EXPRESSION OF HUMAN SQUAMOUS-CELL DIFFERENTIATION MARKER, SPR1, IN TRACHEOBRONCHIAL EPITHELIUM DEPENDS ON JUN AND TRE MOTIFS

Tracheobronchial epithelial (TBE) cells that normally do not express t he squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA), The regulati on of SPR1 gene expression by TPA occurs, in part, at the transcriptio nal level in primary human and monkey TBE cells, Using a transient tra nsfection assay, we observed that TPA stimulates the activity of the r eporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transf ected TBE cells, However, this chloramphenicol acetyltransferase activ ity is cell type specific with significantly less activity in transfor med 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, especi ally c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion an alysis of the promoter region demonstrates that the DNA fragment of th e first 98 base pairs of the 5'-flanking region contains the basal pro moter activity, while the region between -162 and -96 contains the cis -enhancer elements for both the basal and TPA/c-JUN-stimulating promot er activities, This observation is supported by in vivo genomic footpr inting studies that reveal persistent protections in the following mot ifs of this region: -141 TRE, -131 GT, -123 ETS-like, and -111 TRE-lik e 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 TH E-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced b asal transcriptional activity, However, it is primarily the -111 TRE-l ike motif that is involved in the mediation of the enhanced basal prom oter activity of the human SPR1 gene, These results are further suppor ted by gel mobility shift assays that demonstrate the involvement of c -JUN and these TRE motifs in the formation of the DNA-protein complex.