The human PAI-1 and PAI-2 genes are markedly induced by tumour necrosis factor (THF) and the phorbol ester, PMA in HT-1080 fibrosarcoma cells. Treatment of cells with a combination of TNF and PMA results in a selective synergistic induction of PAI-2 mRNA. The fold increase in PAI-2 mRNA levels induced by these agents is not fully reflected at the level of gene transcription as determined by nuclear run-on assays suggesting involvement of posttranscriptional events. The 3′untranslated region (3′UTR) of PAI-2 mRNA harbors a number of AUUUA motifs as well as a UUAUUUAUU nonamer known to confer mRNA instability in other systems. We hypothesized that the profound increase in PAI-2 mRNA in response to TNF and PMA was a result, at least in part, of inducible mRNA stabilization. To study PAI-2 mRNA decay, we inserted the 3'UTR of PAI-2 into the 3'UTR of a rabbit globin gene linked to the cytomegalovirus promoter. The resulting construct (Glo-PAI2.UTR) as well as the wild-type globin construct was stably introduced into HT-1080 cells and the levels of globin mRNA produced in the presence and absence of the transcription inhibitors DRB or actinomycin-D (Act-D) were compared. As a positive control for conferring mRNA decay, we used a globin construct containing the 3'UTR of urokinase (u-PA) (1). Results indicated firstly, that the 3'UTR of both PAI-2 and u-PA resulted in a lowering in basal globin mRNA. Surprisingly. Act-D or DRB treatment of cells abolished globin mRNA produced by the Glo-uPA.UTR vector, but did not alter levels generated by GloPAI2.UTR suggesting that the destabilizing effect of the PAI-2 3'UTR requires on-going transcription. Preliminary results also indicate that Glo-PAI2.UTR expressing cells treated with TNF+PMA produce an increase in globin mRNA suggesting that inducible cytoplasmic factors recognize regions within the PAI2 3'UTR to promote stabilization. Our results indicate that the 3'UTR of PAI-2 harbors regulatory regions which influence basal expression and may also play a role in the stabilization of PAI-2 by TNF and PMA.
|Number of pages||1|
|Issue number||SUPPL. 3|
|Publication status||Published - 1 Dec 1996|