Sp1 Target Research Articles

Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics

Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the MatchTM software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.

In vitro apoptotic effects of methanol extracts of Dianthus chinensis and Acalypha australis L. targeting specificity protein 1 in human oral cancer cells

The aims of this study were to evaluate the apoptotic activities and molecular mechanisms of methanol extracts of Dianthus chinensis (MEDC) and Acalypha australis L. (MEAL) in human oral cancer cells. The apoptotic effects and related molecular mechanisms of MEDC and MEAL on oral cancer cells were evaluated using MTS assay, DAPI staining, immunostaining, Western blotting, and reverse transcriptase-polymerase chain reaction. Sp1 was overexpressed in oral tumor tissues compared with normal oral mucosa. Downregulation of Sp1 inhibited the growth of SCC-15 and YD-15 oral cancer cells. MEDC and MEAL inhibited cell growth and induced apoptosis in both cell lines by decreasing the expression of Sp1. In addition, treatment of cells with MEDC and MEAL decreased Mcl-1 expression, which is a downstream target of Sp1. Our results indicate that MEDC and MEAL are bioactive natural products that can potentially induce apoptosis of tumor cells that overexpress the Sp1 protein.

Hesperidin Induces Apoptosis by Inhibiting Sp1 and Its Regulatory Protein in MSTO-211H Cells

Hesperidin, a flavanone present in citrus fruits, has been studied as potential therapeutic agents that have anti-tumor activity and apoptotic effects in several cancers, but there is no report about the apoptotic effect of hesperidin in human malignant pleural mesothelioma through the specificity protein 1 (Sp1) protein. We investigated whether hesperidin inhibited cell growth and regulated Sp1 target proteins by suppressing the levels of Sp1 protein in MSTO-211H cells. The IC50 value of hesperidin was determined to be 152.3 μM in MSTO-211H cells for 48 h. Our results suggested that hesperidin (0-160 μM) decreased cell viability, and induced apoptotic cell death. Hesperidin increased Sub-G1 population in MSTO-211H cells. Hesperidin significantly suppressed mRNA/protein level of Sp1 and modulated the expression level of the Sp1 regulatory protein such as p27, p21, cyclin D1, Mcl-1, and survivin in mesothelioma cells. Also, hesperidin induced apoptotic signaling including: cleavages of Bid, caspase-3, and PARP, upregulation of Bax, and down-regulation of Bcl-xl in mesothelioma cells. These results show that hesperidin suppressed mesothelioma cell growth through inhibition of Sp1. In this study, we demonstrated that Sp1 acts as a novel molecular target of hesperidin in human malignant pleural mesothelioma.

SP Transcription Factor Paralogs and DNA-Binding Sites Coevolve and Adaptively Converge in Mammals and Birds

Functional modification of regulatory proteins can affect hundreds of genes throughout the genome, and is therefore thought to be almost universally deleterious. This belief, however, has recently been challenged. A potential example comes from transcription factor SP1, for which statistical evidence indicates that motif preferences were altered in eutherian mammals. Here, we set out to discover possible structural and theoretical explanations, evaluate the role of selection in SP1 evolution, and discover effects on coregulatory proteins. We show that SP1 motif preferences were convergently altered in birds as well as mammals, inducing coevolutionary changes in over 800 regulatory regions. Structural and phylogenic evidence implicates a single causative amino acid replacement at the same SP1 position along both lineages. Furthermore, paralogs SP3 and SP4, which coregulate SP1 target genes through competitive binding to the same sites, have accumulated convergent replacements at the homologous position multiple times during eutherian and bird evolution, presumably to preserve competitive binding. To determine plausibility, we developed and implemented a simple model of transcription factor and binding site coevolution. This model predicts that, in contrast to prevailing beliefs, even small selective benefits per locus can drive concurrent fixation of transcription factor and binding site mutants under a broad range of conditions. Novel binding sites tend to arise de novo, rather than by mutation from ancestral sites, a prediction substantiated by SP1-binding site alignments. Thus, multiple lines of evidence indicate that selection has driven convergent evolution of transcription factors along with their binding sites and coregulatory proteins.

Sp1 is a transcription repressor to stanniocalcin-1 expression in TSA-treated human colon cancer cells, HT29

Our previous study demonstrated that, stanniocalcin-1 (STC1) was a target of histone deacetylase (HDAC) inhibitors and was involved in trichostatin A (TSA) induced apoptosis in the human colon cancer cells, HT29. In this study, we reported that the transcriptional factor, specificity protein 1 (Sp1) in association with retinoblastoma (Rb) repressed STC1 gene transcription in TSA-treated HT29 cells. Our data demonstrated that, a co-treatment of the cells with TSA and Sp1 inhibitor, mithramycin A (MTM) led to a marked synergistic induction of STC1 transcript levels, STC1 promoter (1 kb)-driven luciferase activity and an increase of apoptotic cell population. The knockdown of Sp1 gene expression in TSA treated cells, revealed the repressor role of Sp1 in STC1 transcription. Using a protein phosphatase inhibitor okadaic acid (OKA), an increase of Sp1 hyperphosphorylation and so a reduction of its transcriptional activity, led to a significant induction of STC1 gene expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binding on STC1 proximal promoter in TSA treated cells. The binding of Sp1 to STC1 promoter was abolished by the co-treatment of MTM or OKA in TSA-treated cells. Re-ChIP assay illustrated that Sp1-mediated inhibition of STC1 transcription was associated with the recruitment of another repressor molecule, Rb. Collectively our findings identify STC1 is a downstream target of Sp1.

Glutamine stimulates the gene expression and processing of sterol regulatory element binding proteins, thereby increasing the expression of their target genes

Here we show that the larger the amount of glutamine added to the medium, the more the expression of genes related to lipid homeostasis is promoted by the activation of sterol regulatory element binding proteins (SREBPs) at the transcriptional and post-translational levels in human hepatoma HepG2 cells. Glutamine increases the mRNA levels of several SREBP targets, including SREBP-2. The gene expression of SREBP-1a, a predominant form of SREBP-1 in most cultured cells and a target of the general transcription factor Sp1, is significantly augmented by glutamine via an increased binding of Sp1 to the SREBP-1a promoter. In contrast, the increased expression of SREBP targets including SREBP-2 is due to stimulation of the processing of SREBP proteins by glutamine. It is also shown that glutamine accelerates SREBP processing through increased transport of the SREBP/SREBP cleavage-activating protein complex from the endoplasmic reticulum to the Golgi apparatus. The processing of activating transcription factor 6 is activated by the same proteases as SREBPs in the Golgi in response to endoplasmic reticulum stress and is not induced by glutamine. Taken together, these results clearly demonstrate that glutamine brings about not only the induction of SREBP-1a transcription but also the stimulation of SREBP processing, thereby facilitating the gene expression of SREBP targets in cultured cells.

A Novel Specificity Protein 1 (SP1)-like Gene Regulating Protein Kinase C-1 (Pkc1)-dependent Cell Wall Integrity and Virulence Factors in Cryptococcus neoformans

Eukaryotic cells utilize complex signaling systems to detect their environments, responding and adapting as new conditions arise during evolution. The basidiomycete fungus Cryptococcus neoformans is a leading cause of AIDS-related death worldwide and utilizes the calcineurin and protein kinase C-1 (Pkc1) signaling pathways for host adaptation and expression of virulence. In the present studies, a C-terminal zinc finger transcription factor, homologous both to the calcineurin-responsive zinc fingers (Crz1) of ascomycetes and to the Pkc1-dependent specificity protein-1 (Sp1) transcription factors of metazoans, was identified and named SP1 because of its greater similarity to the metazoan factors. Structurally, the Cryptococcus neoformans Sp1 (Cn Sp1) protein was found to have acquired an additional zinc finger motif from that of Crz1 and showed Pkc1-dependent phosphorylation, nuclear localization, and whole genome epistatic associations under starvation conditions. Transcriptional targets of Cn Sp1 shared functional similarities with Crz1 factors, such as cell wall synthesis, but gained the regulation of processes involved in carbohydrate metabolism, including trehalose metabolism, and lost others, such as the induction of autophagy. In addition, overexpression of Cn Sp1 in a pkc1Δ mutant showed restoration of altered phenotypes involved in virulence, including cell wall stability, nitrosative stress, and extracellular capsule production. Cn Sp1 was also found to be important for virulence of the fungus using a mouse model. In summary, these data suggest an evolutionary shift in C-terminal zinc finger proteins during fungal evolution, transforming them from calcineurin-dependent to PKC1-dependent transcription factors, helping to shape the role of fungal pathogenesis of C. neoformans.

Apoptotic effect of tolfenamic acid in androgen receptor‐independent prostate cancer cell and xenograft tumor through specificity protein 1

Tolfenamic acid (Tol) is a non-steroidal anti-inflammatory drug that was reported to exhibit anticancer activity in pancreatic and colorectal cancer models. This study examined the role of Tol in the death regulation of PC-3 and DU145 human androgen-independent prostate cancer cells. The results showed that Tol inhibited cell growth and induced apoptosis, as evidenced by nuclear fragmentation and cleaved caspase 3 and poly(ADP-ribose) polymerase. Tol suppressed the specificity protein 1 (Sp1) protein in both PC-3 and DU145 cells. Tol also attenuated Sp1 mRNA and its promoter activity in DU145 cells, but did not alter them in PC-3 cells, indicating that Tol degrades Sp1 protein in these cells. Tol also downregulated protein levels, mRNA levels and promoter activities of survivin and myeloid cell leukemia-1, which are downstream targets of Sp1. The expressions of survivin and Mcl-1 and cancer cell growth were lower in the PC-3 cells treated with Sp1 interfering RNA and mithramycin A. Moreover, an oral injection of Tol decreased tumor growth and downregulated the Sp1 protein in athymic nude mice bearing DU145 cell xenografts without hepatotoxicity. Overall, Tol downregulates the Sp1 protein to inhibit growth and induce apoptosis in androgen-refractory prostate cancers, both in vitro and in vivo, that show resistance against many chemotherapeutic agents.

Neuronal nitric oxide synthase interacts with Sp1 through the PDZ domain inhibiting Sp1-mediated copper–zinc superoxide dismutase expression

In this report we demonstrate that neuronal nitric oxide synthase (nNOS) is able to interact with Sp1 both in vivo and in vitro. In particular, we show that such interaction is mediated by the N-terminal PDZ domain of full length nNOS (fl-nNOS). In fact nNOS mutant lacking the PDZ domain (ΔnNOS) displays an impaired ability to bind to Sp1, as demonstrated by co-immunoprecipitation experiments. The overexpression of fl-nNOS in SH-SY5Y cells leads to the formation of nNOS/Sp1 heterocomplex and inhibits the binding of Sp1 to DNA. Among the Sp1 target genes we looked at the possible alteration of binding to copper–zinc superoxide dismutase gene ( sod1) promoter. We find that the interaction of nNOS with Sp1 leads to a significant decrease of SOD1 mRNA, protein level and activity. The overexpression of ΔnNOS results in an inability to sequester Sp1 and unaffected Sp1 DNA binding capacity, allowing sod1 to be expressed. The data reported give effort to the possible involvement of nNOS in regulating gene transcription in NO-independent manner giving an additional significance to the expression of specific nNOS splicing variants.

Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation

BackgroundNeuropilin is a transmembrane receptor for vascular endothelial growth factor (VEGF) and is expressed in normal endothelial cells and upregulated in cancer cells. Neuropilin-1 (NRP-1) has been shown to promote tumour cell migration and survival in colon cancer in response to VEGF binding. The expression profiles of neuropilins, associated co-receptors and known ligands have been mapped in three colorectal cell lines: Caco-2, HCT116 & HT29. We have previously shown that butyrate, a naturally occurring histone deacetylase inhibitor (HDACi) produced by fermentation of fibre in the colon, causes apoptosis of colon cancer cell lines.ResultsHere we demonstrate that butyrate down-regulates NRP-1 and VEGF at the mRNA and protein level in colorectal cancer cell lines. NRP-1 is a known transcriptional target of Sp1, whose activity is regulated by acetylation. NRP-1 down-regulation by butyrate was associated with decreased binding affinity of Sp1 for canonical Sp-binding sites in the NRP-1 promoter. siRNA-mediated knock-down of Sp1 implied that Sp1 may have strong DNA binding activity but weak transactivation potential.ConclusionThe downregulation of the key apoptotic and angiogenesis regulator NRP-1 by butyrate suggests a novel contributory mechanism to the chemopreventive effect of dietary fibre.

Suv39H1 Recruitment by Sp1 Causes a Decline in 12(S)‐Lipoxygenase Expression

Sp1 is an important transcription factor that regulates gene expression. Several modifications such as phosphorylation, acetylation, glycosylation, ubiquitination, and sumoylation have been found in Sp1, and these affect its transcriptional activity. In this study, we found that Sp1 can directly interact with the methyltransferase Suv39H1 and that it is also modified by methylation. In addition, to inhibit the lynsine‐specific demethylase 1 with pargyline or Suv39H1 overexpression would increase the Sp1 methylation signal. In the early stages of mitosis, the Suv39H1 protein level increases and co‐localizes with Sp1. Sp1 plays the role of an anchor protein, recruiting Suv39H1 to the chromatin region in order to methylate histone H3, which leads to chromosome packaging during mitosis. In addition, the increase in Sp1 methylation represses the expression of Sp1 target gene, 12(S)‐Lipoxygenase. Taken together, these data indicate that Suv39H1 could be recruited to the genomic region by Sp1 to affect the Sp1‐denpendent gene expression through modulation of the post‐translational modification of Sp1 and histone H3 by methylation.

Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells

Telomeres are essential for the maintenance of genomic integrality, they enablecomplete DNA replication, organize subnuclear domains, and protect chromosome endsagainst fusion. Loss of telomere function in human cells results in the formation ofdicentric chromosomes and other abnormalities such as end-to-end fusions.TRF2 is aubiquitously expressed protein which binds directly to the tandem array of duplextelomeric repeats and is involved in the telomere structure and chromosome endprotection. Inhibition of TRF2 induces end-to-end chromosome fusions and growth arrestor apoptosis in human melanoma cells.Lack of TRF2 in ALT cells also caused PMLdependentp53 activation and loss of telomeric DNA.TRF2 over-expression is found inseveral human tumors such as breast carcinomas, liver hepatocarcinomas, lungcarcinomas and gastric carcinomas. This suggests that TRF2 may have a role intumorigenisis.Cancers of the gastrointestinal tract share numerous common geneticfeatures despite differing widely in incidence and outcome.However, the role andmechanisms that lead to over-expressed TRF2 in colorectal carcinoma are not wellknown. Sp1 is a member of the multigene family with sequence-specific DNA-bindingC-terminal zinc-finger motifs, which plays a critical role in the regulation of expressionof mammalian genes lacking a TATA box, by binding to GC/GT boxes to activatetranscription.Cancer-related Sp1 targets include PDEGF, p21, VEGF, TGF-β, cyclinD1, E2F1, c-fos, transforming growth factor α and osteopontin (OPN).The promoterof TRF2 is GC-rich sequence (70-75%), which lacks an obvious TATA box. It is unclearwhether or not TRF2 is regulated via Sp1, which may in turn be regulated to genomicstability. We tested the expression of TRF2 in colorectal carcinoma tissues and coloncarcinoma SW480 cell line, and studied the role of Sp1 in regulating TRF2 expression incolon carcinoma. Results showed that TRF2 was over-expressed in colorectal carcinomatissues and SW480 cell line. It was also shown that Sp1 transcription factor is involved inthe up-regulation of TRF2 expression.

Profiling and comparing transcription factors activated in non‐metastatic and metastatic nasopharyngeal carcinoma cells

Transcription factors (TFs) are modulators of gene expression that are critically important in the establishment and progression of human cancers. In the current study, the activity profiles of TFs in a normal nasopharyngeal epithelial cell line and in nasopharyngeal carcinoma (NPC) cell lines were studied using oligonucleotide array-based TF assays. Compared to the normal epithelial cell line NP69, nine TFs in the non-metastatic NPC cell line (6-10B) and eight TFs in a metastatic NPC cell line (5-8F) were upregulated. Among upregulated TFs, Sp1, AP2, and ATF/CREB families exhibited relatively high activities in NPC cell lines. Transcription levels of Sp1, ATF-1, ATF-2, AP2alpha, AP2gamma, and CREB1 were higher in 5-8F cells than in 6-10B cells. In addition, higher expression of the Sp1 target genes MMP-9 and VEGF was observed in 5-8F cells. Sp1 silencing reduced VEGF and MMP-9 expression. Inhibition of Sp1 expression and activity in 5-8F cells by mithramycin resulted in downregulated expression and secretion of MMP-9 and VEGF, concomitant with inhibition of cell migration and invasion. These results suggest that dynamic changes in TF activities occur in NPC cells and that these changes may play important roles in regulating the expression of genes associated with the development and progression of NPC.

Red wine polyphenolics have anti‐cancer effects in colon‐cancer and target oncogenic microRNAs as potential underlying mechanisms

Red wine has been identified as a food with anti‐cancer properties. The aim of this study was to investigate the anti‐cancer effect of a Texas red wine from Black Spanish grapes (Vitis labrusca) in colorectal adenocarcinoma cells (HT‐29). A polyphenolic extract prepared from the wine was tested (20‐300 mg/L) regarding anti‐cancer effects and underlying mechanisms related to apoptosis and angiogenesis. Reactive oxygen species (ROS) increased up to 6.5‐fold at 300mg/L and mRNA of p53 was increased up to 1.9‐fold compared to the control and caspase‐3 activity was increased up to 1.8 fold within the same concentration‐range, indicating pro‐apoptotic activity of the wine extract. The oncogenic specificity protein Sp1 was significantly decreased, where the zinc‐finger protein ZBTB10 a transcription factor which has been found to inhibit Sp1 was increased up to 1.3‐fold with the wine extract. Vascular endothelial growth factor (VEGF) mRNA a target of Sp1 was decreased up to 3.7‐fold, indicating anti‐angiogenic effects. Additionally, the pro‐oncogenic microRNAs 21 and 27a were significantly decreased within an extract concentration range of 37‐300 mg/L, and 75‐300mg/L, respectively. In conclusion, since ZBTB10 is a relevant target for miRNA 27a, the reduction of miRNA27a may be responsible for a ZBTB10‐involved reduction of the oncogenic Sp1 causing decrease in VEGF and inducing apoptosis as underlying mechanism.Grant Funding SourceTexas A&M University

Phosphorylation by CDK1 and JNK1 regulate the DNA binding activity of transcription factor Sp1 during mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated at threonine 739 by JNK and CDK1 and not co‐localized with the chromatin at the beginning of mitotic stage. EMSA, DNA affinity precipitation assay, and ChIP assay were performed and revealed that during mitosis the DNA‐binding ability of phosphorylated Sp1was decrease as well as dephosphorylation of Sp1 by phosphatase restored the DNA binding affinity. In addition, the phosphorylation of Sp1 during mitotic stage also suppressed Sp1 to interact with p300, Brg1, and RNA polII and reduced the transcriptional activity of Sp1 target genes. Moreover, in late mitosis, the Pin1 interacted with the high phosphorylation of Sp1 and recruited the PP2A to dephosphorylate Sp1. In Pin1 knockout MEF cells, there was a delay in the relocalization of Sp1 to the nucleus during telophase. Taken together, our results indicated that Sp1 interacts with JNK and CDK1, followed by phosphorylation of Sp1 at early mitosis. Sp1, then, is disassociated from DNA and loses interaction with co‐activator. In late mitosis, Pin1 and PP2A are recruited by phosphorylation of Sp1, leading to the enhancement of the DNA binding‐ability and protein interactional function of Sp1.

Chromatin structure and expression of the AMPA receptor subunit GluR2 in human glioma cells and the role of REST

The AMPA receptors are postsynaptic ion channels that open following stimulation with glutamate. The regulatory region of the gene encoding the GluR2 subtype of AMPA receptors contains a binding site for the transcriptional repressor REST. The cell-type specific microenvironment, in particular the cell type-specific structure of the chromatin, is crucial for the ability of REST to control target gene transcription. Using antibodies directed against methylated lysine residues 4 or 9 (H3K4 or H3K9) of histone H3, we show that the GluR2 gene has an open chromatin configuration in human U87MG glioma cells, with nucleosomes carrying di-and trimethylated H3K4. In contrast, the GluR2 gene is embedded into a repressed chromatin environment in non-neuronal hepatoma cells and keratinocytes. Chromatin immunoprecipitation experiments revealed binding of REST and histone deacetylase-1 to the GluR2 gene under physiological conditions. While overexpression of REST reduced GluR2 mRNA levels, expression of a mutant of REST that contained a transcriptional activation domain enhanced GluR2 gene transcription in U87MG glioma cells. Treatment of the cells with the histone deacetylase inhibitor trichostatin A (TSA) induced an upregulation of GluR2 expression, indicating that the transcription of the GluR2 gene is dependent of the balance between histone acetylation and deactylation. Transcription of the GluR2 gene was also inducible by TSA following inhibition of Sp1 target gene transcription, indicating that the inhibition of Sp1 is not required for the upregulation of GluR2 expression following histone deacetylase inhibition.

Progesterone Receptor Rapid Signaling Mediates Serine 345 Phosphorylation and Tethering to Specificity Protein 1 Transcription Factors

Human progesterone receptors (PR) rapidly activate cytosolic signaling pathways, in addition to their classical function as ligand-activated transcription factors. Using ER+/PR-B+ T47D breast cancer cells, we probed the role of progestin-stimulated rapid PR signaling in the transcriptional regulation of target genes involved in breast cancer cell proliferation. Epidermal growth factor receptor (EGFR) was rapidly activated after a 10-min treatment with R5020. Progestin induced EGFR-, c-Src-, and MAPK-dependent phosphorylation of PR-B on the MAPK consensus site, Ser345. Ser345-phosphorylated PR-B receptors strongly associated with specificity protein 1 (Sp1) transcription factors to regulate PR cell cycle (p21) and growth-promoting (EGFR) target genes whose promoters lack canonical progesterone response element sequences. Inhibitors of EGFR, c-Src, or MAPK activities blocked PR tethering to Sp1 and progestin-stimulated S-phase entry. Mutant PR-B receptors defective for c-Src binding (mPro) were not phosphorylated on Ser345 in response to progestin and failed to interact with Sp1. Hormone-induced complexes containing Sp1 and wild-type PR-B, but not S345A or mPro PR-B, were recruited to Sp1 sites within the endogenous p21 promoter. Progestin-induced S-phase entry was attenuated in T47D cells containing wild-type PR-B and treated with EGFR, c-Src, or MAPK kinase inhibitors or in T47D cells stably expressing mPro or mutant DNA-binding domain PR-B. In sum, rapid progestin-activated PR signaling leads to PR Ser345 phosphorylation and tethering to Sp1. These events are critical for progestin-stimulated regulation of Sp1 target genes and breast cancer cell proliferation. Our data demonstrate the therapeutic potential for PR-targeted breast cancer treatment by exploiting multiple nodes along the PR signaling pathway, including PR-B, EGFR, c-Src, MAPK, or Sp1.

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.

The Herpes Simplex Virus Gene Pol Expressed in Herpes-Associated Erythema Multiforme Lesions Upregulates/Activates SP1 and Inflammatory Cytokines

Background/Aims: Herpes-simplex-virus-associated erythema multiforme (HAEM) is characterized by lesional skin expression of the viral protein Pol and localized inflammation. The objective of this study is to examine the mechanism whereby Pol induces localized inflammation. Methods: A431 cells transfected with Pol or an empty vector and lesional skin from HAEM or drug-induced erythema multiforme patients were examined for expression of the transcription factor SP1 and SP1-regulated genes by immunoblotting, immunohistochemistry and immunofluorescence. Results: SP1, TGF-β, p21^waf1 and Hsp27 were upregulated in A431 cells transfected with Pol but not the empty vector. Expression was further increased by exposure to IFN-γ. Pol+ HAEM lesional skin expressed SP1, Hsp27, TGF-β and p21^waf1. Normal skin and drug-induced erythema multiforme lesional skin were negative. Conclusion: The data indicate that Pol activates SP1, causing upregulation of SP1 target genes (notably TGF-β) involved in localized inflammation. Upregulation is potentiated by IFN-γ.

Wnt-mediated Down-regulation of Sp1 Target Genes by a Transcriptional Repressor Sp5

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.