Activator Protein-1 Motif Research Articles

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs co-modulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCC). However, how inflammatory gene signatures and cREL/p63/p73 targets are co-modulated genome-wide is unclear. Here, we examined how inflammatory factor TNF-α broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome-wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to Activator Protein-1 (AP-1) sites. cREL, ΔNp63α, and TAp73 binding and oligomerization on NF-κB, TP53 or AP-1 specific sequences were independently validated by ChIP-qPCR, oligonucleotide-binding assays, and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53, AP-1, and NF-κB specific response elements, or p21, SERPINE1, and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with co-binding activities for cREL, ΔNp63α, and TAp73 observed upon array profiling and RT-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

Estrogen increases the transcription of human α2-Heremans-Schmid-glycoprotein by an interplay of estrogen receptor α and activator protein-1

The expression of α2-Heremans-Schmid-glycoprotein (AHSG) was estrogen responsive in oophorectomized (OVX) osteopenic rats and HepG2 cells. Estrogen receptor α (ERα) interacted with the c-Jun/c-Fos heterodimer and indirectly associated with the -1488/-1482 activator protein-1 (AP-1) motif of the AHSG promoter. Estrogen increased c-Jun/c-Fos expression via the mitogen-activated protein kinase (MAPK) pathway. AHSG is a hepatic secretory protein implicated in the regulation of bone homeostasis. Serum AHSG in women has been reported to decrease after menopause and increase with estrogen therapy. The detailed regulatory mechanism of estrogen on AHSG is unclear. A postmenopausal osteoporosis model was generated in OVX rats. Skeletal parameters were determined by automatic biochemical analysis and dual X-ray absorptiometry. The expression of AHSG was evaluated by ELISA, real-time PCR, and Western blot. The 1.5-kb 5'-promoter region of AHSG was analyzed by serial truncation and luciferase assays. The putative -1488/-1482 AP-1 responsive element was identified by electrophoresis mobility shift assay (EMSA). Chromatin immunoprecipitation (ChIP), re-ChIP, and co-immunoprecipitation (Co-IP) were used to characterize the interaction of ERα and AP-1 at the -1488/-1482 AP-1 binding site. The MAPK pathway was evaluated using a specific inhibitor and active transfection. The expression of AHSG was estrogen responsive in both OVX rats and estradiol (E2)/ERα-treated HepG2 cells. E2/ERα most prominently increased luciferase activity of a construct with a putative -1488/-1482 AP-1 binding element. ERα interacted with the c-Jun/c-Fos heterodimer and indirectly associated with the -1488/-1482 AP-1 motif of the AHSG promoter. c-Jun/c-Fos expression was increased via the MAPK pathway by E2/ERα. Estrogen activated the transcription of AHSG through an indirect binding of ERα to the -1488/-1482 AP-1 binding element, with the c-Jun/c-Fos heterodimers.

BATF–JUN is critical for IRF4-mediated transcription in T cells

Interferon regulatory factor 4 (IRF4) is an IRF family transcription factor with critical roles in lymphoid development and in regulating the immune response1,2. IRF4 binds DNA weakly due to a C-terminal auto-inhibitory domain, but cooperative binding with factors such as PU.1 or SPIB in B cells increases binding affinity3, allowing IRF4 to regulate genes containing ETS/IRF composite elements (EICEs; 5′-GGAAnnGAAA-3′)1. Here, we show that in CD4+ T cells, where PU.1/SPIB expression is low, and in B cells, where PU.1 is well expressed, IRF4 unexpectedly can cooperate with Activator Protein-1 (AP-1) complexes to bind to AP-1/IRF4 composite (TGAnTCA/GAAA) motifs that we denote as AP-1/IRF composite elements (AICEs). Moreover, BATF/Jun family protein complexes cooperate with IRF4 in binding to AICEs in pre-activated CD4+ T cells stimulated with IL-21 and in Th17 differentiated cells. Importantly, BATF binding was diminished in Irf4−/− T cells and IRF4 binding was diminished in Batf−/− T cells, consistent with functional cooperation between these factors. Moreover, we show that AP-1 and IRF complexes cooperatively promote transcription of the Il10 gene, which is expressed in Th17 cells and potently regulated by IL-21. These findings reveal that IRF4 can signal via complexes containing ETS or AP-1 motifs depending on the cellular context, thus indicating new approaches for modulating IRF4-dependent transcription.

Prenatal Exposure of Mice to Diethylstilbestrol Disrupts T-Cell Differentiation by Regulating Fas/Fas Ligand Expression through Estrogen Receptor Element and Nuclear Factor-κB Motifs

Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effect of prenatal exposure to DES on thymocyte differentiation involving apoptotic pathways. Prenatal DES exposure caused thymic atrophy, apoptosis, and up-regulation of Fas and Fas ligand (FasL) expression in thymocytes. To examine the mechanism underlying DES-mediated regulation of Fas and FasL, we performed luciferase assays using T cells transfected with luciferase reporter constructs containing full-length Fas or FasL promoters. There was significant luciferase induction in the presence of Fas or FasL promoters after DES exposure. Further analysis demonstrated the presence of several cis-regulatory motifs on both Fas and FasL promoters. When DES-induced transcription factors were analyzed, estrogen receptor element (ERE), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein-1 motifs on the Fas promoter, as well as ERE, NF-κB, and NF-AT motifs on the FasL promoter, showed binding affinity with the transcription factors. Electrophoretic mobility-shift assays were performed to verify the binding affinity of cis-regulatory motifs of Fas or FasL promoters with transcription factors. There was shift in mobility of probes (ERE or NF-κB2) of both Fas and FasL in the presence of nuclear proteins from DES-treated cells, and the shift was specific to DES because these probes failed to shift their mobility in the presence of nuclear proteins from vehicle-treated cells. Together, the current study demonstrates that prenatal exposure to DES triggers significant alterations in apoptotic molecules expressed on thymocytes, which may affect T-cell differentiation and cause long-term effects on the immune functions.

PMA up-regulates the transcription of Axl by AP-1 transcription factor binding to TRE sequences via the MAPK cascade in leukaemia cells

Axl is a receptor tyrosine kinase promoting anti-apoptosis, invasion and mitogenesis, and is highly expressed in different solid cancers. Axl basal transcriptional activity is driven by Sp1/Sp3, and overexpression of MZF-1 (myeloid zinc-finger 1) induces Axl transcription and gene expression. Furthermore, Axl expression is epigenetically controlled by CpG hypermethylation; however, little is known about inducible Axl gene expression and Axl regulation in haematopoetic malignancies. In the present study, we studied Axl transcriptional regulation under PMA-stimulated conditions in leukaemia cells. Luciferase analysis with sequential 5'-deletion constructs revealed that the -660/-580 region of the Axl promoter is indispensable for induced promoter activity under PMA stimulation. This region includes AP-1 (activator protein 1)/CREB [CRE (cAMP-response-element)-binding protein] motifs, five times partially overlapping TGCGTG repeats and multiple GT repeats. Mutational, supershift and ChIP (chromatin immunoprecipitation) analysis determined that AP-1 family members bind to AP-1 motifs and to the 5 × TGCGTG overlapping repeats, thus transactivating Axl promoter activity. Furthermore, specific inhibitors of PKC (protein kinase C), ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 reduced Axl expression. Additionally, mithramycin treatment abolished constitutive and PMA-induced Axl expression. Taken together the results of the present study suggest that PMA-induced Axl gene expression in leukaemia cells is mediated by AP-1 motifs and 5 × TGCGTG repeats within the promoter region -660/-580, and through the PKC/ERK1/2/AP-1 or PKC/p-38/AP-1 signalling axis.

The activator protein 1 binding motifs within the human cytomegalovirus major immediate-early enhancer are functionally redundant and act in a cooperative manner with the NF-{kappa}B sites during acute infection.

Human cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Jun, the major subunits of activator protein 1 (AP-1), which in turn have been postulated to activate the viral immediate-early (IE) genes. Accordingly, the major IE promoter (MIEP) enhancer, a critical control region for initiating lytic HCMV infection and reactivation from the latent state, contains one well-characterized AP-1 site and a second candidate interaction site. In this study we explored the role of these AP-1 elements in the context of the infection. We first show that the distal candidate AP-1 motif binds c-Fos/c-Jun heterodimers (AP-1 complex) and confers c-Fos/c-Jun-mediated activity to a core promoter. Site-directed mutagenesis studies indicate that both AP-1 response elements are critical for 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced MIEP activity in transient-transfection assays. In marked contrast to the results obtained with the isolated promoter, disruption of the AP-1 recognition sites of the MIEP in the context of the infectious HCMV genome has no significant influence on the expression of the MIE protein IE1 or viral replication in different cell types. Moreover, a chimeric murine CMV driven by the HCMV MIEP (hMCMV-ES) with the two AP-1 binding sites mutated is not compromised in virulence, is able to grow and disseminate to different organs of the newborn mice as efficiently as the parental virus, and is competent in reactivation. We show, however, that combined inactivation of the enhancer AP-1 and NF-κB recognition sites leads to an attenuation of the hMCMV-ES in the neonatal murine infection model, not observed when each single element is abolished. Altogether, these results underline the functional redundancy of the MIEP elements, highlighting the plasticity of this region, which probably evolved to ensure maximal transcriptional performance across many diverse environments.

An Akt- and Fra-1-Dependent Pathway Mediates Platelet-Derived Growth Factor-Induced Expression of Thrombomodulin, a Novel Regulator of Smooth Muscle Cell Migration

Overdistension of hollow organs evokes pathological changes characterized by smooth muscle remodeling. Mechanical stimuli induce smooth muscle cell (SMC) growth through acute activation of signaling cascades and by increased expression of soluble mitogens. Physical forces have also been implicated in ligand-independent activation of receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) receptor, although the extent to which this occurs in intact tissue is unknown. Previously, we implicated Akt and activator protein-1 (AP-1) as mediators of growth and gene expression in SMC exposed to cyclic stretch or PDGF. Here we show that bladder wall distension leads to PDGFR activation and identify thrombomodulin (TM) as an Akt and AP-1 target in SMC. We demonstrate that TM, also induced by bladder stretch injury, is regulated at the transcriptional level by the AP-1 components c-jun and Fra1. Mutation of an AP-1 motif at -2010/-2004 abolished both AP-1 binding and PDGF responsiveness of the TM promoter. Fra1 silencing diminished PDGF-induced TM expression and SMC cell cycle transit. In contrast, TM knockdown did not affect cell growth but attenuated PDGF-stimulated SMC migration. Taken together, these results reveal new facets of TM regulation in SMC and provide the first demonstration of a role for endogenous TM in PDGF-induced cell migration. Moreover, TM induction on bladder injury suggests that it may be a biomarker for pathological smooth muscle remodeling.

Regulation of CCN2/Connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: Role of Smad and activator protein 1 signaling

To investigate transforming growth factor beta (TGFbeta) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGFbeta-mediated CTGF promoter activity. CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGFbeta treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGFbeta-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFbeta was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGFbeta expression in the degenerated state. TGFbeta, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.

Genomic organization and regulation of the human orexin (hypocretin) receptor 2 gene: identification of alternative promoters

Orexins (hypocretins), acting via their receptors, are involved in the control of feeding behaviour, sleep, arousal and energy homoeostasis. However, regulation of the human orexin receptor 2 (hOX2R) gene remains unknown. We have identified four transcripts arising from alternative splicing from three exons. These exon 1 variants were designated exons 1A, 1B and 1C on the basis of their 5'-3' order. RT (reverse transcription)-PCR demonstrates the differential expression in various human tissues. The alternative 5'-UTRs (untranslated regions) possessed by these isoforms have different translational efficiencies, which regulate the level of protein expression. In the present study, we have demonstrated that the hOX2R gene is regulated by two promoters and the novel transcripts are regulated by the distal promoter located upstream of exon 1A. We have demonstrated that the AP-1 (activator protein 1) motif is critical for sustaining the basal activity of distal promoter. Analysis of the proximal promoter revealed the region regulating promoter activity contained putative binding elements including those for CREB (cAMP-response-element-binding protein), GATA-2 and Oct-1. Using the chromatin immunoprecipitation assay, we demonstrated that CREB, GATA-2 and Oct-1 transcription factors bind to these critical regulatory promoter elements. Mutational studies suggested that these motifs functioned independently, but have a compound effect regulating hOX2R gene transcription. Furthermore, proximal promoter activity is enhanced by both PKA (protein kinase A) and PKC (protein kinase C) pathway activation, via binding of CREB and GATA-2 transcription factors. In conclusion, we have demonstrated that expression of hOX2R is regulated by a complex involving a proximal PKA/PKC-regulated promoter and a distal promoter regulating tissue-specific expression of alternative transcripts which in turn post-transcriptionally regulate receptor levels.

A cis-regulatory site downregulates PTHLH in translocation t(8;12)(q13;p11.2) and leads to Brachydactyly Type E

Parathyroid hormone-like hormone (PTHLH) is an important chondrogenic regulator; however, the gene has not been directly linked to human disease. We studied a family with autosomal-dominant Brachydactyly Type E (BDE) and identified a t(8;12)(q13;p11.2) translocation with breakpoints (BPs) upstream of PTHLH on chromosome 12p11.2 and a disrupted KCNB2 on 8q13. We sequenced the BPs and identified a highly conserved Activator protein 1 (AP-1) motif on 12p11.2, together with a C-ets-1 motif translocated from 8q13. AP-1 and C-ets-1 bound in vitro and in vivo at the derivative chromosome 8 breakpoint [der(8) BP], but were differently enriched between the wild-type and BP allele. We differentiated fibroblasts from BDE patients into chondrogenic cells and found that PTHLH and its targets, ADAMTS-7 and ADAMTS-12 were downregulated along with impaired chondrogenic differentiation. We next used human and murine chondrocytes and observed that the AP-1 motif stimulated, whereas der(8) BP or C-ets-1 decreased, PTHLH promoter activity. These results are the first to identify a cis-directed PTHLH downregulation as primary cause of human chondrodysplasia.

Aromatase Promoter I.f is Regulated by Estrogen Receptor Alpha (ESR1) in Mouse Hypothalamic Neuronal Cell Lines1

Aromatase (CYP19A1) catalyzes the conversion of C(19) steroids to estrogens. Aromatase and its product estradiol (E(2)) are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The regulation of aromatase expression in the brain is not well understood. The aromatase (Cyp19a1) gene is selectively expressed in distinct neurons of the hypothalamus through a distal brain-specific promoter I.f located approximately 36 kb upstream of the coding region. Here, we investigated a short feedback effect of E(2) on aromatase mRNA expression and enzyme activity using estrogen receptor alpha (ESR1; also known as ER alpha)-positive or ESR1-negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. Estradiol regulated aromatase mRNA expression and enzyme activity in a time- and dose-dependent manner, whereas an E(2) antagonist reversed these effects. The nucleotide -200/-1 region of promoter I.f conferred E(2) responsiveness. Two activator protein 1 (AP-1) elements in this region were essential for induction of promoter activity by E(2). ESR1 and JUN (c-Jun) bound to these AP-1 motifs in intact cells and under cell-free conditions. The addition of an ESR1 mutant that interacts with JUN but not directly with DNA enhanced E(2)-dependent promoter I.f activity. Independently, we demonstrated an interaction between ESR1 and JUN in hypothalamic cells. Knockdown of ESR1 abolished E(2)-induced aromatase mRNA and enzyme activity. Taken together, E(2) regulates Cyp19a1 expression via promoter I.f by enhanced binding of an ESR1/JUN complex to distinct AP-1 motifs in hypothalamic cells. We speculate that this mechanism may, in part, regulate gonadotropin secretion and sexual activity.

Glucocorticoid and Growth Factor Synergism Requirement for Notch4 Chromatin Domain Activation

The Notch signaling pathway modulates cell fate in diverse contexts, including vascular development. Notch4 is selectively expressed in vascular endothelium and regulates vascular remodeling. The signal-dependent transcription factor activator protein 1 (AP-1) activates Notch4 transcription in endothelial cells, but other factors/signals that regulate Notch4 are largely unknown. We demonstrate that, unlike the established transrepression mechanism in which the glucocorticoid receptor (GR) antagonizes AP-1, AP-1 and GR synergistically activated Notch4 transcription in endothelial cells. Fibroblast growth factor 2 (FGF-2) and cortisol induced AP-1 and GR occupancy, respectively, at a Notch4 promoter composite response element consisting of an imperfect half-glucocorticoid response element and an AP-1 motif, which mediated signal-dependent activation. Analysis of Notch4 promoter complex assembly provided evidence that GR and AP-1 independently occupy the composite response element, but AP-1 stabilizes GR occupancy. In multipotent 10T1/2 cells, FGF-2 and cortisol induced a histone modification pattern at the Notch4 locus mimicking that present in endothelial cells and reprogrammed Notch4 from a repressed to an active state. These results establish the molecular basis for a novel AP-1/GR-Notch4 axis in vascular endothelium.

Distinct modulation of angiotensin II-induced early left ventricular hypertrophic gene programming by dietary fat type

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.

Insulin-mediated activation of activator protein-1 through the mitogen-activated protein kinase pathway stimulates collagenase-1 gene transcription in the MES 13 mesangial cell line.

The initial stages of diabetic nephropathy are characterized, in part, by expansion of the mesangial matrix and thickening of the glomerular basement membrane which are caused by increased extracellular matrix (ECM) protein synthesis and reduced degradation, a consequence of decreased matrix metalloproteinase (MMP) activity. These changes have been largely attributed to the effects of hyperglycemia such that the potential contribution of impaired insulin action to alterations in the ECM have not been studied in detail. We have shown here that insulin stimulates collagenase-1 fusion gene transcription in the MES 13 mesangial-derived cell line. Multiple collagenase-1 promoter elements are required for the full stimulatory effect of insulin but the action of insulin appears to be mediated through an activator protein-1 (AP-1) motif. Thus, mutation of this AP-1 motif abolishes insulin-stimulated collagenase fusion gene transcription and, in isolation, this AP-1 motif can mediate a stimulatory effect of insulin on the expression of a heterologous fusion gene. This suggested that the other collagenase-1 promoter elements that are required for the full stimulatory effect of insulin probably bind accessory factors that enhance the effect of insulin mediated through the AP-1 motif. In MES 13 cells, the AP-1 motif is bound by Fra-1, Fra-2, Jun B and Jun D. Stimulation of collagenase-1 fusion gene transcription by insulin requires activation of the mitogen-activated protein kinase (MEK) pathway since inhibition of MEK-1 and -2 blocks this effect. The potential significance of these observations with respect to a role for insulin in the pathophysiology of diabetic glomerulosclerosis is discussed.

Up-regulation of matrix metalloproteinase-1 expression in U937 cells by low-density lipoprotein-containing immune complexes requires the activator protein-1 and the Ets motifs in the distal and the proximal promoter regions.

We reported previously that low-density lipoprotein (LDL)-containing immune complexes (LDL-IC) stimulated matrix metalloproteinase-1 (MMP-1) expression in U937 histiocytes through Fc gamma receptor (FcgammaR)-mediated extracellular signal-regulated kinase pathway. The present study has explored the transcriptional mechanisms involved in the stimulation. Deletion analysis showed that LDL-IC stimulated MMP-1 promoter activity in cells transfected with the Construct 1 that contained a 4,334-bp MMP-1 promoter fragment, but had no effect in cells transfected with other constructs that had shorter MMP-1 promoter (2685-bp or less), suggesting that cis-acting elements located between -4334 and -2685 are required for the promoter stimulation. The mutation study further indicated that the activator protein-1 (AP-1) (-3471) or Ets (-3836) motifs in this distal region were essential for the LDL-IC-stimulated MMP-1 expression. Moreover, although above deletion analysis showed that LDL-IC did not stimulate MMP-1 promoter activity in cells transfected with constructs that contained the proximal AP-1 (-72) and Ets (-88) in the promoter fragments that are 2685-bp or less, the mutations of the -72 AP-1 or the -88 Ets motif in the construct 1 abolished the stimulation of MMP-1 expression by LDL-IC, suggesting that a long promoter sequence is required for the -72 AP-1 and -88 Ets motifs to be involved in the stimulation. Finally, electrophoretic mobility shift assay showed that LDL-IC stimulated the activities of transcription factors AP-1 and Ets. In conclusion, the present study shows that both the distal and proximal AP-1 and Ets motifs are required for LDL-IC-stimulated MMP-1 expression in U937 histiocytes.

Transcriptional synergism on the pS2 gene promoter between a p160 coactivator and estrogen receptor-alpha depends on the coactivator subtype, the type of estrogen response element, and the promoter context.

The pS2 gene is estrogen responsive in hepatocarcinoma cells (HepG2) in the presence of estrogen receptor alpha (ERalpha). The estrogenic activity is mediated through an estrogen response element (ERE) in the 5'-flanking region of the pS2 gene; however, an activator protein 1 (AP1) response element located close to the ERE in the pS2 promoter has also proven essential for a maximum response to estrogen. In the present study, we show estrogen-induced synergistic activity by the p160 coactivator steroid receptor coactivator-1 (SRC-1), mediated via the ERE and the AP1 response element in the pS2 promoter. In addition, we present data that support an interaction between the ERE and the AP1 motif via SRC-1. The related but distinct p160 coactivator, transcriptional intermediary factor-2, was a more potent activator of pS2 gene expression. In addition, transcriptional intermediary factor-2 was less dependent on an intact AP1 response element in the pS2 promoter than SRC-1. Furthermore, the type of ERE in the pS2 promoter influenced the potentiation by SRC-1, supported by less dependence on the AP1 motif when the natural ERE was substituted for by a consensus ERE. These results highlight several mechanisms whereby fine-tuning of estrogen responsiveness of an individual gene may be achieved.

Accessory Elements, Flanking DNA Sequence, and Promoter Context Play Key Roles in Determining the Efficacy of Insulin and Phorbol Ester Signaling through the Malic Enzyme and Collagenase-1 AP-1 Motifs

Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-1-CAT fusion gene expression in H4IIE cells through identical activator protein-1 (AP-1) motifs. In contrast, insulin and phorbol esters only stimulate collagenase-1-CAT and not ME-CAT fusion gene expression in HeLa cells. The experiments in this article were designed to explore the molecular basis for this differential cell type- and gene-specific regulation. The results highlight the influence of three variables, namely promoter context, AP-1 flanking sequence, and accessory elements that modulate insulin and phorbol ester signaling through the AP-1 motif. Thus, fusion gene transfection and proteolytic clipping gel retardation assays suggest that the AP-1 flanking sequence affects the conformation of AP-1 binding to the collagenase-1 and ME AP-1 motifs such that it selectively binds the latter in a fully activated state. However, this influence of ME AP-1 flanking sequence is dependent on promoter context. Thus, the ME AP-1 motif will mediate both an insulin and phorbol ester response in HeLa cells when introduced into either the collagenase-1 promoter or a specific heterologous promoter. But even in the context of the collagenase-1 promoter, the effects of both insulin and phorbol esters, mediated through the ME AP-1 motif are dependent on accessory factors.

PS2 Gene expression in HepG2 cells: complex regulation through crosstalk between the estrogen receptor alpha, an estrogen-responsive element, and the activator protein 1 response element.

The pS2 promoter is complex with binding sites for a number of protein factors that may participate in modulating its activity. The pS2 gene was transcriptionally activated by estrogens in HepG2 cells transformed (HepER3) to express the estrogen receptor alpha (ERalpha). The phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated pS2 expression in both HepER3 and the parental, non-ER-expressing HepG2 cells, although its activity was substantially less in HepG2 cells. The use of selective protein kinase inhibitors suggested that the MAPK pathway contributes substantially to estrogen stimulation of the pS2 promoter. The activator protein 1 (AP1) site at -332 to -338 in the pS2 promoter had a dominant role in the response to both estrogens and PMA, although the estrogen response element at -393 to -405 was essential to mediate the response to estrogen. The potentiation of pS2 promoter activity by the AP1 motif in response to estrogen was dependent on the ligand binding domain of ERalpha. Furthermore, the presence of an intact AP1 element in the pS2 promoter sustained suppression of pS2 promoter activity by an LXXLL peptide. In summary, the data suggest that the effect of estrogen is mediated through a cross-talk between the estrogen-responsive element and the AP1 response element and that ERalpha plays a crucial role in mediating the effect of both estrogen and PMA.

Direct left ventricular wall stretch activates GATA4 binding in perfused rat heart: involvement of autocrine/paracrine pathways.

The signaling cascades that activate transcription factors during cardiac hypertrophy are largely unknown. To evaluate the mechanisms for GATA4 and activator protein-1 (AP-1) activation, isolated perfused rat hearts were exposed to elevated wall stretch by inflating a left ventricular balloon. Gel mobility shift assays were used to analyze the transacting factors that interact with the GATA or the AP-1 motifs of the B-type natriuretic peptide (BNP) promoter. Direct wall stretch for 30 min produced a twofold increase ( P<0.001) in left ventricular BNP GATA4- but not GATA5- and GATA6-binding activity. In addition, increased BNP AP-1-binding activity and the presence of c-fos were demonstrated in wall-stretch-stimulated ventricles compared with unloaded ventricles. The mixed endothelin-1 ETA/ETB receptor antagonist bosentan and the angiotensin II (Ang II) type 1 (AT1) receptor antagonist CV-11974 completely inhibited the wall-stretch-induced increase in left ventricular BNP GATA4 and AP-1 activity. Infusions of ET-1 and Ang II in the absence of wall stretch also stimulated BNP GATA4-binding activity ( P<0.01). These results show that ET-1 and Ang II are required for the stimulation of GATA4 and AP-1 DNA-binding activity in response to direct left ventricular wall stretch.