REGULATION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 mRNA IN HUMAN ENDOTHELIAL CELLS

Abstract

Cultured human endothelial cells (HEC) increase their production of plasminogen activator inhibitor (PAI-1) upon stimulation with endotoxin and IL-1, agents that are known to cause an increase in PAI-1 levels in vivo. In order to study the regulation of PAI-1 synthesis at the mRNA level, we isolated a cDNA clone for the human PAI-1 gene from an endothelial expression cDNA library in λ gt 11 by screening with a PAI-1 specific antibody. Three positive cross-hybridizing clones were isolated. The longest insert (1500 bp) was partially sequenced (1000 bp). The sequence was identical to the PAI-1 sequence recently reported by others. The identity of the cDNA clone was further confirmed by comparison with part of the amino acid sequence of PAI-1. For that purpose t-PA-PAI-1 complex was purified from HEC conditioned medium by immunoadsorption to anti-t-PA IgG, and a suitable peptide was sequenced after comparison of the HPLC elution profiles of CNBr digests of t-PA and t-PA-PAI-1 complex. The amino acid sequence (M)FRQFQADFT completely matches the sequence predicted from the cDNA sequence. By hybridization of the cDNA probe to Northern blots of total cellular RNA from human umbilical vein and artery EC (HUVEC, HUAEC), two transcripts of 2.3 and 3 kb were found. Primary HUAEC, incubated for 18 hours in growth medium, produced considerable although variable levels of PAI-1 activity and contained PAI-1 mRNA levels comparable to those found in subcultured HUAEC. When subcultured HUEC were incubated for 6 h with endotoxin, IL-1 or TNF, a 2-fold increase in PAI-1 mRNA was found with each of these mediators. Stimulation of the cells in the presence of cycloheximide resulted in a further increase of the 3 kb PAI-1 transcript. The 3’ end of this transcript contains a 75 bp AT-rich sequence. Similar 3’ AT-rich sequences have been found in mRNA’s for a number of inflammatory mediators and cellular oncogenes, and in some cases it has been shown that removal of the sequence increased mRNA stability. The influence of cyclohex-imid on the larger PAI-1 transcript might be explained by inhibition of synthesis of a specific nuclease that controls the level of mRNA’s harbouring such an AT rich sequence.