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

J. Katsikis, H. Yu, F. Maurer, R. L. Medcalf

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Abstract

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.

Original languageEnglish
Number of pages1
JournalFibrinolysis and Proteolysis
Volume14
Issue numberSUPPL. 1
Publication statusPublished - 1 Dec 2000
Externally publishedYes

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