Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

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CCAAT Displacement Protein (CDP/cut) Recognizes a Silencer Element Within the Lactoferrin Gene Promoter

Blood ◽

10.1182/blood.v90.7.2784 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2784-2795 ◽

Cited By ~ 50

Author(s):

Arati Khanna-Gupta ◽

Theresa Zibello ◽

Sarah Kolla ◽

Ellis J. Neufeld ◽

Nancy Berliner

Keyword(s):

Luciferase Activity ◽

Gene Promoter ◽

Luciferase Reporter ◽

Transcriptional Level ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Ccaat Displacement Protein ◽

Lactoferrin Gene ◽

Mobility Shift Assay ◽

Silencer Element

Abstract Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non–LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing basepair (bp) −387 to bp −726 of the LF promoter, but not in a −916-bp plasmid. Transfection of a −916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the −916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non–LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non–LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor–induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

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An E-box-containing region is involved in the tissue-specific expression of the human MC2R gene

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0320811 ◽

2004 ◽

Vol 32 (3) ◽

pp. 811-823 ◽

Cited By ~ 2

Author(s):

A Blondet ◽

M Doghman ◽

P Durand ◽

M Begeot ◽

D Naville

Keyword(s):

Granulosa Cells ◽

Gene Promoter ◽

Cell Types ◽

Melanocortin Receptor ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Specific Expression ◽

E Box ◽

Mobility Shift Assay

Expression of the melanocortin receptor (MC2R) gene is limited to adrenocortical cells and the aim of this study was to determine the factors responsible for this tissue specificity. We used different fragments of the human (h) MC2R gene promoter, inserted in a vector upstream of the luciferase reporter gene, to transiently transfect either bovine adrenocortical (BAC) cells or granulosa cells from bovine ovaries (B-Gran). Similar promoter activities were obtained in both cell types using constructs containing fragments up to 1017 bp of the hMC2R gene promoter. On the contrary, a 2-fold decrease was obtained after transfection of the B-Gran cells with vectors containing 1069 bp and more of the promoter. Results obtained here using BAC cells confirmed our previous data on human cells showing that steroidogenic factor 1 is the major transactivating factor involved in the basal expression of the hMC2R gene in adrenal cells. However, we showed that this factor did not permit, by itself, the expression of the hMC2R gene in B-Gran cells despite its expression in these cells. This study demonstrated for the first time that an E-box (located at -1020 bp) is involved in the repression of hMC2R gene expression in granulosa cells through interactions with several factors, such as activator protein 4, as suggested by electrophoretic mobility shift assay analyses.

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CCAAT Displacement Protein (CDP/cut) Recognizes a Silencer Element Within the Lactoferrin Gene Promoter

Blood ◽

10.1182/blood.v90.7.2784.2784_2784_2795 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2784-2795 ◽

Cited By ~ 4

Author(s):

Arati Khanna-Gupta ◽

Theresa Zibello ◽

Sarah Kolla ◽

Ellis J. Neufeld ◽

Nancy Berliner

Keyword(s):

Luciferase Activity ◽

Gene Promoter ◽

Luciferase Reporter ◽

Transcriptional Level ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Ccaat Displacement Protein ◽

Lactoferrin Gene ◽

Mobility Shift Assay ◽

Silencer Element

Expression of neutrophil secondary granule protein (SGP) genes is coordinately regulated at the transcriptional level, and is disrupted in specific granule deficiency and leukemia. We analyzed the regulation of SGP gene expression by luciferase reporter gene assays using the lactoferrin (LF) promoter. Reporter plasmids were transiently transfected into non–LF-expressing hematopoietic cell lines. Luciferase activity was detected from reporter plasmids containing basepair (bp) −387 to bp −726 of the LF promoter, but not in a −916-bp plasmid. Transfection of a −916-bp plasmid into a LF-expressing cell line resulted in abrogation of the silencing effect. Sequence analysis of this region revealed three eight-bp repetitive elements, the deletion of which restored wild-type levels of luciferase activity to the −916-bp reporter plasmid. Electrophoretic mobility shift assay and UV cross-linking analysis identified a protein of approximately 180 kD that binds to this region in non–LF-expressing cells but not in LF-expressing cells. This protein was identified to be the CCAAT displacement protein (CDP/cut). CDP/cut has been shown to downregulate expression of gp91-phox, a gene expressed relatively early in the myeloid lineage. Our observations suggest that the binding of CDP/cut to the LF silencer element serves to suppress basal promoter activity of the LF gene in non–LF-expressing cells. Furthermore, overexpression of CDP/cut in cultured myeloid stem cells blocks LF expression upon granulocyte colony-stimulating factor–induced neutrophil maturation without blocking phenotypic maturation. This block in LF expression may be due, in part, to the persistence of CDP/cut binding to the LF silencer element.

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Tumor necrosis factor-α inhibits peroxisome proliferator-activated receptor γ activity at a posttranslational level in hepatic stellate cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00411.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. G722-G729 ◽

Cited By ~ 49

Author(s):

Chin K. Sung ◽

Hongyun She ◽

Shigang Xiong ◽

Hidekazu Tsukamoto

Keyword(s):

Hepatic Stellate Cells ◽

Stellate Cells ◽

Luciferase Reporter ◽

Critical Event ◽

Peroxisome Proliferator ◽

Mrna Levels ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Peroxisome Proliferator Activated Receptor ◽

Tnf Α

Diminished activity of peroxisome proliferator-activated receptor γ (PPARγ) is implicated in activation of hepatic stellate cells (HSC), a critical event in the development of liver fibrosis. In the present study, we investigated PPARγ regulation by TNF-α in an HSC line designated as BSC. In BSC, TNF-α decreased both basal and ligand (GW1929)-induced PPARγ mRNA levels without changing its protein expression. Nuclear extracts from BSC treated with TNF-α showed decreased binding of PPARγ to PPAR-responsive element (PPRE) as determined by electrophoretic mobility shift assay. In BSC transiently transfected with a PPARγ1 expression vector and a PPRE-luciferase reporter gene, TNF-α decreased both basal and GW1929-induced transactivation of the PPRE promoter. TNF-α increased activation of ERK1/2 and JNK, previously implicated in phosphorylation of Ser82 of PPARγ1 and resultant negative regulation of PPARγ transactivity. In fact, TNF-α failed to inhibit transactivity of a Ser82Ala PPARγ1 mutant in BSC. TNF-α-mediated inhibition of PPARγ transactivity was not blocked with a Ser32Ala/Ser36Ala mutant of inhibitory NF-κBα (IκBα). These results suggest that TNF-α inhibits PPARγ transactivity in cultured HSC, at least in part, by diminished PPARγ-PPRE (DNA) binding and ERK1/2-mediated phosphorylation of Ser82 of PPARγ1, but not via the NF-κB pathway.

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GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells

Journal of Molecular Endocrinology ◽

10.1530/jme-13-0089 ◽

2013 ◽

Vol 51 (3) ◽

pp. 313-324 ◽

Cited By ~ 5

Author(s):

Robin L Thomas ◽

Natalie M Crawford ◽

Constance M Grafer ◽

Weiming Zheng ◽

Lisa M Halvorson

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Promoter Activity ◽

Gene Promoter ◽

Proximal Promoter ◽

Mrna Levels ◽

Paracrine Factor ◽

Mobility Shift ◽

Subunit Expression ◽

Mobility Shift Assay

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine–paracrine factor in this tissue. GNRH has been shown to increase pituitaryAdcyap1gene expression through the interaction of CREB and jun/fos with CRE/AP1cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognatecis-elements which regulateAdcyap1gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulateAdcyap1promoter activity via a GATAcis-element located at position −191 in the ratAdcyap1gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation ofAdcyap1gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

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Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

AJP Cell Physiology ◽

10.1152/ajpcell.00444.2001 ◽

2002 ◽

Vol 282 (3) ◽

pp. C518-C527 ◽

Cited By ~ 17

Author(s):

Julia M. Giger ◽

Fadia Haddad ◽

Anqi X. Qin ◽

Kenneth M. Baldwin

Keyword(s):

Gene Promoter ◽

Firefly Luciferase ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Type I ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Slow Type ◽

Responsive Element ◽

Gel Mobility Shift

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.

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Nuciferine Inhibited the Differentiation and Lipid Accumulation of 3T3-L1 Preadipocytes by Regulating the Expression of Lipogenic Genes and Adipokines

Frontiers in Pharmacology ◽

10.3389/fphar.2021.632236 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hanyuan Xu ◽

Linjie Wang ◽

Kemin Yan ◽

Huijuan Zhu ◽

Hui Pan ◽

...

Keyword(s):

Lipid Accumulation ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Mrna Levels ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Aporphine Alkaloid ◽

Lipogenic Genes ◽

Proliferation And Differentiation

Purposes: Nuciferine, a main aporphine alkaloid component found in lotus leaf (Nelumbo nucifera), has been demonstrated to possess the property of reducing fat mass and alleviating dyslipidemia in vivo. The purpose of this study is to explore the effects of nuciferine on the proliferation and differentiation of 3T3-L1 cells and further investigate the possible underlying molecular mechanisms.Methods: 3T3-L1 preadipocytes were treated with 0∼20 μM nuciferine for 24∼120 h, the cell viability was assessed using CCK8. 3T3-L1 preadipocytes and human primary preadipocytes were then induced differentiation and the effects of nuciferine on the lipid metabolism in differentiating and fully differentiated adipocytes were observed by the methods of intracellular triglyceride (TG) assay, Oil Red O staining, RT-qPCR and western blot. Transient transfection and dual luciferase reporter gene methods were used to assess the effects of nuciferine on FAS promoter activities.Results: Nuciferine inhibited the proliferation of 3T3-L1 preadipocytes in a dose- and time-dependent manner. 20 μM nuciferine significantly attenuated lipid accumulation and reduced intracellular TG contents by 47.2, 59.9 and 55.4% on the third, sixth and ninth day of preadipocytes differentiation, respectively (all p < 0.05). Moreover, the mRNA levels of PPARγ, C/EBPα, C/EBPβ, FAS, ACC, HSL and ATGL were notably decreased by 39.2∼92.5% in differentiating preadipocytes when treated with 5∼20 μM nuciferine (all p < 0.05). In fully differentiated adipocytes treated with 20 μM nuciferine for 48 h, the mRNA levels of FAS, ACC and SREBP1 were remarkably downregulated by 22.6∼45.2% compared with the controls (0 μM) (all p < 0.05), whereas the expression of adipokines FGF21 and ZAG were notably promoted by nuciferine. Similarly, in fully differentiated human primary adipocytes, the mRNA levels of FAS, ACC, SREBP1 were decreased and the expression of FGF21 and ZAG were elevated after treated with nuciferine (all p < 0.05). Further mechanism studies showed that 2.5∼20 μM nuciferine significantly decreased FAS promoter activities in 3T3-L1 preadipocytes.Conclusion: Nuciferine inhibited the proliferation and differentiation of 3T3-L1 preadipocytes. The inhibitory effects of nuciferine on adipogenesis might be due to the downregulation of PPARγ, C/EBPα and C/EBPβ, which led to the reduction of intracellular lipid accumulation in 3T3-L1 cells and by downregulating the expression of critical lipogenic enzymes, especially of FAS, which was achieved by inhibiting the FAS promoter activities. Besides, nuciferine promoted the expression of adipokines in fully differentiated adipocytes.

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Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes

International Journal of Molecular Sciences ◽

10.3390/ijms20205066 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5066 ◽

Cited By ~ 3

Author(s):

Yuanyou Li ◽

Jianhong Zhao ◽

Yewei Dong ◽

Ziyan Yin ◽

Yang Li ◽

...

Keyword(s):

Binding Site ◽

Direct Interaction ◽

Bioinformatic Analysis ◽

Fatty Acyl ◽

Luciferase Reporter ◽

Mrna Levels ◽

Mobility Shift ◽

Siganus Canaliculatus ◽

Pufa Biosynthesis ◽

Mobility Shift Assay

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n−6 to 18:3n−6, 18:2n−6 to 20:2n−6, and 18:3n−3 to 20:3n−3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

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Glucose and thyroid hormone co-regulate the expression of the intestinal fructose transporter GLUT5

Biochemical Journal ◽

10.1042/bj3390233 ◽

1999 ◽

Vol 339 (2) ◽

pp. 233-239 ◽

Cited By ~ 26

Author(s):

Miriam MATOSIN-MATEKALO ◽

José E. MESONERO ◽

Thibaut J. LAROCHE ◽

Michel LACASA ◽

Edith BROT-LAROCHE

Keyword(s):

Thyroid Hormone ◽

Reporter Gene ◽

Hormone Receptors ◽

Thyroid Hormone Receptor ◽

Culture Media ◽

Luciferase Reporter ◽

Dependent Manner ◽

Luciferase Reporter Gene ◽

Mobility Shift ◽

Glucose Responsiveness

Expression of the fructose transporter GLUT5 in Caco-2 cells is controlled by the carbohydrate content of the culture media [Mesonero, Matosin, Cambier, Rodriguez-Yoldi and Brot-Laroche (1995) Biochem. J. 312, 757–762] and by the metabolic status of the cells [Mahraoui, Takeda, Mesonero, Chantret, Dussaulx, Bell, and Brot-Laroche (1994) Biochem. J. 301, 169–175]. In this study we show that, in fully differentiated Caco-2/TC7 cells, thyroid hormone and glucose increase GLUT5 mRNA abundance in a dose-dependent manner. Using Caco-2/TC7 cells stably transformed with various fragments of the GLUT5 promoter inserted upstream of the luciferase reporter gene, we localized the sequences that confer 3,3´,5-l-tri-iodothyronine (T3)- and/or glucose-sensitivity to the gene. Glucose responsiveness is conferred by the -272/+41 fragment of the promoter, but it is only with the -338/+41 region that transcription of the luciferase reporter gene is stimulated by T3. This 70 bp fragment from position -338 to -272 of the GLUT5 gene is able to confer T3/glucose-responsiveness to the heterologous thymidine kinase promoter. Electrophoretic-mobility-shift assays demonstrate that thyroid hormone receptors α and β are expressed in Caco-2/TC7 cells. They further show that the -308/-290 region of the GLUT5 promoter binds thyroid hormone receptor/retinoid X receptor heterodimers, and that glucose and/or T3 exert a deleterious effect on the binding of the nuclear protein complex.

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SalA Attenuates Hypoxia-Induced Endothelial Endoplasmic Reticulum Stress and Apoptosis via Down-Regulation of VLDL Receptor Expression

Cellular Physiology and Biochemistry ◽

10.1159/000369671 ◽

2015 ◽

Vol 35 (1) ◽

pp. 17-28 ◽

Cited By ~ 16

Author(s):

Ping Xie ◽

Yingchun Duan ◽

Xianzhi Guo ◽

Lina Hu ◽

Minghua Yu

Keyword(s):

Endothelial Cells ◽

Er Stress ◽

Umbilical Vein ◽

Gene Promoter ◽

Immunoblot Analysis ◽

Receptor Expression ◽

Luciferase Reporter ◽

Mrna Levels ◽

Luciferase Reporter Gene ◽

Vldl Receptor

Background: Salvianolic acid A (SalA) has been shown to display robust protection against endothelial injury. VLDL receptor (VLDLr) is expressed at high levels in the endothelial cells. However its endothelial biological function has not been completely elucidated. Here, we investigated molecular effects of SalA on endothelial VLDLr expression, ER stress, and apoptosis under hypoxia condition. Methods: Human umbilical vein endothelial cells (HUVECs) pretreated with SalA were subjected to hypoxia stimulation. Endothelial ER stress and apoptosis were examined. The mRNA levels were tested by real-time RT-PCR, and the protein levels were determined by immunoblot analysis. Results: Pretreatment of HUVECs with SalA markedly attenuated hypoxia-induced endothelial ER stress and apoptosis. Hypoxia resulted in enhancement of VLDLr expression, which was effectively inhibited by SalA pretreatment. Furthermore, luciferase reporter gene assays indicated that SalA inhibited vldlr gene promoter activity, and ChIP assays showed that hypoxia increase the recruitment of HIF-1α to the vldlr gene promoter, and this process was hampered markedly by pretreatment of SalA. Finally, overexpression of VLDLr abolished SalA-mediated protection of endothelial cells from ER stress and apoptosis. Knockdown of VLDLr mimicked SalA protective effect. Conclusion: These results for the first time demonstrate that SalA protects against hypoxia-induced endothelial ER stress and apoptosis through inhibiting recruitment of HIF-1α to vldlr gene promoter and thus suppressing VLDLr expression.

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