4.6 Article

Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells

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AMER THORACIC SOC
DOI: 10.1165/ajrcmb.26.5.4276

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Funding

  1. NHLBI NIH HHS [F32 HL009573, HL35635] Funding Source: Medline
  2. NIEHS NIH HHS [ES05707, ES06230, ES04699, ES09701] Funding Source: Medline

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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-re porter 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.

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