Am. J. Respir. Cell Mol. Biol.,
Volume 26, Number 5, May, 2002 627-635
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
Yoneda,
Richart
Harper,
and
Reen
Wu
Center for Comparative Respiratory Biology and Medicine; Department of Internal Medicine, School of Medicine; Veterans Affairs
Northern California Health System; and Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine,
University of California at Davis, Davis, California
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-
antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR- nuclear receptor. These
results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.
Abbreviations: base pair, bp; bovine serum albumin, BSA; chloramphenicol
acetyltransferase, CAT; dimethyl sulfate, DMS; dithiothreitol, DTT; electrophoretic mobility shift assay, EMSA; ligation-mediated PCR, LM-PCR;
polymerase chain reaction, PCR; phenylmethyl sulfonyl fluoride, PMSF; purine, Pu; retinoic acid, RA; RA responsive element, RARE; RA receptor,
RAR/RXR; all-trans-retinol, ROH; nucleotide, nt; tracheobronchial epithelial cells, TBE; thymidine kinase, tk; human thioredoxin, Trx; wild-type, wt.
