Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells

Wen Hsing Chang, Sekhar P M Reddy, Yuan Pu Peter Di, Ken Y Yoneda, Richart W Harper, Reen Wu

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Human thioredoxin (Trx) is a 12-kD protein known to be involved in various reduction/oxidation reactions essential for cell growth and cellular injury repair. We previously demonstrated, based on nuclear run-on assay, that retinoic acid (RA) stimulated Trx gene expression in airway epithelial cells at the transcriptional level. Nucleotide sequencing of the 5′-flanking region of the human Trx gene revealed the presence of a TATA box at -28 and four RA response element (RARE)-like half sites at -426, -453, -507, and -626 nt. Transient transfection assays with a Trx promoter-reporter gene, chloramphenicol acetyltransferase (CAT), demonstrated a dose-dependent involvement of these four RARE-like half sites in RA-enhanced promoter activity. When the DNA fragment that flanks these four RARE-like half sites from -357 to -671 nt was introduced into a heterologous promoter of the tk-CAT2 vector, both basal and RA-stimulated CAT activities were observed. A site-directed mutagenesis approach demonstrated an essential role for RARE-I and RARE-II at -426 and -453 nt, respectively, and an auxiliary role for RARE-III at -507 nt in both basal and RA-stimulated CAT activities. Both in vivo and in vitro genomic footprinting experiments further demonstrated specific protein-DNA interactions in these "putative" RARE-I/II/III half sites. Gel electrophoretic mobility shift assays demonstrated specific interactions of these RARE-like half sites with the nuclear extracts obtained from RA-treated cultures. The anti-RAR-α antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR-α nuclear receptor. These results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.

Original languageEnglish (US)
Pages (from-to)627-635
Number of pages9
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume26
Issue number5
StatePublished - 2002

Fingerprint

Thioredoxins
Gene Expression Regulation
Response Elements
Vitamin A
Gene expression
Epithelial Cells
Tretinoin
Chloramphenicol O-Acetyltransferase
Assays
Genes
Gene Expression
Electrophoretic mobility
Mutagenesis
TATA Box
5' Flanking Region
Redox reactions
DNA
Cell growth
Electrophoretic Mobility Shift Assay
Cytoplasmic and Nuclear Receptors

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

Cite this

Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells. / Chang, Wen Hsing; Reddy, Sekhar P M; Di, Yuan Pu Peter; Yoneda, Ken Y; Harper, Richart W; Wu, Reen.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 26, No. 5, 2002, p. 627-635.

Research output: Contribution to journalArticle

@article{890c4c2bce7146b9a16376851923715a,
title = "Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells",
abstract = "Human thioredoxin (Trx) is a 12-kD protein known to be involved in various reduction/oxidation reactions essential for cell growth and cellular injury repair. We previously demonstrated, based on nuclear run-on assay, that retinoic acid (RA) stimulated Trx gene expression in airway epithelial cells at the transcriptional level. Nucleotide sequencing of the 5′-flanking region of the human Trx gene revealed the presence of a TATA box at -28 and four RA response element (RARE)-like half sites at -426, -453, -507, and -626 nt. Transient transfection assays with a Trx promoter-reporter gene, chloramphenicol acetyltransferase (CAT), demonstrated a dose-dependent involvement of these four RARE-like half sites in RA-enhanced promoter activity. When the DNA fragment that flanks these four RARE-like half sites from -357 to -671 nt was introduced into a heterologous promoter of the tk-CAT2 vector, both basal and RA-stimulated CAT activities were observed. A site-directed mutagenesis approach demonstrated an essential role for RARE-I and RARE-II at -426 and -453 nt, respectively, and an auxiliary role for RARE-III at -507 nt in both basal and RA-stimulated CAT activities. Both in vivo and in vitro genomic footprinting experiments further demonstrated specific protein-DNA interactions in these {"}putative{"} RARE-I/II/III half sites. Gel electrophoretic mobility shift assays demonstrated specific interactions of these RARE-like half sites with the nuclear extracts obtained from RA-treated cultures. The anti-RAR-α antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR-α nuclear receptor. These results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.",
author = "Chang, {Wen Hsing} and Reddy, {Sekhar P M} and Di, {Yuan Pu Peter} and Yoneda, {Ken Y} and Harper, {Richart W} and Reen Wu",
year = "2002",
language = "English (US)",
volume = "26",
pages = "627--635",
journal = "American Journal of Respiratory Cell and Molecular Biology",
issn = "1044-1549",
publisher = "American Thoracic Society",
number = "5",

}

TY - JOUR

T1 - Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells

AU - Chang, Wen Hsing

AU - Reddy, Sekhar P M

AU - Di, Yuan Pu Peter

AU - Yoneda, Ken Y

AU - Harper, Richart W

AU - Wu, Reen

PY - 2002

Y1 - 2002

N2 - Human thioredoxin (Trx) is a 12-kD protein known to be involved in various reduction/oxidation reactions essential for cell growth and cellular injury repair. We previously demonstrated, based on nuclear run-on assay, that retinoic acid (RA) stimulated Trx gene expression in airway epithelial cells at the transcriptional level. Nucleotide sequencing of the 5′-flanking region of the human Trx gene revealed the presence of a TATA box at -28 and four RA response element (RARE)-like half sites at -426, -453, -507, and -626 nt. Transient transfection assays with a Trx promoter-reporter gene, chloramphenicol acetyltransferase (CAT), demonstrated a dose-dependent involvement of these four RARE-like half sites in RA-enhanced promoter activity. When the DNA fragment that flanks these four RARE-like half sites from -357 to -671 nt was introduced into a heterologous promoter of the tk-CAT2 vector, both basal and RA-stimulated CAT activities were observed. A site-directed mutagenesis approach demonstrated an essential role for RARE-I and RARE-II at -426 and -453 nt, respectively, and an auxiliary role for RARE-III at -507 nt in both basal and RA-stimulated CAT activities. Both in vivo and in vitro genomic footprinting experiments further demonstrated specific protein-DNA interactions in these "putative" RARE-I/II/III half sites. Gel electrophoretic mobility shift assays demonstrated specific interactions of these RARE-like half sites with the nuclear extracts obtained from RA-treated cultures. The anti-RAR-α antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR-α nuclear receptor. These results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.

AB - Human thioredoxin (Trx) is a 12-kD protein known to be involved in various reduction/oxidation reactions essential for cell growth and cellular injury repair. We previously demonstrated, based on nuclear run-on assay, that retinoic acid (RA) stimulated Trx gene expression in airway epithelial cells at the transcriptional level. Nucleotide sequencing of the 5′-flanking region of the human Trx gene revealed the presence of a TATA box at -28 and four RA response element (RARE)-like half sites at -426, -453, -507, and -626 nt. Transient transfection assays with a Trx promoter-reporter gene, chloramphenicol acetyltransferase (CAT), demonstrated a dose-dependent involvement of these four RARE-like half sites in RA-enhanced promoter activity. When the DNA fragment that flanks these four RARE-like half sites from -357 to -671 nt was introduced into a heterologous promoter of the tk-CAT2 vector, both basal and RA-stimulated CAT activities were observed. A site-directed mutagenesis approach demonstrated an essential role for RARE-I and RARE-II at -426 and -453 nt, respectively, and an auxiliary role for RARE-III at -507 nt in both basal and RA-stimulated CAT activities. Both in vivo and in vitro genomic footprinting experiments further demonstrated specific protein-DNA interactions in these "putative" RARE-I/II/III half sites. Gel electrophoretic mobility shift assays demonstrated specific interactions of these RARE-like half sites with the nuclear extracts obtained from RA-treated cultures. The anti-RAR-α antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR-α nuclear receptor. These results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.

UR - http://www.scopus.com/inward/record.url?scp=0036010531&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036010531&partnerID=8YFLogxK

M3 - Article

C2 - 11970916

AN - SCOPUS:0036010531

VL - 26

SP - 627

EP - 635

JO - American Journal of Respiratory Cell and Molecular Biology

JF - American Journal of Respiratory Cell and Molecular Biology

SN - 1044-1549

IS - 5

ER -