Anti-AP-1 activity of all-trans retinoic acid in glomerular mesangial cells

1994 ◽  
Vol 267 (5) ◽  
pp. F805-F815 ◽  
Author(s):  
M. S. Simonson
Keyword(s):  
Transcription Factors ◽  
Retinoic Acid ◽  
Dna Binding ◽  
Mesangial Cells ◽  
Cell Number ◽  
Immediate Early ◽  
Thymidine Uptake ◽  
Glomerular Mesangial Cells ◽  
Mobility Shift ◽  
Transcriptional Responses

Functional antagonism between retinoic acid (RA) receptors and activator protein-1 (AP-1) transcription factors might regulate expression of genes involved in the response to injury in the kidney. We designed experiments to analyze the mechanisms by which RA inhibits AP-1-directed transcriptional responses in glomerular mesangial cells. RA inhibited serum-stimulated mesangial cell proliferation as assessed by measurements of [3H]thymidine uptake and cell number. In transient transfection assays with a chloramphenicol acetyltransferase reporter, RA completely blocked transcription directed by an AP-1 cis-element in cells stimulated by serum. AP-1 DNA binding was analyzed in electrophoretic gel mobility shift assays using nuclear extracts from control or RA-pretreated cells stimulated with serum. RA did not abolish AP-1 DNA binding activity under the conditions of this assay. The apparent equilibrium dissociation constant, maximal density of binding, and association rate for the AP-1-DNA interaction were similar in serum-stimulated cells or RA-pretreated cells stimulated with serum. RA repressed serum-stimulated induction of the immediate early genes c-fos and c-jun, whose protein products dimerize to form AP-1. Repression was relatively selective for c-fos/c-jun; induction of other immediate early transcription factors (junB, c-myc, and egr-1) was not downregulated by RA. That repression of c-fos by RA might contribute to anti-AP-1 activity was suggested by experiments with an antisense c-fos expression vector, which demonstrated that c-fos induction was required for serum-stimulated AP-1 activity. Together, these data demonstrate that RA antagonizes AP-1-directed transcription without inhibiting AP-1 DNA-binding in mesangial cells. Selective repression of c-fos and c-jun might contribute to the anti-AP-1 activity of RA.

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1065-C1072 ◽  
Author(s):  
Ashish K. Gupta ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Inos Gene ◽  
E Box ◽  
Inos Promoter ◽  
Gel Mobility Shift

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.

Examination of DNA-binding activity of neuronal transcription factors by electrophoretical mobility shift assay

1998 ◽  
Vol 2 (4) ◽  
pp. 243-249 ◽  
Author(s):  
Koichiro Kako ◽  
Hisanori Wakamatsu ◽  
Toshiyuki Hamada ◽  
Marek Banasik ◽  
Keiko Ohata ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Activity ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Mobility Shift Assay

scholarly journals DNA-binding specificity of GATA family transcription factors

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

Glycated albumin activates PAI-1 transcription through Smad DNA binding sites in mesangial cells

2004 ◽  
Vol 287 (4) ◽  
pp. F665-F672 ◽  
Author(s):  
Hyun Soon Lee ◽  
Kyung Chul Moon ◽  
Chi Young Song ◽  
Bong Cho Kim ◽  
Suxia Wang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Mrna Expression ◽  
Binding Sites ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Luciferase Activity ◽  
Glycated Albumin ◽  
Plasminogen Activator Inhibitor 1 ◽  
Mobility Shift ◽  
Pai 1

Amadori-modified glycated albumin stimulates extracellular matrix and transforming growth factor-β (TGF-β) expression in cultured mesangial cells. Smad proteins transduce the TGF-β-mediated signal, and Smad-binding CAGA sequences are present in the plasminogen activator inhibitor-1 (PAI-1) promoter. This study examined whether glycated albumin induces PAI-1 transcription in human mesangial cells (HMC) through Smad-binding sites in the PAI-1 promoter. Quiescent HMC were exposed to 200 μg/ml bovine serum albumin (BSA) or glycated BSA (Gly-BSA) for 12–72 h. At 24 h, Gly-BSA stimulated TGF-β1 and PAI-1 mRNA expression in HMC to 1.8 and 3.2 times that in the BSA-treated control cells. Gly-BSA also activated the PAI-1 promoter luciferase activity 2.3-fold. Gly-BSA-treated cells enhanced Smad2 and Smad3 protein levels 2.5 times the control levels in the nuclei. An electrophoretic mobility shift assay performed using CAGA sequences as a probe showed that Gly-BSA increased DNA/protein complexes. When nuclear extracts were preincubated with 100-fold molar excess of unlabeled CAGA oligonucleotide, the formation of complex was prevented. The DNA-binding protein was shown to be Smad3 by antibody supershift. Transfection of phosphorothioate CAGA oligonucleotide, a CAGA antisense analog, inhibited Gly-BSA-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked Gly-BSA-induced PAI-1 promoter luciferase activity. These results indicate that Gly-BSA increases DNA binding activity of Smad3 and that it stimulates PAI-1 transcription through Smad-binding CAGA sequences in the PAI-1 promoter in HMC. Thus progression of diabetic nephropathy may be promoted by PAI-1 upregulation mediated by the glycated albumin-induced Smad/DNA interactions.

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