# Targeted Interference with USF2 Binding to the SERPINE1 Proximal Promoter E-Box in Dual Mutant p53R282Q,H179Y Human Keratinocytes Inhibits Serum-/TGF-β1-Induced SERPINE1 Expression and Stimulates Epithelial Cell Proliferation

## TL;DR

Blocking USF2 binding to a specific DNA site in skin cells reduces SERPINE1 expression and boosts cell growth.

## Contribution

Demonstrates that USF2 binding to the SERPINE1 promoter regulates cell proliferation in p53 mutant keratinocytes.

## Key findings

- Interfering with USF2 at the PE2 E box reduces SERPINE1 expression in response to serum and TGF-β1.
- Reduced USF2 occupancy increases the number of proliferating keratinocytes.
- Overexpression of USF2 or PAI-1 inhibits HaCaT cell colony growth.

## Abstract

The SERPINE1 gene encodes the serine protease inhibitor plasminogen activator inhibitor type-1 (PAI-1), a major negative regulator of the plasmin-dependent pericellular proteolytic cascade and a crucial determinant in the program of stromal remodeling. Recent omics approaches confirmed that high tumor SERPINE1 levels are prognostic for poor disease outcomes and shorter disease-free survival in various malignancies. Kinetic analysis of biomarkers of cell cycle transit in growth-synchronized p53 dual mutant human keratinocytes confirmed that PAI-1 transcription occurred early after growth activation of quiescent (G0) cells and prior to G1 entry. Previous evidence has confirmed that differential residence of USF family members (USF1→USF2 switch) at the PE2 region hexanucleotide E box motif (CACGTG) in the SERPINE1 proximal promoter characterizes the G0→G1 transition period and the transcriptional status of the SERPINE1 gene. A consensus PE2 E box motif (5′-CACGTG-3′) at nucleotides −566 to −561 is required for USF occupancy of the PE2 E box and serum-stimulated SERPINE1 transcription. Interference with USF2 occupancy of the PE2 E Box site by a double-stranded PE2 “decoy”, or induced expression of a dominant-negative USF (A-USF) construct, attenuate serum- and TGF-β1-stimulated SERPINE1 synthesis. Tet-Off activation of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of proliferating (Ki-67+ cells). Conversely, overexpression of USF2 or adenoviral delivery of a PAI-1 vector inhibited HaCaT colony expansion. These findings are discussed in this review and collectively suggest that the USF1→USF2 transition at the PE2 E box site and subsequent SERPINE1 transcription impact serum-stimulated keratinocyte growth and, likely, cell cycle progression.