Molecular basis for the aberrant expression of the PAI-2 gene in THP-1 monocytes

Plasminogen activator inhibitor type 2 (PAI-2) is a urokinase inhibitor that is expressed primarily in monocytes. THP-1 monocytes, however, contain a unique defect in the production of PAI-2 in that the PAI-2 transcript is truncated and the expressed protein inactive. ' In addition, treatment of THP-1 cells with either phorbol ester, LPS or TNF causes a reduction in the level of expression of PAI-2 mRNA which is contrary to the regulation reported elsewhere. We designed experiments to determine the basis of this mutation. Southern blot analysis of THP-1-derived genomic DNA indicated that there were no obvious deletions in the structure of the PAI-2 gene. However, assessment of the THP-1-derived PAI-2 transcript by RT-PCR indicated that only exons 7 and 8 of the normal PAI2 mRNA could be detected. Cloning of the 5' region of the PAI-2 mRNA by 5'-RACE indicated that the PAI-2 cDNA derived from THP-1 cells comprised of 180 nt of genomic sequence derived from intron 5 of the PAI-2 gene, followed by sequences corresponding to exons 7 and 8 of the normal PAI-2 mRNA. The presence of the intron 5 fragment in endogenous THP-1 derived PAI-2 mRNA was confirmed by Northern blotting. The complete absence of any wild-type PAI-2 mRNA in these cells suggests that one copy of the PAI-2 allele has been deleted. The remaining allele producing the truncated mRNA appears to have undergone a translocation event that has removed genomic DNA downstream of a position within intron 5 and probably includes part of the PAI-2 promoter. In addition, at least one other mutation has occurred that has disrupted the splicing of the PAI-2 primary transcript permitting the PAI-2 intron 5 fragment to be juxtaposed to exon 7. We are presently determining whether the introduction of wild-type active PAI-2 into THP-1 cells can influence the behaviour of these cells in general and their ability respond to differentiation inducing agents in particular.