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

doi:10.1074/jbc.270.44.26451

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

From the (1) California Regional Primate Research Center,
(2) Department of Internal Medicine, School of Medicine, University of California at Davis, Davis, California 95616, and
(3) Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033

** To whom all correspondence should be addressed. Tel.: 916-752-2648; Fax: 916-752-2880.

↵^¶ Present address: Biological Dosimetry Group, Lawrence Livermore National Laboratory, Livermore, CA 94551.

Abstract

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.

Footnotes

↵^§ Contributed equally to this paper.
↵* Supported in part by National Institutes of Health Grants DE10742, HL35635, ES06230, and ES06553 and California Tobacco-related Disease Research Program Grant 3RT-0409. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SPR

small proline-rich protein

TPA

12-O-tetradecanoylphorbol-13-acetate

TBE

tracheobronchial epithelium

TRE

TPA-responsive element

CRE

cAMP-responsive element

tk

thymidine kinase

CAT

chloramphenicol acetyltransferase

DMS

dimethyl sulfate

GT

GGTGG motif

ETS

E-26 transformation specific

HSV

herpes simplex virus

PCR

polymerase chain reaction.
- Received June 2, 1995.
- Revision received August 31, 1995.