase in mRNA expression, secretion and promoter activity of ET-1. Fusion plasmids with decreasing length of 5'-flanking sequence of ET-1 from -566 bpLuc to -250 bpLuc displayed increased luciferase activity after 24 h of oxLDL treatment. Interestingly, fusion plasmid from -233 and -185 bpLuc significantly reduced the luciferase activity in control and oxLDL-treated HUVECs. In addition, transfection of the reporter construct -250Luc, which contains a 2 bp mutation at activator protein-1 site, abolished both basal and oxLDL-stimulated ET-1 promoter activities. Collectively, our data favor the notion that oxLDL stimulates ERK phosphorylation via ROS accumulation, which in turn stimulates vascular endothelial transcriptional factor activator protein-1 and ET-1 expression as well as secretion.

Elevated intracellular calcium increases ferritin H expression through an NFAT-independent post-transcriptional mechanism involving mRNA stabilization

An increase in intracellular Ca2+ is one of the initiating events in T-cell activation. A calcium-mediated signalling cascade in T-cells involves activation of calcineurin and the dephosphorylation and translocation of NFAT (nuclear factor of activated T-cells), resulting in the transcriptional activation of target genes such as IL-2 (interleukin-2). In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an ARE (antioxidant-responsive element). The facts that the ferritin H ARE contains a composite AP-1 (activator protein 1) site and that NFAT collaborates with AP-1 transcription factors led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T-cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Although NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter after ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT-binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability after actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through post-transcriptional stabilization of the ferritin H mRNA.

Activator Protein-1 Contributes to High NaCl-induced Increase in Tonicity-responsive Enhancer/Osmotic Response Element-binding Protein Transactivating Activity

Tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1-4 and NF-kappaB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-kappaB.AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE.AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE.AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCl-induced increase of TonEBP/OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE.AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity.

Regulation of NGFI‐B/Nur77 gene expression in the rat ovary and in leydig tumor cells MA‐10

NR4A1, also called NGFI-B in the rat, Nur77 in the mouse and TR3 in humans, belongs to the orphan nuclear steroid hormone receptor superfamily and is one of the immediate-early genes. In the endocrine organs, including the gonads, NGFI-B/Nur77 gene expression is rapidly induced by pituitary hormones. NGFI-B/Nur77 expression was found to be rapidly reduced by an estrogenic endocrine disrupter, diethylstilbestrol (DES) in theca interna cells of immature rat ovaries. DES treatment also triggered a rapid decrease of serum luteinizing hormone (LH) levels, suggesting that DES acts on the hypothalamo-pituitary axis to suppress LH secretion from the pituitary. The transcriptional regulation of NGFI-B/Nur77 by LH/human chorionic gonadotropin (hCG) or 8-bromoadenosine 3'-5'-cyclic monophosphate (8 Br-cAMP) was examined in mouse Leydig tumor cells MA-10. Luciferase assays using NGFI-B/Nur77 promoter constructs and electric mobility shift assays (EMSA) showed that NGFI-B/Nur77 gene expression was mediated through three of the four activator protein-1 (AP-1)-like sites, namely the -233 AP-1, -213 AP-1 and -69 AP-1 sites adjacent to the transcription start site of the NGFI-B/Nur77 promoter. We also demonstrated here that both the Jun family and cAMP-responsive element binding (CREB) proteins bind to the -233 AP-1 site, whereas the main binding protein to the -213 AP-1 site was CREB, and Jun family protein to the -69 AP-1 site, respectively. The rapid induction of NGFI-B/Nur77 gene expression by LH/hCG in MA-10 cells appears to be mediated by both CREB and Jun family proteins through the cAMP-protein kinase A (PKA) pathway.

Aberrant expression of Fra-2 promotes CCR4 expression and cell proliferation in adult T-cell leukemia

Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Primary ATL cells frequently express CCR4 at high levels. Since HTLV-1 Tax does not induce CCR4 expression, transcription factor(s) constitutively active in ATL may be responsible for its strong expression. We identified an activator protein-1 (AP-1) site in the CCR4 promoter as the major positive regulatory element in ATL cells. Among the AP-1 family members, Fra-2, JunB and JunD are highly expressed in fresh primary ATL cells. Consistently, the Fra-2/JunB and Fra-2/JunD heterodimers strongly activated the CCR4 promoter in Jurkat cells. Furthermore, Fra-2 small interfering RNA (siRNA) or JunD siRNA, but not JunB siRNA, effectively reduced CCR4 expression and cell growth in ATL cells. Conversely, Fra-2 or JunD overexpression promoted cell growth in Jurkat cells. We identified 49 genes, including c-Myb, BCL-6 and MDM2, which were downregulated by Fra-2 siRNA in ATL cells. c-Myb, BCL-6 and MDM2 were also downregulated by JunD siRNA. As Fra-2, these proto-oncogenes were highly expressed in primary ATL cells but not in normal CD4+ T cells. Collectively, aberrantly expressed Fra-2 in association with JunD may play a major role in CCR4 expression and oncogenesis in ATL.

Expression Pattern, Regulation, and Functions of Methionine Adenosyltransferase 2β Splicing Variants in Hepatoma Cells

Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2beta) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5'-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2. Studies were conducted using RNA from normal human tissues, resected hepatocellular carcinoma specimens, and cell lines. Gene expression, promoter and nuclear binding activities, growth, and apoptosis were measured by routine assays. MAT2beta is expressed in most but not all tissues, and the 2 variants are differentially expressed. The messenger RNA levels of both variants are markedly increased in hepatocellular carcinoma. Tumor necrosis factor (TNF)-alpha, which induces MAT2A in HepG2 cells, also induced V1 (but not V2) expression. TNF-alpha induced the promoter activity of MAT2beta V1, likely via nuclear factor kappaB and activator protein 1. Both variants regulate growth, but only V1 regulates apoptosis. Reduced expression of V1 led to c-Jun-N-terminal kinase (JNK) activation, apoptosis, and sensitized HepG2 cells to TNF-alpha-induced apoptosis, while overexpression of V1 was protective. However, blocking JNK1 or JNK2 activation did not prevent apoptosis induced by V1 knockdown. V1 (but not V2) knockdown also leads to apoptosis in a colon cancer cell line, suggesting these variants play similar roles in many cell types. Different variants of MAT2beta regulate growth and death, which broadens their importance in biology.

Induction of Apoptosis by Vinblastine via c-Jun Autoamplification and p53-Independent Down-Regulation of p21WAF1/CIP1

Vinblastine treatment in all cell lines examined causes a robust increase in c-Jun protein expression and phosphorylation and a corresponding increase in activator protein-1 (AP-1) transcriptional activity. We show in KB-3 carcinoma cells that this is due to a strong autoamplification loop involving the proximal AP-1 site in the c-Jun promoter, resulting in highly increased c-Jun mRNA and c-Jun protein. Inhibitors of RNA transcription and protein translation blocked both vinblastine-induced c-Jun expression and apoptotic cell death, suggesting that apoptosis is dependent, at least in part, on transcription/translation. Small interfering RNA (siRNA) to c-Jun was used to interrupt the amplification cycle and was found to be highly effective, reducing vinblastine-induced c-Jun expression at both the mRNA and protein levels by 90%. Apoptosis and caspase-3 activation were significantly inhibited in c-Jun siRNA-treated cells. To uncover potential mechanisms of c-Jun-mediated cell death and protection by c-Jun siRNA, candidate target genes were examined. Chromatin immunoprecipitation revealed preferential association of c-Jun with the p21 (cyclin-dependent kinase inhibitor) gene promoter after vinblastine treatment. In KB-3 cells, which have compromised p53 function, and in p53-null cells but not in p53 wild-type cells, vinblastine caused down-regulation of p21 expression concomitant with increased c-Jun expression, suggesting a role for c-Jun in negative regulation of the p21 promoter independent of p53. These results provide strong evidence that c-Jun induction in response to vinblastine plays a proapoptotic role in part via down-regulation of p21, promoting cycling and subsequent cell death of mitotically impaired cells.