| Research Abstract |
I have investigated the structure and the functions of plasminogen activator inhibitor type 2 (PAI-2) from 1990 to 1992, which was supported in part by Grant-in-Aid for Scientific Research (C) from The Japanese Ministry of Education, Science and Culture. PL-21 is a promyelocytic leukemia cell line that produces PAI-2. Differentiation-linked expression of PAI- 2 was investigated by adding cell-differentiation promoting agents such as phorbol myristate acetate (PMA), retinoic acid (RA), dexamethasone (Dex), and recombinant cytokines, including tumor necrosis factor (TNF), transforming growth factor (TGF), granulocyte-colony stimulating factor (G-CSF), and interleukin-6 (IL-6)into the culture medium of PL-21 cells. PAI activity both in the cultured medium and in the cell lysate increased approximately 70-fold after exposure to PMA. Dex also increased the intracellular PAI activity approximately 6-fold, parallel with PAI-2 antigen. AS with the case of PMA, TNF and IL-6 induced PL-21 cells
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to macrophage-like cells, but did not affect the PAI activity. Other cytokines examined did not increase the PAI activity. Dex has various effects on the fibrinolytic system because it has been shown that the mRNA of PAI-2 in human fibrosarcoma cell line decreases in response to Dex.
The regulation of urokinase (u-PA) production in a human pre-B cell lymphoma line, RC-K8, by Dex and PMA was investigated. PMA up-regulated the u-PA secretion without inducing PAIs and the down-regulation of u-PA secretion by Dex resulted from the inhibition of the expression of u-PA itself but not from the induction of PAIs.
The effects of the agents which raise intracellular cyclic AMP (cAMP) and protein kinase C activators on PAI-2 production in PL-21 cells were investigated. The agents which raise intracellular cAMP little increased the PAI-2 production when tested alone, but showed synergistic effects with PMA. To clarify the mechanism, the gene expression of PAI-2 induced by PMA and/or cAMP was investigated by Northern blot hybridization technique using a PAI-2 cDNA probe cloned from human placenta cDNA library. Steady-state level of PAI-2 mRNA markedly increased during PMA-stimulation, reaching a maximum in 9 h. The induction was inhibited by inhibition of protein synthesis with cycloheximide (CHX). PAI-2 mRNA levels slightly increased during cAMP-stimulation, but contrary to the case of PMA, the increase still lasted after 24 h. Moreover, the increase was not inhibited by CHX, rather enhanced. Nuclear run on assay revealed that PAI-2 gene transcription markedly increased in PMA-treated cells, but not clearly increased in cAMP- or CHX-treated cells. The apparent half lives of PAI-2 mRNA induced by PMA and cAMP were approximately 9 h and 3 h, respectively. CHX stabilized PAI-2 mRNA induced by either PMA or cAMP. These data suggest that PAI-2 mRNA expression induced by PMA requires de novo protein synthesis and it is regulated through transcriptional and post-transcriptional mechanism. Whereas, the effects of cAMP may be due to a weak activation of a transcriptional factor(s) in which de novo protein synthesis is not required. Less
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