Regulation of u-PA gene expression in human prostate cancer.
u-PA contributes to CaP progression, especially in the metastatic androgen-insensitive state. In vitro, u-PA is expressed by androgen-insensitive, but not androgen-sensitive, CaP cell lines. We hypothesized that in androgen-sensitive CaP an activated ARE represses u-PA expression but in androgen-insensitive CaP this repression is lost and u-PA is upregulated through MAP kinase signaling pathways. To determine whether binding of the DHT-AR complex to AREs in the u-PA promoter region represses u-PA transcription in androgen-sensitive CaP, we studied 2 PC3 androgen-insensitive human CaP cell lines stably transfected with AR [PC3(AR)(2) and PC3(AR)(13)] and 1 mock-transfected cell line [PC3(M)]. In the presence of the synthetic androgen mibolerone, both PC3(AR)(2) and PC3(AR)(13), but not PC3(M), cells showed decreased u-PA expression as assayed by Western and Northern blotting. The AR inhibitor flutamide abrogated mibolerone's effect. Androgen regulation of a second gene, PSA, was also demonstrated in the PC3(AR)(2) cell line. To explore the pathway stimulating u-PA expression in CaP, we performed transient transfections in PC3(AR)(2) cells using u-PA promoter-regulated CAT reporter constructs. Compared to full-length u-PA promoter-CAT constructs, either deletion or mutation of the 5' AP-1 or PEA3 site reduced CAT expression. The location of androgen responsiveness in the u-PA promoter was not identified through the combination of promoter search and transient transfection assays, indicating that a more complicated mechanism is involved in the AR-mediated downmodulation of u-PA expression.
Evans
C P
CP
Department of Urology, University of California, Davis, School of Medicine, Sacramento, CA 95817, USA. cpevans@ucdavis.edu
Stapp
E C
EC
Dall'Era
M A
MA
Juarez
J
J
Yang
J C
JC
D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D015536:Down-Regulation; D005485:Flutamide / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D020935:MAP Kinase Signaling System; D008297:Male; D009154:Mutation; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics*
D004396:Coloring Agents; D011993:Recombinant Fusion Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D012313:RNA; D005485:Flutamide; D015500:Chloramphenicol O-Acetyltransferase; D014568:Urokinase-Type Plasminogen Activator; C022616:thiazolyl blue