Requirement of an upstream AP-1 motif for the constitutive and phorbol ester-inducible expression of the urokinase-type plasminogen activator receptor gene.
The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix proteolysis by accelerating plasmin formation at the cell surface. The present study was undertaken to identify elements in the u-PAR promoter required for the elevated expression of this binding site. Toward this end, we used two cultured colon cancer cell lines; one (RKO) has a transcriptionally activated u-PAR gene, and the other (GEO) overexpresses the receptor only after phorbol ester treatment. A chloramphenicol acetyltransferase (CAT) reporter driven by 398 nucleotides of 5' regulatory sequence of the u-PAR gene was strongly activated in the RKO cells, which displays approximately 3 x 10(5) receptors/cell. A region of this promoter between -197 and -8 was required for optimal expression, as indicated using a CAT reporter driven by 5' deleted fragments. DNase I footprinting revealed three protected regions (I, -190 to -171; II, -148 to -124; and III, -99 to -70) in this part of the promoter. Mutation of an AP-1 binding site at -184 within region I reduced activation of the promoter by 85%. Deletion of either region II or III also reduced promoter activity by over 60%. An oligonucleotide spanning the AP-1 motif at -184 bound, specifically, nuclear factors from RKO cells, and antibodies specific for Jun-D, c-Jun, or Fra-1 proteins supershifted the complex indicating the presence of these proteins. The amount of these factors was reduced in GEO cells in which the u-PAR gene is only weakly transcriptionally activated. Expression of a vector encoding a wild-type Jun-D cDNA increased u-PAR promoter activity in GEO cells. Conversely, transfection of RKO cells with a transactivation domain-lacking Jun-D expression construct resulted in a dose-dependent decrease in u-PAR promoter activity. Treatment of GEO cells with phorbol ester increased u-PAR mRNA and the activity of a CAT reporter driven by the wild-type but not the AP-1 (-184)-mutated u-PAR promoter, and this was associated with a strong induction in the amount of Jun-D, c-Jun, and c-Fos. Methylation interference studies using a fragment of the u-PAR promoter (spanning -201 to -150) bound with nuclear extracted proteins from RKO cells, and phorbol 12-myristate 13-acetate-treated and -untreated GEO cells showed that the contact points corresponded to the AP-1 binding site at -184. Thus, the elevated expression of u-PAR in RKO cells, which constitutively produces this binding site, as well as in phorbol 12-myristate 13-acetate-stimulated GEO cells requires an AP-1 motif located 184 bp upstream of the transcriptional start site.
Lengyel
E
E
Department of Obstetrics and Gynecology, Technical University of Munich, 81675 Munich, Germany.
Wang
H
H
Stepp
E
E
Juarez
J
J
Wang
Y
Y
Doe
W
W
Pfarr
C M
CM
Boyd
D
D
D001483:Base Sequence; D001665:Binding Sites; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics*; D055293:Receptors, Urokinase Plasminogen Activator; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D018808:Transcription Factor AP-1; D013755:Tetradecanoylphorbol Acetate