Dissection analysis of the negative regulation of the human urokinase-type plasminogen activator (uPA) gene

The urokinase-type plasminogen activator (uPA) is a serine protease involved in processes such as cell migration and invasion through the activation of the extracellular protease plasmin responsible of the degradation of the proteins of the extracellular matrix. The ability of the cells to migrate and to invade the surrounding tissues plays a fundamental role in many normal and pathological processes such as fibrinolysis, wound healing, angiogenesis, embryogenesis, gametogenesis, ovulation, mammary gland involution and tumor metastasis. Due to its high destructive potential, localized activation of plasminogen is regulated by a complex network of molecular interactions involving both specific inhibitors (PAI-1 and PAI-2), and cell bound receptor (uPAR) and the synthesis of every component of the plasminogen activator system is tightly regulated by a number of factors like hormones, growth factors and cytokines. On the other hand, the restricted expression of uPA in the organism to a very few cell types (kidney and lung), its inducibility by different stimuli and its overexpression in tumours and several transformed and tumoral cell lines, indicate that uPA regulation occurs also at the level of gene expression. The gene encoding uPA and its 5' flanking region have been sequenced and characterized in human, mouse and pig. Both in vivo and in vitro studies of progressive 5'deletions of the regulatory region of uPA, have revealed the presence of positive and negative cis-acting sequences and of specific contributions of the proximal regulatory regions to cell-type specific expression of the gene, suggesting the presence of multiple array of cis-acting sequences specific for different transcription factors that direct uPA expression in a celltype specific manner. In the human uPA promoter the enhancer is located between -2100 and -1870 from the transcriptional start site and contains two binding sites for the transcription factor API and PE A3 which are important for both the constitutive and regulated expression of the gene. Furthermore, the cooperation between the two API sites is required for TPA inducibility and is mediated by other proteins called Upstream Enhancer Factors (UEF). A negative cis-acting element has been localized between -1870 and -1570 and contains a binding site for a multiproteic complex (NF-kB/c-rel) that mediates the inducibility by TPA in HeLa and HepG2 cell lines. Much evidence, however, indicates that the regulation of uPA is mainly negative. The studies described in this thesis have led to the identification of negative regulatory elements that may play an important role in the establishment of a silenced phenotype in a cell-type specific manner. The analysis of promoter activity in cell lines not expressing (HeLa and CVl) and expressing uPA (PC3) allowed the identification of at least three regions that play different roles in the silencing of the uPA gene in different cell lines, named SI (-1870/-1428), 82 (-787/-537) and S3 (-537/-86). Of the three only S2 shows cell-type specificity, as it is active only in cells that do not express uPA; S1 and S3, on the other hand, could act as modulators of uPA gene expression in those cells that express uPA. All of them negatively regulate the activity of the minimal promoter, suggesting that they can interfere with the formation of a competent pre-initiation complex at the start site of transcription. A dissection analysis of silencer S2 detected the presence of multiple silencing units, although the deletion of a single one does not have any effect on the activity of the promoter. Transcription from a heterologous promoter is affected only when more than one copy of a single unit is cloned in front of it, suggesting that in the context of the whole S2 region each unit acts synergistically with the others in the silencing of the gene. DNase I footprinting analysis of S2 showed that this region is extensively protected by nuclear extract from uPA producing (PC-3) and not producing (HeLa) cell lines, with few differences between them. However, the LMSA analysis of the complexes indicated that their molecular composition may be different in the two cell lines tested. In particular, in HeLa cell extract, a single strand binding activity seems to bind S2 and it may be responsible for the assembly of a negative-acting complex that would prevent the loading of a competent transcription initiation complex at the start site of transcription. Furthermore, the presence of two binding sites for proteins belonging to the HMG-box containing protein family, suggested that the assembly of a stereospecific complex is required for the activity of silencer S2, although it is not clear if their activity is required for the silencing of the gene or for the inactivation of the inhibition in those cells that express uPA as in HepG2 cell line. In these cells uPA is expressed at a very low basal level, but its expression is inducible by TPA. In this cell line S2 is not active; on the contrary its presence is required for basal expression of uPA. The presence in S2 of a sequence matching the transforming growth factor β inihibitory element (TIE), described to be important for mediating the inhibition of TPA induction by TGF-βl of the stromelysin gene, has led me to investigate the role of this element on S2 activity in HepG2 cells. Transient transfection analysis of uPA promoter deletions showed that, at least in this system, the TIE is not involved in the regulation of uPA expression. Preliminary results, shown in appendix 2, have suggested another interesting aspect of the negative regulation of uPA expression by the p53 tumor suppressor gene product, although no p53 binding to uPA promoter has been shown so far. In conclusion, the studies presented in this thesis have showed that a complex array of regulatory sequences, in addition to the enhancer and minimal promoter, appear to regulate uPA transcription, some of which are cell-type specific.