Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells.
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-alpha antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR-alpha nuclear receptor. These results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.
Chang
Wen-Hsing
WH
Center for Comparative Respiratory Biology and Medicine, University of California at Davis, Davis, California 95616, USA.
Reddy
Sekhar P-M
SP
Di
Yuan-Pu Peter
YP
Yoneda
Ken
K
Harper
Richart
R
Wu
Reen
R
D024506:5' Flanking Region / Q000502:physiology; D002478:Cells, Cultured; D018983:DNA Footprinting; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020218:Response Elements / Q000502:physiology; D000072482:Retinoic Acid Receptor alpha; D017422:Sequence Analysis, DNA; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014801:Vitamin A / Q000494:pharmacology*
C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D014801:Vitamin A; D013879:Thioredoxins