Growth state-dependent binding of USF-1 to a proximal promoter E box element in the rat plasminogen activator inhibitor type 1 gene

Induced PAI-1 gene expression in renal epithelial (NRK-52E, clone EC-1) cel ls occurs as part of the immediate-early response to serum. PAI-1 transcrip ts are maximally expressed early in G(1) (within 4 h of serum addition to q uiescent EC-1 cells) and then subsequently decline to basal levels prior to entry into DNA synthetic phase, Comparative analysis of PAI-1 mRNA abundan ce and de novo-synthesized thiolated RNA in quiescent cells, as well as at 4 h (early G(1)) and 20 h (late G(2)) postserum addition, in conjunction wi th RNA decay measurements indicated that PAI-1 gene regulation upon growth activation was predominantly transcriptional. An E box motif (CACGTG), impo rtant in the induced expression of some growth state-dependent genes, mappe d to nucleotides -160 to -165 upstream of the transcription start site in t he PAI-1 proximal promoter. Mobility-shift assessments, using a 18-bp deoxy oligonucleotide construct containing the E box within the context of PAI-1- specific flanking sequences, confirmed binding of EC-1 nuclear protein(s) t o this probe and, specifically, to the E box hexanucleotide site. The speci ficity of this protein-probe interaction was verified by competition analys es with double-stranded DNA constructs that included E box deoxyoligonucleo tides with non-PAI-l flanking bases, mutant E box sequences incapable of bi nding NRK nuclear proteins, and unrelated (i.e,, AP-1) target motifs, Extra ct immunodepletion and supershift/complex-blocking experiments identified o ne PAI-1 E box-binding protein to be upstream stimulatory factor-1 (USF-1), a member of the HLH family of transcription factors, Mutation of the CACGT G; site to TCCGTG in an 18-bp PAI-1 probe inhibited the formation of USF-l- containing complexes confirming that an intact E box motif at -160 to -165 bp in the PAI-1 promoter and, in particular, the CA residues at -165 and -1 64 are essential for USF-1 binding. Incorporation of this 2 bp change into a reporter construct containing 764 bp of the proximal PAI-1 "promoter" lig ated to a CAT gene effectively reduced (by 74%) CAT activity in cycling cel ls, An intact E box motif at nucleotides -160 to -165 in the PAI-1 promoter , thus, is an important functional element in the regulation of PAI-1 trans criptional activity in renal cells. (C) 2000 Academic Press.