Attenuation of plasminogen activator inhibitor type-1 promoter activity in serum-stimulated renal epithelial cells by a distal 5? flanking region

Abstract

Urokinase plasminogen activator (u‐PA) and its fast acting type‐1 inhibitor (PAI‐1) localize to cellular focal adhesive structures and the ajoining proximal undersurface region, respectively (Kutz et al., J. Cell. Physiol. 176:8–18, 1997). PAI‐1 may function in this locale to modulate pericellular proteolytic activity, cell‐to‐substrate adhesion, or matrix‐dependent motility. While PAI‐1 synthesis is regulated in an immediate‐early response manner in growth “activated” renal cells coincident with cytoskeletal restructuring, adhesive influences both repress the amplitude and prolong the time course of serum‐induced PAI‐1 transcription (Ryan et al., Biochem. J. 314:1041–1046, 1996). To identify potential adhesion‐responsive elements within the PAI‐1 gene that function in this complex mode of expression control, reporter constructs containing defined directionally deleted PAI‐1 5′ genomic fragments cloned upstream of a CAT gene were employed in transient transfection assays. A 483‐bp distal PAI‐1 flanking segment (corresponding to nucleotides −2395 to −1912) conferred significant adhesion‐dependent attenuation on serum‐induced PAI‐1 transcription. This 483‐bp distal PAI‐1 segment functioned as a repressor of reporter (CAT) activity under both adhesive and suspension culture conditions, however, when ligated upstream of either an SV40 promoter/enhancer or a minimal PAI‐1 promoter. These data suggest that repressor elements located between −2395 and −1912 bp interact with more proximal adhesion‐dependent regulatory elements to affect PAI‐1 expression attenuation during serum stimulation of adherent renal epithelial cells. Cell Motil. Cytoskeleton 44:168–176, 1999. © 1999 Wiley‐Liss, Inc.

TGF-β1-induced PAI-1 expression is E box/USF-dependent and requires EGFR signaling

“…Recovered plasmids were transformed into XL-1 Blue bacteria and mutations confirmed by loss of E box-specific endonuclease sensitivity, using 1 μl BbrPI (10 U/μl) which cleaves the palindromic E box motif CAC↓GTG, and by sequencing. Cells were transfected with either the wild-type or mutant reporter plasmids, cultured in serum-free medium for 3 days then stimulated with TGF-β1 for 24 h prior to CAT activity assay [32].…”

Epithelial monolayer wounding stimulates binding of USF-1 to an E-box motif in the plasminogen activator inhibitor type 1 gene

“…Multiple promoter elements mediate stimulus-specific controls on PAI-1 transcription (e.g. Westerhausen et al, 1991;Ryan et al, 1996;Slack and Higgins, 1999). One prominent regulatory sequence is the hexanucleotide E-box motif (CACGTG) that is recognized by several members of the helix-loop-helix family of transcription factors (e.g.…”