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) cells occurs as part of the immediate-early response to serum. PAI-1 transcripts are maximally expressed early in G(1) (within 4 h of serum addition to quiescent EC-1 cells) and then subsequently decline to basal levels prior to entry into DNA synthetic phase. Comparative analysis of PAI-1 mRNA abundance 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 with RNA decay measurements indicated that PAI-1 gene regulation upon growth activation was predominantly transcriptional. An E box motif (CACGTG), important in the induced expression of some growth state-dependent genes, mapped to nucleotides -160 to -165 upstream of the transcription start site in the PAI-1 proximal promoter. Mobility-shift assessments, using a 18-bp deoxyoligonucleotide construct containing the E box within the context of PAI-1-specific flanking sequences, confirmed binding of EC-1 nuclear protein(s) to this probe and, specifically, to the E box hexanucleotide site. The specificity of this protein-probe interaction was verified by competition analyses with double-stranded DNA constructs that included E box deoxyoligonucleotides with non-PAI-1 flanking bases, mutant E box sequences incapable of binding NRK nuclear proteins, and unrelated (i.e., AP-1) target motifs. Extract immunodepletion and supershift/complex-blocking experiments identified one 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 CACGTG site to TCCGTG in an 18-bp PAI-1 probe inhibited the formation of USF-1-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 -164 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" ligated to a CAT gene effectively reduced (by 74%) CAT activity in cycling cells. 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 transcriptional activity in renal cells.

White LA ，Bruzdzinski C ，Kutz SM ，Gelehrter TD ，Higgins PJ ... -  《EXPERIMENTAL CELL RESEARCH》

Upstream stimulatory factor regulates E box-dependent PAI-1 transcription in human epidermal keratinocytes.

Certain growth factors (e.g., TGF-beta1) initiate a "plastic" response in human keratinocytes (HaCaT cells) characterized by changes in gene expression and increased cell motility. While microarray analyses identified a number of involved genes, plasminogen activator inhibitor type 1 (PAI-1) is among the subset most highly responsive to TGF-beta1. Previous antisense attenuation of PAI-1 synthesis confirmed an essential role for this protease inhibitor in cell motility (Providence et al., 2002, J Cell Sci 115:3767-3777; Providence and Higgins, 2004, J Cell Physiol 200:297-308). It was important, therefore, to clarify molecular mechanisms underlying PAI-1 expression control in human keratinocytes. A consensus E box motif (5'-CACGTG-3') at nucleotides -566 to -561 in the PE2 region of the PAI-1 gene was required for TGF-beta1-induced transcription of a PAI-1 promoter-driven luceriferase reporter. Truncation of the PE2 E box or mutation of the CACGTG hexanucleotide to CAATTG inhibited growth factor-stimulated promoter function confirming the importance of this site in inducible expression. A similar mutation at the PE1 E box (nucleotides -682 to -677), in contrast, did not result in reduced luciferase activity. Competing CACGTG-containing DNAs, regardless of the presence or absence of PAI-1-specific flanking sequences or lacking accessory sequences (i.e., Smad-binding sites, AAT trinucleotide spacer), inhibited complex formation between HaCaT cell nuclear factors and a 45-mer PE2 region probe. A deoxyoligonucleotide that differed from the consensus E box by a CG --> AT substitution (the same base change incorporated into the PAI-1p806-lucerifase reporter by site-directed mutagenesis) but with random (i.e., non-PAI-1) flanking sequences also failed to compete with the PE2 region probe for protein binding whereas the same construct with an intact CACGTG motif was an effective competitor. The major protein/DNA interactions in the PE2 segment, therefore, are E box-dependent. USF-1, a member of the upstream stimulatory factor family, bound the PE2 construct suggesting a role for USF proteins in E box residence and PAI-1 gene expression. Chromatin immunoprecipitation, using primers designed to amplify a 300-bp PE2-associated promoter fragment and containing no other E box motifs except the target CACGTG at nucleotides -566 to -561, confirmed that this site was occupied by USF-1 or a USF-1-containing complex in both quiescent and TGF-beta1-stimulated cells. Transfection of a dominant-negative USF construct effectively attenuated serum- and TGF-beta1-induced PAI-1 synthesis as well as TGF-beta1-stimulated Matrigel barrier invasion. Dominant-negative USF-expressing keratinocytes, moreover, specifically had a reduced capacity for Matrigel barrier invasion. USF elements, therefore, are important regulators of growth factor-initiated PAI-1 transcription (as predicted from the identification of PAI-1 as a direct USF target gene) and the associated epithelial migratory response.

Allen RR ，Qi L ，Higgins PJ 《JOURNAL OF CELLULAR PHYSIOLOGY》

Antisense targeting of c-fos transcripts inhibits serum- and TGF-beta 1-stimulated PAI-1 gene expression and directed motility in renal epithelial cells.

Plasminogen activator inhibitor type-1 (PAI-1), the major regulator of pericellular plasmin generation, and the c-FOS transcription factor are expressed by migrating cells in response to monolayer wounding. Induced c-fos and PAI-1 transcripts were evident within 30 min and 2 h, respectively, of scrape injury to confluent, growth-arrested, cultures of NRK epithelial cells. Since c-FOS/AP-1 DNA-binding activity modulates both basal and inducible modes of PAI-1 gene control, and AP-1 motif binding factors were present in quiescent as well as stimulated NRK cells, a model of directionally regulated cell movement (migration into scrape-denuded "wounds") was used to assess the consequences of c-fos transcript targeting on PAI-1 expression and cell motility. This in vitro model of epithelial injury closely approximated in vivo wound repair with regard to the spatial and temporal emergence of cohorts of cells involved in migration, proliferation, and PAI-1 expression. Stable cell lines (NRKsof) were generated by transfection of parental NRK cells with a c-fos antisense expression vector. Serum-inducible c-fos transcripts and PAI-1 protein levels were significantly attenuated in NRKsof transfectants relative to parental controls or cells transfected with a neo(R) vector without the sof insert. NRKsof cells had a markedly impaired ability to repair scrape-generated monolayer wounds under basal, serum-stimulated, or TGF-beta 1-supplemented culture conditions. Since injury closure and PAI-1 induction were attenuated in c-fos antisense cells, it was important to clarify the role of specific AP-1 sites in serum-mediated PAI-1 transcription. PAI-1 "promoter"-driven CAT reporter expression was assessed within the real time of serum-stimulated PAI-1 induction. A segment of the PAI-1 promoter corresponding to nucleotides -533 to -764 upstream of the transcription start site functioned as a prominent serum-responsive region (SSR). The 9-fold increase in CAT mRNA levels attained with the -533 to -764 bp PAI-1 SRR ligated to a minimal PAI-1 promoter (i.e., 162 bp of 5' flanking sequence containing the basal transcription complex) closely approximated the serum-induced transcriptional activity of a fully responsive (1,230 bp) PAI-1 promoter construct as well as the endogenous PAI-1 gene. AP-1-like, CTF/NF-1-like, and AP-2 sites were identified in the SRR. The SRR AP-1 motif was homologous to the sequence TGACACA that mapped between nucleotides -740 and -703 in the human PAI-1 gene, a region essential for growth factor-inducible PAI-1 transcription. While the functionality of this AP-1 site in wound-regulated PAI-1 synthesis remains to be determined, antisense c-fos transcripts effectively attenuated PAI-1 induction and basal as well as growth factor-stimulated cell locomotion, indicating that expression of both the PAI-1 and c-fos genes is necessary for wound-initiated NRK cell migration.

Kutz SM ，Providence KM ，Higgins PJ 《cell motility and the cytoskeleton》

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

Kutz SM ，Higgins CE ，Samarakoon R ，Higgins SP ，Allen RR ，Qi L ，Higgins PJ ... -  《EXPERIMENTAL CELL RESEARCH》

USF in the Lytechinus sea urchin embryo may act as a transcriptional repressor in non-aboral ectoderm cells for the cell lineage-specific expression of the LpS1 genes.

Seid CA ，George JM ，Sater AK ，Kozlowski MT ，Lee H ，Govindarajan V ，Ramachandran RK ，Tomlinson CR ... -  《JOURNAL OF MOLECULAR BIOLOGY》