Egr-1 Binding Site Research Articles

Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-κB and other signal transcription factors in head and neck squamous cell carcinoma

Microarray profiling of ten head and neck cancer lines revealed novel p53 and NF-κB transcriptional gene expression signatures which distinguished tumor cell subsets in association with their p53 status.

Cutting Edge: TCR-Induced NAB2 Enhances T Cell Function by Coactivating IL-2 Transcription

TCR engagement leads to the up-regulation of genetic programs that can both activate and inhibit T cell function. The early growth receptor (Egr) proteins Egr-2 and Egr-3 have recently been identified as TCR-induced negative regulators of T cell function. NAB2 (NGFI-A-binding protein 2) is both a coactivator and a corepressor of Egr-mediated transcription and has been implicated in regulating Schwann cell myelination. In this report we demonstrate that NAB2 is induced by TCR engagement and that its expression is enhanced by the presence of costimulation. The overexpression of NAB2 enhanced IL-2 production while small interfering RNA to NAB2 markedly inhibited IL-2 expression. Mechanistically, we demonstrate that NAB2 enhances IL-2 transcription by acting as a coactivator for Egr-1. Indeed, chromatin immunoprecipitation analysis reveals that NAB2 is recruited to the Egr-1 binding site of the IL-2 promoter. Taken together, our findings identify NAB2 as a novel coactivator of T cell function.

A network of p73, p53 and Egr1 is required for efficient apoptosis in tumor cells

p73, a transcription factor rarely mutated in cancer, regulates a subset of p53 target genes that cause cells to respond to genotoxic stress by growth arrest and apoptosis. p73 is produced in two main forms; only TAp73 reiterates the roles of p53, while DeltaNp73 can be oncogenic in character. We show that the TAp73 form produced by TP73 P1 promoter has five distinct Egr1-binding sites, each contributing to the transcriptional upregulation of TAp73 by Egr1 in several cell types. In contrast, TP73 P2 promoter transcribes DeltaNp73, is not induced by Egr1, but is induced by TAp73 and p53. Induction of TAp73 by genotoxic stress requires Egr1 in mouse in vivo. Newly discovered non-consensus p53-binding sites in p73, p53 and Egr1 promoters reveal inter-regulating networks and sustained expression by feedback loops in response to stress, resulting in prolonged expression of the p53 family of genes and efficient apoptosis.

Nerve Growth Factor Regulates Adrenergic Expression

The mechanism by which nerve growth factor (NGF) regulates adrenergic expression was examined in PC-12 cells transfected with a rat phenylethanolamine N-methyl-transferase (PNMT) promoter-luciferase reporter gene construct pGL3RP893. NGF treatment increased PNMT promoter-driven luciferase activity in a dose- and time-dependent manner. Induction was attenuated by inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase (MAPK) pathway ( approximately 60%) but not by inhibition of the protein kinase A (PKA), protein kinase C, phosphoinositol kinase, or p38 MAPK pathways. Deletion PNMT promoter-luciferase reporter gene constructs showed that the NGF-responsive sequences lay within the proximal -392 base pairs (bp) of PNMT promoter, wherein binding elements for Egr-1 (-165 bp) and Sp1 (-48 bp) reside. Western analysis further showed that NGF increased nuclear levels of Egr-1, but not Sp1 or the catalytic subunit of PKA. Gel mobility shift assays showed increased potential for Egr-1, but not Sp1, protein-DNA binding complex formation. Mutation of either the Egr-1 or Sp1 binding sites in the PNMT promoter attenuated NGF activation. NGF, combined with pituitary adenylyl cyclase-activating protein (PACAP), another PNMT transcriptional activator, cooperatively stimulated PNMT promoter driven-luciferase activity beyond levels observed with either neurotrophin alone. Finally, post-transcriptional control seems to be another important mechanism by which neurotrophins regulate the adrenergic phenotype. NGF, PACAP, and a combination of the two stimulated both intron-retaining and intronless PNMT mRNA and PNMT protein, but to different extents.

In vivo detection of Egr2 binding to target genes during peripheral nerve myelination

Egr2/Krox20 is a zinc finger transactivator that regulates a diverse array of genes required for peripheral nerve myelination. Although several studies have elucidated the Egr2-regulated gene network, it is not clear if Egr2 regulates its target genes directly or indirectly through induction of other transactivators. Moreover, very few Egr2 binding sites have been identified in regulatory elements of myelin genes. To address this issue, we have successfully adapted chromatin immunoprecipitation assays to test if Egr2 binds directly to target genes in myelinating rat sciatic nerve. These experiments demonstrate direct binding of Egr2 to previously described binding sites within the Schwann cell enhancer of the myelin basic protein gene. Furthermore, we show Egr2 binding to a conserved site within the myelin-associated glycoprotein gene. Finally, our experiments provide the first evidence that Egr2 directly regulates expression of desert hedgehog, which is critically involved in development, maintenance and regeneration of multiple nerve elements including myelinated fibers. Surprisingly, this analysis has identified an apparent preponderance of Egr2 binding sites within conserved intron sequences of several myelin genes. Application of chromatin immunoprecipitation analysis to myelination in vivo will prove to be a valuable asset in assaying transcription factor binding and chromatin modifications during activation of myelin genes.

Differential role played by the MEK/ERK/EGR-1 pathway in orthopoxviruses vaccinia and cowpox biology

Appropriation of signalling pathways facilitates poxvirus replication. Poxviruses, as do most viruses, try to modify the host cell environment to achieve favourable replication conditions. In the present study, we show that the early growth response 1 gene (egr-1) is one of the host cell factors intensely modulated by the orthopoxviruses VV (vaccinia virus) and CPV (cowpox virus). These viruses stimulated the generation of both egr-1 mRNA and its gene product, throughout their entire replication cycles, via the requirement of MEK [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway. We showed that, upon VV infection, EGR-1 translocates into the nucleus where it binds to the EBS (egr-1-binding site) positioned at the 5' region of EGR-1-regulated genes. In spite of both viruses belonging to the same genus, several lines of evidence, however, revealed a remarkable contrast between them as far as the roles played by the MEK/ERK/EGR-1 pathway in their biological cycles are concerned. Hence (i) the knocking-down of egr-1 by siRNA (small interfering RNA) proved that this transcription factor is of critical relevance for VV biology, since a decrease of about one log cycle in virus yield was verified, along with a small virus plaque phenotype, whereas the gene silencing did not have a detrimental effect on either CPV multiplication or viral plaque size; (ii) while both pharmacological and genetic inhibition of MEK/ERK resulted in a significant decrease in VV yield, both approaches had no impact on CPV multiplication; and (iii) CPV DNA replication was unaffected by pharmacological inhibition of MEK/ERK, but phosphorylation of MEK/ERK was dependent on CPV DNA replication, contrasting with a significant VV DNA inhibition and VV DNA replication-independence to maintain ERK1/2 phosphorylation, observed under the same conditions.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

NAB2 Represses Transcription by Interacting with the CHD4 Subunit of the Nucleosome Remodeling and Deacetylase (NuRD) Complex

Early growth response (EGR) transactivators act as critical regulators of several physiological processes, including peripheral nerve myelination and progression of prostate cancer. The NAB1 and NAB2 (NGFI-A/EGR1-binding protein) transcriptional corepressors directly interact with three EGR family members (Egr1/NGFI-A/zif268, Egr2/Krox20, and Egr3) and repress activation of their target promoters. To understand the molecular mechanisms underlying NAB repression, we found that EGR activity is modulated by at least two repression domains within NAB2, one of which uniquely requires interaction with the CHD4 (chromodomain helicase DNA-binding protein 4) subunit of the NuRD (nucleosome remodeling and deacetylase) chromatin remodeling complex. Both NAB proteins can bind either CHD3 or CHD4, indicating that the interaction is conserved among these two protein families. Furthermore, we show that repression of the endogenous Rad gene by NAB2 involves interaction with CHD4 and demonstrate colocalization of NAB2 and CHD4 on the Rad promoter in myelinating Schwann cells. Finally, we show that interaction with CHD4 is regulated by alternative splicing of the NAB2 mRNA.

The phyto-chemical (−)-epigallocatechin gallate suppresses gene expression of epidermal growth factor receptor in rat hepatic stellate cells in vitro by reducing the activity of Egr-1

Hepatic stellate cells (HSC) are the major effectors in hepatic fibrogenesis. During liver injury, HSC become activated and proliferative. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are the potent mitogens for many cell types. We previously demonstrated that (−)-epigallocatechin gallate (EGCG), the major and active component in green tea extracts, inhibited HSC growth, including reducing cell proliferation, and inducing apoptosis. We have reported that EGCG interrupts PDGF signaling by reducing receptor tyrosine phosphorylation and gene expression of PDGF-β receptor. Additional experiments are necessary to elucidate the effect of EGCG on EGF signaling in activated HSC. The aims of this study are to evaluate the effect of EGCG on the expression of EGFR and to elucidate the underlying molecular mechanisms in activated HSC. We hypothesize that EGCG might interrupt EGF signaling by suppressing gene expression of EGF receptor (EGFR) in activated HSC, which, together with the interruption of PDGF signaling, might collectively result in the inhibition of HSC growth. The present report demonstrates that the phyto-chemical dose-dependently suppresses gene expression of EGFR in activated HSC in vitro. The Egr-1 binding site located in the egfr promoter is found to be cis-activating element in regulating the promoter activity of the gene. EGCG inhibits the trans-activation activity of Egr-1 in activated HSC by suppressing gene expression of the transcription factor. The interruption of the ERK signaling pathway by EGCG reduces the trans-activation activity of Egr-1 and the promoter activity of EGFR gene in HSC. Taken together, our results demonstrate that EGCG suppresses gene expression of EGFR in rat activated HSC in vitro mediated by reducing the trans-activation activity of Egr-1.

Involvement of Cyclic AMP Response Element (CRE) in Exendin‐4 Induction of Rat Egr‐1 Gene

We recently observed that exendin-4 (EX-4), a potent agonist of GLP-1, induced cyclin D1 expression through the activation of putative cAMP-responsive element (CRE) site and Egr-1 binding site on cyclin D1 promoter (J Endocrinol, 2006; Diabetologia, 2006, both in press). In the present study, induction of Egr-1 expression by EX-4 and the cis-acting elements involved in the transcriptional activation of Egr-1 gene were investigated in INS-cells. EX-4 (10 nM) rapidly and transiently induced the expressions of Egr-1 mRNA and protein. These inductions were completely inhibited by a specific PKA inhibitor, H-89, suggesting the involvement of cAMP/PKA signaling pathway. In promoter assay using serial-deletion constructs of rat Egr-1 promoter, EX-4 induced the highest promoter activity with full-length constructs (−462 to +27) and the activity of similar degree with the construct deleted up to −124. The construct deleted to −73, which was without serum response elements (SREs) site known to be involved in regulating Egr-1 transcription, still responded to EX-4 moderately. However, further deletion to −46 failed to respond to EX −4. Thus, in examining the role of the nucleotide region from −73 to −46, mutation of the putative CRE site located at −69 resulted in 52% reduction in promoter activity in response to EX-4. This study indicates that the putative CRE site as well as the SRE site is required for the induction of Egr-1 gene by EX-4. This research was grant-supported by Korea Science and Engineering Foundation (R-2004-000-10127-0), Republic of Korea.

A novel chimeric promoter that is highly responsive to hypoxia and metals

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

Gonadotroph-specific expression of the human follicle stimulating hormone β gene in transgenic mice

A paucity of in vitro models has hampered studies of molecular mechanisms of FSH subunit gene expression. Consequently, we used an in vivo transgenic strategy to map the location of regulatory elements in the cloned 10 kb human FSHβ gene. Analyses of transgenic mouse lines revealed that successive 5′ truncations of the hFSHβ promoter region to −350 bp relative to the transcriptional initiation site retained gonadotroph-specific expression and the sexually dimorphic pattern of male greater than female FSHβ mRNA levels found normally in rodent pituitary. Truncation of the 3′ flanking sequences from positions +3142 to +2138 bp relative to the translational stop codon in exon 3 resulted in a complete loss of transgene expression, suggesting the presence of critical regulatory elements mapping to the 1 kb genomic segment downstream of position +2138, in addition to the proximal 5′ promoter elements. In silico phylogenetic comparisons of mammalian FSHβ genes revealed five islands of highly conserved sequence homology corresponding precisely to the proximal 5′ promoter region, exon 2, the 5′ translated region of exon 3, and two regions at the 3′ untranslated end of exon 3 that include putative polyadenylation and transcriptional termination signals. Sequence analyses of the 5′ proximal promoter revealed the presence of several putative homeodomain binding sites as well as GATA, SMAD, AP-1, NF-1, NF-Y and steroid hormone transcription factor binding sites within the highly conserved −350 bp promoter region. Notably absent from these 5′ sequences, however, are consensus binding sites for either Egr-1 or Lim-2 transcription factors known to be critical for the gonadotroph-specific expression of the LHβ gene. These findings support the hypothesis that one of the mechanisms underlying the differential regulation of the LHβ, FSHβ, and common α-gonadotropin subunits within pituitary gonadotrophs may be differences in sequence-specific binding requirements for distinct combinations of transcription factors.

Egr-1 Induces the Expression of Its Corepressor Nab2 by Activation of the Nab2 Promoter Thereby Establishing a Negative Feedback Loop

The transcription factor Egr-1 regulates the expression of numerous genes involved in differentiation, growth, and in response to environmental signals. Egr-1 activity is modulated in part through the binding of corepressors Nab1 and Nab2. Nab2 appears crucial for controlling Egr-1-mediated transactivation because it is a delayed early response gene, induced by the same stimuli that induce the immediate early gene Egr-1. To identify important elements regulating Nab2 expression, we cloned the human Nab2 gene and investigated the 5'-region. The TATA- and initiator-less Nab2 promoter, located from -679 to -74 bp, contains a total of 11 Egr binding sites, including a cluster of multiple overlapping Egr/Sp1 sites between -329 and -260 bp. This region is critical for basal promoter activity as well as for maximum induction by phorbol esters. Electromobility shifts show that Sp1 binds to this region in normal and stimulated cells, whereas stimulation induces binding of Egr-1. In addition Egr-1 activates the Nab2 promoter in a pattern similar to phorbol esters, suggesting that Egr-1 is a major inducer of protein kinase C-mediated Nab2 induction. Depletion of Egr-1 by each of two distinct Egr-1 short-interfering RNAs reduces Nab2 expression and inducibility, confirming that Egr-1 is an important regulator of Nab2 expression. Transfection experiments show that Egr-1-induced Nab2 promoter activity is itself repressed by Nab2. These results indicate that Egr-1 mediates the induction of its own repressor, thereby preventing a permanent transactivation of Egr-1 target genes and a damaging overreaction in response to environmental signals.

Simvastatin enhances bone morphogenetic protein receptor type II expression

Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP–BMPR2 signaling thereby enhancing endothelial differentiation and function.

Vascular Endothelial Growth Factor Induction by Prostaglandin E2 in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP2/EP4 Receptors and SP-1 Transcription Factor Binding Sites

Prostaglandin E2 (PGE2) can increase endothelial vascular endogrowth factor A (VEGF-A) production but the mechanisms involved are unclear. Here we characterized the transcriptional mechanisms involved in human airway smooth muscle cells (HASMC). PGE2 increased VEGF-A mRNA and protein but not mRNA stability. PGE2 stimulated the activity of a transiently transfected 2068-bp (-2018 to +50) VEGF-A promoter-driven luciferase construct. Functional 5' deletional analysis mapped the PGE2 response element to the 135-bp sequence (-85/+50) of the human VEGF-A promoter. PGE2-induced luciferase activity was reduced in cells transfected with a 135-bp VEGF promoter fragment containing mutated Sp-1 binding sites but not in cells transfected with a construct containing mutated EGR-1 binding sites. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed binding of Sp-1 to the VEGF promoter. PGE2 increased phosphorylation of Sp-1 and luciferase activity of a transfected Sp-1 reporter construct. PGE receptor agonists EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) mimicked the effect of PGE2, and reverse transcription-PCR, Western blotting, and flow cytometry confirmed the presence of EP2 and EP4 receptors. VEGF protein release and Sp-1 reporter activity were increased by forskolin and isoproterenol, which increase cytosolic cAMP, and the cAMP analogue, 8-bromoadenosine-3',5'-cyclophosphoric acid. These studies suggest that PGE2 increases VEGF transcriptionally and involves the Sp-1 binding site via a cAMP-dependent mechanism involving EP2 and EP4 receptors.

Genetic and pharmacological inactivation of adenosine A2Areceptor reveals an Egr-2-mediated transcriptional regulatory network in the mouse striatum

The adenosine A2A receptor (A2AR) is highly expressed in the striatum, where it modulates motor and emotional behaviors. We used both microarray and bioinformatics analyses to compare gene expression profiles by genetic and pharmacological inactivation of A2AR and inferred an A2AR-controlled transcription network in the mouse striatum. A comparison between vehicle (VEH)-treated A2AR knockout (KO) mice (A2AR KO-VEH) and wild-type (WT) mice (WT-VEH) revealed 36 upregulated genes that were partially mimicked by treatment with SCH-58261 (SCH; an A2AR antagonist) and 54 downregulated genes that were not mimicked by SCH treatment. We validated the A2AR as a specific drug target for SCH by comparing A2AR KO-SCH and A2AR KO-VEH groups. The unique downregulation effect of A2AR KO was confirmed by comparing A2AR KO-SCH with WT-SCH gene groups. The distinct striatal gene expression profiles induced by A2AR KO and SCH should provide clues to the molecular mechanisms underlying the different phenotypes observed after genetic and pharmacological inactivation of A2AR. Furthermore, bioinformatics analysis discovered that Egr-2 binding sites were statistically overrepresented in the proximal promoters of A2AR KO-affected genes relative to the unaffected genes. This finding was further substantiated by the demonstration that the Egr-2 mRNA level increased in the striatum of both A2AR KO and SCH-treated mice and that striatal Egr-2 binding activity in the promoters of two A2AR KO-affected genes was enhanced in A2AR KO mice as assayed by chromatin immunoprecipitation. Taken together, these results strongly support the existence of an Egr-2-directed transcriptional regulatory network controlled by striatal A2ARs.

1052. A Versatile Chimeric Enhancer Vector That Is Highly Inducible by Hypoxia and Metals

To develop a potent hypoxia-inducible vector, we evaluated the usefulness of chimeric combinations of the early growth response factor-1 (Egr-1)-binding site (EBS) from the Egr-1 promoter, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 (PGK1) gene. In transient trasfection assays, combination of three copies of HRE (3xHRE) with either EBS or MRE resulted in a significant increase in hypoxia-responsiveness. With three-enhancer combination such as the EBS-MRE-3xHRE (E-M-H), a hypoxia induction ratio of 69 was achieved. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus (HCMV) promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. The E-M-H was also tested for induction by hypoxia mimetics and other stresses such as heat, low glucose, low pH, and hydrogen peroxide. Co2+, deferoxamine, and Ni2+turned out to be potent inducers whereas hydrogen peroxide worked as a moderate inducer. Gel shift assay together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1|[alpha]| (HIF-1|[alpha]|), metal transcription factor-1 (MTF-1), and Egr-1 may be associated with the high inducibility of E-M-H enhancer. In vivo evaluation of the E-M-H vector in ischemic calf muscle revealed that luciferase expression was significantly higher in E-M-H group than in control, H, or HCMV group. With its high induction capacity and versatile means of modulation, the novel chimeric enhancer should find wide application in the treatment of ischemic diseases and cancer.

Early Growth Response-1 Regulates Lipopolysaccharide-induced Suppressor of Cytokine Signaling-1 Transcription

Suppressor of cytokine signaling (SOCS)-1 is a critical regulator of lipopolysaccharide (LPS) tolerance and LPS-induced cytokine production. The mechanisms regulating the transcription of SOCS-1 in response to LPS are not entirely understood. Functional analysis of the SOCS-1 promoter demonstrates that early growth response-1 (Egr-1) is an important transcriptional regulator of SOCS-1. Two Egr-1 binding sites are present within the SOCS-1 promoter as shown by EMSA and supershift analysis. Further, mutation of the Egr-1 binding sites significantly reduces both the basal and LPS-induced transcriptional activity of the promoter. Chromatin immunoprecipitation experiments confirm LPS-induced binding of Egr-1 to the SOCS-1 promoter in vivo. Additionally, Egr-1(-/-) macrophages show reduced levels of LPS-induced SOCS-1 expression in comparison with macrophages derived from Egr-1(+/+) littermate controls. These results demonstrate an important role for Egr-1 in regulating both the basal and LPS-induced activity of the SOCS-1 promoter.

Stretch-inducible Expression of the Angiogenic Factor CCN1 in Vascular Smooth Muscle Cells Is Mediated by Egr-1

CCN1 is an angiogenic factor that promotes cell adhesion, proliferation, and differentiation. CCN1-deficient mice suffer embryonic death because of vascular defects, demonstrating that CCN1 is required for vessel development. Because mechanical stretch may act as a trigger for vessel development, we investigated the impact of mechanical stretch on the regulatory mechanism of CCN1 expression. Mechanical stretch rapidly enhances CCN1 expression and release in vascular smooth muscle cells (VSMC) in vitro and CCN1 expression in murine aortic segments in vivo. Transfection experiments of VSMC with deletion constructs of the CCN1 promoter revealed the regulatory region responsible for the stretch-induced CCN1 expression in the approximately 200-bp promoter region upstream of the TATA-box containing potential binding sites for early growth response-1 (Egr-1), nuclear factor of activated T-cells and cAMP response element binding protein. Decoy oligonucleotides to Egr-1, but not to nuclear factor of activated T-cells or cAMP response element binding protein, abolished the stretch-induced transcription of CCN1. In addition, mutagenesis of the Egr-1 binding site within the CCN1 promoter completely blunted the stretch-induced activation of the promoter. Furthermore, mechanical stretch induced the expression and DNA-binding activity of Egr-1 in VSMC as demonstrated by Western blot and electromobility shift assay. Moreover, a pressure overload-dependent de novo synthesis of Egr-1 was observed after aortic banding. These findings indicate that mechanical stretch leads to enhanced expression of CCN1 via the mechanosensitive transcription factor Egr-1, suggesting a central role for mechanical stretch in the regulation of CCN1-dependent pro-angiogenic potency.

Specificity of transcriptional regulation by the zinc finger transcription factors Sp1, Sp3, and Egr‐1

The transcription factors Sp1, Sp3, and Egr-1 bind with their zinc finger DNA-binding domains to GC-rich sequences in the regulatory regions of their target genes. The similarity of the DNA-binding sites of Sp1, Sp3, and Egr-1 has triggered the hypothesis that they compete for the same DNA-binding site. We have investigated the specificity of transcriptional regulation by Sp1, Sp3, and Egr-1 using dominant-negative mutants that block the DNA-binding site of Sp1, Sp3, or Egr-1, respectively. The results show that constitutive transcription of Sp1 regulated reporter genes, containing Sp1 sites derived from the aldolase C and p21WAF1/Cip1 genes, or the long terminal repeat of HIV-1, was impaired by dominant-negative mutants of Sp1 and Sp3, but not by a dominant-negative Egr-1. Transcription mediated by Egr-1 was induced by transfection of expression vectors encoding wild-type or mutated Egr-1 or by stimulation of the extracellular signal-regulated protein kinase pathway via an inducible B-Raf-estrogen receptor fusion protein. In all cases transcription of Egr-1-regulated reporter genes, containing Egr-1 binding sites derived from the Egr-1 or the synapsin I gene was impaired by a dominant-negative Egr-1, but not by dominant-negative Sp1 or Sp3 mutants. These results show that there are genuine Sp1/Sp3 or Egr-1 controlled genes showing no cross-regulation of Sp1/Sp3 and Egr-1 through the same DNA-binding site. This does not exclude the existence of composite Sp1/Sp3/Egr-1 binding sites, where competition for a common DNA-binding site occurs.