Sp1 cooperates with Sp3 to upregulate MALAT1 expression in human hepatocellular carcinoma.

Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as nuclear-enriched transcript 2 (NEAT2), is highly conserved among mammals and highly expressed in the nucleus. It was first identified in lung cancer as a prognostic marker for metastasis but is also associated with several other solid tumors. In hepatocellular carcinoma (HCC), MALAT1 is a novel biomarker for predicting tumor recurrence after liver transplantation. The mechanism of overexpression in tumor progression remains unclear. In the present study, we investigated the role of specificity protein 1/3 (Sp1/3) in regulation of MALAT1 transcription in HCC cells. The results showed a high expression of Sp1, Sp3 and MALAT1 in HCC vs. paired non-tumor liver tissues, which was associated with the AFP level (Sp1, r=7.44, P=0.0064; MALAT1, r=12.37, P=0.0004). Co-silencing of Sp1 and Sp3 synergistically repressed MALAT1 expression. Sp1 binding inhibitor, mithramycin A (MIT), also inhibited MALAT1 expression in HCC cells. In conclusion, the upstream of MALAT1 contains five Sp1/3 binding sites, which may be responsible for MALAT1 transcription. Inhibitors, such as MIT, provide a potential therapeutic strategy for HCC patients with MALAT1 overexpression.

Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the E1b far upstream promoter.

Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The upstream E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues, including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide -300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.

The PP2A-Aß gene is regulated by multiple transcriptional factors including Ets-1, SP1/SP3, and RXRα/ß.

Protein phosphatase-2A (PP-2A) is a major serine/threonine phosphatase abundantly expressed in eukaryotes. PP-2A is a heterotrimer that contains a 65 kD scaffold A subunit, a 36 kD catalytic C subunit, and a regulatory B subunit of variable isoforms ranging from 54-130 kDs. The scaffold subunits, PP2A-Aα/ß, act as platforms for both the C and B subunits to bind, and thus are key structural components for PP-2A activity. Mutations in both genes encoding PP2A-Aα and PP2A-Aß lead to carcinogenesis and likely other human diseases. Our previous work showed that the gene coding for PP2A-Aα is positively regulated by multiple transcription factors including Ets-1, CREB, and AP-2α but negatively regulated by SP-1/SP-3. In the present study, we have functionally dissected the promoter of the mouse PP2A-Aß gene. Our results demonstrate that three major cis-elements, including the binding sites for Ets-1, SP1/SP3, and RXRα/ß, are present in the proximal promoter of the mouse PP2A-Aß gene. Gel mobility shifting assays reveal that Ets-1, SP1/SP3, and RXRα/ß all bind to PP2A-Aß gene promoter. In vitro mutagenesis and reporter gene activity assays demonstrate that while Ets-1 displays negative regulation, SP1/SP3 and RXRα/ß positively regulate the promoter of the PP2A-Aß gene. Co-expression of the cDNAs encoding Ets-1, SP1/SP3, or RXRα/ß and the luciferase reporter gene driven by PP2A-Aß promoter further confirm their control over the PP2A-Aß promoter. Finally, ChIP assays demonstrate that Ets-1, SP1/SP3, and RXRα/ß can all bind to the PP2A-Aß gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aß gene. Moreover, our results provide important information explaining why PP2A-Aα and PP2A-Aß display distinct expression levels.

Transcription factors Sp1 and Sp3 regulate basal transcription of the human IRF-3 gene.

Interferon regulatory factor 3 (IRF-3) plays a crucial role in initiation and development of the IFN antiviral response. The expression level of human IRF-3 is thought to be closely related to antiviral state of cells. However, the mechanisms of the transcription regulation of IRF-3 have remained largely unknown. We previously reported that transcription factor E2F1 negatively regulates the basal transcriptional activity of IRF-3. Here we demonstrate that transcription factors Sp1 and Sp3 up-regulate the basal transcriptional activity of IRF-3 and increase IRF-3 expression at mRNA level. By transient transfection analysis we revealed that mutation of Sp1/NRF-1 binding site resulted in a profound reduction of IRF-3 promoter activity. Overexpression of Sp1 and Sp3, but not NRF-1, transactivated the IRF-3 promoter activity in reporter gene assays while knocking-down of endogenous Sp1 and Sp3 by a shRNA strategy markedly inhibited IRF-3 promoter activity. Chromatin immunoprecipitation (ChIP) assays showed that Sp1 and Sp3 interact with the IRF-3 promoter in vivo. These results suggest that basal expression level of IRF-3 is regulated by transcription factors Sp1 and Sp3.

Extracellular signal-regulated kinase mitogen-activated protein kinase-dependent SOCS-3 gene induction requires c-Jun, signal transducer and activator of transcription 3, and specificity protein 3 transcription factors.

SOCS-3 gene induction by cAMP-elevating agents or the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), in primary HUVECs was found to require PKCη- and PKCε-dependent extracellular signal-regulated kinase (ERK) activation. The minimal, ERK-responsive element of the SOCS-3 promoter was localized to a region spanning nucleotides -107 to the transcription start site and contains conserved binding sites for AP-1 and SP1/SP3 transcription factors, as well as proximal and distal signal transducer and activator of transcription (pSTAT and dSTAT) binding elements. All three classes of transcription factor were activated in response to ERK activation. Moreover, representative protein components of each of these transcription factor binding sites, namely c-Jun, STAT3, and SP3, were found to undergo ERK-dependent phosphorylation within their respective transactivation domains. Mutational analysis demonstrated an absolute requirement for the SP1/SP3 binding element in controlling basal transcriptional activity of the minimal SOCS-3 promoter. In addition AP-1, pSTAT, and SP1/SP3 binding sites were required for ERK-dependent, PMA-stimulated SOCS-3 gene activation. The dSTAT site seems to be important for supporting activity of the AP-1 site, because combined deletion of both sites completely blocks transcriptional activation of SOCS-3 by PMA. Together these results describe novel, ERK-dependent regulation of transcriptional activity that requires codependent activation of multiple transcription factors within the same region of the SOCS-3 gene promoter.

Sp3 transcription factor is crucial for transcriptional activation of the human NOX4 gene.

NOX is the catalytic subunit of NADPH oxidase, the superoxide-generating enzyme. Among several isoforms of NOX, NOX4 is abundantly expressed in various tissues. To clarify the mechanisms of constitutive and ubiquitous expression of NOX4, the promoter activities of the human NOX4 gene were analyzed by reporter assays. The 5'-flanking and non-coding regions of the human NOX4 gene are known to contain multiple GC bases. Among them, three GC-boxes containing putative Sp/Klf-binding sites, which were not found in rodent genes, were suggested to be essential for the basal expression of the NOX4 gene in SH-SY5Y and HEK293 cells. Electrophoresis mobility shift assays demonstrated that Sp1 and Sp3 could bind to GC-boxes at positions -239/-227 and +69/+81 in these cells. Chromatin immunoprecipitation assays showed that Sp1 and Sp3 could also bind to GC-boxes at positions -239/-227 and +69/+81 in vivo. The promoter activity of the NOX4 gene was reduced in SH-SY5Y and HEK293 cells by transfection of an anti-Sp3 short hairpin RNA-expression plasmid. Taken together, these results suggest that Sp3 plays a key role in the expression of NOX4 in various cell lineages in humans.

The role of Sp1 and Sp3 in normal and cancer cell biology.

Sp1 and Sp3 are transcription factors expressed in all mammalian cells. These factors are involved in regulating the transcriptional activity of genes implicated in most cellular processes. Dysregulation of Sp1 and Sp3 is observed in many cancers and diseases. Due to the amino acid sequence similarity of the DNA binding domains, Sp1 and Sp3 recognize and associate with the same DNA element with similar affinity. However, others and our laboratory demonstrated that these two factors possess different properties and exert different functional roles. Both Sp1 and Sp3 can interact with and recruit a large number of proteins including the transcription initiation complex, histone modifying enzymes and chromatin remodeling complexes, which strongly suggest that Sp1 and Sp3 are important transcription factors in the remodeling chromatin and the regulation of gene expression. In this review, the role of Sp1 and Sp3 in normal and cancer cell biology and the multiple mechanisms deciding the functional roles of Sp1 and Sp3 will be presented.

Regulation of plasma-membrane-associated sialidase NEU3 gene by Sp1/Sp3 transcription factors.

Gene expression of the human plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is relatively high in brain and is modulated in response to many cellular processes, including neuronal cell differentiation and tumorigenesis. We demonstrated previously that NEU3 is markedly up-regulated in various human cancers and showed that NEU3 transgenic mice developed a diabetic phenotype and were susceptible to azoxymethane-induced aberrant crypt foci in their colon tissues. These results suggest that appropriate control of NEU3 gene expression is required for homoeostasis of cellular functions. To gain insights into regulation mechanisms, we determined the gene structure and assessed transcription factor involvement. Oligo-capping analysis indicated the existence of alternative promoters for the NEU3 gene. Transcription started from two clusters of multiple TSSs (transcription start sites); one cluster is preferentially utilized in brain and another in other tissues and cells. Luciferase reporter assays showed further that the region neighbouring the two clusters has promoter activity in the human cell lines analysed. The promoter lacks TATA, but contains CCAAT and CAAC, elements, whose deletions led to a decrease in promoter activity. Electrophoretic mobility-shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp (specificity protein) 1 and Sp3 to the promoter region. Down-regulation of the factors by siRNAs (short interfering RNAs) increased transcription from brain-type TSSs and decreased transcription from other TSSs, suggesting a role for Sp1 and Sp3 in selection of the TSSs. These results indicate that NEU3 expression is diversely regulated by Sp1/Sp3 transcription factors binding to alternative promoters, which might account for multiple modulation of gene expression.

Differential control of Notch1 gene transcription by Klf4 and Sp3 transcription factors in normal versus cancer-derived keratinocytes.

In specific cell types like keratinocytes, Notch signaling plays an important pro-differentiation and tumor suppressing function, with down-modulation of the Notch1 gene being associated with cancer development. Besides being controlled by p53, little else is known on regulation of Notch1 gene expression in this context. We report here that transcription of this gene is driven by a TATA-less "sharp peak" promoter and that the minimal functional region of this promoter, which extends from the -342 bp position to the initiation codon, is differentially active in normal versus cancer cells. This GC rich region lacks p53 binding sites, but binds Klf4 and Sp3. This finding is likely to be of biological significance, as Klf4 and, to a lesser extent, Sp3 are up-regulated in a number of cancer cells where Notch1 expression is down-modulated, and Klf4 over-expression in normal cells is sufficient to down-modulate Notch1 gene transcription. The combined knock-down of Klf4 and Sp3 was necessary for the reverse effect of increasing Notch1 transcription, consistent with the two factors exerting an overlapping repressor function through their binding to the Notch1 promoter.

Proximal human FOXP3 promoter transactivated by NF-kappaB and negatively controlled by feedback loop and SP3.

Forkhead box protein 3 (Foxp3) is indispensable for the development of CD4(+)CD25(+) regulatory T cells (Tregs). Here we analyzed three prominent evolutionary conserved regions (ECRs) upstream of the transcription start site of the human FOXP3 gene. We show that ECR2 and ECR3 fragments derived from positions -1.3 to -2.0 kb and -5.0 to -6.0 kb, respectively, display basal transcriptional activity. Reporter constructs derived from ECR1, located between -0.6 and +0.23 kb and thus the most proximal ECR in respect of transcription initiation, remained almost inactive. However, ECR1 was transactivated by the NF-kappaB subunit p65 in HEK 293 cells. In Jurkat and primary T cells, in addition to p65, a second stimulus delivered by either T-cell receptor stimulation or addition of PMA was needed. Co-expression of I kappaB alpha inhibited p65-mediated FOXP3 proximal promoter transactivation, and the NF-kappaB inhibitor curcumin reduced Foxp3 neoexpression in IL-2/CD3/CD28/TGF-beta stimulated PBMCs. Moreover, proximal FOXP3 promoter transactivation was inhibited by Foxp3 and the SP transcription factor family member SP3. Thus, the human proximal FOXP3 promoter is controlled by activation through the TCR involving PKC and the NF-kappaB subunit p65 and by inhibition through a negative feedback loop and SP3.

HDAC3 represses the expression of NKG2D ligands ULBPs in epithelial tumour cells: potential implications for the immunosurveillance of cancer.

The expression of the NKG2D ligands on cancer cells leads to their recognition and elimination by host immune responses mediated by natural killer and T cells. UL16-binding proteins (ULBPs) are NKG2D ligands, which are scarcely expressed in epithelial tumours, favouring their evasion from the immune system. Herein, we investigated the epigenetic mechanisms underlying the repression of ULBPs in epithelial cancer cells. We show that ULBP1-3 expression is increased in tumour cells after exposure to the inhibitor of histone deacetylases (HDACs) trichostatin A (TSA), which enhances the natural killer cell-mediated cytotoxicity of HeLa cells. Our experiments showed that the transcription factor Sp3 is crucial in the activation of the ULBP1 promoter by TSA. Furthermore, by small interfering RNA-mediated knockdown and overexpression of HDAC1-3, we showed that HDAC3 is a repressor of ULBPs expression in epithelial cancer cells. Remarkably, TSA treatment caused the complete release of HDAC3 from the ULBP1-3 promoters. HDAC3 is recruited to the ULBP1 promoter through its interaction with Sp3 and TSA treatment interfered with this association. Together, we describe a new mechanism by which cancer cells may evade the immune response through the epigenetic modulation of the ULBPs expression and provide a model in which HDAC inhibitors may favour the elimination of transformed cells by increasing the immunogenicity of epithelial tumours.

Sp1 and Sp3 regulate transcription of the cyclin-dependent kinase 5 regulatory subunit 2 (p39) promoter in neuronal cells.

Cyclin-dependent kinase 5 (cdk5) activity is critical for development and function of the nervous system. Cdk5 activity is dependent on association with the regulators p35 and p39 whose expression is highly regulated in the developing nervous system.We have identified a small 200 bp fragment of the p39 promoter that is sufficient for cell type-specific expression in neuronal cells. Mutational analysis revealed that a cluster of predicted binding sites for Sp1, AP-1/CREB/ATF and E box-binding transcription factors is essential for full activity of the p39 promoter. Electrophoretic mobility shift assays revealed that Sp1 and Sp3 bound to sequences required for p39 promoter function and chromatin immunoprecipitation assays confirmed binding of these proteins to the endogenous p39 promoter. Furthermore, depletion of either Sp1 or Sp3 by siRNA reduced expression from the p39 promoter. Our data suggest that the ubiquitously expressed transcription factors Sp1 and Sp3 regulate transcription of the cdk5 regulator p39 in neuronal cells, possibly in cooperation with tissue-specific transcription factors.

Effect of SP3 silencing on cytokeratin expression pattern in HPV-positive cells.

In an attempt to understand the molecular factors underlying squamous cell carcinogenesis in HPV-infected oral and cervical tissues, we examined the Sp3-dependent cytokeratin expression in HPV-positive CaSki cells. Two sets of cytokeratins were examined: the simple epithelial CK 7, 8, 18, 19, and 20, which are generally expressed in simple epithelia and CK4, 10, 13, and 17, which are expressed in squamous epithelia. Two additional CK pairs, i.e. CK6/CK16 and CK4/CK13 were analyzed as controls of the proliferation/differentiation cell status, respectively. We report that Sp3 gene silencing specifically hits CK18 and CK19, which are markers of oral and cervical squamous tumors. These data may be of help in immunopathological definition of squamous carcinogenesis.

Cellular transcription factors Sp1 and Sp3 suppress varicella-zoster virus origin-dependent DNA replication.

The varicella-zoster virus (VZV) origin of DNA replication (oriS) contains a 46-bp AT-rich palindrome and three consensus binding sites for the VZV origin binding protein (OBP) encoded by VZV ORF51. All three OBP binding sites are upstream of the palindrome in contrast to the sequence of the herpes simplex virus oriS, which has required OBP binding sites upstream and downstream of the AT-rich region. We are investigating the roles that sequences downstream of the palindrome play in VZV oriS-dependent DNA replication. Computer analysis identified two GC boxes, GC box 1 and GC box 2, in the downstream region which were predicted to be binding sites for the cellular transcription factor Sp1. Electrophoretic mobility shift assay and supershift assays showed that two members of the Sp family (Sp1 and Sp3) stably bind to GC box 1, but not to GC box 2. A predicted binding site for the cellular factor Yin Yang 1 (YY1) that overlaps with GC box 2 was also identified. Supershift and mutational analyses confirmed the binding of YY1 to this site. Mutation of GC box 1 resulted in loss of Sp1 and Sp3 binding and an increase in origin-dependent replication efficiency in DpnI replication assays. In contrast, mutation of the YY1 site had a statistically insignificant effect. These results suggest a model where origin-dependent DNA replication and viral transcription are coupled by the binding of Sp1 and Sp3 to the downstream region of the VZV replication origin during lytic infection. They may also have implications regarding establishment or reactivation of viral latency.

Transcription of PRDM1, the master regulator for plasma cell differentiation, depends on an SP1/SP3/EGR-1 GC-box.

The positive regulatory domain containing 1, encoded by the PRDM1 gene, is a transcriptional repressor considered as a master regulator that is required and sufficient for plasma cell differentiation. In the present study we have performed sequence analysis of the upstream region of the human PRDM1 gene to detect the minimal promoter region necessary for PRDM1 gene transcription. This region comprises the region upstream of the initiation site, as well as the first exon. Collectively, deletion and mutation analysis in conjunction with luciferase reporter assays, EMSA and supershift assays identified a phylogenetically conserved GC-box as an essential element for PRDM1 expression. This GC-box element matches to a binding site for multiple transcription factors such as SP1 and SP3 isoforms as well as early growth response 1. Chromatin immunoprecipitation assays confirmed the in vivo binding capability of these factors to the human PRDM1 promoter. These studies together characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC-box.

The human receptor tyrosine kinase Axl gene--promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation.

Axl is a receptor tyrosine kinase which promotes anti-apoptosis, mitogenesis, invasion, angiogenesis and metastasis, and is highly expressed in cancers. However, the transcriptional regulation of this important gene has never been characterized. The present study was initiated to characterize the promoter, cis-acting elements and promoter methylation driving expression of Axl. The 2.4 kb sequence upstream of the translational start site, and sequential 5'-deletions were cloned and revealed a minimal GC-rich region (-556 to +7) to be sufficient for basal Axl promoter activity in Rko, HCT116 and HeLa cells. Within this minimal region, five Sp (specificity protein)-binding sites were identified. Two sites (Sp a and Sp b) proximal to the translation start site were indispensable for Axl promoter activity, whereas mutation of three additional upstream motifs (Sp c, Sp d and Sp e) was of additional relevance. Gel-shift assays and chromatin immunoprecipitation identified that Sp1 and Sp3 bound to all five motifs, and mutation of all motifs abolished binding. Mithramycin, which inhibits binding of Sp factors to GC-rich sites, dramatically reduced Axl promoter activity and Axl, Sp1 and Sp3 expression. In Drosophila Schneider SL2-cells, exogenous expression of Sp1/Sp3 increased Axl promoter activity. Use of Sp1/Sp3 siRNAs (small interfering RNAs) significantly reduced Axl promoter activity and protein levels in Rko and HeLa cells. Methylation-bisulfite sequencing detected methylated CpG sites within three Sp motifs (Sp a, Sp b and Sp c) and GC-rich flanking sequences, and demethylation by 5-aza-2'-deoxycytidine up-regulated Axl and Sp3 expression in low-Axl-expressing Colo206f/WiDr cells, but not in high-Axl-expressing Rko cells. The results of the present study suggest that Axl gene expression in cancer cells is (1) constitutively driven by Sp1/Sp3 bound to five core promoter motifs, and (2) restricted by methylation within/around Sp-binding sites. This might enhance the understanding and treatment of essential mechanisms associated with cancer and other diseases.

Contributions of specificity protein-1 and steroidogenic factor 1 to Adcy4 expression in Y1 mouse adrenal cells.

The type 4 adenylyl cyclase, Adcy4, is the least abundant of five different adenylyl cyclase isoforms expressed in the Y1 mouse adrenocortical cell line and is deficient in a Y1 mutant with impaired steroidogenic factor 1 (SF1) activity. This study examines the contributions of SF1 and other DNA promoter/regulatory elements to Adcy4 expression in the Y1 cell line and its derivative Adcy4-deficient mutant. Primer extension and in silico analyses indicate that Adcy4 transcription initiates from multiple sites just downstream of a GC-rich sequence. Luciferase reporter gene assays identify a 124-bp sequence, situated 19 bp upstream of the major transcription start site and highly conserved among several mammalian species, as the major determinant of Adcy4 expression in Y1 cells and as a site of compromised activity in the Adcy4-deficient mutant. EMSAs using competitor nucleotides and specific antibodies indicate that this conserved region contains three specificity protein (Sp)-1/Sp3-binding sites and one SF1-binding site. As determined by site-specific mutagenesis, the 5'-most Sp1/Sp3-site enhances promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress Adcy4 promoter activity. In the Adcy4-deficient mutant, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with small interfering RNAs increases Adcy4 expression, confirming the contribution of SF1 to the mutant phenotype. These studies demonstrate roles for Sp1/Sp3 and SF1 in Adcy4 expression in Y1 cells and establish a repressor function for SF1 in certain promoter contexts.

Transcription factors sp1 and sp3 regulate expression of human extracellular superoxide dismutase in lung fibroblasts.

The molecular mechanisms that govern the transcription of human extracellular superoxide dismutase (EC-SOD), the major extracellular antioxidant enzyme, are largely unknown. To elucidate the mechanisms involved in human EC-SOD gene regulation and expression, we localized multiple transcription start sites to a finite region located 3.9 kb upstream of the ATG initiation codon. Within this segment, we subcloned a 2.7-kb fragment upstream of a luciferase reporter gene; the resulting construct exhibited strong in vivo promoter activity in two lung-derived cell lines. Deletion analysis of the EC-SOD 5'-flanking sequences identified a minimal 0.3-kb region that had strong basal promoter activity. Computer sequence analysis revealed a putative Sp1-like binding site within the EC-SOD proximal promoter region that lacked a TATA-box and showed a high frequency of GC nucleotides. Binding of Sp1 and Sp3 transcription factors to the EC-SOD promoter was confirmed by DNase I footprint analysis, electophoretic mobility shift assay, and competition and supershift assays. Cotransfection of the EC-SOD promoter-luciferase reporter constructs with plasmids encoding Sp1 and Sp3 into Sp-deficient insect SL2 cells showed strong activation of luciferase gene expression. The occupancy of the EC-SOD promoter by Sp1/Sp3 and RNA polymerase II in vivo was determined by chromatin immunoprecipitation assay and correlated well with levels of EC-SOD expression in lung epithelial cells (A549) and pulmonary fibroblasts (MRC5). Collectively, our results demonstrate the important role Sp1 and Sp3 plays in regulating the expression of human EC-SOD in the lung.

Retinoic acid (RA) regulates 17beta-hydroxysteroid dehydrogenase type 2 expression in endometrium: interaction of RA receptors with specificity protein (SP) 1/SP3 for estradiol metabolism.

CONTEXT: The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (HSD17B2) exerts a local antiestrogenic effect by metabolizing biologically active estradiol to inactive estrone in endometrial epithelial cells. Retinoic acid (RA) induces HSD17B2 expression, but the underlying mechanism is not known. OBJECTIVE: Our objective was to elucidate the molecular mechanisms responsible for HSD17B2 expression in human endometrial cells. METHOD: Human endometrial Ishikawa and RL95-2 cell lines were cultured in the presence or absence of RA to analyze endogenous HSD17B2 expression, transcription factor complex formation, and promoter activity. RESULTS: RA induced HSD17B2 mRNA levels in a dose- and time-dependent manner in endometrial cells. The RA antagonist ANG11273 abolished RA-induced HSD17B2 expression. Small interfering RNA ablation of RA receptor (RAR)alpha or retinoid X receptor (RXR)alpha completely blocked RA-induced HSD17B2 gene expression. Analysis of serial deletion and site-directed mutants of the HSD17B2 promoter fused to a reporter gene indicated that RA induction requires a cis-regulatory sequence that binds the specificity protein (SP) class of transcription factors. Chromatin-immunoprecipitation-PCR and gel-shift assays showed that RARalpha/RXRalpha and SP1/SP3 interact with this HSD17B2 promoter sequence. Small interfering RNA ablation of SP1 and SP3 expression markedly decreased HSD17B2 basal expression and blocked RA-induced expression. Finally, immunoprecipitationimmunoblotting demonstrated RA-induced interactions between RARalpha/RXRalpha and SP1/SP3 in intact endometrial cells. CONCLUSIONS: In endometrial epithelial cells, RA stimulates formation of a multimeric complex comprised of RARalpha/RXRalpha tethered to transcription factors SP1 and SP3 on the HSD17B2 promoter. Assembly of this transcriptional complex is necessary for RA induction of HSD17B2 expression and may be an important mechanism for local estradiol inactivation in the endometrium.

Fibroblast growth factor receptor 1 gene expression is required for cardiomyocyte proliferation and is repressed by Sp3.

Fibroblast growth factor receptor 1 (FGFR1) is the only high-affinity FGFR in the vertebrate myocardium. FGFR1 is a tyrosine kinase receptor and has a non-redundant role in proliferation and differentiation of cardiomyocytes during embryogenesis. Results presented here demonstrate that FGFR1 gene expression declines as neonatal cardiomyocytes develop into adult cardiomyocytes. Furthermore, silencing FGFR1 gene expression reduced neonatal cardiomyocyte proliferation, indicating that FGFR1 gene expression is required for the optimal proliferative capacity of cardiomyocytes. To determine the mechanism that governs FGFR1 gene expression in cardiomyocytes, sequence analysis of the proximal mouse FGFR1 promoter identified a potential binding site for Sp transcription factors. Mutation of this site increased FGFR1 promoter activity compared to the wild-type promoter, indicating the presence of a negative transcriptional regulator of the FGFR1 promoter at this site in cardiomyocytes. Sp3 expression in neonatal cardiomyocytes and Drosophila SL2 cells reduced FGFR1 promoter activity in a dose-dependent manner. Western blots and immunocytochemistry indicated that Sp3 was present in the nuclear and cytoplasmic compartments of neonatal cardiomyocytes. Chromatin-immunoprecipitation studies verified that endogenous Sp3 in cardiomyocytes interacts with the FGFR1 promoter. Transient chromatin-immunoprecipitation studies using wild-type and mutated FGFR1 promoter constructs in SL2 cells identified the specific Sp3 binding site within the FGFR1 promoter. These studies implicate Sp3 as a negative transcriptional regulator of FGFR1 promoter activity in cardiomyocytes and as a suppressor of cardiomyocyte proliferation.