Induction of fibrinolytic factors in vascular endothelial cells through GC box moties

Understanding the molecular mechanisms of controlling fibrinolysis and matrix homeostasis is essential for elucidating the basis of tissue repair. Recently, we described two novel transcriptional regulations of urokinase plasminogen activator (u-PA) via GC box motifs in the vascular endothelial cells. The GC box motifs were identified in many mammalian and viral genes as cognate binding sites for general transcription factor, Spl. However, now, it is known that increasing numbers of Krüppel-like factors bind to GC box motifs and regulate their target genes expression. We revealed that retinoic acid receptors (RAR/RXR) physically interact with Sp 1 and potentiate its binding to GC box motifs within the human uPA promoter, leading to the enhanced transactivation of u-PA promoter'. Increased transactivation of the target genes through physical interaction with Spl has been also reported by other groups for the other members of nuclear hormone receptor family. We next suggested a role of a Kriippel-like factor, Zf9 (KLF6) in the injury response by enhancing u-PA transcription and eventual activation of latent TGF-.2 Until now, GC box motifs have been thought to be important only for constitutive transcription by Sp 1 whose cellular levels are fairly constant. Now, we and others have suggested that GC box motifs are also important for injury response reaction at least in part via physical interaction between Sp 1 and stimuli-induced nuclear hormone receptors, as well as stimuli-induced Kruppel-lilce factors such as Zf9. Here, we investigated the specificity of GC box motifs. In addition to u-PA, promoters of transglutaminase, TGFl, and TGF- receptors types I and II, could be transactivated through a similar mechanism. Functional mapping suggested the presence of both functional and unfunctional GC boxes. We analyzed their difference employing the u-PA promoter containing three contiguous GC boxes. Reporter assays, gel shift and footprinting studies revealed that the middle and 3' site GC boxes were functional; they have, respectively, high and medium affinity to Spl and their bindings were potentiated by RAR/RXR. In contrast, the 5' site GC box was unfunctional; it showed extremely low affinity to Spl or Zf9 and the binding was barely potentiated by RAR/RXR. This difference could be partly ascribed to the presence of five tandem purine or pyrimidine bases at the immediate 3' flanking region of the hexanucleotide GGGCGG core motif. Together with the previous findings, we suggest an importance of GC box motifs in increased expression of fibrinolytic factors and other key factors in the injured vascular endothelial cells or hepatic stellate cells.