Tumor Extracellular Matrix Stiffness Promptly Modulates the Phenotype and Gene Expression of Infiltrating T Lymphocytes.

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4+ and CD8+ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8+ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4+ and CD8+ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.

Targeted inhibition of Axl receptor tyrosine kinase ameliorates anti-GBM-induced lupus-like nephritis.

Glomerulonephritis (GN) is a typical lesion in autoantibody and immune complex disorders, including SLE. Because the Gas6/Axl pathway has been implicated in the pathogenesis of many types of GN, targeting this pathway might ameliorate GN. Consequently, we have studied the efficacy and mechanism of R428, a potent selective Axl inhibitor, in the prevention of experimental anti-GBM nephritis. Axl upregulation was investigated with Sp1/3 siRNA in the SV40-transformed mesangial cells. For Axl inhibition, a daily dose of R428 (125 mg/kg) or vehicle was administered orally. GN was induced with anti-GBM sera. Renal disease development was followed by serial blood urine nitrogen (BUN) determinations and by evaluation of kidney histology at the time of sacrifice. Axl-associated signaling proteins were analyzed by Western blotting and inflammatory cytokine secretion was analyzed by Proteome array. SiRNA data revealed the transcription factor Sp1 to be an important regulator of mesangial Axl expression. Anti-GBM serum induced severe nephritis with azotemia, protein casts and necrotic cell death. R428 treatment diminished renal Axl expression and improved kidney function, with significantly decreased BUN and glomerular proliferation. R428 treatment inhibited Axl and significantly decreased Akt phosphorylation and renal inflammatory cytokine and chemokine expression; similar effects were observed in anti-GBM antiserum-treated Axl-KO mice. These studies support a role for Axl inhibition in glomerulonephritis.

Sinomenine relieves oxygen and glucose deprivation-induced microglial activation via inhibition of the SP1/miRNA-183-5p/IκB-α signaling pathway.

Studies have shown that the inflammatory activation of miroglia (MG) and nuclear factor kappa B ( NF-κB ) play a dominant role in inflammatory response. Previous studies have shown that sinomenine, an anti-inflammatory agent extracted from Sinomenium acutum, can directly protect neurons against cerebral ischemia injury. However, there are no reports on its effect on ischemia/reperfusion-induced inflammatory activation of MG. In the present study, an in vitro ischemia/reperfusion model was developed with mouse BV-2 microglia cells, a model of oxygen-glucose deprivation/reperfusion (OGD/R), and the inhibitory effect of sinomenine pretreatment on inflammatory activation was confirmed through measurement of inflammatory indicators. Mechanistically, sinomenine suppressed OGD/R-induced inflammatory activation through the SP1/miRNA-183-5p/IκB-α pathway. In conclusion, this study shows that sinomenine effectively inhibits OGD/R-induced inflammatory activation in MG by suppressing the activation of transcription specificity protein 1 (SP 1). This finding is of significance for the clinical use of sinomenine in treating cerebral ischemia/reperfusion injury.

LPS depletes PHLPP levels in macrophages through the inhibition of SP1 dependent transcriptional regulation.

We have previously reported that bacterial endotoxin LPS attenuates expression of PHLPP, a ser/thr phosphatase, at both transcript and protein levels in different immune cells, however the underlying molecular mechanism is unknown and is of significant interest. Here, in line with the decreased transcript levels upon LPS treatment, we observed that LPS caused significant reduction in PHLPP promoter activity. We observed that SP1, a transcription factor frequently associated with inflammation, was recruited to the PHLPP promoter region. Ectopic expression of SP1 enhanced both transcript and protein levels of PHLPP while knockdown of SP1 or pharmacological inhibition of SP1 DNA binding by mithramycin reduced PHLPP expression. Moreover, over-expression of SP1 co-activators CBP/p300 augmented SP1 driven PHLPP promoter activity. Of note, LPS treatment depleted SP1 and CBP protein levels due to which recruitment of SP1 to PHLPP promoter was reduced. Further, we found that re-introduction of SP1 restored promoter activity and transcript levels of PHLPP in LPS stimulated cells. Collectively, our data revealed the molecular mechanism underlying the regulation of PHLPP expression during LPS induced macrophage inflammatory response.

A stimulation-dependent alternate core promoter links lymphotoxin α expression with TGF-ß1 and fibroblast growth factor-7 signaling in primary human T cells.

Lymphotoxin (LT)-α regulates many biologic activities, yet little is known of the regulation of its gene. In this study, the contribution to LTA transcriptional regulation of the region between the transcription and translation start sites (downstream segment) was investigated. The LTA downstream segment was found to be required for, and alone to be sufficient for, maximal transcriptional activity in both T and B lymphocytes. The latter observation suggested that an alternate core promoter might be present in the downstream segment. Characterization of LTA mRNAs isolated from primary and from transformed human T cells under different stimulation conditions identified eight unique transcript variants (TVs), including one (LTA TV8) that initiated within a polypyrimidine tract near the 3' end of the downstream segment. Further investigation determined that the LTA downstream segment alternate core promoter that produces the LTA TV8 transcript most likely consists of a stimulating protein 1 binding site and an initiator element and that factors involved in transcription initiation (stimulating protein 1, TFII-I, and RNA polymerase II) bind to this LTA region in vivo. Interestingly, the LTA downstream segment alternate core promoter was active only after specific cellular stimulation and was the major promoter used when human T cells were stimulated with TGF-ß1 and fibroblast growth factor-7. Most importantly, this study provides evidence of a direct link for crosstalk between T cells and epithelial/stromal cells that has implications for LT signaling by T cells in the cooperative regulation of various processes typically associated with TGF-ßR and fibroblast growth factor-R2 signaling.

Nuclear factor κB2 p52 protein has a role in antiviral immunity through IκB kinase epsilon-dependent induction of Sp1 protein and interleukin 15.

In this study we describe a previously unreported function for NFκB2, an NFκB family transcription factor, in antiviral immunity. NFκB2 is induced in response to poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of IκB kinase ε (IKKε) and the transactivating potential of RelA/p65. We identify a novel NFκB2 binding site in the promoter of the transcription factor Sp1 that is required for Sp1 gene transcription activated by poly(I:C). We show that Sp1 is required for IL-15 induction by both poly(I:C) and respiratory syncytial virus, a response that also requires NFκB2 and IKKε. Our study identifies NFκB2 as a target for IKKε in antiviral immunity and describes, for the first time, a role for NFκB2 in the regulation of gene expression in response to viral infection.

The intracellular DNA sensor IFI16 gene acts as restriction factor for human cytomegalovirus replication.

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between -54 and -43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor.

Nifetepimine, a dihydropyrimidone, ensures CD4+ T cell survival in a tumor microenvironment by maneuvering sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA).

Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) have long been known to regulate intracellular Ca(2+) homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. In this context it remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Here, we show that human CD4(+) T cells in the presence of tumor conditions manifested an up-regulation of SERCA3 expression that resulted in development of endoplasmic reticulum stress leading to CD4(+) T cell apoptosis. Prostaglandin E(2) produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that an increase in SERCA expression also resulted in subsequent inhibition of PKCα and -θ and retention of NFκB in the cytosol; however, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine), protected the CD4(+) T cells from tumor-induced apoptosis. In fact, nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB, thereby ensuring its survival. Studies further undertaken in a tumor-bearing mice model revalidated the immunoprotective role of nifetepimine. Our present study thus strongly suggests that imbalance in cellular calcium homeostasis is an important factor leading to CD4(+) T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.

17ß-Estradiol suppresses MHC class I chain-related B gene expression via an intact GC box.

Major histocompatibility complex class I chain-related B (MICB) is a membrane-bound glycoprotein involved in both innate and adaptive immunity through its interaction with NKG2D receptors present on γδ T, αß CD8(+) T, and natural killer cells. Factors known to upregulate MICB expression include heat shock, viral or bacterial infection, and tumorigenesis, and here, we explored the effect of 17ß-estradiol (E2) on MICB regulation. Physiological concentrations of E2 were found to suppress MICB mRNA and surface protein levels and this effect was antagonized by the antiestrogen ICI 182780. The inhibitory effect of E2 was also observed for other NKG2D ligands, MICA and ULBPs. Evaluation of promoter fragments from the common MICB*00502 allele revealed that inhibition of transcription by E2 required the GC box at -87. The electrophoretic mobility shift assay and supershift analysis established the presence of SP1, SP3, or estrogen receptor α recognition sites within the MICB promoter sequence and interaction of these factors in situ was confirmed by chromatin immunoprecipitation. We conclude that E2 upon forming a complex with its cognate receptor suppresses MICB expression through binding with SP1/SP3 sites within the MICB promoter GC box. These results suggest that the partial benefit of 17ß-estradiol on autoimmune diseases may be mediated by reducing the immune NKG2D ligands like MICB.

Sp1 induced gene TIMP1 is related to immune cell infiltration in glioblastoma.

Tumor immune microenvironment exerts a profound effect on the population of infiltrating immune cells. Tissue inhibitor of matrix metalloproteinase 1 (TIMP1) is frequently overexpressed in a variety of cells, particularly during inflammation and tissue injury. However, its function in cancer and immunity remains enigmatic. In this study, we find that TIMP1 is substantially up-regulated during tumorigenesis through analyzing cancer bioinformatics databases, which is further confirmed by IHC tissue microarrays of clinical samples. The TIMP1 level is significantly increased in lymphocytes infiltrating the tumors and correlated with cancer progression, particularly in GBM. Notably, we find that the transcriptional factor Sp1 binds to the promoter of TIMP1 and triggers its expression in GBM. Together, our findings suggest that the Sp1-TIMP1 axis can be a potent biomarker for evaluating immune cell infiltration at the tumor sites and therefore, the malignant progression of GBM.

Identification of a novel cell type-specific intronic enhancer of macrophage migration inhibitory factor (MIF) and its regulation by mithramycin.

The aim of this study was to determine the genetic regulation of macrophage migration inhibitory factor (MIF). DNase I hypersensitivity was used to identify potential hypersensitive sites (HS) across the MIF gene locus. Reporter gene assays were performed in different human cell lines with constructs containing the native or mutated HS element. Following phylogenetic and transcription factor binding profiling, electrophoretic mobility shift assay (EMSA) and RNA interference were performed and the effects of incubation with mithramycin, an antibiotic that binds GC boxes, were also studied. An HS centred on the first intron of MIF was identified. The HS acted as an enhancer in human T lymphoblasts (CEMC7A), human embryonic kidney cells (HEK293T) and human monocytic cells (THP-1), but not in a fibroblast-like synoviocyte (FLS) cell line (SW982) or cultured FLS derived from rheumatoid arthritis (RA) patients. Two cis-elements within the first intron were found to be responsible for the enhancer activity. Mutation of the consensus Sp1 GC box on each cis-element abrogated enhancer activity and EMSA indicated Sp1 binding to one of the cis-elements contained in the intron. SiRNA knock-down of Sp1 alone or Sp1 and Sp3 together was incomplete and did not alter the enhancer activity. Mithramycin inhibited expression of MIF in CEMC7A cells. This effect was specific to the intronic enhancer and was not seen on the MIF promoter. These results identify a novel, cell type-specific enhancer of MIF. The enhancer appears to be driven by Sp1 or related Sp family members and is highly sensitive to inhibition via mithramycin.

The expression of heparin-binding epidermal growth factor-like growth factor by regulatory macrophages.

We previously described a population of regulatory macrophages that produced high levels of IL-10 and low levels of IL-12/23. We now describe and characterize the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) by these macrophages. HB-EGF has previously been associated with a number of physiological and pathological conditions, including tumor growth and angiogenesis. The induction of HB-EGF in regulatory macrophages is due to new transcription and not to increased mRNA stability. The transcription factor Sp1 is a major factor in HB-EGF production, and knockdown of Sp1 substantially diminishes HB-EGF production. Sp1 was recruited to three sites within the first 2 kb of the HB-EGF promoter following stimulation, and the site located at -83/-54 was required for HB-EGF promoter activity. These regions of the promoter become more accessible to endonuclease activity following macrophage activation, and this accessibility was contingent on activation of the MAPK, ERK. We show that several experimental manipulations that give rise to regulatory macrophages also result in HB-EGF production. These observations indicate that in addition to the secretion of the anti-inflammatory cytokine IL-10, another novel characteristic of regulatory macrophages is the production of angiogenic HB-EGF.

Angiotensin II-inducible platelet-derived growth factor-D transcription requires specific Ser/Thr residues in the second zinc finger region of Sp1.

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein-DNA interactions, protein-protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-zeta. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-zeta and have generated novel anti-peptide antibodies recognizing the PKC-zeta-phosphorylated form of Sp1. Angiotensin II, which activates PKC-zeta phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-zeta-phospho-Sp1 axis and angiotensin II-inducible gene expression.

The Sp1-Responsive microRNA-15b Negatively Regulates Rhabdovirus-Triggered Innate Immune Responses in Lower Vertebrates by Targeting TBK1.

As is known to all, the production of type I interferon (IFN) plays pivotal roles in host innate antiviral immunity, and its moderate production play a positive role in promoting the activation of host innate antiviral immune response. However, the virus will establish a persistent infection model by interfering with the production of IFN, thereby evading the organism inherent antiviral immune response. Therefore, it is of great necessity to research the underlying regulatory mechanisms of type I IFN appropriate production under viral invasion. In this study, we report that a Sp1-responsive miR-15b plays a negative role in siniperca chuatsi rhabdovirus (SCRV)-triggered antiviral response in teleost fish. We found that SCRV could dramatically upregulate miiuy croaker miR-15b expression. Enhanced miR-15b could negatively regulate SCRV-triggered antiviral genes and inflammatory cytokines production by targeting TANK-binding kinase 1 (TBK1), thereby accelerating viral replication. Importantly, we found that miR-15b feedback regulates antiviral innate immune response through NF-κB and IRF3 signaling pathways. These findings highlight that miR-15b plays a crucial role in regulating virus-host interactions, which outlines a new regulation mechanism of fish's innate immune responses.

Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis.

Natural killer (NK) cells are important effectors in the immune response to tumors. A number of cell-surface inhibitory and activating receptors on NK cells tightly regulate their interaction with target cell ligands. In particular, the strength of an anti-tumor immune response appears to depend critically on surface levels of one activating receptor, NKG2D. Correspondingly, expression of NKG2D ligands on target cells is a requirement for effective tumor immunosurveillance and the elimination of pathogen-infected cells. Sodium butyrate, a potent repressor of histone deacetylase (HDAC), has recently been proposed as a potential agent in cancer treatment based on its ability to modify, in several cancer cell types, the expression of a variety of genes related to cell cycle regulation and apoptosis. Here we report that, in the HeLa and HepG2 tumor cell lines, sodium butyrate upregulated the expression of the MHC class I-related chain molecules A and B (MICA and MICB) at both the mRNA and protein levels, resulting in an enhanced susceptibility of cells in both lines to NK lysis. It also led to an elevated expression of heat shock protein 70 (HSP70) and transcription factor Sp1, and increased the binding of transcription factors Sp1 and heat shock transcription factor 1 (HSF1) to the MICA/B promoter, resulting in increased expression of MICA and MICB. siRNA targeting Sp1 significantly attenuate the enhancement of MICA expression by sodium butyrate. These results suggest that sodium butyrate and other HDAC inhibitors may have therapeutic potential by enhancing the immune response to cancer.

Inhibition of Rac1 GTPase downregulates vascular endothelial growth factor receptor-2 expression by suppressing Sp1-dependent DNA binding in human endothelial cells.

RhoA, Rac1 and CDC42 are small GTP-binding proteins of the Rho family that play a crucial role in regulation of the actin-based cytoskeleton. In addition to cell growth regulation, they are implicated in transcriptional activation, oncogenic transformation and angiogenesis. The small Rho-GTPases have been linked to vascular endothelial growth factor (VEGF)-induced signalling pathways, but their role has not yet been elucidated. As signalling via the VEGF receptor-2 (VEGFR2) pathway is critical for angiogenic responses in cancer, wound repair and ischaemic and inflammatory diseases, we investigated whether the small Rho-GTPase Rac1 influences VEGFR2 expression in human endothelial cells. In this study, we show that a dominant negative Rac1 expression vector led to a pronounced decrease in VEGFR2 mRNA and protein expression. To identify minimal promoter requirements and potential applications of the small Rho-GTPases, we used VEGFR2 promoter-reporter gene constructs containing various deletions. The inhibitory effects of dominant negative Rac1 on the transcriptional activity of the VEGFR2 promoter localized to an element between -77 and -60 that contains an Sp1 transcription factor binding site. Electrophoretic mobility shift assays demonstrated that constitutive Sp1-dependent DNA binding decreased with Rac1 inhibition. Hence, repression of the small Rho GTPase Rac1 seems to be an additional critical molecular mechanism in the regulation of VEGFR2 expression.

The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction.

Mutations of DOCK8 in humans cause a combined immunodeficiency characterized by atopic dermatitis with high serum IgE levels. However, the molecular link between DOCK8 deficiency and atopic skin inflammation is unknown. Here we show that CD4+ T cells from DOCK8-deficient mice produce large amounts of IL-31, a major pruritogen associated with atopic dermatitis. IL-31 induction critically depends on the transcription factor EPAS1, and its conditional deletion in CD4+ T cells abrogates skin disease development in DOCK8-deficient mice. Although EPAS1 is known to form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) and control hypoxic responses, EPAS1-mediated Il31 promoter activation is independent of ARNT, but in collaboration with SP1. On the other hand, we find that DOCK8 is an adaptor and negative regulator of nuclear translocation of EPAS1. Thus, EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction in CD4+ T cells.

Sp1 downregulates proinflammatory cytokine­induced catabolic gene expression in nucleus pulposus cells.

During the pathogenesis of intervertebral disc degeneration, pro­inflammatory cytokines, including tumor necrosis factor­α (TNF­α), stimulate the degradation of the extracellular matrix (ECM) of intervertebral discs via the activity of catabolic enzymes including matrix metalloproteinases (MMPs), disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs), and cyclooxygenase 2 (Cox2). The transcriptional promoters of the human catabolic enzymes MMPs, ADAMTS, Cox2 and Syndecan 4 contain at least one specificity protein­1 (Sp1) transcription factor­binding site. The present study investigated the role of Sp1 in the regulation of the mRNA and protein expression of the aforementioned catabolic enzyme genes in nucleus pulposus cells, using reverse transcription­quantitative polymerase chain reaction, western blot, transfection and RNA interference. The data demonstrated that Sp1 transcription factor protein expression is induced by TNF­α and interleukin­1ß. Specific inhibitors of Sp1 DNA binding to its GC­rich consensus site, WP631 and mithramycin A, partially suppressed TNF­α­induced catabolic enzyme expression and activity. Genetic inhibition of Sp1 by small interfering RNA­mediated Sp1 knockdown partially inhibited catabolic enzyme induction by TNF­α. In addition, Sp1 transcription factor inhibitors decreased the activity of MMP3, ADAMTS4 and ADAMTS5 promoters. Furthermore, chromatin immunoprecipitation revealed functional Sp1 binding sites at ­577/­567 bp within the ADAMTS4 promoter and ­718/­708 bp within the ADAMTS5 promoter. These results provide pharmacological and genetic evidence of the importance of Sp1 in catabolic enzyme gene regulation during TNF­α stimulation. Thus, Sp1 may represent an effective target in reducing intervertebral disc­associated ECM loss.

Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression.

Stimulation of NIH3T3 cells with platelet-derived growth factor (PDGF)-BB enhances expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a key mediator of tumor angiogenesis. Here, we identified cis-acting VEGF promoter elements and trans-acting factors which are involved in PDGF-stimulated VEGF expression. By 5'-deletion and transient transfection analysis, a G + C-rich region at -85 to -50 of the human VEGF promoter was shown to be necessary and sufficient for both PDGF inducible and basal expression. The region contains three potential recognition sites for Sp1 transcription factors, which overlap with two Egr-1 sites. Mutations that abolish the ability of Sp1 to interact with the VEGF promoter element also abrogate expression induced by PDGF. Mutations of the potential Egr-1 binding sites did not affect PDGF responsiveness. Gel shift and antibody supershift analyses showed that Sp1 and Sp3 interact constitutively with the VEGF promoter element. Our data strongly suggest that enhanced VEGF gene expression in PDGF-induced NIH3T3 cells is mediated by Sp1 and/or Sp3 transcription factors bound to the -85 to -50 promoter region of the VEGF gene.

Sp1 as G1 cell cycle phase specific transcription factor in epithelial cells.

Sp1 binding sites have been identified in enhancer/promoter regions of several growth and cell cycle regulated genes, and it has been shown that Sp1 is increasingly phosphorylated in G1 phase of the cell cycle. Interactions of Sp1 with proteins involved in control of cell cycle and tumor formation have been reported. Here we show that expression of Sp1 protein predominates in the G1 phase of the cell cycle in epithelial cells. This is achieved by proteasome-dependent degradation. Inhibition of endogeneous Sp1 activity by a dominant-negative Sp1 mutant was associated with a cell cycle arrest in G1 phase, a strongly reduced expression of cyclin D1, the EGF-receptor and increased levels of p27Kip1. We have thus identified Sp1 as an important regulator of the cell cycle in G1 phase.