Expression of Human Squamous Cell Differentiation Marker, SPR1, in Tracheobronchial Epithelium Depends on JUN and TRE Motifs (∗)

• Published in: The Journal of biological chemistry Vol. 270; no. 44; pp. 26451 - 26459
• Main Authors: Reddy, Sekhar P.M., Chuu, Yue-Jen, Lao, Paul N., Donn, Jerry, Ann, David K., Wu, Reen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.11.1995; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Biomarkers; Bronchi - metabolism; Cell Differentiation; Cell Nucleus - metabolism; Cells, Cultured; Chloramphenicol O-Acetyltransferase - biosynthesis; Consensus Sequence; Cornified Envelope Proline-Rich Proteins; Epithelium - metabolism; Gene Expression - drug effects; Haplorhini; Humans; Membrane Proteins; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-jun - biosynthesis; Proto-Oncogene Proteins c-jun - metabolism; Recombinant Fusion Proteins - biosynthesis; Sequence Deletion; Tetradecanoylphorbol Acetate - pharmacology; Trachea - metabolism; Transcription, Genetic; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.44.26451

Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by cotransfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5′-flanking region contains the basal promoter activity, while the region between −162 and −96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: −141 TRE, −131 GT, −123 ETS-like, and −111 TRE-like motifs and in the enhanced protections in −141 TRE and −111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both −141 TRE and −111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the −111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex.