scholarly journals Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein α (C/EBPα) DNA binding

2000 ◽  
Vol 350 (3) ◽  
pp. 791-796 ◽  
Author(s):  
Sivaprakasam BALASUBRAMANIAN ◽  
Chapla AGARWAL ◽  
Tatiana EFIMOVA ◽  
George R. DUBYAK ◽  
Eric BANKS ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Binding Protein ◽  
Regulatory Region ◽  
Keratinocyte Differentiation ◽  
Calcium Stores ◽  
Mrna Levels ◽  
Promoter Activation ◽  
Protein Levels ◽  
Enhancer Binding Protein ◽  
Differentiation Marker

Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10nM and maximal at 100nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPα-dependent promoter activation via a mechanism that involves inhibition of C/EBPα binding to DNA without changing C/EBPα protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.

scholarly journals Epidermal Growth Factor-Induced GnRH-II Synthesis Contributes to Ovarian Cancer Cell Invasion

2009 ◽  
Vol 94 (9) ◽  
pp. 3618-3618
Author(s):  
Song Ling Poon ◽  
Gareth T. Hammond ◽  
Peter C. K. Leung
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Promoter Activity ◽  
Binding Protein ◽  
Ovarian Cancer Cells ◽  
Mrna Levels ◽  
Upstream Regulator ◽  
Enhancer Binding Protein ◽  
Responsive Element ◽  
Interfering Rna

GnRH-II modulates ovarian cancer cells invasion and is expressed in normal ovary and ovarian epithelial cancer cells; however, the upstream regulator(s) of GnRH-II expression in these cells remains unclear. We now demonstrate that epidermal growth factor (EGF) increases GnRH-II mRNA levels in several human ovarian carcinoma cell lines and up-regulates GnRH-II promoter activity in OVCAR-3 cells in a dose-dependent manner, whereas an EGF receptor inhibitor (AG148) abolishes EGF-induced increases in GnRH-II promoter activity and GnRH-II mRNA levels. EGF increases the phosphorylation of cAMP-responsive element-binding protein (p-CREB) and its association with the coregulator, CCAAT/enhancer binding protein β, whereas blocking the EGF-induced ERK1/2 phosphorylation with MAPK inhibitors (PD98059/U0126) markedly reduced these effects. Moreover, depletion of CREB using small interfering RNA attenuated EGF-induced GnRH-II promoter activity. Chromatin immunoprecipitation assays demonstrated that EGF induces p-CREB binding to a cAMP responsive-element within the GnRH-II promoter, likely in association with CCAAT/enhancer binding protein β, and mutagenesis of this cAMP responsive-element prevented EGF-induced GnRH-II promoter activity in OVCAR-3 cells. Importantly, GnRH-II acts additively with EGF to promote invasion of OVCAR-3 and CaOV-3 cells, but not SKOV-3 cells that express low levels of GnRH receptor (GnRHR). Treatment with GnRHR small interfering RNA also partially inhibited the EGF-induced invasion of OVCAR-3 and CaOV-3 cells. Furthermore, EGF treatment transiently increases GnRHR levels in OVCAR-3 and CaOV-3, which likely accentuates the effects of increase GnRH-II production on cell invasion. These results provide evidence that EGF is an upstream regulator of the autocrine actions of GnRH-II on the invasive properties of ovarian cancer cells.

Exchange protein directly activated by cAMP (EPAC) promotes transcriptional activation of the decidual prolactin gene via CCAAT/enhancer-binding protein in human endometrial stromal cells

2018 ◽  
Vol 30 (11) ◽  
pp. 1454
Author(s):  
Kazuya Kusama ◽  
Kazuhiro Tamura ◽  
Hanako Bai ◽  
Toshihiro Sakurai ◽  
Hirotaka Nishi ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Binding Protein ◽  
Luciferase Assay ◽  
Mrna Levels ◽  
Reporter Activity ◽  
Camp Analogue ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Decidual Prolactin ◽  
Exchange Protein

Protein kinase A (PKA) signalling accompanies elevated intracellular cAMP levels during endometrial stromal cell (ESC) decidualisation. Exchange protein directly activated by cAMP (EPAC), an alternate mediator of cAMP signalling, promotes PKA analogue-induced decidualisation; however, the precise mechanism by which EPAC and PKA co-operatively stimulate decidualisation has not been characterised. To examine the role of CCAAT/enhancer-binding protein (C/EBP) in EPAC- and PKA-mediated decidualisation of primary human ESCs, a reporter plasmid containing the 332 bp region upstream from the transcription initiation site of the decidual prolactin (dPRL) gene was generated and the promoter activity was evaluated using a luciferase assay. The dPRL promoter activity was increased by treatment of transfected ESCs with the PKA-selective cAMP analogue N6-phenyl-cAMP (Phe) and enhanced further by co-treatment with the EPAC-selective cAMP analogue 8-(4-chlorophenyltio)-2′-O-methyl cAMP (CPT). Treatment with forskolin, an adenylyl cyclase activator, had a similar effect on reporter activity. Site-directed mutagenesis of the C/EBPβ- and/or C/EBPδ-binding site in the dPRL promoter abolished Phe/CPT-mediated elevation of the reporter activity. EPAC2 knockdown markedly reduced Phe-stimulated C/EBPβ and C/EBPδ mRNA levels, as well as forkhead box O1 (FOXO1) protein levels. These results suggest that EPAC signalling enhances PKA-mediated dPRL expression in ESCs by acting on C/EBP response elements in the promoter region of the gene.

scholarly journals Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

2018 ◽  
Vol 314 (1) ◽  
pp. G14-G21 ◽  
Author(s):  
Saminathan Muthusamy ◽  
Jong Jin Jeong ◽  
Ming Cheng ◽  
Jessica A. Bonzo ◽  
Anoop Kumar ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Core Promoter ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Protein Levels ◽  
Hepatocyte Nuclear Factor 4Α

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

Overexpression of CCAAT Enhancer-Binding Protein α Inhibits the Growth of K562 Cells via the Foxo3a-Bim Pathway

2016 ◽  
Vol 136 (2) ◽  
pp. 65-70 ◽  
Author(s):  
Guili Zhang ◽  
Fei Dong ◽  
Caifu Luan ◽  
Xia Zhang ◽  
Huiyuan Shao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Binding Protein ◽  
K562 Cells ◽  
Cell Counting ◽  
Stable Cell Line ◽  
Mrna Levels ◽  
Forkhead Transcription Factor ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Blood Specimens

We aimed to investigate the role of CCAAT enhancer-binding protein α (C/EBPα) in the pathogenesis of chronic myeloid leukemia (CML) and the mechanism underlying its effect. Bone marrow specimens from 50 patients with CML and peripheral blood specimens from 20 healthy individuals were collected. K562 cells were treated with imatinib. Subsequently, a stable cell line, K562-C/EBPα, was constructed. Cell proliferation was assayed with cell counting kit-8, and mRNA levels of C/EBPα, forkhead transcription factor FKHRL1 (Foxo3a) and Bim were detected by semiquantitative PCR. The correlation of C/EBPα and BCR-ABL was assessed by Spearman's correlation analysis. The results showed that C/EBPα mRNA levels were significantly reduced in CML patients compared with healthy subjects (p < 0.001) and were negatively correlated with BCR-ABL1 (r = -0.5046, p < 0.01). Additionally, imatinib enhanced the expression of C/EBPα in K562 cells compared with untreated cells (p < 0.05). Overexpression of C/EBPα significantly decreased cell proliferation and upregulated the expressions of the apoptosis-related genes Foxo3a (p < 0.01) and Bim (p < 0.05) in K562 cells. In conclusion, C/EBPα expression was decreased in patients with CML. Imatinib enhances the expression of C/EBPα in K562 cells, and the overexpression of C/EBPα inhibits cell proliferation and increases apoptosis via the Foxo3a-Bim pathway.

scholarly journals CCAAT/Enhancer-Binding Protein β Mediates Oxygen-Induced Retinal Neovascularization via Retinal Vascular Damage and Vascular Endothelial Growth Factor

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Tingting Li ◽  
Xuan Cai ◽  
Xiangning Wang ◽  
Xueyan Zhang ◽  
Hui Zhang ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Western Blot ◽  
Binding Protein ◽  
Retinal Function ◽  
Vascular Damage ◽  
Vascular Endothelial ◽  
Protein Levels ◽  
Enhancer Binding Protein ◽  
Endothelial Growth Factor

Objective. To evaluate the role of CCAAT/enhancer-binding protein β (C/EBP β) in retinal neovascularization (RNV) in an oxygen-induced retinopathy (OIR) model. Methods. Rats with OIR were exposed to alternating hypoxic and hyperopic conditions for 14 days. Then, the rats with OIR were assigned randomly to groups that received intravitreal injections of either shRNA lentiviral particles targeting C/EBP β (LV.shC/EBP β) or control particles (LV.shScrambled). The effectiveness of transduction using intravitreal injection of C/EBP β shRNA was examined in rats with OIR. The retinal vascular damage and accumulation of RNV were determined by retinal fluorescein-dextran perfusion, retinal ADPase staining, and periodic acid-Schiff (PAS) staining. Retinal function was recorded by electroretinogram responses to full-field light flashes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analyses were used to measure mRNA and protein levels of C/EBP β and vascular endothelial growth factor (VEGF). The expression of p-C/EBP β was also examined by western blot analyses. The location of C/EBP β expression in the retina was determined by immunohistochemistry. Results. In OIR rats, the expression levels of C/EBP β and VEGF were significantly increased at both the mRNA and protein levels (P<0.01). The p-C/EBP β expression was consistent with the level of C/EBP β. C/EBP β was predominantly localized to the ganglion cell layer (GCL) and the inner nuclear layer (INL). The retinal C/EBP β level was significantly reduced in tissues from rats with OIR transduced with LV.shC/EBP β compared with tissues from those transduced with LV.shScrambled (P<0.01). Compared with those of the rats with OIR in the LV.shScrambled group, nonperfused retinal areas, neovascular tufts, pericyte death, and the ratio of endothelial cells to pericytes in the LV.shC/EBP β group were significantly reduced. In OIR rats, retinal function was impaired. There was no significant change in retinal dysfunction between the LV.shC/EBP β group and the LV.shScrambled group. The levels of VEGF mRNA and protein in the LV.shC/EBP β group were also decreased significantly compared with those of the rats with OIR in the LV.shScrambled group (P<0.01). Conclusions. C/EBP β shRNA inhibits RNV in OIR. A potential mechanism may be that the activity of C/EBP β increases with its overexpression, which in turn aggravates the amount of the retinal vascular damage and promotes transcription of VEGF. C/EBP β might be a new therapeutic target for preventing RNV.

scholarly journals Intestinal maturation in mice lacking CCAAT/enhancer-binding protein α (C/EPBα)

1998 ◽  
Vol 330 (3) ◽  
pp. 1165-1171 ◽  
Author(s):  
J. Thomas OESTERREICHER ◽  
L. Lucy LEEPER ◽  
J. Milton FINEGOLD ◽  
J. Gretchen DARLINGTON ◽  
J. Susan HENNING
Keyword(s):  
Glucose Transporter ◽  
Binding Protein ◽  
Fatty Acid Binding Proteins ◽  
Mrna Levels ◽  
Fatty Acid Binding ◽  
Proliferative Zone ◽  
Glucose Transporter 2 ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Morphological Maturation

In rodents, there is a surge of intestinal expression of CCAAT/enhancer-binding protein α (C/EBPα) in the late fetal phase just before morphological maturation and the onset of expression of numerous epithelial genes. To investigate directly the hypothesis that C/EBPα plays a causal role in the latter phenomena, we have assessed both structural and functional maturation in neonatal intestine from C/EBPα-null mice and their littermates. No effects of C/EBPα genotype were observed on mucosal architecture or on the size of the proliferative zone in the intestinal crypts. Likewise, the mRNA levels for the glucose transporter 2 (GLUT2), intestinal and liver fatty acid-binding proteins, and apolipoprotein A-IV in newborn intestine were similar in all genotypes. Paradoxically, Na+/glucose co-transporter (SGLT1), lactase phlorizin-hydrolase and apolipoprotein B mRNAs were more abundant in the C/EBPα-deficient animals. In wild-type intestines, C/EBPβ and C/EBPΔ mRNAs were detectable throughout the late fetal period and increased toward term in parallel with C/EBPα mRNA. In newborn intestine, there was no compensatory up-regulation of these isoforms in the C/EBPα-deficient mice. We conclude that C/EBPα has no essential role in morphological maturation of the intestine, the pattern of proliferation of the epithelium, or the onset of expression of this cluster of epithelial mRNAs. However, since other C/EBP isoforms are present in the developing intestine, it is possible that there is a generic requirement for a member of the C/EBP family.

scholarly journals CCAAT/Enhancer Binding Protein β2 Is Involved in Growth Hormone-Regulated Insulin-Like Growth Factor-II Gene Expression in the Liver of Rainbow Trout (Oncorhynchus mykiss)

Endocrinology ◽  
2010 ◽  
Vol 151 (5) ◽  
pp. 2128-2139 ◽  
Author(s):  
Jay H. Lo ◽  
Thomas T. Chen
Keyword(s):  
Gene Expression ◽  
Rainbow Trout ◽  
Binding Protein ◽  
Regulatory Region ◽  
Negative Regulator ◽  
Proximal Promoter ◽  
Gh Treatment ◽  
Enhancer Binding Protein ◽  
Histone H4 Acetylation ◽  
Ccaat Enhancer Binding Protein

Previously, we showed that levels of different CCAAT/enhancer binding protein (C/EBP) mRNAs in the liver of rainbow trout were modulated by GH and suggested that C/EBPs might be involved in GH-induced IGF-II gene expression. As a step toward further investigation, we have developed monospecific polyclonal antibodies to detect rainbow trout C/EBPα, -β1, -β2, and -δ2 isoform proteins. Injection of GH into adult rainbow trout resulted in a significant increase of C/EBPβ1, C/EBPβ2, and C/EBPδ2 proteins in the liver. Chromatin immunoprecipitation analysis revealed that C/EBPβ2 binds to multiple sites at the 5′ promoter/regulatory region, introns, and the 3′ untranslated region of the IGF-II gene. GH treatment reduced C/EBPβ2 binding to several of these regions at 6 h after injection. The decreased occupancy of C/EBPβ2 coincided well with an increase of histone H4 acetylation at the proximal promoter and elevation of the IGF-II mRNA level. Immunoblotting analysis showed that C/EBPβ2 existed predominately as a truncated form in the liver, and cotransfection analysis further showed that the truncated C/EBPβ2 acted as a negative regulator on IGF-II proximal promoter. GH treatment caused deacetylation of C/EBPβ2 in the liver. In addition, we observed a GH-dependent interaction of C/EBPβ2 with a complex involving histone H1. All together, these results suggest that C/EBPβ2 was regulated at multiple levels by GH, and C/EBPβ2 may play a suppressive role in mediating GH-induced IGF-II expression in the liver of rainbow trout.

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