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 Scopus citations

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

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

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