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 language | English (US) |
|---|---|
| Pages (from-to) | 627-635 |
| Number of pages | 9 |
| Journal | American Journal of Respiratory Cell and Molecular Biology |
| Volume | 26 |
| Issue number | 5 |
| State | Published - 2002 |
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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 journal › Article
}
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.
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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 -