Ets Sites Research Articles

The 2G single nucleotide polymorphism (SNP) in the MMP-1 promoter contributes to high levels of MMP-1 transcription in MCF-7/ADR breast cancer cells.

Degradation of stromal collagens in the extracellular matrix is mediated largely by matrix metalloproteinase-1 (MMP-1; collagenase-1), and high constitutive levels of MMP-1 in breast cancer correlate with a poor prognosis and invasive disease. MMP-1 expression is, in part, controlled by the mitogen-activated protein kinase (MAPK) pathway(s), which may target several activator protein-1 (AP-1) and polyoma enhancing activity-3/E26 virus (PEA3/ETS) sites within the promoter. An additional ETS site in the MMP-1 promoter is conferred by a single nucleotide polymorphism (SNP) at -1607 bp, when two guanines (5'-GGAT-3'; '2G allele/SNP') are present instead of one guanine (5'-GAT-3'; '1G allele/SNP'). This SNP is adjacent to an AP-1 site at -1602 bp, and in the presence of the 2G allele (ETS site), these sites cooperate to induce higher levels of transcription. ERK 1/2 is one component of the MAPK pathway and is constitutively active in MCF-7/ADR breast cancer cells, which are 1G/2G heterozygotes. This study demonstrates that when these cells are treated with PD098059, an ERK-specific inhibitor, MMP-1 mRNA levels are significantly decreased, suggesting that high constitutive expression of MMP-1 in these cells results from continuous ERK 1/2 activation. Using transient transfection, we determined that this signaling pathway targets different AP-1/ETS sites, depending upon which allele is present. Furthermore, in these cells, the AP-1 site at -1602 bp enhances transcription in the presence of the 2G SNP, but represses transcription from the 1G SNP. Finally, inhibiting ERK signaling and MMP-1 expression blocks type I collagen degradation and reduces the invasive ability of the MCF-7/ADR cells. We conclude that ERK 1/2 signaling and the 2G SNP mediate high levels of MMP-1 expression, which may contribute to the invasive potential of these breast cancer cells.

HER2/Neu- and TAK1-mediated Up-regulation of the Transforming Growth Factor β Inhibitor Smad7 via the ETS Protein ER81

The cytokine transforming growth factor beta (TGF-beta) plays an important role in preventing tumor formation by blocking cell cycle progression. Accordingly, many cancers demonstrate mutations in TGF-beta signaling components or enhanced expression of inhibitors of the TGF-beta pathway such as Smad7. In this report we show that the oncoprotein HER2/Neu is able to collaborate with the ETS transcription factor ER81 to activate Smad7 transcription in breast, endometrial, and ovarian cancer cell lines. ER81 binds to two ETS sites within the Smad7 promoter, and mutation of one of these ETS sites greatly decreases Smad7 induction by HER2/Neu and ER81. Furthermore, we show that Smad7 activation involves the processing of signals from HER2/Neu to ER81 via the ERK mitogen-activated protein kinase pathway. Thus, we have uncovered a novel mechanism by which oncogenic HER2/Neu, in collaboration with ER81, can induce carcinogenesis through Smad7 up-regulation. Moreover, we show that TAK1, a TGF-beta-activated protein kinase, stimulates ER81 via the p38 mitogen-activated protein kinase pathway and thereby induces the Smad7 promoter. This suggests that attenuation of TGF-beta signaling by activating Smad7 transcription may proceed not only through TGF-beta receptor-regulated Smad proteins but also through an independent pathway involving ER81 and TAK1.

Ets motifs are necessary for endothelial cell-specific expression of a 723-bp Tie-2 promoter/enhancer in Hprt targeted transgenic mice.

Tie-2 is an endothelial cell-specific receptor tyrosine kinase that is involved in the remodeling of blood vessels and angiogenesis. Our goal was to characterize Tie-2 promoter function as a means of providing insight into the mechanisms of endothelial cell-specific gene regulation. When targeted to the Hprt locus of mice, a small Tie-2 promoter fragment (containing a 300-bp intronic enhancer coupled upstream to a 423-bp core promoter) (T-short) directed widespread endothelial cell expression in vivo. The T-short promoter contains 2 clusters of Ets sites, one in the first exon, the other in the intronic enhancer. In cultured endothelial cells, a combined mutation of the Ets motifs resulted in a significant reduction in promoter activity. Consistent with these results, the same Ets mutations resulted in a loss of detectable expression of the T-short promoter in all vascular beds with the notable exception of the brain. These results suggest that the T-short promoter contains information for widespread expression in the vascular tree, Ets sites are necessary for in vivo promoter activity, and the shorter Tie-2 fragment may be useful as a tool to direct heterologous gene expression within the intact endothelium.

Fra-1 targets the AP-1 site/2G single nucleotide polymorphism (ETS site) in the MMP-1 promoter.

The matrix metalloproteinase (MMP) family degrades the extracellular matrix. One member of this family, MMP-1, initiates the breakdown of interstitial collagens. The expression of MMP-1 is controlled by the mitogen activated protein kinase (MAPK) pathway(s) via the activity of activator protein-1 (AP-1) and polyoma enhancing activity-3/E26 virus (PEA3/ETS) transcription factors through consensus binding sites present in the promoter. Another ETS site in the MMP-1 promoter is created at -1607 bp by a single nucleotide polymorphism (SNP), which contains two guanines (5'-GGAT-3'; '2G SNP'), rather one guanine (5'-GAT-3'; '1G SNP'), adjacent to an AP-1 binding site at -1602 bp. The 2G SNP displays greater transcriptional activity than the 1G SNP, and AP-1 and Ets families of transcription factors cooperate to increase transcription. The 2G SNP has been linked to the incidence and the progression of several cancers and is also associated with non-neoplastic diseases; although the underlying mechanism(s) has yet to be elucidated. In this study we demonstrate that the expression of Fos-like region antigen (Fra-1), an AP-1 transcription factor component that also correlates strongly with neoplastic disease, is necessary for MMP-1 transcription in A2058 melanoma cells. The inhibition of Fra-1 expression preferentially downregulates transcription from the MMP-1 promoter DNA containing the 2G SNP, compared to DNA containing the 1G SNP. This study provides evidence that, in cooperation with the 2G DNA polymorphism, the AP-1 family member, Fra-1, contributes to the high constitutive expression of MMP-1 in melanoma cells.

Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene.

The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins.

Identification of an enhancer of the human activating protein-2alpha gene that contains a critical Ets1 binding site.

Deletion analysis studies of the 5'-flanking region of the activating protein-2alpha (AP-2alpha) gene indicates that the proximal 152 bp are essential for minimal promoter activity and that a 140-bp fragment from nucleotides -1279 to -1139 acts as an enhancer of basal transcriptional activity. Ligation of the 140-bp fragment to a minimal AP-2alpha promoter or a heterologous simian virus 40 promoter luciferase reporter plasmid conferred enhancer activity in trophoblast cells. In deoxyribonuclease I footprint studies, nuclear extracts from trophoblast cells protected two regions of the 140-bp fragment, E2 and E3. Site-directed mutagenesis of an Ets1-binding site in E2 significantly inhibited AP-2alpha enhancer activity, whereas mutagenesis of two putative polyomavirus enhancer activator-3-binding sites on E2 and an insulin upstream factor 1-binding site in E3 had no effect on enhancer activity. Gel shift and supershift assays indicated that Ets1 binds to the Ets site in E2, and overexpression of Ets1 in transfection studies induced AP-2alpha promoter activity. As the transcription factor Ets1 is abundant in trophoblast cells, these investigations strongly suggest that AP-2alpha gene expression in the placenta is enhanced by a cis-acting element at nucleotides -1279 to -1139 that contains a critical Ets1-binding site.

Characterization and analysis of the proximal Janus kinase 3 promoter.

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase essential for signaling via cytokine receptors that comprise the common gamma-chain (gammac), i.e., the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Jak3 is preferentially expressed in hemopoietic cells and is up-regulated upon cell differentiation and activation. Despite the importance of Jak3 in lymphoid development and immune function, the mechanisms that govern its expression have not been defined. To gain insight into this issue, we set out to characterize the Jak3 promoter. The 5'-untranslated region of the Jak3 gene is interrupted by a 3515-bp intron. Upstream of this intron and the transcription initiation site, we identified an approximately 1-kb segment that exhibited lymphoid-specific promoter activity and was responsive to TCR signals. Truncation of this fragment revealed that core promoter activity resided in a 267-bp fragment that contains putative Sp-1, AP-1, Ets, Stat, and other binding sites. Mutation of the AP-1 sites significantly diminished, whereas mutation of the Ets sites abolished, the inducibility of the promoter construct. Chromatin immunoprecipitation assays showed that histone acetylation correlates with mRNA expression and that Ets-1/2 binds this region. Thus, transcription factors that bind these sites, especially Ets family members, are likely to be important regulators of Jak3 expression.

Orphan Nuclear Receptor Nur77 Is Involved in Caspase-independent Macrophage Cell Death

Activation-induced cell death in macrophages has been observed, but the mechanism remains largely unknown. Activation-induced cell death in macrophages can be independent from caspases, and the death of activated macrophages can even be triggered by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD). Here, we show that this type of macrophage death can occur in the septic mouse model and that toll-like receptor (TLR)-2 or TLR4 signaling is required in this process. We conclude that Nur77 is involved in the macrophage death because Nur77 expression correlates with cell death, and cell death is reduced significantly in Nur77-deficient macrophages. The extracellular signal–regulated kinase pathway, which is downstream of TLR2 or TLR4, and myocyte-specific enhancer binding factor 2 (MEF2) transcription factor activity, which is up-regulated by zVAD, are required for Nur77 induction and macrophage death. Reporter gene analysis suggests that Nap, Ets, Rce, and Sp1 sites in the Nur77 promoter are regulated by TLR4 signaling and that MEF2 sites in the Nur77 promoter are regulated by zVAD treatment. MEF2 transcription factors are constitutively expressed and degraded in macrophages, and zVAD increases MEF2 transcription factor activity by preventing the proteolytic cleavage and degradation of MEF2 proteins. This paper delineates the dual signaling pathways that are required for Nur77 induction in macrophages and demonstrates a role of Nur77 in caspase-independent cell death.

Coactivator CBP in the regulation of conceptus IFNtau gene transcription.

Studies of ovine interferon-tau (oIFNtau) gene regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, have been carried out, but a definitive mechanism for its spatial-temporal transcription has not been elucidated. Recently, specific binding regions for transcription factors AP-1 and Ets-2 on the oIFNtau gene were identified; however, a molecular mechanism by which these factors regulate oIFNtau gene transcription has not been characterized. In the present study, we investigated the potential relationship between AP-1 and Ets-2, and their association with a coactivator, cAMP-response element binding protein-binding protein (CBP), on oIFNtau gene transcription in a transient transfection system using human choriocarcinoma JEG3 cells. The oIFNtau gene promoter/enhancer (-654 to + 1 bases, wild type)-luciferase reporter construct (pGL3-654) or its mutant at the AP-1 or Ets-2 site was cotransfected with CBP (pRc/RSV-CBP) construct along with c-jun, c-fos, and/or Ets-2 expression plasmid. CBP enhanced transcription of the wild type oIFNtau-reporter construct; however, this coactivator had no effect on the oIFNtau-reporter construct with mutated AP-1 or Ets-2 site. Cotransfection of CBP with c-jun and/or Ets-2, but not with c-fos, further increased oIFNtau gene transactivation although amounts of c-jun and c-fos expression, resulting from expression vectors, were similar. In addition, CBP inhibitor adenovirus 12S E1A (E1A), but not the mutant of E1A without CBP binding domain (Delta2-36), suppressed oIFNtau gene transcription. These observations suggest that c-jun and Ets-2 are the most probable binding partners for CBP in the potentiation of oIFNtau gene transcription. Mol. Reprod. Dev. 65: 23-29, 2003.

Activation of the early B-cell-specific mb-1 (Ig-alpha) gene by Pax-5 is dependent on an unmethylated Ets binding site.

Methylation of cytosine in CpG dinucleotides promotes transcriptional repression in mammals by blocking transcription factor binding and recruiting methyl-binding proteins that initiate chromatin remodeling. Here, we use a novel cell-based system to show that retrovirally expressed Pax-5 protein activates endogenous early B-cell-specific mb-1 genes in plasmacytoma cells, but only when the promoter is hypomethylated. CpG methylation does not directly affect binding of the promoter by Pax-5. Instead, methylation of an adjacent CpG interferes with assembly of ternary complexes comprising Pax-5 and Ets proteins. In electrophoretic mobility shift assays, recruitment of Ets-1 is blocked by methylation of the Ets site (5'CCGGAG) on the antisense strand. In transfection assays, selective methylation of a single CpG within the Pax-5-dependent Ets site greatly reduces mb-1 promoter activity. Prior demethylation of the endogenous mb-1 promoter is required for its activation by Pax-5 in transduced cells. Although B-lineage cells have only unmethylated mb-1 genes and do not modulate methylation of the mb-1 promoter during development, other tissues feature high percentages of methylated alleles. Together, these studies demonstrate a novel DNA methylation-dependent mechanism for regulating transcriptional activity through the inhibition of DNA-dependent protein-protein interactions.

Cloning and Analysis of the Thrombopoietin-induced Megakaryocyte-specific Glycoprotein VI Promoter and Its Regulation by GATA-1, Fli-1, and Sp1

The exposure of collagen fibers at sites of vascular injury results in the adherence of platelets and their subsequent activation. The platelet collagen receptor glycoprotein (GP)(1) VI plays a crucial role in platelet activation and thrombus formation and decreased levels or defective GPVI may lead to excessive bleeding. In addition, elevated levels of collagen receptors may predispose individuals to coronary heart disease or strokes. GPVI expression is restricted to platelets and their precursor cell, the megakaryocyte. In this study we investigate the regulation of GPVI expression and show that thrombopoietin induces its expression in the megakaryocytic cell line UT-7/TPO. A 5'-region flanking the transcription start point of the GPVI gene was cloned (-694 to +29) and we report that this putative GPVI promoter bestows megakaryocye-specific expression. Deletion analyses and site-directed mutagenesis identified Sp1(227), GATA(177), and Ets(48) sites as essential for GPVI expression. We show that transcription factors GATA-1, Fli-1, and Sp1 can bind to and activate this promoter. Finally, GPVI mRNA was detected only in megakaryocytic cell lines expressing both Fli-1 and GATA-1, and we show that overexpression of Fli-1 in a stable cell line (which expresses endogenous GATA-1 and Sp1) results in expression of the endogenous GPVI gene.

IRF-8/ICSBP and IRF-1 cooperatively stimulate mouse IL-12 promoter activity in macrophages

IRF-8/ICSBP and IRF-1 are IRF family members whose expression is induced in response to IFN-γ in macrophages. IL-12 is a cytokine produced in macrophages that plays a critical role in host defense. IFN-γ and bacterial lipopolysaccharide (LPS) induce IL-12p40 transcription, which is necessary for the production of IL-12. We have previously shown that IL-12p40 expression is impaired in ICSBP-deficient mice and that transfection of ICSBP together with IRF-1 can activate IL-12p40 expression in mouse macrophage cells. To further study the role of ICSBP and IRF-1, we investigated murine IL-12p40 promoter activity in the macrophage cell line RAW 264.7. We show here that co-transfection of ICSBP and IRF-1 synergistically stimulates IL-12 promoter activity to a level comparable to that induced by IFN-γ/LPS. Mutation of the Ets or NFκB site previously shown to be important for IL-12p40 transcription did not abolish the activation by ICSBP and IRF-1. However, mutation of the ISRE-like site found downstream from the NFκB and C/EBP sites abrogated the activation by ICSBP and IRF-1. Together, these results indicate that ICSBP and IRF-1 cooperatively stimulate murine IL-12 transcription through a novel regulatory element in the murine promoter.

Cement gland-specific activation of the Xag1 promoter is regulated by co-operation of putative Ets and ATF/CREB transcription factors.

The cement gland marks the extreme anterior ectoderm of the Xenopus embryo, and is determined through the overlap of several positional domains. In order to understand how these positional cues activate cement gland differentiation, the promoter of Xag1, a marker of cement gland differentiation, was analyzed. Previous studies have shown that Xag1 expression can be activated by the anterior-specific transcription factor Otx2, but that this activation is indirect. 102 bp of upstream genomic Xag1 sequence restricts reporter gene expression specifically to the cement gland. Within this region, putative binding sites for Ets and ATF/CREB transcription factors are both necessary and sufficient to drive cement gland-specific expression, and cooperate to do so. Furthermore, while the putative ATF/CREB factor is activated by Otx2, a factor acting through the putative Ets-binding site is not. These results suggest that Ets-like and ATF/CREB-like family members play a role in regulating Xag1 expression in the cement gland, through integration of Otx2 dependent and independent pathways.

A Highly Active Homeobox Gene Promoter Regulated by Ets and Sp1 Family Members in Normal Granulosa Cells and Diverse Tumor Cell Types

One mechanism by which normal cells become converted to tumor cells involves the aberrant transcriptional activation of genes that are normally silent. We characterize a promoter that normally exhibits highly tissue- and stage-specific expression but displays ubiquitous expression when cells become immortalized or malignant, regardless of their lineage or tissue origin. This promoter normally drives the expression of the Pem homeobox gene in specific cell types in ovary and placenta but is aberrantly expressed in lymphomas, neuroblastomas, retinoblastomas, carcinomas, and sarcomas. By deletion analysis we identified a region between nucleotides -80 and -104 that was absolutely critical for the expression from this distal Pem promoter (Pem Pd). Site-specific mutagenesis and transfection studies revealed that this region contains two consensus Ets sites and a single Sp1 site that were necessary for Pem Pd expression. Gel shift analysis showed that Ets and Sp1 family members bound to these sites. Transfection studies demonstrated that the Ets family members Elf1 and Gabp and the Sp1 family members Sp1 and Sp3 transactivated the Pem Pd. Surprisingly, we found that Sp3 was a more potent activator of the Pem Pd than was Sp1; this is unusual, because Sp3 is either a weak activator or a repressor of most other promoters. Activation by either Elf1 or Gabp required an intact Sp1 family member binding site, suggesting that Ets and Sp1 family members cooperate to activate Pem Pd transcription. Expression from the Pem Pd (either transiently transfected or endogenous) depended on the Ras pathway, which could explain both its Ets- and Sp1-dependent expression in normal cells and its aberrant expression in tumor cells, in which ras protooncogenes are frequently mutated. We suggest that the Pem Pd may be a useful model system to understand the molecular mechanism by which a tissue-specific promoter can be corrupted in tumor cells.

The Xenopus laevis βTrCP gene: genomic organization, alternative splicing, 5′ and 3′ region characterization and comparison of its structure with that of human βTrCP genes

βTrCP plays a relevant role in the control of stability of several key protein factors. In Xenopus, βTrCP acts as an inhibitor of Wnt signaling and dorsal axis formation. We determined the primary structure of the frog βTrCP gene, which consists of 14 exons and 13 introns, spanning over 34 kb. Isoforms of x-βTrCP have been found, which show differences in the NH 2 and COOH regions. NH 2 isoforms differ for the presence or absence of a 30 aa sequence, coded by exon III. In COOH isoforms, 19 C-terminal amino acids are replaced by three different amino acids. Occurrence of two 5′ splice donor sites for splicing of intron XIII provides an explanation for these isoforms, based on alternative splicing. The DNA region of the putative βTrCP promoter contains several TATA elements, one GCCAAT box, and putative binding sites for Ets, Tcf/Lef and NF-κB transcription factors. Two transcription initiation sites have been mapped downstream of TATA boxes proximal to ATG for start of translation. Comparison of the Xenopus and human βTrCP genes indicates high conservation of exon nucleotide and amino acid sequences, size and organization; differences are limited to exons coding for N- and C-terminal regions.

Opposing functions of the Ets factors NERF and ELF-1 during chicken blood vessel development.

The purpose of this study was to evaluate the role of the Ets factor NERF in the regulation of the Tie1 and Tie2 genes during chicken blood vessel development. We have isolated the full-length cDNA for the chicken homologue of the human Ets factor NERF2 (cNERF2). Northern blot analysis and in situ hybridization demonstrate that cNERF2 is enriched in the developing blood vessels of the chicken chorioallantoic membrane. Interestingly, cNERF2 functions as a competitive inhibitor of a highly related Ets factor cELF-1, which we have previously shown to be enriched in chicken blood vessel development. Although in vitro-translated cELF-1 and cNERF2 can bind equally well to conserved Ets binding sites in the promoters of the Tie1 and Tie2 genes, cELF-1 preferentially binds to the Ets sites in these promoters during early stages of chicken blood vessel development, suggesting that cNERF may bind during later stages of blood vessel development and vascular remodeling. cNERF2 is enriched during embryonic and extraembryonic blood vessel development in the chicken and facilitates tight control of Tie1 and Tie2 gene regulation.

Macrophage-restricted and Interferon γ-inducible Expression of the Allograft Inflammatory Factor-1 Gene Requires Pu.1

Expression of allograft inflammatory factor-1 (Aif-1), a 17-kDa protein bearing an EF-hand Ca(2+) binding motif, increases markedly in monocytes and macrophages participating in allo- and autoimmune reactions, including the perivascular inflammation in transplanted hearts, microglial infiltrates in experimental autoimmune neuritis, and the inflamed pancreas of prediabetic BB rats. To investigate the mechanism of this regulation, we isolated the mouse aif-1 gene and determined its genomic organization. The gene has six exons distributed over 1.6 kilobases, an interferon gamma-inducible DNase I-hypersensitive site near -900, and flanking sequences on either side predicted to associate with nuclear matrix. Reporter gene analyses identified sequences between -902 and -789, including consensus Ets and interferon regulatory factor elements, required for macrophage-specific and interferon gamma-inducible transcriptional activity. Pu.1 bound to the Ets site in electromobility shift assay and forced expression of Pu.1 activated the aif-1 promoter in 3T3 fibroblasts, in which it is normally inactive. However, the transcriptional activity of a concatamer of the Ets site alone did not increase with interferon gamma treatment. Cooperation between Pu.1 and proteins binding to the interferon regulatory factor element appears to be necessary for both macrophage-specific and interferon gamma-inducible expression of the aif-1 gene.

Cloning and Characterization of the Human Lung Endothelial-Cell-Specific Molecule-1 Promoter

Endothelial-cell-specific molecule-1 (ESM-1) is a cysteine-rich protein that is expressed primarily in endothelial cells of the lung, kidney and gut. In the present study, we have cloned and sequenced 3,888 bp of the 5′ flanking region of the human ESM-1 gene. The full-length promoter directed high-level expression of the luciferase reporter gene in bovine lung microvascular endothelial cells and bovine aortic endothelial cells, but not in nonendothelial cell types. In 5′ deletion analyses, a region spanning –81 to +58 was shown to contain information for endothelial-cell-specific expression. Mutational analysis in transient transfection assays uncovered an important role for an Ets-binding motif located between –77 and –74 and a cAMP-response-element (CRE)-like motif located between –68 and –62 in mediating high-level expression in endothelial cells. A second Ets site (–63 to –60) as well as a novel 6-bp palindromic sequence (–58 to –53) were found to inhibit expression. In DNase footprint analyses, both the Ets-binding motifs were protected specifically in endothelial cells, while the CRE-like element and palindrome were protected in endothelial and nonendothelial cells alike. Taken together, these results provide an important foundation for studying endothelial-cell-subtype-specific gene regulation.

Functional characterization of Ets-binding sites in the sea urchin embryo: three base pair conversions redirect expression from mesoderm to ectoderm and endoderm

Because of the limited knowledge of target genes for the ets family of transcription factors, it is yet unclear how specificity of biological function among different members is achieved in this class of proteins. In the present study, we compared two Ets-binding sites in two differentially expressed genes of the sea urchin embryo. The first gene examined is the cytoskeletal actin CyIIa, which is transiently expressed in skeletogenic and secondary mesenchyme and in its terminal and permanent phase in the gut. The second one encodes the hatching enzyme gene of Strongylocentrotus purpuratus, and is regulated cell-autonomously and asymmetrically along the maternally determined animal–vegetal axis. The Ets sites within the regulatory regions of these two genes interact and form different binding complexes with proteins present in the nuclei of mesenchyme blastula embryos. We also demonstrated that the DNA binding specificity of the CyIIa Ets-binding site can be converted to the other type of Ets site, as in the hatching enzyme promoter, by changing only three base pairs near the Ets core sequence. Switching of these three base pairs near the central GGA trinucleotide motif characteristic of all Ets-binding targets was also sufficient to redirect expression of a reporter gene construct containing a heterologous basal promoter from mesenchyme to non-mesenchyme cell type in transgenic sea urchin embryos. These observations suggest that binding affinity of ets transcription factors plays an important role in determining cell type-specific gene expression.

Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells

Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-κB (NF-κB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-κB-dependent expression was not affected. The AP-1/Ets (−891 to −908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression.