Regulation of Thioredoxin Gene Expression by Vitamin A in Human Airway Epithelial Cells

• Published in: American journal of respiratory cell and molecular biology Vol. 26; no. 5; pp. 627 - 635
• Main Authors: Chang, Wen-Hsing, Reddy, Sekhar P.-M, Di, Yuan-Pu Peter, Yoneda, Ken, Harper, Richart, Wu, Reen
• Format: Journal Article
• Language: English
• Published: United States Am Thoracic Soc 01.05.2002; American Thoracic Society
• Subjects: 5' Flanking Region - physiology; Cells, Cultured; DNA Footprinting; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Epithelial Cells - cytology; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Gene Expression Regulation - drug effects; Genes, Reporter; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Receptors, Retinoic Acid - metabolism; Respiratory Mucosa - cytology; Respiratory Mucosa - drug effects; Respiratory Mucosa - metabolism; Response Elements - physiology; Retinoic Acid Receptor alpha; Sequence Analysis, DNA; Thioredoxins - genetics; Thioredoxins - metabolism; Transfection; Vitamin A - pharmacology
• ISSN: 1044-1549; 1535-4989
• DOI: 10.1165/ajrcmb.26.5.4276

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.