Sp1 Binding Activity Research Articles

Identification of regulatory elements in the human adipose most abundant gene transcript-1 ( apM-1) promoter: role of SP1/SP3 and TNF-alpha as regulatory pathways.

The human adipocyte-specific apM-1 (adipose most abundant gene transcript-1) gene encodes for a secretory protein of the adipose tissue that seems to play a role in the pathogenesis of obesity-related insulin resistance and its expression is inhibited by TNF-alpha. Our aim was to characterize the tissue-specific regulation of the recently cloned apM-1 promoter and the mechanisms of TNF-alpha-induced downregulation of the apM-1 gene. We characterised the apM-1 gene by electrophoretic mobility shift assays (EMSA) and luciferase reporter gene assays (LRA). Although several putative binding sites for transcription factors known to be involved in adipogenesis such as C/EBP and PPARgamma are present in the promoter, we could not detect any binding of these nuclear proteins from differentiated adipocytes. However, a proximal SP1 binding site specifically binds both, recombinant SP1 protein and SP1 derived from adipocyte nuclear extracts. Since the expression of SP1 during adipocyte differentiation has not yet been analysed, we could show by using EMSA, that binding activity of SP1 is increased during adipocyte differentiation. The stimulatory activity of SP1 was confirmed in LRA by cotransfection experiments in S2 Schneider cells lacking endogenous SP factors. An inhibitory activity of SP3 on the stimulatory effect of SP1 could be confirmed in LRA by contransfection experiments in adipocytes. Nuclear extracts from adipocytes incubated with TNF-alpha showed a reduced binding activity of SP1. SP1 is expressed and its binding activity is enhanced during adipocyte differentiation. SP1 has stimulatory effects, SP3 has inhibitory effects on apM-1 promoter activity, mediated by a proximal SP1 binding site. The mechanism of TNF-alpha-induced inhibition of apM-1 gene expression is, at least in part, due to a decrease of transcriptional SP1 binding activity caused by TNF-alpha and thus provides a new mechanism of TNF-alpha-dependent signalling.

Involvement of PKA and Sp1 in the induction of p27 Kip1 by tamoxifen

We have previously shown that tamoxifen (Tam) inhibits proliferation of estrogen receptor-negative human non-small cell lung cancer cells and this inhibition is associated with induction of p27 Kip1. In this study, we investigated the mechanism by which Tam increases p27 Kip1 expression. Because intracellular p27 Kip1 protein level is mainly controlled via posttranslational regulation, we first tested whether Tam might affect protein stability of p27 Kip1. Metabolic labeling and pulse chase assays showed that Tam did not affect the half-life of this protein. We next examined whether Tam enhanced p27 Kip1 expression through transcriptional activation. Our results demonstrated that Tam directly stimulated the p27 Kip1 promoter in lung cancer cells. Deletion and mutation analysis revealed that two Sp1 consensus sites located between −545 and −532 bp from the transcription start site were crucial for the induction of p27 Kip1 by Tam. Conversely, mutation in a CTF site (−525/−520) nearby these two Sp1 sites had little effect. Electromobility shift assays showed that Sp1 transcription factor bound to these consensus sites and the DNA binding activity of Sp1 was enhanced by Tam. Our data also demonstrated that induction of p27 Kip1 by Tam was inhibited by protein kinase A inhibitor H89, but not by protein kinase C inhibitor calphostin C and mitogen-activated kinase kinase inhibitor PD98059. Taken together, our results suggest that Tam transcriptionally activates p27 Kip1 expression via the Sp1 consensus sites in the p27 Kip1 promoter and PKA is involved in this process.

Kinetics of lipopolysaccharide-induced transcription factor activation/inactivation and relation to proinflammatory gene expression in the murine spleen

Bacterial lipopolysaccharide (LPS) elicits inflammation and endotoxic shock by inducing proinflammatory cytokine gene expression. The purpose of this study was to test the hypothesis that differential activation of transcription factor binding in the spleen correlates with proinflammatory cytokine gene expression in mice exposed to LPS. When proinflammatory cytokine expression in spleen was evaluated in mice injected ip with 4 mg/kg LPS over an 8-h period, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 mRNAs were elevated up to 5-, 6-, and 300-fold, respectively, over vehicle controls. Both TNF- α and IL-6 mRNA peaked at 2 h and begin to decline thereafter, whereas IL-1β mRNA remained elevated from 2 to 8 h. The capacities of splenic nuclear proteins to bind to six different consensus transcriptional control motifs associated with proinflammatory cytokine promoters were also measured over 8 h. Electrophoretic mobility shift assay (EMSA) revealed that binding activity was markedly increased at 0.5 to 8 h for activator protein-1 (AP-1) as were CCAAT enhancer-binding protein (C/EBP) and nuclear factor κB (NF-κB) at 0.5 to 1.5 h. At 0.5 h, cyclic AMP response element (CRE)-binding protein (CREB) and binding was slightly elevated, whereas activator protein- 2 (AP-2) and specificity protein 1 (Sp1) binding were not affected. Antibody supershift EMSA and Western blot analysis confirmed that increased binding of these factors correlated with LPS-induced increases in nuclear concentrations of AP-1 (c-Jun, phosphorylated c-Jun, Jun D, and Jun B), C/EBPβ, NF-κB (p50, p65, and c-Rel), CREB (CREB-1, CREB-2, and ATF-2), and AP-2α proteins. Remarkably, after 8 h, C/EBP, CREB, AP-2, and Sp1 binding activities were greatly depleted relative to both naive and corresponding vehicle controls. When mice were exposed to a second dose of LPS, 8 h after a 4 mg/kg priming dose, TNF-α and IL-6 mRNA responses were markedly impaired, suggesting that the mice were endotoxin tolerant at this time point. Taken together, the quiescent, active, and suppressive phases of transcription factor binding observed in this model were highly consistent with the rapid transient nature of LPS-induced proinflammatory cytokine expression in vivo as well as tolerance to secondary LPS exposure.

Palmitate-induced activation of the hexosamine pathway in human myotubes: increased expression of glutamine:fructose-6-phosphate aminotransferase.

The nutrient sensing capacity of the hexosamine biosynthetic pathway (HBP) has been implicated in the development of insulin resistance of skeletal muscle. To study the molecular mechanism of the free fatty acid (FFA)-induced activation of the HBP myotubes obtained from muscle biopsies of metabolically characterized, subjects were stimulated with different fatty acids for 20 h. Incubation with the saturated fatty acids palmitate and stearate (0.5 mmol/l) resulted in a three- to fourfold increase in mRNA expression of glutamine:fructose-6-phosphate aminotransferase (GFAT), the key and rate-limiting enzyme of the hexosamine pathway. Unsaturated fatty acids or 30 mmol/l glucose had little or no effect. Palmitate increased the amount of GFAT protein nearly two-fold, and subsequently, the concentration of UDP-N-acetylglucosamine, the end product of the HBP, was 1.3-fold enhanced in the palmitate-stimulated myotubes. The nonmetabolized fatty acid bromopalmitate had no effect. The DNA binding activity of the transcription factor Sp1, a target downstream of the HBP, was increased by palmitate and completely lost after enzymatic removal of O-GlcNAc. No correlation was found between the palmitate-induced increase in GFAT protein and the insulin resistance in the respective subjects. The findings reveal a new mechanism for how FFAs induce the activation of the HBP.

Mechanisms regulating the cadmium-mediated suppression of Sp1 transcription factor activity in alveolar epithelial cells

This study demonstrates that in vitro exposure of adult rat alveolar epithelial cells to CdCl 2 decreases DNA binding activity of specificity protein 1 (Sp1), a zinc-finger transcription factor known to play a key role in eukaryotic gene expression, maintenance of homeostasis, cell cycle control, terminal differentiation, and apoptosis. Suppression of Sp1 function, as assessed by electrophoretic mobility shift assays (EMSAs), is dependent upon cadmium (Cd) dose and duration of exposure. A 45% decrease of Sp1 activity occurs as early as 30 min after Cd addition. By 2 h, Sp1 activity is reduced even further with no loss of cell viability, suggesting that Sp1 inactivation precedes cell death. If Cd is removed from cultures during these early periods of exposure, inhibition of Sp1 binding activity is reversed. Sp1 inactivation does not appear to be a generalized, non-selective response to Cd as other transcription factors are up-regulated under the same conditions. Phosphorylation is involved in Sp1 down-regulation, as evidenced by the finding that alkaline phosphatase treatment of nuclear extracts from cells exposed to Cd for 2 h helps restore Sp1 binding activity. A broad spectrum Protein Kinase C (PKC) inhibitor, GF109203X, substantially reduces the Cd-mediated effect on Sp1 suggesting that a member of the PKC family is required for Sp1 phosphorylation. More prolonged Cd exposure promotes Sp1 degradation with the appearance of cleavage products (40 and 50 kDa), as detected by Western blotting. Changes in the integrity of the Sp1 protein are accompanied by a corresponding decline in cell survival. Cd-induced cell death is substantially attenuated if cells are pretreated with antagonists of PKC activity which implies that a PKC isoform is also a participant in this process.

The Sp1 transcription factor contributes to the tumor necrosis factor-induced expression of the angiogenic factor thymidine phosphorylase in human colon carcinoma cells.

Thymidine phosphorylase (TP; also known as platelet-derived endothelial cell growth factor, PD-ECGF) is an angiogenic factor that is chemotactic for endothelial cells and has been found to induce neovascularization in vivo. TP is frequently overexpressed in human solid tumors, where its expression has been correlated with increased tumor microvessel density, invasion, and metastasis, and shorter patient survival. In this report, TP activity in the WiDr colon carcinoma cell line was found to be induced 100-fold by tumor necrosis factor (TNFalpha), a secretory product of activated macrophages that has indirect angiogenic activities. Increased TP activity was accompanied by increased TP mRNA levels and without an increase in mRNA stability. TNFalpha-induced TP mRNA levels were reduced by mithramycin, a DNA-binding transcription inhibitor specific for GC-rich sequences. Transcriptional regulation by TNFalpha was confirmed by transient transfection of WiDr with upstream TP sequences in a luciferase reporter construct. Deletion analysis of the reporter pinpointed two regions of the TP promoter with regulatory elements for both TNFalpha-inducible and basal expression, and they contained, respectively, three and one consensus binding sites for the Sp1-family of transcription factors. One additional region contributed only to basal TP expression, and it contained three Sp1 sites. TNFalpha-induced TP expression decreased when point mutations were made in three of the four Sp1 sites postulated to contribute to both basal and TNFalpha-inducible expression. Electrophoretic mobility shift assays further demonstrated binding of nuclear Sp1 to these three sites. Sp1-binding activity was also increased in cells treated with TNFalpha. These studies establish a role for Sp1 in the regulation of expression of the angiogenic factor TP in colon cancer WiDr cells.

Interleukin-10 differently regulates monocyte chemoattractant protein-1 gene expression depending on the environment in a human monoblastic cell line, UG3

Interleukin (IL)-4, IL-10, and IL-13 affect monocyte/macrophage functions including regulation of cytokine production. We analyzed the regulatory effects of these cytokines on cytokine production using a human monoblastic cell line, UG3. It is interesting that IL-10 up-regulated, whereas IL-4 and IL-13 down-regulated monocyte chemoattractant protein-1 (MCP-1) production by unstimulated UG3 cells. IL-10-induced expression of MCP-1 mRNA occurred without de novo protein synthesis at transcriptional and post-transcriptional levels. The enhancement of binding activity of nuclear factor Sp1 (Sp-1) and signal transducer and activators of transcription (STAT)1 and 3 but not nuclear factor kappaB (NF-kappaB) was associated with this IL-10-induced MCP-1 expression. Furthermore, IL-10 suppressed lipopolysaccharide (LPS)-induced NF-kappaB binding but not Sp-1. The present results suggest IL-10 has two contrasting actions on the MCP-1 production of monocytes/macrophages, between the resting and activated conditions. The combination of activated Sp-1 and STATs is important for IL-10-induced MCP-1 expression in resting monocytes/macrophages, and the inhibition of LPS-induced NF-kappaB binding is crucial for down-regulation of MCP-1 by IL-10 in stimulated monocytes/macrophages.

Role of Sp1 in insulin regulation of gene expression.

Sp1 is a ubiquitous nuclear factor that plays a key role in maintaining basal transcription of 'house-keeping' genes. However, recent evidence points to a more important function for Sp1 in mediating 'cross-talk' between selected signaling cascades to regulate the target genes that respond to these pathways. The role of Sp1 in mediating the actions of the peptide hormone insulin is of specific interest and serves as a model for detailing effects of intracellular signaling on Sp1 activity. This review summarizes studies suggesting that changes in Sp1 phosphorylation provide one potential mechanism for manipulating activity of this protein. A growing body of evidence reveals that the DNA binding and transcription activity of Sp1 may increase or decrease in response to changes in phosphorylation. This enables 'fine-tuning' of Sp1 activity for regulation of gene transcription. Several mechanisms exist by which Sp1 alters gene activity in response to insulin. These include independent Sp1 activity as well as collaboration or competition with others factors. This review points to an ever-increasing role for Sp1 in regulating the transcription of genes in response to extracellular signals such as insulin.

A GC-box within the proximal promoter region of the rat cathepsin L gene activates transcription in Sertoli cells of sexually mature rats.

It has been proposed that stage-specific gene expression in Sertoli cells results from sequential activation and repression of transcription. However, the exact molecular mechanisms are unknown. As a first step in addressing this fundamental issue, we recently demonstrated that a 3-kilobase (kb) genomic fragment immediately upstream of the rat cathepsin L translation start site directed stage-specific expression of a reporter gene only in Sertoli cells of transgenic mice in a manner comparable to that of the endogenous gene (predominantly in stages VI-VIII tubules). Supporting the activation/repression model of regulation, an upstream domain that mediated an inhibitory effect by male germ cells was identified within this 3-kb promoter region. In the present study, we localized and characterized the regulatory elements that activate transcription. Analyses of a series of 5' deletion constructs demonstrated that a 120-base pair (bp) region that spans the transcription start site of the rat cathepsin L gene was sufficient to activate transcription in Sertoli cells isolated from sexually mature rats. Within this region, electrophoretic mobility shift assays showed that one member of the Sp/XKLF family of factors, Sp3, specifically bound to a GC-box. Furthermore, Sp1-binding activity was not detected in nuclear extracts from Sertoli cells of sexually mature rats. Finally, the GC-box was shown to be essential for promoter activity since mutating this binding motif abolished promoter activity. Collectively, these results suggest that the GC-box is a critical regulatory element for the cathepsin L promoter in mature Sertoli cells.

Down-regulation of Human Type II Collagen Gene Expression by Transforming Growth Factor-β1 (TGF-β1) in Articular Chondrocytes Involves SP3/SP1 Ratio

Although transforming growth factor beta1 (TGF-beta1) is generally considered as a stimulator of type I collagen production in smooth organs, we found that it can inhibit type II collagen biosynthesis in primary rabbit articular chondrocytes (RAC) at transcriptional levels. Constructs of promoter and first intron sequences associated with the luciferase reporter gene were used to delineate the gene sequences involved in TGF-beta1 control of human COL2A1 gene transcription. Cotransfection of these DNA fragments with a TbetaRII/I cDNA hybrid receptor, capable of inducing a TGF-beta1 dominant negative effect, showed that TGF-beta1 inhibits specifically COL2A1 gene transcription in RAC by a 63-bp proximal promoter. Footprint and gel retardation analyses revealed that the TGF-beta1-induced inhibition effect exerted through the 63-bp promoter sequence implies a multimeric complex that binds to the -41/-33 sequence and involves Sp1 and Sp3 transcription factors. Transfection of decoy Sp-binding oligonucleotides corroborated the implication of the proximal promoter in the TGF-beta1-induced inhibition of COL2A1 gene transcription. In addition, TGF-beta1 was found to increase the expression of Sp3 without significant changes to its binding level, but repressed both the biosynthesis and binding activity of Sp1. In functional assays, Sp3 inhibited the 63-bp promoter activity and prevented Sp1 induction of transcription. These findings suggest that TGF-beta1 inhibition of COL2A1 gene transcription in RAC is mediated by an increase of the Sp3/Sp1 ratio and by the repression of Sp1 transactivating effects on that gene.

Mitochondrial Transcription Factor A and Its Downstream Targets Are Up-regulated in a Rat Hepatoma

Mitochondrial transcription factor A is a key regulator involved in mitochondrial DNA transcription and replication. In a poorly differentiated rat hepatoma, Morris hepatoma 3924A, the mRNA and protein levels of this factor were elevated about 10- and 11-fold, respectively, relative to the host liver. The mRNA levels for the hepatoma cytochrome c oxidase I, II, and NADH dehydrogenase 5, 6, the downstream targets of Tfam, were augmented 10-, 8-, 5-, and 3-fold, respectively. Interestingly, Tfam was also found in the hepatoma nucleus. The mRNA levels for nuclear respiratory factor 1 and 2 (NRF-1 and -2), the proteins that are known to interact with specific regulatory elements on human TFAM promoter, were 5- and 3-fold higher, respectively, in the hepatoma relative to the host liver. Unlike the human promoter, the rat Tfam promoter did not form a specific complex with the NRF-1 in the liver or hepatoma nuclear extracts, which is consistent with the absence of an NRF-1 consensus sequence in the proximal rat promoter. A single specific complex formed between the rat promoter and the NRF-2 protein was comparable in the two extracts. The DNA binding activity of Sp1 in the hepatoma nuclear extract was 4-fold greater than that in the liver extract. In vivo genomic footprinting showed occupancy of NRF-2 and Sp1 consensus sites on the promoter of rat Tfam gene. Tfam was also up-regulated in other hepatoma cells. Together, these results show up-regulation of Tfam in some tumors, particularly the liver tumors. Further, the relatively high level of Sp1 binding to the promoter in the hepatoma could play a major role in the up-regulation of Tfam in these tumor cells.

Effect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation

The silencer factor NRSF/REST has been reported to restrict expression to neurons of a variety of genes, including that encoding NMDA receptor subunit type 1 (NR1), by suppressing transcription in nonneuronal cells. However, we recently reported that in addition to the absence of NRSF/REST-binding activity, another neuron-specific mechanism is necessary for high level expression of the NR1 gene in neurons. In this study, we explored the mechanism of induction of NR1 promoter activity during neuronal differentiation of the P19 cell line. We identified a 27 base pair GC-rich region in the promoter as an important element responsible for induction of the NR1 gene after neuronal differentiation. We found that the ubiquitous transcription factors SP1 and MAZ bind to this GC-rich region. Surprisingly, the binding activities of SP1 and MAZ are not remarkably changed after neuronal differentiation. Mutations in the SP1 and MAZ sites impair binding of SP1 and MAZ proteins and also decrease NR1 promoter activity. These findings suggest that SP1 and MAZ mediate enhancement of NR1 promoter activity during neuronal differentiation despite the fact that their binding activity does not change.

Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase.

Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells. Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation). In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.

Lipopolysaccharide Down-regulates Sp1 Binding Activity by Promoting Sp1 Protein Dephosphorylation and Degradation

We examined the in vivo effect of lipopolysaccharide (LPS) on Sp1 (promoter-selective transcription factor 1) DNA binding activity and studied the mechanisms involved in mouse lungs. The Sp1 DNA complex displayed a major band composed of Sp1, Sp2, and Sp3 trimer and a minor band composed of Sp3 homodimer. Compared with control, nuclear proteins from lungs challenged with LPS for 60, 90, 120, 150, 180, and 240 min, respectively, showed a markedly reduced Sp1 binding activity. Down-regulation of Sp1 binding activity was accompanied by a reduced expression of two Sp1-dependent genes (endothelial nitric oxide synthase and cyclooxygenase-1). Immunoprecipitation-Western blot experiments demonstrated that LPS dephosphorylated Sp1 protein at serine and threonine residues but not at the tyrosine residue. Dephosphorylation of Sp1 protein in vitro significantly reduced Sp1 DNA binding activity. Deglycosylation of Sp1 protein also reduced Sp1 binding activity. However, LPS did not cause Sp1 deglycosylation. LPS markedly reduced nuclear Sp1 protein level but had no significant effect on Sp1 mRNA abundance and on Sp1 protein nuclear translocation. Both Sp1 protein dephosphorylation and Sp1 protein degradation are temporally correlated to the reduced Sp1 binding activity. Our results demonstrate that challenge of mice with LPS in vivo down-regulates Sp1 DNA binding activity through promoting Sp1 protein dephosphorylation and degradation.

Methamphetamine activates DNA binding of specific redox-responsive transcription factors in mouse brain.

Cellular oxidative stress and alterations in redox status can be implicated in methamphetamine (METH)-induced neurotoxicity. To elucidate the molecular signaling pathways of METH-induced neurotoxicity, we investigated the effects of a single intraperitoneal injection of METH (1.0, 10, or 20 mg/kg) on DNA-binding activity of specific redox-sensitive transcription factors in mouse brain. Transcription factors studied included activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), cAMP-responsive element-binding protein (CREB), SP-1, and signal transducers and activators of transcription (STAT1 and STAT3). Significant and dose-dependent inductions of AP-1 and CREB DNA-binding activities were observed in four different regions (striatum, frontal cortex, hippocampus, and cerebellum) isolated from the brains of mice injected with METH. However, injections with METH did not affect DNA binding activities of NF-kappaB, SP-1, STAT1, and STAT3. These results suggest that METH-induced oxidative stress may trigger the molecular signaling pathways via specific and selective activation of AP-1 and CREB.

POZ Domain Transcription Factor, FBI-1, Represses Transcription of ADH5/FDH by Interacting with the Zinc Finger and Interfering with DNA Binding Activity of Sp1

The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

Phosphorylation of transcription factor Sp1 during herpes simplex virus type 1 infection.

The expression of most herpes simplex virus type 1 (HSV-1) immediate-early (IE) and early (E) genes decreases late in productive infection. IE and E promoters contain various binding sites for cellular activators, including sites for Sp1, upstream of the TATA box, while late gene promoters generally lack such sites. To address the possibility that Sp1 function may be altered during the course of infection, the modification state and activity of Sp1 were investigated as a function of infection. Sp1 was quantitatively phosphorylated in HSV-1-infected cells without a significant change in abundance. The kinetics of accumulation of phosphorylated Sp1 immediately preceded the decline in E gene (thymidine kinase gene [tk]) mRNA abundance. Phosphorylation of Sp1 required ICP4; however, the proportion of phosphorylated Sp1 was reduced during infection in the presence of phosphonoacetic acid or in the absence of ICP27. While the DNA binding activity of Sp1 was not greatly affected by phosphorylation, the ability of phosphorylated Sp1 isolated from HSV-infected cells to activate transcription in vitro was decreased. These studies suggest that modification of Sp1 may contribute to the decrease of IE and E gene expression late in infection.

A Novel Role of Sp1 and Sp3 in the Interferon-γ-mediated Suppression of Macrophage Lipoprotein Lipase Gene Transcription

The regulation of macrophage lipoprotein lipase by cytokines is of potentially crucial importance in the pathogenesis of atherosclerosis. We have shown previously that macrophage lipoprotein lipase expression is suppressed by interferon-gamma (IFN-gamma) at the transcriptional level. We investigated the regulatory sequence elements and the transcription factors that are involved in this response. We demonstrated that the -31/+187 sequence contains the minimal IFN-gamma-responsive elements. Electrophoretic mobility shift assays showed that the binding of proteins to two regions in the -31/+187 sequence was reduced dramatically when the cells were exposed to IFN-gamma. Both competition electrophoretic mobility shift assays and antibody supershift assays showed that the interacting proteins were composed of Sp1 and Sp3. Mutations of the Sp1/Sp3-binding sites in the minimal IFN-gamma-responsive elements abolished the IFN-gamma-mediated suppression of promoter activity, whereas multimers of the sequence were able to impart the response to a heterologous promoter. Western blot analysis showed that IFN-gamma reduced the steady state levels of Sp3 protein. In contrast, the cytokine decreased the DNA binding activity of Sp1 without affecting the protein levels. These studies therefore reveal a novel mechanism for IFN-gamma-mediated regulation of macrophage gene transcription.

Sp1 transcription factor as a molecular target for nitric oxide-- and cyclic nucleotide--mediated suppression of cGMP-dependent protein kinase-Ialpha expression in vascular smooth muscle cells.

cGMP-dependent protein kinase (PKG) expression is highly variable and decreases in cultured vascular smooth muscle cells (VSMCs), exposure of cells to nitric oxide (NO), or in response to balloon catheter injury in vivo. In this study, the mechanisms of human type I PKG-alpha (PKG-Ialpha) gene expression were examined. Three structurally unrelated NO donors decreased PKG-Ialpha promoter activity after transfection of a promoter/luciferase construct in VSMCs. Promoter deletion analysis demonstrated that (1) a 120-bp promoter containing tandem Sp1 sites was sufficient to drive basal PKG-Ialpha promoter activity, and (2) NO was inhibitory at this site. Cyclic nucleotide analogues also suppressed PKG-Ialpha promoter activity with cAMP being more potent than cGMP. The effects of cyclic nucleotides to suppress PKG-Ialpha promoter activity were attenuated by a specific cAMP-dependent protein kinase (PKA) inhibitor. Single or double mutation of Sp1 binding sites abolished PKG-Ialpha expression. Moreover, Sp1 binding activity on the PKG-Ialpha promoter was detected in A7r5 cells, and this binding was inhibited by NO and cyclic nucleotides. These results indicate that PKG-Ialpha gene expression is driven by an Sp1 transcription mechanism, and that NO and cAMP inhibit Sp1-mediated PKG-Ialpha gene expression through separate mechanisms.

Transplacental effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the temporal modulation of Sp1 DNA binding in the developing cerebral cortex and cerebellum

Gestational exposure to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, (dioxin) often leads to neurodevelopmental and neurobehavioral deficits, which clearly suggest the involvement of the central nervous system. The objective of this study was to determine the effects of an acute, gestational exposure to dioxin on the developmental expression profile of a transcription factor (Sp1) that is involved in growth and differentiation in the developing brain of F, generation pups. Timed-pregnant Harlan Sprague Dawley rats were exposed to single oral doses of 0.25, 0.5, and 1.0 microg dioxin/kg body weight or vehicle corn oil on gestation day 15. Electrophoretic mobility shift assays (EMSA) of the nuclear extracts from the cerebral cortex and cerebellum of the resulting pups, on postnatal days (PND) 3.5, 10, 15, 20 and 30, with an Sp1 consensus oligonucleotide sequence revealed modulations in the DNA binding activity of Sp1 as a result of the transplacental dioxin exposure. In the cerebral cortex, a dose-dependent premature peak of Sp1 DNA-binding on PND 3 was observed when compared with the control animals where Sp1 DNA-binding was maximal on PND 10. In the postnatally-developing cerebellum, a dose-dependent premature peak of Sp1 DNA binding on PND 5 was observed when compared with the control animals where Spl DNA-binding was maximal on PND 15. The mean birth index, body weight gain, and gross brain weight of the pups were not statistically different from the control animals over the 30 day postnatal period studied. The data obtained on the Sp1 developmental expression profiles in the brain indicate that (1) Sp I DNA-binding is developmentally regulated and expressed very highly in actively developing brain regions, and (2) a potential consequence of the transplacental effect of dioxin to the fetus is in utero neurotoxicity.