A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells

Manjunath B. Joshi, Danila Ivanov, Maria Philippova, Emmanouil Kyriakakis, Paul Erne, Thérèse J. Resink

Research output: Contribution to journalArticle

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Abstract

T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H2O2) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from -99 to -2304 bp, located the minimal promoter region of T-cad within -285 bp from the translation start site. Reporter activity in ECs transfected with the -285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to -156 to -203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to -156 to -203 bp after streptavidin-agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.

Original languageEnglish
Pages (from-to)271-280
Number of pages10
JournalBiochemical Journal
Volume416
Issue number2
DOIs
Publication statusPublished - 01-12-2008

Fingerprint

Thioredoxins
Endothelial cells
Oxidation-Reduction
Endothelial Cells
Modulation
Oxidative stress
Oxidative Stress
Nucleoproteins
Up-Regulation
Cardiovascular system
Electrophoretic mobility
Glycosylphosphatidylinositols
Proteins
Acetylcysteine
Electrophoretic Mobility Shift Assay
Cadherins
Cardiovascular System
H-cadherin
Reporter Genes
Immunoprecipitation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Joshi, Manjunath B. ; Ivanov, Danila ; Philippova, Maria ; Kyriakakis, Emmanouil ; Erne, Paul ; Resink, Thérèse J. / A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells. In: Biochemical Journal. 2008 ; Vol. 416, No. 2. pp. 271-280.
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A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells. / Joshi, Manjunath B.; Ivanov, Danila; Philippova, Maria; Kyriakakis, Emmanouil; Erne, Paul; Resink, Thérèse J.

In: Biochemical Journal, Vol. 416, No. 2, 01.12.2008, p. 271-280.

Research output: Contribution to journalArticle

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AU - Ivanov, Danila

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AU - Resink, Thérèse J.

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AB - T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H2O2) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from -99 to -2304 bp, located the minimal promoter region of T-cad within -285 bp from the translation start site. Reporter activity in ECs transfected with the -285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to -156 to -203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to -156 to -203 bp after streptavidin-agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.

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