The Structure and the Function of Plasminogen Activator Inhibitor 2

我从1990年到1992年研究了纤溶酶原激活物抑制物2(PAI-2)的结构和功能，该研究得到了日本文部科学省科学研究补助金(C)的部分支持。PL-21是一种产生PAI-2的早幼粒细胞白血病细胞株。将细胞分化促进剂佛波酯(PMA)、维甲酸(RA)、地塞米松(Dex)和重组细胞因子肿瘤坏死因子(TNF)、转化生长因子(TGF)、粒细胞集落刺激因子(G-CSF)、白介素6(IL-6)加入到PL-21细胞的培养上清液中，观察PAI-2的分化相关表达。经PMA处理后，培养上清液和细胞裂解液中PAI活性均增加约70倍。地塞米松还使细胞内PAI活性增加约6倍，与PAI-2抗原平行。与PMA一样，肿瘤坏死因子和IL-6诱导PL-21细胞…多为巨噬细胞样细胞，但不影响PAI活性。检测的其他细胞因子不能增加PAI活性。地塞米松对纤维肉瘤细胞系纤溶系统有多种影响，因为地塞米松对人纤维肉瘤细胞系PAI-2的信使核糖核酸表达降低。PMA在不诱导PAI的情况下上调u-PA的分泌，地塞米松下调u-PA的分泌是由于抑制u-PA的表达，而不是PAI的诱导。单独实验时，升高细胞内cAMP的药物对PAI-2的产生影响不大，但与PMA有协同作用。为阐明PAI-2的作用机制，采用Northern印迹杂交技术，从人胎盘c DNA文库克隆了PAI-2基因探针，研究了PMA和/或cAMP对PAI-2基因表达的影响。在PMA刺激过程中，PAI-2mRNA的稳态表达显著增加，在9h达到最大值，这种诱导作用可被放线菌酮(CHX)抑制蛋白质合成所抑制。PAI-2mRNA水平在cAMP刺激时略有升高，但与PMA相反，这种增加在24小时后仍持续，而且CHX不能抑制这种增加，相反，CHX反而增强了这种增加。Run-on实验显示，PMA处理的细胞PAI-2基因转录显著增加，而cAMP或CHX处理的细胞PAI-2基因转录没有明显增加。PMA和cAMP诱导的PAI-2mRNA表观半衰期分别约为9h和3h。ChX能稳定PMA或cAMP诱导的PAI-2mRNA表达。这些结果表明，PMA诱导的PAI-2mRNA表达需要从头合成蛋白质，并通过转录和转录后机制进行调节。然而，cAMP的作用可能是由于转录因子(S)的微弱激活，在该转录因子中不需要从头合成蛋白质。较少

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 … More 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