Transcriptional regulation of tissue- and urokinase-type plasminogen activator genes by thrombin in human fetal lung fibroblasts
Hayakawa, Y.; Tazawa, S.; Ishikawa, T.; Niiya, K.; Sakuragawa, N.
Thrombosis and Haemostasis 74(2): 704-710
1995
ISSN/ISBN: 0340-6245 PMID: 8585010 Document Number: 444600
The mechanism of thrombin induction of tissue- and urokinase-type plasminogen activator (t-PA and u-PA) biosynthesis was investigated in cultured human fetal lung fibroblast cells, IMR-90. Northern blot analysis of total RNA from thrombin-treated cells showed marked accumulations of both t-PA and u-PA mRNA during 24 h. Nuclear run-on experiments showed that the transcription rates of both genes were increased in the thrombin-treated cells. These thrombin effects were inhibited by cycloheximide (CHX), an inhibitor of protein biosynthesis. Treatment of IMR-90 cells with CHX alone caused an increase in u-PA mRNA but not in t-PA mRNA. CHX, however, did not affect the transcription rates of both genes in the cells. Thus, on-going protein synthesis is required for increased accumulations of both t-PA and u-PA mRNA by thrombin but not for the constitutive expression of u-PA gene in IMR-90 cells. Therefore, we conclude that the accumulations of t-PA and u-PA mRNA due to thrombin result mainly from increased rates of their gene transcriptions, and that this influence is exerted in part by proteins synthesized by thrombin stimulation. Thrombin also increased plasminogen activator inhibitor type-1 (PAI-1) in the levels of both antigen and mRNA more rapidly than it increased t-PA in IMR-90 cells. In conditioned medium, most of the secreted PAI-1 seemed to form a complex with t-PA. Northern blot analysis using a PAI-2 cDNA probe showed that the levels of PAI-2 mRNA were markedly increased in response to thrombin. PAI-2 appeared not to have been secreted by the cells because the antigen was rarely detectable in the conditioned medium. Thus, thrombin increases not only plasminogen activators, but their inhibitors as well. Furthermore, the accumulations of t-PA, u-PA, PAI-I and PAI-2 mRNA are caused, to some extent, by a synthetic peptide (SFLLRNPNDKYEPF) that is a full agonist for thrombin receptor activation. This suggests that the effects of thrombin on IMR-90 cells are in part mediated by the thrombin receptor which exists on the surface of the cells.