Role Of Sp1 Research Articles

Role of Sp1 and AP-2 in the transcriptional regulation of the CHRNA7 gene and the link to schizophrenia

Role of Sp1 and AP-2 in the transcriptional regulation of the CHRNA7 gene and the link to schizophrenia

P-57: The role of Sp1 in the diet-induced regulation of the rat fatty acid synthase (FAS) gene

P-57: The role of Sp1 in the diet-induced regulation of the rat fatty acid synthase (FAS) gene

Sp1-dependent activation of KLF4 is required for PDGF-BB-induced phenotypic modulation of smooth muscle

There is clear evidence that the phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs to alter their phenotype in response to extracellular stimuli and is hallmarked by the loss of SMC marker gene expression. The transcription factor Krüppel-like factor 4 (KLF4) is a known powerful negative regulator of SMC marker gene expression that works, in part, by decreasing the expression of the serum response factor (SRF) myocardin. KLF4 is not expressed in healthy adult SMCs but is increased in SMCs in response to vascular injury in vivo or PDGF-BB treatment in vitro. The aim of the present study was to determine the molecular mechanisms that regulate the expression of KLF4 in phenotypically modulated SMCs. The results demonstrated that the transcription factor stimulating protein-1 (Sp1) regulated the expression of KLF4 in SMCs. The KLF4 promoter contains three consensus Sp1 binding sites. Using a series of truncated KLF4 promoters, we showed that only fragments containing these Sp1 sites could be activated by PDGF-BB. In addition, overexpression of Sp1 alone was sufficient to increase the activity of the KLF4 promoter. Moreover, inhibiting Sp1 expression with small-interfering RNA attenuated the effects of PDGF-BB on KLF4 expression. Mutation of the three Sp1 sites within the KLF4 promoter abolished both baseline and PDGF-BB-induced activity. Finally, the results demonstrated enhanced Sp1 binding to the KLF4 promoter in SMCs treated with PDGF-BB in vitro and following vascular injury in vivo. Taken together, the results suggest a novel role for Sp1 in increasing the expression of KLF4 in phenotypically modulated SMCs.

The role of Sp1 in IL-1β andH. pylori-mediated regulation of H,K-ATPase gene transcription

Helicobacter pylori infection of the gastric body induces transient hypochlorhydria and contributes to mucosal progression toward gastric carcinoma. Acid secretion is mediated by parietal cell H,K-ATPase, in which the catalytic alpha-subunit (HKalpha) promoter activity in transfected gastric epithelial [gastric adenocarcinoma (AGS)] cells is repressed by H. pylori through NF-kappaB p50 homodimer binding to the promoter. IL-1beta, an acid secretory inhibitor whose mucosal level is increased by H. pylori, upregulates HKalpha promoter activity in AGS cells. Because IL-1beta also activates NF-kappaB signaling, we investigated disparate HKalpha regulation by H. pylori and IL-1beta, testing the hypothesis that IL-1beta-induced HKalpha promoter activation is mediated by the transcription factor Sp1. DNase I footprinting revealed Sp1 binding to the HKalpha promoter at -56 to -39 bp. IL-1beta stimulated the activity of three HKalpha promoter constructs containing NF-kappaB and Sp1 sites transfected into AGS cells and also stimulated a construct containing only an Sp1 site. This stimulation was abrogated by mutating the HKalpha promoter Sp1 binding site. Gelshift assays showed that IL-1beta increased Sp1 but not p50 binding to cognate HKalpha probes and that Sp1 also interacts with an HKalpha NF-kappaB site when bound to its cognate HKalpha cis-response element. H. pylori did not augment Sp1 binding to an HKalpha Sp1 probe, and small interfering RNA-mediated knockdown of Sp1 expression abrogated IL-1beta-induced HKalpha promoter stimulation. We conclude that IL-1beta upregulates HKalpha gene transcription by inducing Sp1 binding to HKalpha Sp1 and NF-kappaB sites and that the H. pylori perturbation of HKalpha gene expression is independent of Sp1-mediated basal HKalpha transcription.

P53-dependent suppression of the human calcyclin gene (S100A6): the role of Sp1 and of NFkappaB.

Calcyclin (S100A6) is believed to participate in cell cycle control. It was, however, unclear if its expression depends on p53, a key regulator of apoptosis and cell cycle. We therefore performed transcription regulation assays in HeLa cells and found that wild type p53 suppressed the S100A6 promoter up to 12-fold in a dose-dependent manner. In contrast, the well-characterized V143A, R175H, R249S, and L344A p53 mutants cloned from human cancers suppressed this promoter with a 6 to 9-fold lower efficiency. All the sites mediating the p53-dependent suppression were contained in the -167 to +134 fragment of the S100A6 promoter. Separate overexpression of either Sp1 or of NFkappaB only partially counteracted the p53 inhibitory effect on the S100A6 promoter, while simultaneous overexpression of both these transactivators resulted in a complete abolishment of the p53 inhibitory effect on this promoter. Sp1 and NFkappaB binding to the probes resembling their putative binding sites present in the S100A6 promoter was decreased in the presence of wild type p53. We propose that the suppression of S100A6 is yet another mechanism by which p53 inhibits proliferation. Insufficient suppression of this gene by p53 mutants could well be responsible for calcyclin overexpression and cell cycle deregulation observed in cancer tissues.

Functional characterization of cis-acting elements involved in basal transcription of the human Tbx2 gene: A new insight into the role of Sp1 in transcriptional regulation

Tbx2, a member of the T-box family of genes that encode developmentally important transcription factors, plays a critical role in development and has been linked to cancer. Here a 5′-flanking region of the human Tbx2 gene was cloned and characterized. While we identify a Sp1 element and a reverse CCAAT box to be essential for basal Tbx2 promoter activity, a physical interaction between Sp1 and NF-Y does not seem to be required for mediating their effect. Furthermore, our data reveal a downstream promoter element (DPE) in the Tbx2 promoter which significantly influences basal activity.

P53-dependent repression of the human MCL-1 gene encoding an anti-apoptotic member of the BCL-2 family: the role of Sp1 and of basic transcription factor binding sites in the MCL-1 promoter

p53 regulates transcription of one anti-apoptotic and four pro-apoptotic members of the BCL-2 family, but nothing is known about the regulation of MCL-1, another antiapoptotic member of this family, by p53. Confocal microscopic analysis of COS1, HEK 293 and HeLa cells transfected with a p53 expression plasmid demonstrated a decrease in the signal of endogenous MCL-1 compared to neighboring non-transfected cells. Transcription regulation assays showed that the 1826 bp human MCL-1 promoter fragment was repressed up to 30-fold by wild-type p53 in a dose-dependent manner. As shown by electrophoretic mobility shift assays, Sp1 binding to the sites located in the -295 to +16 MCL-1 promoter fragment was decreased in the presence of p53. However, the MCL-1 promoter devoid of all Sp1 binding sites was still repressed by p53, albeit 2-fold weaker than the wild-type promoter. Overexpression of Sp1 reduced p53-dependent repression of the MCL-1 promoter only up to 2.2-fold. Transcription regulation assays performed with MCL-1 promoter deletion mutants showed that most of the p53 inhibitory effect was mediated by the -41 to +16 bp promoter fragment containing binding sites only for TATA-binding protein and other basal transcription factors. We propose a novel, promoter-based mechanism by which p53 down-regulates expression of the antiapoptotic MCL-1 protein.

Role of Sp1 and HNF1 transcription factors in SGLT1 regulation during chronic intestinal inflammation

In a rabbit model of chronic intestinal inflammation, we previously demonstrated that the activity of Na-glucose cotransporter (SGLT1), SLC5A1, is inhibited. This inhibition is secondary to a decrease in the number of cotransporters, indicating that the regulation of SGLT1 during chronic inflammation is at the level of transcription. However, the regulation of SGLT1 expression and the transcription factors involved in the regulation are not yet known. In this report, we describe the cloning and characterization of rabbit SGLT1 promoter and the identification of transcription factors affected in villus cells during chronic intestinal inflammation. The promoter sequence for SGLT1 gene was identified by using the publicly available rabbit genomic sequence. Even though rabbit SGLT1 promoter did not have considerable overall homology with other mammalian SGLT1 promoters, two specificity protein 1 (Sp1) and a hepatocyte nuclear factor 1 (HNF1) binding sites were highly conserved among the species. Rabbit SGLT1 cDNA was encoded by 15 exons. Minimal promoter region determination showed that 196 nucleotides upstream of the transcription start site were sufficient for optimal promoter activity. This region encompassed two transcription factor binding sites, Sp1 and HNF1. For maximal SGLT1 promoter activity, these two transcription factor binding sites were essential, and their effect was synergistic, indicating that two separate regulatory pathways might be involved in their regulation. Using mobility shift assays, we further demonstrated that the binding of both Sp1 and HNF1 transcription factors to SGLT1 promoter regions were affected during chronic intestinal inflammation. Thus this report demonstrates that Sp1 and HNF1 transcription factors act in concert to regulate SGLT1 transcription in the chronically inflamed intestine.

Association of polymorphisms of the MMP-2 and TIMP-2 genes with the risk of endometriosis in North Chinese women

We investigated whether three polymorphisms in the matrix metalloproteinase-2 (MMP-2; -1306C-->T and -735C-->T) and tissue inhibitor of metalloproteinase-2 (TIMP-2; -418G-->C) genes were related to the risk of endometriosis in reproductive-aged women with and without endometriosis. Our results indicate that the TIMP-2 -418C/C homozygote may be a protective factor against the development of endometriosis in North Chinese women.

Role of Sp1 in Transcription of Human ATP2A2 Gene in Keratinocytes

The ATP2A2 gene encodes Ca2+-dependent ATPase, the dysfunction of which causes Darier disease. In this study, we analyzed the promoter structure of the human ATP2A2 gene using primary normal human keratinocytes (NHK). Reporter assays showed that deletion of -550/-529, -488/-472, -390/-362, or -42/-21 resulted in a significant decrease in human ATP2A2 promoter activity. Electrophoretic mobility shift assay (EMSA) showed that Sp1 is a transcription factor that binds to the -550/-529 and -488/-472 regions of the promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1, but not Sp3, binds to the promoter region of the ATP2A2 gene in NHK cells in vivo. Knockdown of Sp1 expression by small interfering RNA resulted in a marked reduction in ATP2A2 promoter activity and ATP2A2 mRNA levels in NHK, suggesting that Sp1 positively transactivates the ATP2A2 promoter in NHK. This is early evidence demonstrating that Sp1 plays an important and positive role in ATP2A2 gene expression in NHK in vivo and in vitro.

Phosphorylation of Sp1 in Response to DNA Damage by Ataxia Telangiectasia-Mutated Kinase

Sp1, a transcription factor that regulates expression of a wide array of essential genes, contains two SQ/TQ cluster domains, which are characteristic of ATM kinase substrates. ATM substrates are transducers and effectors of the DNA damage response, which involves sensing damage, checkpoint activation, DNA repair, and/or apoptosis. A role for Sp1 in the DNA damage response is supported by our findings: Activation of ATM induces Sp1 phosphorylation with kinetics similar to H2AX; inhibition of ATM activity blocks Sp1 phosphorylation; depletion of Sp1 sensitizes cells to DNA damage and increases the frequency of double strand breaks. We have identified serine 101 as a critical site phosphorylated by ATM; Sp1 with serine 101 mutated to alanine (S101A) is not significantly phosphorylated in response to damage and cannot restore increased sensitivity to DNA damage of cells depleted of Sp1. Together, these data show that Sp1 is a novel ATM substrate that plays a role in the cellular response to DNA damage.

Therapeutic inhibition of Sp1 expression in growing tumors by mithramycin a correlates directly with potent antiangiogenic effects on human pancreatic cancer

Human pancreatic cancer over expresses the transcription factor Sp1. However, the role of Sp1 in pancreatic cancer angiogenesis and its use as target for antiangiogenic therapy remain unexplored. Archived human pancreatic cancer specimens were used to assess gene expression and microvessel density (MVD) status by immunohistochemistry: Small-interfering RNA (siRNA) was used to determine the impact of altered Sp1 expression on tumor growth and angiogenesis, and mithramycin A (MIT) was used to evaluate Sp1-targeted antiangiogenic treatment of human pancreatic cancer in animal models. The expression level of Sp1 was correlated directly with the MVD status (P < .001) and the expression level of vascular endothelial growth factor (VEGF) (P < .05). Knockdown of Sp1 expression did not affect the growth of pancreatic cancer cells in vitro but inhibited their growth and metastasis in mouse models. This antitumor activity was consistent with the in vitro and in vivo antiangiogenic activity resulting from Sp1 knockdown. Subcutaneous and intraperitoneal injection of MIT significantly suppressed the growth of human pancreatic cancer in mouse models. This tumor suppression was correlated with the suppression of Sp1 expression in growing tumors but not in normal tissues. Moreover, treatment with MIT reduced tumor MVD, which was consistent with the down-regulation of VEGF, platelet-derived growth factor, and epidermal growth factor receptor. Both clinical and experimental evidence indicated that Sp1 is a critical regulator of human pancreatic cancer angiogenesis and the antitumor activity of MIT is a result, at least in part, of the suppression of Sp1 expression and consequent down-regulation the downstream targets of Sp1 that are key to angiogenesis.

Sp1 expression is disrupted in schizophrenia; a possible mechanism for the abnormal expression of mitochondrial complex I genes, NDUFV1 and NDUFV2.

BackgroundThe prevailing hypothesis regards schizophrenia as a polygenic disease, in which multiple genes combine with each other and with environmental stimuli to produce the variance of its clinical symptoms. We investigated whether the ubiquitous transcription factor Sp1 is abnormally expressed in schizophrenia, and consequently can affect the expression of genes implicated in this disorder.Methodology/Principal FindingsmRNA of Sp1 and of mitochondrial complex I subunits (NDUFV1, NDUFV2) was analyzed in three postmortem brain regions obtained from the Stanley Foundation Brain Collection, and in lymphocytes of schizophrenic patients and controls. Sp1 role in the transcription of these genes was studied as well. Sp1 was abnormally expressed in schizophrenia in both brain and periphery. Its mRNA alteration pattern paralleled that of NDUFV1 and NDUFV2, decreasing in the prefrontal cortex and the striatum, while increasing in the parieto-occipital cortex and in lymphocytes of schizophrenic patients as compared with controls. Moreover, a high and significant correlation between these genes existed in normal subjects, but was distorted in patients. Sp1 role in the regulation of complex I subunits, was demonstrated by the ability of the Sp1/DNA binding inhibitor, mithramycin, to inhibit the transcription of NDUFV1 and NDUFV2, in neuroblastoma cells. In addition, Sp1 activated NDUFV2 promoter by binding to its three GC-boxes. Both activation and binding were inhibited by mithramycin.Conclusions/SignificanceThese findings suggest that abnormality in Sp1, which can be the main activator/repressor or act in combination with additional transcription factors and is subjected to environmental stimuli, can contribute to the polygenic and clinically heterogeneous nature of schizophrenia.

Transcriptional control of human T‐BET expression: The role of Sp1

Murine T-bet (T-box expressed in T cells) is a master regulator of IFN-gamma gene expression in NK and T cells. T-bet also plays a critical role in autoimmunity, asthma and other diseases. However, cis elements or trans factors responsible for regulating T-bet expression remain largely unknown. Here, we report on our discovery of six Sp1-binding sites within the proximal human T-BET promoter that are highly conserved among mammalian species. Electrophoretic mobility shift assays demonstrate a physical association between Sp1 and the proximal T-BET promoter with a direct dose response between Sp1 expression and T-BET promoter activity. Ectopic overexpression of Sp1 also enhanced T-BET expression and cytokine-induced IFN-gamma secretion in NK cells and T cells. Mithramycin A, which blocks the binding of Sp1 to the T-BET promoter, diminished both T-BET expression and IFN-gamma protein production in monokine-stimulated primary human NK cells. Collectively, our results suggest that Sp1 is a positive transcriptional regulator of T-BET. As T-BET and IFN-gamma are critically important in inflammation, infection, and cancer, targeting Sp1, possibly with mithramycin A, may be useful for preventing and/or treating diseases associated with aberrant T-BET or IFN-gamma expression.

Role of Sp1 and SREBP-1a in the insulin-mediated regulation of glucokinase transcription in the liver of gilthead sea bream ( Sparus aurata)

Insulin induction of glucokinase (GCK) transcription in the liver is essential for maintaining glucose homeostasis. To study the molecular mechanism underlying the regulation of hepatic GCK expression in the carnivorous fish gilthead sea bream ( Sparus aurata), we analysed the role of sterol regulatory element binding protein-1a (SREBP-1a) and specificity protein (Sp) 1 in insulin-dependent GCK transcription. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays allowed us to identify a cis-element in the proximal region of GCK promoter implicated in transactivation by SREBP-1a. Consistently, mutations in the SRE binding site completely abolished the enhancing effect of SREBP-1a. These results and previous findings suggest that SREBP-1a plays a role in the transcriptional regulation of key enzymes in glycolysis–gluconeogenesis. Since SREBP-1a and Sp1 may mediate insulin action on S. aurata GCK transcription, we analysed the effect of insulin on HepG2 cells transfected with GCK promoter reporter constructs carrying intact or mutated SRE or Sp boxes. Insulin transactivated GCK irrespective of the presence of an intact or mutated SRE box. However, insulin failed to induce GCK transcription when using reporter constructs that had either a mutated Sp site or no Sp site. Our findings indicate that Sp1, rather than SREBP-1a, mediates the insulin-dependent induction of S. aurata GCK.

Structure of the human 5-HT 7 receptor gene and characterization of its promoter region

The molecular mechanism regulating serotonin 5-HT 7 receptor expression is still unclear. In this study we provide evidence that transcription of the 5-HT 7 gene is at least partly regulated by Sp1 and Sp3. We isolated and sequenced > 3000 bp of the upstream sequences and identified by RACE a number of transcriptional initiation sites over a region of 300 bp upstream of the coding region. This region has a high GC content, but contains no obvious TATA or a CAAT box. Besides a Sp1/Sp3 consensus motif, regulatory elements for AP2, Egr-1 and MAZ are also present. Transient transfection assays using deletion variants indicated that the GC-rich region is essential for full promoter activity. The role of Sp1 in this was confirmed by transient overexpression of both wild type Sp1 or dominant-negative forms. By gel shift and supershift analyses, targeting the Sp1 consensus sequence and the GC-rich region just upstream of the transcription initiation sites, binding of Sp1 and Sp3 was demonstrated. Both in vitro as well as in vivo experiments, using a cell line which endogenously expresses the 5-HT 7 receptor, indicated that mithramycin A, an inhibitor of Sp1/3 transcription factor binding, was able to inhibit 5-HT 7 promoter activity. Taken together these results support the essential role of Sp factors in regulating 5-HT 7 promoter activity.

Role of SP-1 in SDS-Induced Adipose Differentiation Related Protein Synthesis in Human Keratinocytes

Skin irritation is a complex phenomenon, and keratinocytes play an important role in it. We have recently characterized the expression and protective role of adipose differentiation related protein (ADRP) in skin irritation. In particular, ADRP expression is induced to recover functional cell membrane following the cell damage caused by skin irritants. The purpose of this study was to characterize in a human keratinocyte cells line (NCTC 2544) the biochemical events that lead to ADRP expression following SDS treatment, and in particular, to investigate the role of transcription factor SP-1. Analysis of ADRP promoter region revealed the presence of a potential binding site for the transcription factor SP-1 close to the start site. Evaluated by measuring the DNA binding activity, we found that SDS induced a dose and time related SP-1 activation, which was correlated with SDS-induced ADRP mRNA expression. Furthermore, SDS-induced SP-1 activation, ADRP mRNA expression and lipid droplets accumulation could be modulated by mithramycin A, an antibiotic that selectively binds to the GC box preventing SP-1 binding and gene expression. This demonstrated that SDS-induced ADRP expression was mediated in part through the transcription factor SP-1. In addition, SDS-induced SP-1 activation and ADRP expression could be modulated by the calcium chelator BAPTA, indicating a role of calcium in ADRP-induction. Thus, every time an irritant perturbs the membrane barrier, it renders the membrane leaky and allows extracellular calcium to enter the cells, an event that provides the upstream mechanisms initiating the signaling cascade that triggers the activation of SP-1 and culminates in the enhancement of ADRP expression, which helps to restore the normal homeostasis and ultimately repairs the to membrane.

The role of Sp1 and Sp3 in the constitutive DPYD gene expression

Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in the 5-fluorouracil (5-FU) catabolic pathway, has been implicated as one of the factors determining the efficacy and toxicity of the anticancer agent 5-FU. Studies have attributed variation in DPD activity partially to alterations at the transcriptional level of DPYD gene. We investigated the transcription factors implicated in the constitutive expression of DPYD by utilizing a 174-bp fragment of the DPYD promoter region in which three consensus Sp protein binding sites (SpA, SpB and SpC) were predicted. The binding of Sp1 and Sp3 transcription factors to this region was detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. By ectopically expressing human Sp1 and Sp3 in Sp-deficient Drosophila S2 cells, we demonstrated that Sp1 is a strong activator, while Sp3 by its own is a weak activator of the DPYD promoter. Moreover, Sp3 may serve as a competitor of Sp1, thus decreasing the Sp1 induced promoter activity. SpA, SpB and SpC sites are all Sp1 inducible. In the full activation of the DPYD promoter in human cell lines, the SpB site is essential; the SpC site works cooperatively with SpB, while SpA has minor promoter activity. These studies provide further insight into the molecular mechanisms underlying the heterogeneity of DPD activity, and may facilitate the efficacy and safety of 5-FU-based chemotherapy.

Control of Glut1 promoter activity under basal conditions and in response to hyperosmolarity: role of Sp1

We previously identified (Hwang DY and Ismail-Beigi F. Am J Physiol Cell Physiol 281: C1365-C1372, 2001) a 44-bp GC-rich segment of the rat proximal glucose transporter (Glut)1 promoter, located at -104 to -61, as necessary for basal transcription of the Glut1 gene. Using deletion and mutational analysis and expression of transfected reporter constructs, we report in the present study that mutation of the Sp1 site located within this segment of the promoter leads to a marked ( approximately 4-fold) decrease in basal promoter activity. Double mutations located in the Sp1 site and in a second downstream GC-rich region (-71 to -51) did not cause a further decrease in promoter activity. Gel shift and supershift assays verified the importance of the Sp1 site. Exposure of cells to trichostatin A resulted in increased expression of the endogenous Glut1 as well as the transfected wild-type construct. Finally, the presence of the Sp1 site was found to be essential for the positive response of the promoter to hyperosmolarity. We conclude that the consensus Sp1 site located in the rat proximal Glut1 promoter is necessary and sufficient for basal expression of the Glut1 gene, as well as for its response to hyperosmolarity.

The roles of Sp1, Sp3, USF1/USF2 and NRF-1 in the regulation and three-dimensional structure of the Fragile X mental retardation gene promoter.

Expansion of a CGG.CCG-repeat tract in the 5'-untranslated region of the FMR1 (Fragile X mental retardation 1) gene causes its aberrant transcription. This produces symptoms ranging from premature ovarian failure and Fragile X associated tremor and ataxia syndrome to FMR syndrome, depending on the size of the expansion. The promoter from normal alleles shows four protein-binding regions in vivo. We had previously shown that in mouse brain extracts two of these sites are bound by USF1/USF2 (upstream stimulatory factors 1 and 2) heterodimers and NRF-1 (nuclear respiratory factor-1). We also showed that these sites are involved in the positive regulation of FMR1 transcription in neuronally derived cells. In the present study, we show that Sp1 (specificity protein 1) and Sp3 are also strong positive regulators of FMR1 promoter activity. We also show that, like Sp1 and E-box-binding proteins such as USF1 and USF2, NRF-1 causes DNA bending, in this case producing a bend of 57 degrees towards the major groove. The combined effect of the four protein-induced bends on promoter geometry is the formation of a highly compact arch-like structure in which the 5' end of the promoter is brought in close proximity to the 3' end. We had previously shown that while point mutations in the GC-boxes decrease promoter activity, deletion of either one of them leads to an increase in promoter activity. We can reconcile these observations with the positive effect of Sp1 and Sp3 if protein-induced bending acts, at least in part, to bring together distally spaced factors important for transcription initiation.