Sp1 Binding Elements Research Articles

R5020 and RU486 Act as Progesterone Receptor Agonists to Enhance Sp1/Sp4-Dependent Gene Transcription by an Indirect Mechanism

It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type alpha, p27, thymidine kinase 1 and p21 genes reveals a different mechanism. The FR-alpha P4 promoter and the endogenous FR-alpha gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Overexpression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR-alpha promoter by PR, knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared with its activation of a classical glucocorticoid response element-driven promoter and activation of both the promoter and the endogenous FR-alpha gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR-alpha P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, thymidine kinase 1, and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of one or more PR target genes whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.

Specificity Protein 1 (Sp1) Consensus Binding Sequences Flank Many Functional GATA-1 Binding Sites and Are Sufficient To Allow GATA-1 Binding to a WGATAR Sequence in Nuclear Chromatin.

GATA-1 is an hematopoietic transcription factor known for its roles in erythropoiesis and erythroid gene expression. In vitro, GATA-1 readily binds to WGATAR and closely related sequences. However, in vivo, GATA-1 appears to bind only a small subset of potential sites - those associated with gene regulatory elements. It is currently unclear what allows stable binding of GATA-1 at these sites in nuclear chromatin. We hypothesized that the binding of factors adjacent to WGATAR sites may “open” the local chromatin region to allow GATA-1 access to binding sites, while other factors may stabilize GATA-1 binding. To begin to test this model, we examined 11 well-characterized regulatory elements which are known to bind GATA-1 to determine whether there were any common associated binding sites. To do this we used the Transcription Element Search Software (TESS). These sites were from the human β-globin LCR HSs 2–4, human and mouse β-globin promoters, human γ-globin, NF-E2, EKLF and SCl/TALI (proximal and distal) promoters and the chicken 3′ β-globin enhancer. This analysis revealed that in each case, Sp1 consensus binding sites were found flanking the WGATAR elements. While this association between Sp1 and GATA binding motifs has been noted before, the reason for this association has not been determined. On average, the 5′ Sp1 motifs were located 18 +/− 8 bp upstream of the WGATAR elements, and the 3′ Sp1 motifs were located 30 +/− 17 bp downstream. To determine whether these flanking Sp1 elements were sufficient to allow GATA-1 binding in chromatin, we developed a new approach based on the Bouhassira lab's recombinase mediated cassette exchange (RMCE) system. This system allows us to design oligonucleotides up to ~120 bp in length containing clusters of test binding sites and then insert them as single copies into a defined genomic location in MEL cells. ChIP experiments, analyzed by quantitative RT-PCR, can then be used to characterize factor binding and local histone modification. The positive internal control for GATA-1 binding and histone H3 acetylation was the −2.8G region of the mGATA-1 promoter. The negative control was the murine necdin gene promoter. As an additional positive control, we inserted the 100 bp LCR HS4 core element and found, as expected, that it was able to mediate GATA-1 binding. We then inserted 3 different GATA-1 binding elements and two derivative sites which have been shown to bind GATA-1 with high affinity in vitro. None of these sites alone was able to bind GATA-1 in our test system. We next placed one of the high affinity sites, a GATA-PAL motif from the murine gata-1 gene promoter, between two exact Sp1 consensus sequences (GGGGCGGGG) located 18 bp upstream and 30 bp downstream of the WGATAR element. When tested in our system, the flanking Sp1 elements were sufficient to allow GATA-1 binding to the test site at levels equivalent to those seen for the positive control site. As expected, no GATA-1 bound when the Sp1 sites were used alone, but H3 histones in the region did become hyperacetylated, indicating that factors interacting with the Sp1 direct localized histone acetylation and mediate GATA-1 binding. These results begin to provide a mechanism for GATA-1 targeting to sites in nuclear chromatin and provide an explanation for the observed association of WGATAR and Sp1 binding elements within erythroid gene regulatory elements.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene ( VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor.

Evidence that the mouse insulin receptor substrate-1 belongs to the gene family on which the promoter is activated by estrogen receptor α through its interaction with Sp1

In the present study, the molecular mechanism underlying the up-regulatory effect of estradiol (E2) on mouse insulin receptor substrate-1 (IRS-1) promoter was investigated in CHO cells on which the same promoter had first been functionally characterized. The mouse IRS-1 promoter bears four consensus half Estrogen Responsive Elements (ERE) sequences and thirteen AP-1- and ten Sp1-binding elements. We performed molecular dissection of this promoter gene providing 3' different deleted constructs, containing the same AP-1 rich region with a progressively increased number of ERE half sites located downstream. None of these constructs was responsive to E2, while a downstream region (nt -1420 to -160) rich in GC elements was induced by E2. However, the latter region lost its intrinsic E2 responsiveness when the whole IRS-1 promoter was mutated for deletion in all four ERE half sites. Deletion analysis of the ERE half sites demonstrated that only ERE located at the position -1500 to -1495, close to the GC-rich region, was able to maintain the induced activatory effect of E2 on the IRS-1 gene. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the region containing the half ERE/Sp1 (nt -1500 to -1477) as the one conferring E2 responsiveness to the whole promoter. This effect occurs through the functional interaction between E2/ERalpha and Sp1.

Yin yang 1 (YY1) represseshistidine decarboxylasegene expression with SREBP-1a in part through an upstream Sp1 site

Histidine decarboxylase (HDC) is the enzyme that converts histidine to histamine, a bioamine that plays an important role in many physiological aspects including allergic responses, inflammation, neurotransmission, and gastric acid secretion. In previous studies, we demonstrated that Kruppel-like factor 4 represses HDC promoter activity in a gastric cell line through both an upstream Sp1-binding GC box (GGGCGG sequence) and downstream gastrin-responsive elements. In the current study, Yin Yang 1 (YY1), a pleiotropic transcriptional factor, was also shown in cotransfection assays to repress HDC promoter activity through the upstream GC box. DNA affinity purification assay demonstrated that YY1 was pulled down specifically by the upstream GC box. In addition, sterol-responsive element-binding protein 1a (SREBP-1a), a transcriptional factor that binds YY1, represses the HDC promoter. Interestingly, deletion analysis and cotransfection assays indicated that mutation of the upstream GC box or truncation of downstream gastrin-responsive elements in the HDC promoter disrupted the inhibitory effect of YY1 and SREBP-1a in an identical fashion. Furthermore, quantitative real-time PCR analysis indicated that gastrin treatment downregulated SREBP-1a gene expression and reduced the DNA binding activity of SREBP in EMSAs. Taken together, these results suggest that YY1 and SREBP-1a form a complex to inhibit HDC gene expression through both the upstream GC box and downstream gastrin-responsive elements and gastrin-induced activation of HDC gene expression is mediated at least partly through downregulation of transcriptional repressors such as SREBPs.

Sp1 Is a Co-activator with Ets-1, and Net Is an Important Repressor of the Transcription of CTP:Phosphocholine Cytidylyltransferase α

Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT) encoded by the Pcyt1a and Pcyt1b genes. Previously, we identified an Ets-1-binding site located at -49/-47 in the promoter of Pcyt1a as an important transcriptional element involved in basal CTalpha transcription (Sugimoto, H., Sugimoto, S., Tatei, K., Obinata, H., Bakovic, M., Izumi, T., and Vance, D. E. (2003) J. Biol. Chem. 278, 19716-19722). In this study, we determined whether or not there were other important elements and binding proteins for basal CTalpha transcription in the Pcyt1a promoter, and if other Ets family proteins bind to the Ets-1-binding site. The results indicate the formation of a ternary complex with Ets-1 binding at -49/-47 and Sp1 binding at -58/-54 of the Pcyt1a promoter that is important for activating CTalpha transcription. When nuclear extracts of COS-7 cells expressing various Ets family repressors were incubated with DNA probes, binding of Net to the probes was observed. Net dose-dependently depressed the promoter-luciferase activity by 98%, even when co-expressed with Ets-1. RNA interference targeting Net caused an increase of endogenous CTalpha mRNA. After synchronizing the cell cycle in NIH3T3 cells, CTalpha mRNA increased at the S-M phase corresponding to an increase of Ets-1 mRNA and a decrease of Net mRNA. These results indicated that Net is an important endogenous repressor for CTalpha transcription.

TNF Family Protein Regulation in Megakaryocytes and Platelets.

Activated platelets express and release several TNF family proteins with diverse immunological and inflammatory functions. While the biological effects of these potent immunological modifiers are an area of intense study, there are no studies to date that have investigated the mechanism of TNF protein regulation in megakaryocytes. Understanding the mechanisms of TNF protein regulation in megakaryocytes may allow for the design of new therapeutic strategies for the treatment of chronic inflammatory and autoimmune diseases. Our data show that CD40 Ligand, Fas Ligand and TNF-Related Apoptosis Inducing Ligand (TRAIL) expression are induced during megakaryocyte-differentiation of hematopoietic precursor cells. Promoter analysis and expression studies reveal multiple mechanisms of TNF family protein regulation. Megakaryocyte-specific regulation of TRAIL and Fas Ligand are primarily dependent on Sp1 and Sp3 transcription factor binding to proximal Sp1 binding elements within the respective promoter regions. Our preliminary data demonstrates differentiation-induced expression of CD40 Ligand on the other hand, is mediated via an Ets-1 site within 50 nucleotides of the transcription start site. In addition, endogenous CD40 Ligand but not TRAIL or Fas Ligand is constitutively expressed with ectopic expression of Fli-1, in the absence of differentiation-stimuli. Fli-1 is an Ets-1 family transcription factor that modulates several megakaryocyte-restricted genes. Further regulation studies reveal megakaryocyte expression of CD40 Ligand is diminished by the peroxisome proliferator-activated receptor (PPAR) agonist prostaglandin J2 and enhanced by 9-cis retinoic acid. Conversely, TRAIL and Fas Ligand expression are enhanced by prostaglandin J2 and diminished by retinoic acid. Taken from these data is there are differential mechanisms of differentiation-driven and induced regulation of the TNF family proteins. Continuing studies include the determination of the mechanisms of PPAR agonist, retinoic acid and differentiation-modulated TNF protein expression. Our studies demonstrate novel TRAIL and Fas Ligand regulatory pathways in the context of megakaryocyte differentiation and represent the first investigation into megakaryocyte-associated regulatory mechanisms for the platelet-derived CD40 Ligand, Fas Ligand, and TRAIL.

Analysis of Human UDP-Glucose Dehydrogenase Gene Promoter: Identification of an Sp1 Binding Site Crucial for the Expression of the Large Transcript

UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, which is required in liver for the excretion of toxic compounds, and for the biosynthesis of complex carbohydrates, such as hyaluronan, in many cell types. Analysis of a human EST database, as well as the results of a 5'-RACE experiment, have revealed the presence of two transcription start sites approximately 160 bp apart in the human UGDH gene confirming previous Northern hybridization results. To delineate the regions in the UGDH promoter required for regulating the expression of the gene, in particular the synthesis of the large transcript, serial deletions of the 2.1-kb UGDH promoter region were constructed and their activities determined by the firefly luciferase reporter gene assay. Our results indicate that the region from nucleotide position -486 to -632 relative to the start of the small transcript contains positive regulatory elements that contribute to gene expression. Mithramycin A, an inhibitor of transcription factor Sp1, abrogates the promoter activity, suggesting the involvement of this specific protein in UGDH expression. By using site-directed mutagenesis, we analyzed the functional contribution of three putative Sp1 binding elements within this region. A mutation at position -564 demonstrated that this site serves as an enhancing element in both HepG2 and HeLa cells. The complex formation pattern revealed by an electrophoretic mobility shift assay as well as an anti-Sp1 antibody-mediated supershift assay confirmed the identity of this GC box as an Sp1 binding motif. Our results thus identify an alternative transcription start site on the UGDH promoter, and locate the cis-element that greatly enhances the basal transcriptional activity of UGDH gene.

Enhancement of Folate Receptor α Expression in Tumor Cells Through the Glucocorticoid Receptor: A Promising Means to Improved Tumor Detection and Targeting

The utility of the folate receptor (FR) type alpha, in a broad range of targeted therapies and as a diagnostic serum marker in cancer, is confounded by its variable tumor expression levels. FR-alpha, its mRNA and its promoter activity were coordinately up-regulated by the glucocorticoid receptor (GR) agonist, dexamethasone. Optimal promoter activation which occurred at <50 nmol/L dexamethasone was inhibited by the GR antagonist, RU486, and was enhanced by coactivators, supporting GR mediation of the dexamethasone effect. The dexamethasone response of the FR-alpha promoter progressed even after dexamethasone was withdrawn, but this delayed effect required prior de novo protein synthesis indicating an indirect regulation. The dexamethasone effect was mediated by the G/C-rich (Sp1 binding) element in the core P4 promoter and was optimal in the proper initiator context without associated changes in the complement of major Sp family proteins. Histone deacetylase (HDAC) inhibitors potentiated dexamethasone induction of FR-alpha independent of changes in GR levels. Dexamethasone/HDAC inhibitor treatment did not cause de novo FR-alpha expression in a variety of receptor-negative cells. In a murine HeLa cell tumor xenograft model, dexamethasone treatment increased both tumor-associated and serum FR-alpha. The results support the concept of increasing FR-alpha expression selectively in the receptor-positive tumors by brief treatment with a nontoxic dose of a GR agonist, alone or in combination with a well-tolerated HDAC inhibitor, to increase the efficacy of various FR-alpha-dependent therapeutic and diagnostic applications. They also offer a new paradigm for cancer diagnosis and combination therapy that includes altering a marker or a target protein expression using general transcription modulators.

Sp1-associated activation of macrophage inflammatory protein-2 promoter by CpG-oligodeoxynucleotide and lipopolysaccharide

Macrophage inflammatory protein-2 (MIP-2) is a C-X-C chemokine that is important in recruiting neutrophils to inflammatory sites. Our previous reports demonstrated that lipopolysaccharide (LPS) or CpG-oligode-oxynucleotide (CpG-ODN) rapidly induce MIP-2 gene expression in the macrophage cell line, RAW 264.7. Here, we show that the DNA sequence of the MIP-2 promoter between -114 and +14 is sufficient for strong promoter activity in LPS- or CpG-ODN-stimulated RAW 264.7 cells. Importantly, comprehensive mutant analysis reveals that an Sp1 element in the promoter region between -114 and -94 is essential for synergistic MIP-2 promoter activation by NF-kappaB and c-Jun regardless of the presence of an AP-1 site. By combining deletion or site-specific mutant analysis with immunocomplex assays, we also confirmed that Sp1 mediates the recruitment of transcription factors NF- kappaB and c-Jun in LPS- or CpG-ODN-treated RAW 264.7 cells. Several lines of experimental evidence imply that the Sp1-binding element is an important determinant of MIP-2 promoter activity, and that NF-kappaB, c-Jun and Sp1 can functionally cooperate to elicit maximal activation of the promoter.

Vascular smooth muscle cell-specific regulation of cyclin-dependent kinase inhibitor p21(WAF1/Cip1) transcription by Sp1 is mediated via distinct cis-acting positive and negative regulatory elements in the proximal p21(WAF1/Cip1) promoter.

Smooth muscle cells (SMC) play a central role in common vascular pathologies such as atherosclerosis and restenosis. Understanding the molecular regulation of SMC proliferation at a transcriptional level may provide important clues for the targeted control of vascular hyperplasia. We recently reported the capacity of the transcription factor Sp1 to down-regulate p21(WAF1/Cip1) production thereby reducing p21(WAF1/Cip1)-cyclin D1-Cdk4 complex formation and inhibiting vascular SMC proliferation (Kavurma and Khachigian [2003] J. Biol. Chem. 278, 32537-32543). We have now localized the Sp1-response elements in the p21(WAF1/Cip1) promoter responsible for p21(WAF1/Cip1) repression in WKY12-22 SMCs. The proximal region of the p21(WAF1/Cip1) promoter contains five distinct Sp1-binding elements that we have termed A, B, C, D, and E. Electrophoretic mobility shift analysis revealed that SMC nuclear Sp1 interacts with all five Sp1-binding sites, and each of these sites is critical for Sp1 repression of the p21(WAF1/Cip1) promoter, since mutation in any one element ablates repression, and in some cases results in activation. In contrast, only elements C, D, and E are bound by Sp1 in endothelial cells. Sp1 overexpression activates the p21(WAF1/Cip1) promoter in this cell type. Furthermore, mutation in any of these five elements is not sufficient to prevent activation of the p21(WAF1/Cip1) promoter by Sp1 in endothelial cells. Surprisingly, double mutations of elements C and E facilitates superactivation by Sp1 in both cell types, whereas triple mutations of C, D, and E inactivate the promoter. These findings demonstrate cell type-specific regulation of p21(WAF1/Cip1) transcription by Sp1 via distinct cis-acting positive and negative regulatory elements in the proximal p21(WAF1/Cip1) promoter.

Transcriptional regulation of type B human natriuretic Peptide receptor gene promoter: dependence on Sp1.

The type B natriuretic peptide receptor (NPR-B) is the cognate receptor for the C-type natriuretic peptide and, as such, is responsible for signaling growth-suppressant activity in vascular smooth muscle cells. Here we report the isolation and characterization of the human (h) NPR-B gene promoter. Using 5' rapid amplification of cDNA ends analysis, we have identified the 5' terminus of the hNPR-B gene transcript approximately 732 base pairs upstream from the presumed translation start site of the protein. We generated a series of 5' deletion mutants linked to a luciferase reporter and introduced these constructs into rat aortic smooth muscle cells or neonatal rat cardiac fibroblasts. Maximal expression was seen with a construct harboring 441 base pairs of 5' flanking sequence. Site-directed mutagenesis of the proximal promoter revealed a series of GC-rich sequences, 5 of which contributed modestly (approximately 25%) to basal hNPR-B promoter activity. Mutation of a sixth GC-rich sequence led to a >90% reduction in promoter activity. This sequence was shown to associate with Sp1 and Sp3 in vitro. The same mutation that resulted in loss of functional activity also resulted in loss of binding activity in vitro. Overexpression of Sp1 or Sp3 in Drosophila Schneider cells resulted in an increase in hNPR-B promoter activity that was completely nullified with the Sp1 binding site mutation described above. These studies provide the first description and characterization of the NPR-B gene promoter and suggest that this promoter's activity is dominated by a single cluster of Sp1-binding elements in the proximal 5' flanking sequence of the gene.

Strict regulation of CAIX(G250/MN) by HIF-1alpha in clear cell renal cell carcinoma.

Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIXG250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250MN promoter constructs revealed strong promoter activity in G250MN -positive RCC cell lines, but no activity in G250MN -negative cell lines. DNase-I footprint and band-shift analysis demonstrated that Sp1 and HIF-1alpha proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1alpha expression and G250MN expression was observed. In contrast, no relationship between HIF-2alpha expression and G250MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1alpha and Sp1, in combination with CBP/p300, are crucial elements for G250MN expression in ccRCC, and CAIXG250 can be regarded as a unique HIF-1alpha target gene in ccRCC.

193. Hypoxia-Inducible VEGF Gene Therapy Using the RTP801 Promoter

Background; The angiogenic gene therapy using VEGF is a new potential treatment for ischemic disease. Gene expression in ischemic tissue must be locally enhanced and regulated to be safe and effective. Previously, it was reported that the RTP801 promoter was strongly induced under hypoxic conditions. This strong induction of the RTP801 promoter suggests its applicability to the regulation of VEGF gene in the VEGF gene therapy. In this study, we evaluated the role of the RTP801-VEGF plasmid as a therapeutic plasmid in vitro and in vivo study. Method; To identify the cis-regulatory element, the RTP801 promoter was analyzed by various methods. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter upstream of the VEGF cDNA. pEpo-SV-VEGF and pSV-VEGF were used as positive controls and WSLP only injection without plasmid was used as a negative control the in vivo model. In vitro cell transfection study, polyethylenimine (PEI, 25,000 Da) was used as a gene carrier and 293T cells were used. In an in vivo VEGF expression study, we used water-soluble lipopolymer (WSLP) as a gene carrier. The circumflex artery of a New Zealand white rabbit was ligated with a 6-0 Prolene suture and WSLP-plasmid complex (150 ug/750 ul) was injected into the infarcted myocardium using a 30-guage needle. The hearts were harvested 4 days after the injections and homogenized in lysis buffer. The expression level of the VEGF gene was measured by ELISA. Result; In luciferase assay, the region between -495 and -445 was responsible for the hypoxia-induced transcription. In this region, there was a potential Sp1 binding element. The mutation of the Sp1 site reduced the hypoxia-induced transcription of the RTP801 promoter. In addition, co-transfection with antisense Sp1 oligonucleotide decreased the promoter activity. These results suggest that the hypoxia induction of the RTP801 promoter is mediated by Sp1. The pRTP801-VEGF had stronger VEGF expression than pEpo-SV-VEGF, which contained the Epo (erythropoietin) enhancer and the SV 40 promoter system. In addition, the VEGF expression by pRTP801-VEGF was induced under hypoxic condition. In the animal study, the expression level of VEGF by pRTP801-VEGF in the ischemic myocardium was 5.7 times higher than controls (p < 0.001) and 2.5 times higher than the pSV-VEGF (p < 0.01) and 2 times higher than the pEpo-SV-VEGF (p < 0.05). Conclusion; Therefore pRTP801-VEGF will be useful for the VEGF gene therapy for ischemic heart disease with strong induction under hypoxic condition.

Krüppel-Like Factor 4 Is Transactivated by Butyrate in Colon Cancer Cells

High-fiber diets decrease the incidence of colorectal cancers, and SCFA, derived from dietary fiber, are involved in the regulation of cell growth, differentiation, and apoptosis of the colonic epithelium. The mediators of these effects remain poorly defined. Krüppel-like factor-4 (KLF4/GKLF) is a zinc-finger transcription factor that exhibits some physiologic properties similar to those of SCFA in the colon. The present study was undertaken to examine the role of KLF4 in the butyrate-mediated effect in colon cancer HT-29 cells. Butyrate induced KLF4 mRNA expression and stimulated KLF4 promoter activity in a dose- and time-dependent manner in HT-29 cells. Similar effects were observed in SCFA possessing different carbon lengths (C3-C7), but not in branched isobutyric acid, indicating that the stimulatory properties of SCFA were related to fatty acid structure. Transfection studies using 5' deletion and mutant constructs of the KLF4 promoter demonstrated that the butyrate-responsive element was located at a putative stimulatory protein (Sp)1-binding site. Electrophoretic mobility shift assays using an oligonucleotide containing a consensus Sp1-binding element revealed a DNA-protein complex that was enhanced by butyrate treatment and supershifted by the Sp1 antiserum. Furthermore, the effects of butyrate on cell growth and KLF4 mRNA expression were the same as those of trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC1). Overexpression of HDAC1 significantly attenuated transcriptional activation of the KLF4 promoter by butyrate or TSA. These results suggest that KLF4 may function as one of the downstream effectors of butyrate that mediates its growth arrest effect in the colon. Moreover, transactivation of KLF4 by butyrate appears to be mediated through interaction with a Sp1-binding domain on the promoter and is also likely to involve histone acetylation.

TGF-β1 induction of the adenine nucleotide translocator 1 in astrocytes occurs through Smads and Sp1 transcription factors

BackgroundThe adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation. We recently showed that rodent Ant1 is upregulated by transforming growth factor-beta (TGF-β) in reactive astrocytes following CNS injury. In the present study, we describe the molecular mechanisms by which TGF-β1 regulates Ant1 gene expression in cultured primary rodent astrocytes.ResultsTranscription reporter analysis verified that TGF-β1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. A 69 basepair TGF-β1 responsive element of the Ant1 promoter was also identified. Electrophoretic mobility shift assays demonstrated that astrocyte nuclear proteins bind to this response element and TGF-β1 treatment recruits additional nuclear protein binding to this element. Antibody supershift and promoter deletion analyses demonstrated that Sp1 consensus binding sites in the RE are important for TGF-β1 regulation of Ant1 in astrocytes. Additionally, we demonstrate that Smad 2, 3 and 4 transcription factors are expressed in injured cerebral cortex and in primary astrocyte cultures. TGF-β1 activated Smad transcription factors also contribute to Ant1 regulation since transcription reporter assays in the presence of dominant negative (DN)-Smads 3 and 4 significantly reduced induction of Ant1 by TGF-β1.ConclusionThe specific regulation of Ant1 by TGF-β1 in astrocytes involves a cooperative interaction of both Smad and Sp1 binding elements located immediately upstream of the transcriptional start site. The first report of expression of Smads 2, 3 and 4 in astrocytes provided here is consistent with a regulation of Ant1 gene expression by these transcription factors in reactive astrocytes. Given the similarity in TGF-β1 regulation of Ant1 with other genes that are thought to promote neuronal survival, this interaction may represent a general mechanism that underlies the neuroprotective effects of TGF-β1.

Fibroblast Growth Factor-2 Represses Platelet-derived Growth Factor Receptor-α (PDGFR-α) Transcription via ERK1/2-dependent Sp1 Phosphorylation and an Atypical cis-Acting Element in the Proximal PDGFR-α Promoter

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs) whose biological activity is mediated via its high affinity interaction with specific cell surface receptors. The molecular mechanisms governing the expression of PDGF receptor-alpha (PDGFR-alpha) are poorly understood. Here we demonstrate that PDGFR-alpha protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. Electrophoretic mobility shift, competition, and supershift analysis revealed the existence of an atypical G-rich Sp1-binding element located in the PDGFR-alpha promoter -61 to -52 bp upstream of the transcriptional start site. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGFR-alpha promoter. PDGFR-alpha transcription, mRNA, and protein expression were repressed in SMCs exposed to fibroblast growth factor-2 (FGF-2). This inhibition was rescued by the blockade of extracellular signal-regulated kinase-1/2 (ERK1/2). FGF-2 repression of PDGFR-alpha transcription was abrogated upon mutation of this Sp1-response element. FGF-2 stimulated Sp1 phosphorylation in an ERK1/2- but not p38-dependent manner, the growth factor enhancing Sp1 interaction with the PDGFR-alpha promoter. Mutation of residues Thr(453) and Thr(739) in Sp1 (amino acids phosphorylated by ERK) blocked FGF-2 repression of PDGFR-alpha transcription. These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-alpha transcription via the -61/-52 element in the PDGFR-alpha promoter. Phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-alpha transcription, a finding previously unreported in any Sp1-dependent gene.

Activation of CTP:Phosphocholine Cytidylyltransferase α Expression during the S Phase of the Cell Cycle Is Mediated by the Transcription Factor Sp1

An essential step during cell division is induction of phosphatidylcholine biosynthesis. In this pathway, CTP:phosphocholine cytidylyltransferase alpha (CT alpha) plays an important regulatory role. Previous studies (Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692) demonstrated that CT alpha mRNA accumulates during S phase in preparation for cellular mitosis. We now demonstrate that increased binding of the transcription factor Sp1 to the proximal promoter of CT alpha is responsible for increased transcription during the S phase. The Sp1 binding element present in position -67/-62 is essential for activation, and the Sp1 site in position -31/-9 is required to enhance transcription. Inhibition of Sp1 expression by RNA interference abolished the enhanced expression of CT alpha. Immunoprecipitation studies demonstrated that Sp1 interacts with cyclin E, cyclin A, and cyclin-dependent kinase 2 during the S phase. We conclude that Sp1 binding to the CT alpha proximal promoter is necessary to enhance transcription during the S phase. This is the first elucidation of a mechanism by which expression of a key enzyme in phospholipid biosynthesis is regulated during the cell cycle.

Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RARβ-RXRα heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter

AbstractThe present work investigated the mechanism for down-regulation of thrombomodulin (TM), an anticoagulant glycoprotein, on cultured umbilical vein endothelial cells (HUVECs) exposed to lipid extracts from oxidized low-density lipoprotein (ox-LDL). HUVECs exposed to phospholipid extracts, but not to free cholesterol, triglyceride, or cholesterol ester, isolated from ox-LDL reduced TM mRNA levels to nearly the same extent as native ox-LDL. Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), but not native PAPC or a reduced form of ox-PAPC, markedly decreased TM mRNA levels. The apparent half-life (t 1/2 = 2.7 hours) of TM mRNA in control cells was not significantly different from that in cells exposed to ox-LDL or ox-PAPC. TM mRNA levels were regulated by transcriptional activation via a retinoid receptor β (RARβ). The binding activities of nuclear proteins from HUVECs treated with ox-LDL or ox-PAPC to the DR4 or stimulatory protein 1 (Sp1) sequence in the TM promoter were significantly reduced with decreased expression of RARβ, retinoid X receptor α (RXRα), Sp1, and Sp3 in the nuclei. The promoter activity in HUVECs transfected with a reporter plasmid expressing the TM promoter with targeted deletions in the DR4 and Sp1 binding elements was decreased to about 20% of that with the wild-type construct. Treatment of the cells with ox-PAPC had no additional effect on the promoter activity. These results suggest that oxidized phospholipids in ox-LDL inhibit transcription of the TM gene in HUVECs by inhibiting the binding of RARβ-RXRα heterodimer and Sp, including Sp1 and Sp3, to the DR4 element and Sp1 binding element, respectively, in the TM promoter with reduced expression of RARβ, RXRα, and Sp1 and Sp3 in the nuclei.

Metastatic lymph node 64 (MLN64), a gene overexpressed in breast cancers, is regulated by Sp/KLF transcription factors.

MLN64, is invariably coamplified and coexpressed with erbB-2 in breast cancers. The human MLN64 and ERBB2 genes are positioned at less than 50 kb from each other, on chromosome 17q12. To understand the molecular basis of MLN64 overexpression in cancer, the genomic region containing the MLN64 and ERBB2 genes was isolated and mapped. The two genes, DARPP32 and Telethonin, flanking MLN64 respectively on its centromeric and telomeric sides, although coamplified, are not overexpressed in breast cancer cells, indicating that gene amplification is not sufficient to allow overexpression. The MLN64 minimal promoter was isolated and found to be a housekeeping gene promoter containing four potential Sp1 binding elements. Using Sp1-deficient Drosophila SL2 cells, MLN64 promoter activity was induced in a dose-dependent manner by exogenous Sp1 addition. Furthermore, mutation of each individual Sp1 element resulted in a significant decrease in reporter gene activity, indicating that all the Sp1 binding elements are functional and act together to promote gene expression. Since the ERBB2 promoter is also positively regulated by Sp1, this study indicates that MLN64 and ERBB2 genes share common transcriptional controls together with a physical link on chromosome 17q. We speculate that, in addition to the oncogenic potential of erbB-2 overexpression, the unbalanced action of MLN64 contributes to the poor clinical outcome of breast tumors bearing this amplified region.