Expression of PAI-1 mRNA and PAI-1 reporter studies.

(A) Relative quantitative PCR (Q-PCR) analysis of PAI-1 mRNA expression. HUVECs were stimulated with TNFα for the indicated periods and harvested. Relative expression of PAI-1 was normalized to the expression of β-2 microglobulin. The inset shows nuclear run-on data. HUVECs were either untreated or treated for 4 hours with TNFα, and the RNA isolated after the run-on reaction was hybridized to PAI-1 and GAPDH probes that were immobilized on membranes. (B) Reporter gene analysis. A series of deletions in the PAI-1 promoter were fused to a luciferase reporter gene, transfected into HUVECs, and measured by luminometry. HUVECs expressing the reporter gene constructs were induced with TNFα. (C) EMSAs of overlapping parts of the PAI-1 promoter. Nuclear extracts were obtained from HUVECs and incubated with labeled double-stranded oligonucleotides representing the nt −280 to −260, −270 to −250, and −260 to −240 of the PAI-1 promoter, respectively. Specific binding activity of nuclear protein complexes is indicated by black arrows. (D) Overview of the region of the PAI-1 promoter containing the NBRE. Oligonucleotide sequences that were used for EMSAs and for the yeast screens are shown. Light gray indicates care consensus sequence; dark gray, 2 additional adenines required for binding; and black, nucleotides exchanged.