Sp1 Transcription Factor Activity Research Articles

Susceptibility of DNA damage recognition activities linked to nucleotide excision and mismatch repair in zebrafish (Danio rerio) early and mid-early embryos to 2.5 to 4.5°C heat stress.

Fish at early life stages are sensitive to temperature change because of their narrower temperature tolerance ranges. Initiated by damage detection, DNA mismatch repair (MMR) and nucleotide excision repair (NER) maintain genome integrity respectively by eliminating mismatched nucleotides and helix-distorting DNA lesions. As discharge of heated effluent from power plants may elevate water temperatures to only 2 to 6°C higher than ambient, this study explored if temperatures within this range affected MMR and NER-linked damage detection activities in fish embryos using zebrafish (Danio rerio) embryo as a model organism. Exposure of early embryos at 10h post fertilization (hpf) to a warmer temperature at + 4.5°C for 30min enhanced damage recognition activities targeting UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) that distorted helical structures. Conversely, photolesions sensing activities were inhibited in 24 hpf mid-early embryos under the same stress conditions. A much higher temperature at + 8.5°C imposed similar effects on UV damage detection. A mild heat stress at + 2.5°C for 30min, however, repressed both CPD and 6-4PP binding activities in 10 and 24 hpf embryos. Inhibition of damage recognition under mild heat stress impeded the overall NER capacity evidenced by a transcription-based repair assay. Warmer water temperatures at + 2.5 and + 4.5°C also inhibited G-T mismatch binding activities in 10 and 24 hpf embryos, but G-T recognition was more sensitive to + 4.5°C stress. Inhibition of G-T binding partially correlated with a downregulation of Sp1 transcription factor activity. Our results showed the potential of water temperature elevation within 2 to 4.5°C to disturb DNA damage repair in fish at embryonic stages.

Abstract 2847: Human CYP1B1 induces cancer cell metastasis through Sp1-mediated activation of uPA-uPAR signaling pathway

Abstract Human CYP1B1 is known as a major metabolizer for estrogen and shows tumor-specific hyper-expression. To explore the role of CYP1B1 on metastasis of human cancer cells, we studied the effects of CYP1B1 inhibition and activation in MCF-10A, MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 significantly induced expression of urokinase-type plasminogen activator receptor (uPAR) as well as uPA. Treatment with DMBA, a well-known CYP1B1 inducer, enhanced uPAR expression while CYP1B1 siRNA and TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, suppressed uPAR. We also found that CYP1B1 activated uPAR signaling through regulation of related factors including integrin β1 and α5. Interestingly, uPAR overexpression only caused a significant increase of integrin β1 and α5 in protein levels, indicating protein degradation may play an important role in regulating integrin protein level. Surprisingly, CYP1B1 down-regulated p53 expression through MDM2 activation and nutlin-3a, an inhibitor for MDM2, blocked the promoting effects of DMBA on uPAR, which explains that CYP1B1 induced uPAR expression through regulation of MDM2-p53 system. In addition, we revealed that CYP1B1 regulates uPAR through activation of Sp1 transcription factor. CYP1B1 and its enzymatic product, 4-OHE2, promote Sp1 expression levels in both mRNA and protein levels and subsequently Sp1 elevates the transcriptional activity of uPAR through binding to promoter region of uPAR. Taken together, our data suggest that CYP1B1 promotes cancer cell metastasis via activating uPAR pathway which is one of the targets of p53 and Sp1 signaling. Citation Format: Yeo-Jung Kwon, Sangyun Shin, Young-Jin Chun. Human CYP1B1 induces cancer cell metastasis through Sp1-mediated activation of uPA-uPAR signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2847.

Abstract 4668: Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375

Abstract Human cytochrome P450 1B1 (CYP1B1) is an important enzyme that catalyzes metabolism of 17β-estradiol (E2) to catechol estrogens, including carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). CYP1B1 may play a crucial role in estrogen-mediated carcinogenesis since CYP1B1 shows tumor-specific expression pattern in various hormone-responsive tissues. However, the detailed mechanism of CYP1B1-dependent carcinogenesis remains still unclear. Here, we investigated the association between TRAIL, DR4, and CYP1B1 expression in human tumor cells, including MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 can inhibit the expression and release of TRAIL simultaneously with the expression of the death receptor DR4/5, known as TRAIL receptors, to prevent apoptosis. Furthermore, we found that CYP1B1 represses the transcription of DR4 by inducing DNA methylation in the promoter region of DR4. In addition, we revealed that CYP1B1 regulates TRAIL and DR4 through activation of Sp1 transcription factor. CYP1B1 and its enzymatic product, 4-OHE2, promote Sp1 expression levels in both mRNA and protein levels. Interestingly, CYP1B1 elevated Sp1 through downregulation of miR-375, which is a candidate tumor suppressive microRNA and transcriptional inhibitor of Sp1. Taken together, our data suggest that CYP1B1 inhibits TRAIL-DR4-mediated cancer cell apoptosis through induction of Sp1 through repression of miR-375, which promotes cancer cell survival. Citation Format: Yeo-Jung Kwon, Dong-Jin Ye, Huoung-Seok Baek, Sangyun Shin, Tae-Uk Kwon, Eunah Cho, Hong-Gyu Ahn, Youngjin Chun. Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4668.

Regulatory T cells as a biomarker for response to adalimumab in rheumatoid arthritis

Regulatory T cells as a biomarker for response to adalimumab in rheumatoid arthritis

Downregulation of lipolysis-stimulated lipoprotein receptor promotes cell invasion via claudin-1-mediated matrix metalloproteinases in human endometrial cancer.

Lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecule present at tricellular contacts which recruits tricellulin (TRIC), a molecular component of tricellular tight junctions (tTJs). LSR and TRIC are colocalized with the bicellular tight junction (bTJ) protein claudin (CLDN)-1-based tight junction strands at tricellular corners. Knockdown of LSR in normal epithelial cells affects tTJ formation and the epithelial barrier function. In cancer cells knockdown of LSR has been demonstrated to increase cell invasion. However, the detailed mechanisms of how the downregulation of LSR enhances cell invasion in cancer remain unclear. In the present study, knockdown of LSR by small interfering RNA (siRNA) in Sawano human endometrial adenocarcinoma cells induced cell invasion. In LSR-knockdown Sawano cells, upregulation of CLDN-1 protein, which contributes to the cell invasion via matrix metalloproteinases (MMPs), was observed compared with the control group by western blotting and immunostaining. Knockdown of LSR significantly induced Sp1 transcription factor activity in the CLDN-1 promoter region. In LSR-knockdown Sawano cells, DNA microarray analysis demonstrated that MMP-1, MMP-2 and MMP-10 mRNA levels were increased, and the protein levels of membrane-type 1-MMP, MMP-2, MMP-9 and MMP-10 were shown to be increased on western blots. Knockdown of CLDN-1 with siRNA prevented the upregulation of cell invasion induced by the knockdown of LSR in Sawano cells. On the invasive front of human endometrial carcinoma tissue samples, a decrease in LSR and increase in CLDN-1 protein levels were observed using immunohistochemical methods. In conclusion, the results indicate that the downregulation of LSR promotes cell invasion of human endometrial cancer via CLDN-1 mediation of MMPs. This mechanism is important for studying the association of tTJs with the cellular invasion of cancer.

DD-03 * THE NATURALLY OCCURRING STEROID, WITHAFERIN A, IN SYNERGISTIC CONCERT WITH HER2/EGFR INHIBITORS ABROGATES PROLIFERATION OF HUMAN GLIOBLASTOMA CELL CULTURES AT NANOMOLAR CONCENTRATIONS

INTRODUCTION: Withaferin A, a steroidal lactone that is isolated from the winter cherry (Withania sominifera), confers abrogatory effects on proliferating cancer cells. This is achieved, partly though Withaferin A's ability to retard angiogenesis along with VEGF and calcium signaling. At the level of transcription, Withaferin A inhibits both Nfkb and Sp1 Transcription factor activity. In glioblastomas, Withaferin A promotes cytotoxicity by impinging on Akt/mTOR-associated transduction pathways. In this report, we probe the relationship of Withaferin A with Her2- and EGFR-directed signaling (through the use of receptor-targeted inhibitors such as Afatinib and Lapatinib) in human glioblastomas. METHODS: Human parietal-cortical glioblastoma cells (pcGBM2, GBM39, LN229) were derived from primary tumors. Genomic material (both RNA and DNA) was isolated via standardized methods so as to study the mutational and expression status of relevant gene targets (PTEN, p53, MGMT, EGFRvIII). Proliferative potential of these cells was assessed by the alamar blue bioassay. RESULTS: The human glioblastomas studied displayed both wild-type and mutant p53. Both MGMT and PTEN expressions were usually suppressed. Expression of EGFRvIII was highly variegated with both wild type and vIII receptors being present. GBM39 expressed EGFRvIII levels 20-fold higher than pcGBM2. Growth of GBM39 and pcGBM2 were inhibited by Withaferin A with an IC50 of 0.3 + /-0.05 µM. The EGFR inhibitors, Afatinib and Lapatinib, abrogated glioblastoma growth at IC50s of 4.5/-1.0 uM and 18.0 + /-2.0 uM respectively. Inclusion of 0.15 µM, 0.3 µM and 0.6 µM of Withaferin A on top of Afatinib or Lapatinib resulted in dose-dependent, 2 to 4-fold sensitization of glioblastomas to the Her2/EGFR inhibitors. Addition of the Akt inhibitor, BKM120 to Afatinib and Withaferin A did not add further to the sensitization. CONCLUSIONS: The results suggest that Withaferin A exerts an inhibitory effect on glioblastoma proliferation and that it provides a synergistic effect on inhibition when co-administered with Afatinib or Lapatinib.

Induction of IL-6 gene expression in a CF bronchial epithelial cell line by Pseudomonas aeruginosa is dependent on transcription factors belonging to the Sp1 superfamily

Cystic fibrosis (CF) is a genetic disease characterized by chronic bacterial lung infection, most commonly sustained by Pseudomonas aeruginosa. Upon infection, elevated concentrations of pro-inflammatory cytokines (i.e. IL-6 and IL1β) and chemokines (i.e. IL-8 and GROγ) are found in the bronchoalveolar fluid of CF patients. We report in this paper that: (a) IL-8, IL-6, IL-1β, ICAM-1, and GRO-γ genes are upregulated following infection of CF bronchial epithelial IB3-1 cells with P. aeruginosa; (b) Sp1 transcription factor activity is induced following infection of the cystic fibrosis IB3-1 and CuFi-1 cell lines; (c) inhibition of Sp1 activity using transcription factor decoy molecules leads to inhibition of the expression of IL-6 gene. From the theoretical point of view, our results demonstrate that Sp1 transcription factor activity is induced following infection of CF cells with P. aeruginosa, and that this effect is important in the activation of IL-6 gene transcription. From the practical point of view, our data sustain the potential use of decoy molecules targeting the transcription factor Sp1 to control a relevant molecule involved in the inflammatory process associated with the cystic fibrosis airway pathology.

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

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

The p38 Mitogen-activated Kinase Pathway Regulates the Human Interleukin-10 Promoter via the Activation of Sp1 Transcription Factor in Lipopolysaccharide-stimulated Human Macrophages

Interleukin-10 (IL-10), a pleiotropic cytokine that inhibits inflammatory and cell-mediated immune responses, is produced by a wide variety of cell types including T and B cells and monocytes/macrophages. Regulation of pro- and anti-inflammatory cytokines has been suggested to involve distinct signaling pathways. In this study, we investigated the regulation of the human IL-10 (hIL-10) promoter in the human monocytic cell line THP-1 following activation with lipopolysaccharide (LPS). Analysis of hIL-10 promoter sequences revealed that DNA sequences located between base pairs -652 and -571 are necessary for IL-10 transcription. A computer analysis of the promoter sequence between base pairs -652 and -571 revealed the existence of consensus sequences for Sp1, PEA1, YY1, and Epstein-Barr virus-specific nuclear antigen-2 (EBNA-2)-like transcription factors. THP-1 cells transfected with a plasmid containing mutant Sp1 abrogated the promoter activity, whereas plasmids containing the sequences for PEA1, YY1, and EBNA-2-like transcription factors did not influence hIL-10 promoter activity. To understand the events upstream of Sp1 activation, we investigated the role of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases by using their specific inhibitors. SB202190 and SB203580, the p38-specific inhibitors, inhibited LPS-induced IL-10 production. In contrast, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinases, failed to modulate IL-10 production. Furthermore, SB203580 inhibited LPS-induced activation of Sp1, as well as the promoter activity in cells transfected with a plasmid containing the Sp1 consensus sequence. These results suggest that p38 mitogen-activated protein kinase regulates LPS-induced activation of Sp1, which in turn regulates transcription of the hIL-10 gene.

IL-4-induced Oxidative Stress Upregulates VCAM-1 Gene Expression in Human Endothelial Cells

Vascular cell adhesion molecule-1 (VCAM-1) is expressed in early stages of atherosclerosis; however, the mechanisms of its upregulation are not fully understood. In the present study, we examined the effects of interleukin-4 (IL-4) on VCAM-1 gene expression and its transcriptional regulatory mechanism in human umbilical vein endothelial cells (HUVEC). Reverse transcription-polymerase chain reaction showed that VCAM-1 mRNA was induced in IL-4-treated HUVEC in a time- and dose-dependent manner. Among known transcription factors that have binding sites in the promoter region of the VCAM-1 gene, IL-4 activated only SP-1. In contrast, nuclear factor- κ B (NF- κ B), activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), which also have consensus binding sequences in the 5-flanking region of the human VCAM-1 gene, were not activated. The role of SP-1 in IL-4-induced VCAM-1 expression was confirmed in HUVEC transfected with a reporter construct of the VCAM-1 promoter with mutated SP-1 binding site. As IL-4 treatment of HUVEC enhanced the intracellular oxidizing potential, as indicated by an increase in 2,7-dichlorofluorescein (DCF) fluorescence, we studied the effect of antioxidants on IL-4-induced VCAM-1 expression. Pretreatment of HUVEC with pyrrolidine dithiocarbamate (PDTC) or N-acetylcysteine (NAC) completely prevented IL-4-induced VCAM-1 expression. In addition, PDTC inhibited IL-4-related activation of SP-1. These results suggest that IL-4-induced oxidative stress upregulates the expression of VCAM-1 gene in HUVEC at transcriptional levels via activation of SP-1 transcription factor. In contrast, NF- κ B, AP-1 or IRF-1 do not appear to be involved in the signal transduction cascade.