Activator Protein-1 Binding Site Research Articles

Regulation of chemokine mRNA expression in a rat model of vanadium-induced pulmonary inflammation.

Environmental and occupational exposure to vanadium dusts results in toxic effects mainly confined to the respiratory system. Using a rat model of acute lung inflammation induced by intratracheal instillation of sodium metavanadate (NaVO3) at the dose of 200 microg V/kg, we investigated the relationship between the cytologic characterization of pulmonary inflammation and the expression of chemokine mRNA. Significant polymorphonuclear leukocyte (PMN) influx (P < 0.01) into the lung was noted 4 h after NaVO3 instillation, whereas alveolar macrophages (AMs) in bronchoalveolar lavage (BAL) cells appeared to decrease significantly. In contrast, neither PMNs nor AMs changed substantially 1 h after NaVO3 instillation. By Northern analysis, macrophage inflammatory protein (MIP)-2 mRNA in BAL cells increased markedly 1 h after NaVO3 instillation and reduced a little bit at 4 h, whereas MIP-1alpha mRNA in BAL cells was expressed relatively high 1 h after NaVO3 instillation, although a basal expression was detected in control group, and returned rapidly nearly to control level at 4 h. Since MIP-2 is a potent PMN chemoattractant and MIP-1alpha is a potent macrophage/monocyte chemoattractant has been well known. The facts that PMN influx was preceded by increased MIP-2 mRNA expression, suggesting that MIP-2 is involved in the development of NaVO3-induced pulmonary inflammation, whereas increased MIP-1alpha mRNA expression was followed by decreased AMs in BAL cells, suggesting AMs might be activated by MIP-1alpha, adherent to the lining surface of the airways and then resistant to be washed out. To delineate the mechanisms of transcriptional activation, we recently cloned the 5'-flanking region of the MIP-2 gene. The promotor region contains consensus binding sites for transcription factor nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1). Using electrophoretic mobility shift assay, increased nuclear NF-kappaB, not AP-1, binding activity was detected 1 h after NaVO3 instillation, which correlated with the induction of MIP-2 mRNA. p65 (Rel A) and p50 protein appears to be involved in MIP-2 NF-kappaB binding. Taken together, our studies suggest that MIP-2 is an important mediator of NaVO3-induced pulmonary inflammation in the rat model. In addition, elevated MIP-2 mRNA levels are accompanied by increased NF-kappaB binding activity in BAL cells, suggesting possible MIP-2 transcriptional regulation through NF-kappaB.

Regulation of Vascular Endothelial Growth Factor Expression in Human Keratinocytes by Retinoids

Vascular endothelial growth factor (VEGF) is overexpressed in hyperproliferative diseases, such as psoriasis and cancers, which are characterized by increased angiogenesis. Experimentally, VEGF overexpression can be induced by the treatment of cell cultures and biological tissues with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Using normal human keratinocytes in conventional cultures and skin grafted onto nude mice in vivo, we show that retinoids can inhibit TPA-mediated VEGF gene induction at the transcriptional level. Because retinoids are biologically active either by interacting with the nuclear retinoic acid receptors or by interfering with the activator protein 1 (AP1) transcription factor, we studied the effect of the retinoic acid derivative CD 2409, which exhibits strong anti-AP1 activity but does not bind to the known retinoic acid receptors in vitro. The results demonstrate that the inhibition of VEGF expression by retinoids only depends on their anti-AP1 activity and does not require gene transactivation via retinoic acid response elements. Because the VEGF promoter contains four potential AP1 binding sites, we used different promoter constructs to identify the functional site responsible for TPA induction and retinoid inhibition. This site turned out to be localized at position -621 of the 5' flanking region of the VEGF gene.

Differential Effects of Flavonoid Compounds on Tumor Promoter-Induced Activation of the Human CYP1A2 Enhancer

Flavonoids, a family of naturally occurring polyphenolic compounds found in many fruits, nuts, vegetables, and beverages, appear to inhibit tumor promotion as part of their chemopreventive properties. To investigate at the molecular level the ability of flavonoids to inhibit tumor-promoting activity, we developed a cell line designed to screen for flavonoids that block the tumor promoter-mediated induction of activator protein-1 (AP-1) transcriptional activity. This cell line, T2Luc, is a HepG2-derived cell line stably integrated with a region of the human CYP1A2 5′-flanking gene containing two AP-1 binding sites linked to the thymidine kinase promoter-driven firefly luciferase reporter gene. Treatment of T2Luc with a commercial extract of green tea alone had no effect on luciferase activity, but did block the induction of luciferase when cells were further challenged with the tumor promoter phorbol 12-O-tetradecanoate 13-acetate (TPA). In contrast, treatment of cells with the flavonoid quercetin alone activated luciferase activity in a concentration-dependent manner and enhanced the TPA-induced transcription of luciferase. Gel mobility shift assays using nuclear extracts from cells treated with green tea extracts or TPA alone revealed induced binding of AP-1 proteins to the CYP1A2 3′AP-1 site. Pretreatment with green tea extracts did not inhibit the TPA-induced formation of AP-1 complexes. Quercetin treatment alone slightly enhanced binding of AP-1 complexes to this site. Our results suggest that these dietary chemopreventive agents may work through different pathways to modulate gene expression.

JEM-1, a novel nuclear co-factor: localisation and functional interaction with AP-1.

JEM-1 is a novel gene whose mRNA expression in acute promyelocytic leukemia (APL) is induced by retinoid treatments. The gene product, a 45 kDa basic nuclear factor containing a leucine repeat, was transiently expressed in HeLa or COS-7 cells and immunocharacterized within the nuclei in fine punctuated structures which increase in size after cell transfection. Jem-1 was not expressed in the nucleoli. Experimental deletion of peptide domains of Jem-1 (JemDelta331-400 and Jem DeltaL179-206) showed that its C-terminal sequence (Thr331 --> Leu400) is required for nuclear translocation, while the leucine repeat domain (Arg179 --> Glu206) has no influence on subcellular localization. The Jem-1 protein was not detected in the PML-containing nuclear bodies or in speckled structures containing the splicing factor SC-35. In contrast it was localized in the nucleus in structures containing activator protein-1 (AP-1). DNA mobility shift assays showed that the in vitro translated Jem protein interacts neither with the DNA binding site of AP-1, nor directly with in vitro co-translated c-Fos or/and c-Jun proteins bound to this specific sequence. Interestingly, Jem-1-1 increased substantially the transcriptional activity of c-Jun (three-fold) and more strongly that of ectopically co-expressed c-Fos and c-Jun (five- to six-fold), as measured by a CAT reporter gene driven by a heterologous promoter containing the AP-1 binding site of the human collagenase gene. These synergistic effects were strongly Jem-1 dose-dependent. However, Jem-1 alone showed no activity on the collagenase promoter. A deletion of the leucine repeat of Jem-1 (Arg179 --> Glu206) did not diminish the enhancer capacity of Jem-1 on AP-1 activity. In contrast, the enhanced AP-1 activity was abrogated when Jem-1 was deleted of its C-terminus (Thr331 --> Leu400). We conclude that the 45 kDa nuclear product of the JEM-1 gene has features of a novel transcription cofactor, which is enhancing AP-1 activity without directly interacting with c-Jun or c-Fos proteins. Possible implications of these findings for APL cell maturation are discussed.

Multiple binding sites for nuclear proteins of the anterior pituitary are located in the 5′-flanking region of the porcine follicle-stimulating hormone (FSH) β-subunit gene

Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), are synthesized specifically in the gonadotropes of the anterior pituitary. The aim of this study was to investigate nuclear factors that bind specifically to the porcine FSH β-subunit gene. We examined nuclear protein binding to 2.75 kilobase pairs (kbp) of DNA adjacent to the porcine FSH β-subunit gene: about 2.32 kbp of upstream DNA and 0.43 kbp of downstream DNA. The upstream region contains only TATA box, CACCC element, and some imperfect sequences of cAMP-responsive element, activator protein-1 binding site, and activator protein-2 binding site. Gel mobility shift assay using nuclear proteins extracted from the porcine anterior pituitary revealed that the proteins bound to a limited region of DNA, 107 bp long (designated as Fd2), located about −800 bp upstream from the transcription initiation site. Competitive binding assays demonstrated that the protein binding was sequence specific; the addition of excess amounts of several putative regulatory sequences and plasmid (non-homologous) DNA fragments did not reduce the binding. Furthermore, all five subfragments of Fd2 were also bound by the pituitary nuclear proteins, showing that the entire region of Fd2 is involved in this interaction. Southwestern blotting demonstrated that at least seven protein species of 110, 98, 78, 63, 52, 42, and 35 kDa recognize Fd2. Nuclear proteins from several other porcine tissues were also able to bind to the Fd2 fragment but the gel shift patterns were different and the bindings were weak, although only the cerebellum showed a pattern of binding that was similar to that of the anterior pituitary. These data suggest that multiple proteins of the anterior pituitary recognize a specific region of the porcine FSH β-subunit gene.

Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis.

The primary structure of the cDNA and metallothionein (MT) genomic sequences of the tropical green mussel (Perna viridis) was determined. The complete cDNA sequences were obtained using degenerate primers designed from known metallothionein consensus amino acid sequences from the temperate species Mytilus edulis. The amino acid sequences of P. viridis metallothionein deduced from the coding region consisted of 72 amino acids with 21 cysteine residues and 9 Cys-X-Cys motifs corresponding to Type I MT class of other species. Two different genomic sequences coding for the same mRNA were obtained. Each putative gene contained a unique 5'UTR and two unique introns located at the same splice sites. The promoters for both genes were different in length and both contained metal responsive elements and active protein-binding sites. The structures of the genomic clones were compared with those of other species. J. Exp. Zool. 284:445-453, 1999.

AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells.

Mechanical induction of growth factor synthesis may mediate adaptive responses of smooth muscle cells (SMC) to increases in physical load. We previously demonstrated that cyclic mechanical stretch induces expression of the SMC, fibroblast, and epithelial cell mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bladder SMC, an observation that suggests that this growth factor may be involved in compensatory bladder hypertrophy. In the present study we provide evidence that the activator protein-1 (AP-1) transcription factor plays a critical role in this mechanoinduction process. Rat bladder SMC were transiently transfected with a series of 5' deletion mutants of a promoter-reporter construct containing 1. 7 kb of the mouse HB-EGF promoter that was previously shown to be stretch responsive. The stretch-mediated increase in promoter activity was completely ablated with deletion of nucleotide positions -1301 to -881. Binding of AP-1, as evaluated by electrophoretic mobility shift assay, to a synthetic oligonucleotide containing an AP-1 binding site increased in response to stretch, and binding was inhibited by excess unlabeled DNA corresponding to nucleotides -993 to -973 from the HB-EGF promoter, a region that contains a previously recognized composite AP-1/Ets site. Stretch-induced promoter activity was significantly inhibited by site-directed mutagenesis of the AP-1 or Ets components of this site. Consistent with the promoter and gel-shift studies, curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA induction after stretch. Stretch also specifically increased mRNA levels for matrix metalloproteinase (MMP)-1, the promoter of which contains a functional AP-1 element, but not for MMP-2, the promoter of which does not contain an AP-1 element. The stretch response of the MMP-1 gene was also completely inhibited by curcumin. Collectively, these findings indicate that AP-1-mediated transcription plays an important role in the regulation of gene expression in bladder muscle in response to mechanical forces.

Genomic Structure and Functional Characterization of NBPhox (PMX2B), a Homeodomain Protein Specific to Catecholaminergic Cells That Is Involved in Second Messenger-Mediated Transcriptional Activation

Homeodomain proteins play essential roles in basic processes during embryogenesis and development. NBPhox, a vertebrate paired-like homeodomain protein specific to catecholaminergic cells, may have a fundamental role in determining and maintaining the catecholaminergic phenotype. Here we describe the human and mouse genomic structures and the functional characterization of NBPhox. The genomic structure of NBPhox is highly conserved at the nucleotide level between human and mouse and is also quite similar to that of other paired-like homeodomain proteins, Arix/Phox2a, CRX, OTX1, and OTX2. The human NBPhox gene (HGMW-approved symbol PMX2B) maps to chromosomal region 5p12–p13. When the NBPhox expression plasmid was introduced into PC12h cells, the transcription from the promoter of the dopamine β-hydroxylase (DBH) gene was slightly stimulated. However, NBPhox substantially enhances second messenger-mediated activation of the DBH promoter by forskolin and/or phorbol ester. Furthermore, we found that NBPhox can also enhance second messenger-mediated activation of the c-fos promoter and several enhancers, including cyclic AMP-response element, the binding site for activator protein 1, and serum-response element. Our findings provide strong evidence that a homeodomain protein is involved in the activation of several genetic regulatory elements responsive to second messenger-mediated signals. These data suggest that this family of proteins may be involved in determining and maintaining a cell-specific phenotype through regulation of certain genes responsive to second messenger-mediated signals.

Regulation of mouse SP-B gene promoter by AP-1 family members.

The regulatory role of activator protein-1 (AP-1) family members in mouse surfactant protein (SP) B (mSP-B) promoter function was assessed in the mouse lung epithelial cell line MLE-15. Expression of recombinant Jun B and c-Jun inhibited mSP-B promoter activity by 50-75%. Although c-Fos expression did not alter mSP-B transcription, Jun D enhanced mSP-B promoter activity and reversed inhibition of mSP-B by c-Jun or Jun B. A proximal AP-1 binding site (-18 to -10 bp) was identified that overlaps a thyroid transcription factor-1 binding site. Mutation of this proximal AP-1 site blocked both Jun B inhibition and Jun D enhancement and partially blocked c-Jun inhibition of promoter activity. Promoter deletion mutants were used to identify additional sequences mediating the inhibitory effects of c-Jun in the distal region from -397 to -253 bp. The AP-1 element in this distal site (-370 to -364 bp) is part of a composite binding site wherein AP-1, cAMP response element binding protein, thyroid transcription factor-1, and nuclear factor I interact. Point mutation of the distal AP-1 binding site partially blocked c-Jun-mediated inhibition of the SP-B promoter. Both stimulatory (Jun D) and inhibitory (c-Jun/Jun B) effects of AP-1 family members on mSP-B promoter activity are mediated by distinct cis-acting elements in the mSP-B 5'-flanking region.

Angiotensin II-induced transactivation of epidermal growth factor receptor regulates fibronectin and transforming growth factor-beta synthesis via transcriptional and posttranscriptional mechanisms.

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of a growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein-coupled receptors. Here, we report the involvement of EGF-R in Ang II-induced synthesis of fibronectin and transforming growth factor-beta (TGF-beta) in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose dependently, with a maximal increase (approximately 5-fold) observed after 12 hours of incubation. Fibronectin synthesis induced by Ang II or calcium ionophore was completely abolished by tyrosine kinase inhibitors and intracellular Ca2+ chelating agents. Ang II-induced fibronectin mRNA was not affected by protein kinase C inhibitors or protein kinase C depletion, whereas specific inhibition of EGF-R function by a dominant negative EGF-R mutant and tyrphostin AG1478 abolished induction of fibronectin mRNA. We isolated the rat fibronectin gene, including the 5'-flanking region, and found that the activator protein-1 (AP-1) binding site present in the promoter region was responsible for the Ang II responsiveness of this gene. A gel retardation assay revealed the binding of nuclear protein to the AP-1 site, which was supershifted with anti-c-fos and anti-c-jun but not anti-activating transcription factor (ATF)-2 antibodies. Conditioned medium from Ang II-treated cells contained TGF-beta bioactivity, and addition of neutralizing TGF-beta antibody modestly (46%) inhibited induction of fibronectin. Ang II-induced synthesis of TGF-beta was also abolished by inhibition of EGF-R function. The effect of TGF-beta was exerted by stabilizing fibronectin mRNA without affecting the promoter activity and required de novo protein synthesis. We concluded that Ang II-induced expression of fibronectin and TGF-beta is mediated by downstream signaling of EGF-R transactivated by Ca2+-dependent tyrosine kinase and that Ang II-induced fibronectin mRNA expression is regulated by 2 different mechanisms, which are transcriptional control by binding of the c-fos/c-jun complex to the AP-1 site and posttranscriptional control by mRNA stabilization due to autocrine or paracrine effects of TGF-beta. Thus, this study suggests that the action of Ang II on extracellular matrix formation should be interpreted in association with the EGF-R signaling cascade.

Comparison of the effects of dopamine D 1 and D 2 receptor antagonists on nerve growth factor mRNA expression

Regulation of the expression of the nerve growth factor (NGF) gene has been reported previously to be mediated by the interaction of c- fos with an activator protein-1 (AP-1) binding site present in the first intron on the NGF gene. Using an RNase protection assay and in situ hybridization, we examined the effects of dopamine D 1 and D 2 receptor antagonists on NGF mRNA. Haloperidol (0.1–8 mg/kg) and (−)-sulpiride (10–100 mg/kg), induced NGF mRNA in a dose-dependent fashion in the hippocampus, piriform cortex, striatum and nucleus accumbens. The haloperidol (1 mg/kg)- and (−)-sulpiride (20 mg/kg)-induced NGF mRNA expression attained a maximum level 120 min after injection and returned to control levels 24 h later. Prior administration of the protein synthesis inhibitor cycloheximide blocked the haloperidol- and (−)-sulpiride-mediated induction of NGF mRNA. In contrast, R-(−)-8-chloro-2,3,4,5-tetrahydro-3,1-methyl-5-phenyl-11-3-benzyoepine-7-ol (SCH23390) did not induce NGF mRNA expression in either a dose-dependent or time-dependent manner. Our previous studies have shown that haloperidol and (−)-sulpiride induce the expression of c- fos and c- jun mRNAs and increase their AP-1 DNA binding activities. Thus, the data suggest that neuroleptics induce NGF gene expression by increasing AP-1 DNA binding activity.

Transforming Growth Factor β-1 Up-Regulates Clusterin Synthesis in Thyroid Epithelial Cells

Porcine epithelial cells in primary culture seeded on plastic substratum form a monolayer containing pseudo-vesicles. When cultured in the presence of thyreotropin (TSH) thyrocytes adopted a follicular-like structure and formed clusters. Transforming growth factor β-1 (TGFβ1) induced a rapid spreading of the TSH-treated cells only. At the same time, TGFβ1enhanced clusterin protein and mRNA expression. The increase of clusterin synthesis was proportional to the TGFβ1concentration in the culture medium. Tunicamycin abolished the up-regulation of whole clusterin synthesis and morphological changes. The activator protein-1 binding site partly directed the TGFβ1-stimulated clusterin expression. Phorbol ester caused rapid spreading of the cells with disappearance of vesicular and follicular structures. It decreased clusterin mRNA and protein expression, but increasedMr45,000 protein secretion in both TSH-treated and nontreated cells. Up-regulation of clusterin expression may be a marker of TGFβ-mediated thyrocyte dedifferentiation.

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.

Polyamine depletion is associated with an increase in JunD/AP-1 activity in small intestinal crypt cells.

Activator protein 1 (AP-1) is a group of dimeric transcription factors composed of protooncogene (Jun and Fos) subunits that bind to a common DNA site, the AP-1 binding site. The proteins of c-Jun, JunB, and Fos are essential for initiation of the cell cycle. Conversely, the activation of the junD gene slows cell growth in some cell types. The current study tests the hypothesis that polyamines influence cell growth by altering the balance of positive and negative Jun/AP-1 activities in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor for polyamine synthesis, for 4 and 6 days completely depleted cellular polyamine levels, while AP-1 binding activity was significantly increased. Spermidine, when given together with DFMO, restored AP-1 binding activity toward normal. The increased AP-1 complexes in polyamine-deficient cells were dramatically supershifted by the anti-JunD antibody but not by antibodies against c-Jun, JunB, or Fos proteins. There were significant increases in JunD mRNA and protein in DFMO-treated cells, although expression of the c-fos, c-jun, and junB genes decreased. The increase in JunD/AP-1 activity in DFMO-treated cells was associated with a significant decrease in cell division. Exposure of control quiescent cells to 5% dialyzed serum increased c-Jun/AP-1 but not JunD/AP-1 activities. DFMO prevented the stimulation of c-Jun/AP-1 activity induced by 5% dialyzed serum. These results indicate that 1) polyamine depletion is associated with an increase in AP-1 binding activity and 2) the increase in AP-1 activity in the DFMO-treated cells was primarily contributed by an increase in the JunD/AP-1. These findings suggest that polyamines regulate cell growth at least partially by modulating the balance of positive and negative Jun/AP-1 activities in the intestinal mucosa.

Cell Type-specific Transcription of the α1(VI) Collagen Gene: ROLE OF THE AP1 BINDING SITE AND OF THE CORE PROMOTER

Analysis of the chromatin of different cell types has identified four DNase I-hypersensitive sites in the 5'-flanking region of the alpha1(VI) collagen gene, mapping at -4.6, -4.4, -2.5, and -0.1 kilobase (kb) from the RNA start site. The site at -2.5 kb was independent from, whereas the other three sites could be related to, alpha1(VI) mRNA expression. The site at -0.1 kb was present in cells expressing (NIH3T3 and C2C12) but absent in cells not expressing (EL4) the mRNA; the remaining two sites were apparently related with high levels of mRNA. DNase I footprinting and gel-shift assays with NIH3T3 and C2C12 nuclear extracts have located a binding site for transcription factor AP1 (activator protein 1) between nucleotides -104 and -73. When nuclear extracts from EL4 lymphocytes were used, the AP1 site-containing sequence was bound by proteins not related to AP1. The existence of the hypersensitive site at -0.1 kb may be related to the binding of AP1 and of additional factors to the core promoter (Piccolo, S., Bonaldo, P., Vitale, P., Volpin, D., and Bressan, G. M. (1995) J. Biol. Chem. 270, 19583-19590). The function of the AP1 binding site and of the core promoter in the transcriptional regulation of the Col6a1 gene was investigated by expressing several promoter-reporter gene constructs in transgenic mice and in cell cultures. The results indicate that regulation of transcription of the Col6a1 gene by different cis-acting elements (core promoter, AP1 binding site and enhancers) is not completely modular, but the final output depends on the specific interactions among the three elements in a defined cell type.

UVB Irradiation-induced Activator Protein-1 Activation Correlates with Increased c-fos Gene Expression in a Human Keratinocyte Cell Line

The effects of UVB irradiation on transcription factor activator protein-1 (AP-1) DNA binding and AP-1 transactivation were studied in a human keratinocyte cell line, HaCaT. UVB-induced AP-1 binding to a consensus AP-1 binding site was observed by gel shift assays with maximum stimulation at 12 h after UVB irradiation. A promoter region of the human collagenase-1 gene containing the same AP-1 binding sequence linked to a luciferase reporter gene was stably transfected into HaCaT cells. UVB irradiation significantly increased luciferase activity in these stably transfected cells, with maximum activity observed at 24 h after UVB irradiation. c-Fos and Jun D were identified by antibody clearing assays as the main components of the bound AP-1 complexes. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide significantly abrogated the effect of UVB on AP-1 DNA binding, indicating that transcription and translation were required for AP-1 activation. Northern and Western analyses revealed a correlation between increased AP-1 activity and accumulation of c-fos mRNA and c-Fos protein after UVB irradiation. UVB irradiation increased c-fos transcription in HaCaT cells stably transfected with a plasmid containing the human c-fos promoter driving a luciferase reporter gene. These results suggest that increased c-fos expression may play an important role in UVB-induced AP-1 activation in HaCaT cells.

Adrenocorticotropic hormone stimulates CYP11B1 gene transcription through a mechanism involving AP-1 factors.

To elucidate the mechanism(s) by which adrenocorticotropic hormone (corticotropin) stimulates transcription of the steroid 11beta-monooxygenase gene (CYP11B1) in adrenocortical cells, the 5'-flanking region of rat CYP11B1 was analyzed using transient transfection and protein-binding assays with mouse adrenocortical Y1 cells. The results indicated that both basal and corticotropin-induced transcriptional activation of CYP11B1 required a common regulatory element containing a binding site for activator protein-1 (AP-1) transcription factors (dimers of the Jun and Fos family proteins) in the 5'-flanking region. Other DNA-binding protein(s) such as transcription factor Ad4BP was not required for either basal or corticotropin-induced transcriptional activation. Corticotropin stimuli were found to induce expression of a subset of the jun and fos family gene products in Y1 cells significantly, while total amounts of AP-1 factors capable of binding to its site in the CYP11B1 promoter did not change greatly. Treatment of rats with corticotropin had similar effects on mRNA levels of the jun and fos family genes in the adrenocortical zona fasciculata cells together with an enhancing effect on the level of CYP11B1 mRNA in the tissue. The effects of corticotropin on mRNA levels of the jun and fos family genes as well as transcription of CYP11B1 in Y1 cells were mimicked by treatment of the cells with dibutyryl cAMP. Furthermore, when components of AP-1 factors were overexpressed by transfecting Y1 cells with their expression vectors, a paired expression of AP-1 components such as c-Jun and c-Fos, which were inducible by corticotropin, transactivated the CYP11B1 promoter more strongly in the absence of corticotropin than other combinations such as JunD and Fra-2 expressed constitutively. These results suggest that corticotropin regulates transcription of the CYP11B1 gene by causing compositional changes in AP-1 transcription factors in the adrenocortical cells via a cAMP-dependent pathway.

Thioredoxin Peroxidase (Natural Killer Enhancing Factor) Regulation of Activator Protein-1 Function in Endothelial Cells

Thioredoxin peroxidase-1 (TxP-1), originally cloned as natural killer enhancing factor-B, belongs to a highly conserved antioxidant family. Tumor necrosis factor-α (TNF) activates the expression of activator protein-1 (AP-1) responsive genes. We show here that over-expression of TxP-1 blocks TNF-induced AP-1 activation in endothelial ECV304 cells, which was demonstrated by three independent experimental protocols: electromobility shift assay with AP-1 oligonucleotide probe; reporter gene expression with AP-1 binding site, and interleukin-8 production, which is dependent on AP-1. These results are consistent with the role of TxP-1 as an antioxidant and the previous reports that TNF-induced reactive oxygen species were responsible for AP-1 activation.

Identification of putative regulatory signals including the HAP1 binding site in the upstream sequence of the Aspergillus nidulans cytochrome c gene (cycA).

We speculate that a HAP1-like protein, similar to those which regulate oxygen transcriptional activation of many yeast respiratory genes, will probably also regulate the A. nidulans cytochrome c (cycA) gene. As part of a study to investigate the significance of a putative HAP1 (Haem Activator Protein) binding site in the regulatory region of the cycA gene, routine sequencing revealed an error in the published sequence (Raitt et al. 1994 Mol. Gen. Genet. 242: 17-22). Examination of the corrected sequence, including RT-PCR analysis of cycA mRNA, showed that an extra intron was present, and that the published translational start site was incorrect. This meant that the putative HAP1-binding site proposed by Raitt et al. could not be a regulatory element. However, further sequence analysis of the upstream sequence of the corrected cycA gene revealed putative regulatory signals, including possible HAP1 binding sites which are a closer match to recently reported yeast consensus sequences (Ha et al. 1996 Nucl. Acids Res. 24: 1453-1459).

Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein.

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.