SYNERGISTIC TRANSCRIPTIONAL ACTIVATION OF THE MOUSE UROKINASE PLASMINOGEN-ACTIVATOR (UPA) GENE AND OF ITS ENHANCER ACTIVATOR PROTEIN-1 (AP1) SITE BY CAMP AND RETINOIC ACID

We have investigated the mechanism whereby all-trans retinoic acid (tR A) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA d id not alter the cellular content of cAMP-dependent protein kinase reg ulatory subunits I and II. In agreement with this, nuclear run-on anal ysis in the presence of the translational inhibitor puromycin demonstr ated that the effect of 8-BrcAMP and its potentiation by tRA were inde pendent of protein synthesis. A transiently transfected 6.6 kb uPA 5'- flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mi micked the response of the endogenous uPA. gene. Thus 1 mM 8-BrcAMP in duced a 100-200 % increase in CAT content, 100 nM tRA had no effect an d 100 nM tRA + 1 mM 8-BrcAMP induced a 300-500 % increase in cells co- transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5' -deleted constructs showed that the tRA effect required at least two c is regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region conta ined a tRA-response element-like motif. Because tRA receptor and 9-cis -RA receptor interact with activator protein 1 (API), we tested whethe r tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA-CAT fusion gene was responsive to I mM 8-BrcAMP (100 %, CAT increase), not respon sive to 100 nM tRA, and synergistically responsive to 100 nM tRA + 1 m M 8-BrcAMP (240 % CAT increase) in cells co-transfected with Fos and J un. Synergistic activation of the same construct and of the 6.6 kb uPA -CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclu de that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription.