The Structure and the Function of Plasminogen Activator Inhibitor 2

纤溶酶原激活剂抑制剂2的结构和功能

• 批准号: 02671129
• 负责人: NIIYA Kenji
• 金额: $ 1.6万
• 依托单位: Toyama Medical & Pharmaceutical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02671129/
• 关键词: PAI-2; Protein kinase C activator; cyclic AMP; Leukemia cell line; Northern blot; Transcription factor; Dexamethasone; Urokinase; 転写活性; 転写因子AP-1; Cーfos; junーB; プラスミノゲン・アクチベ-タ・インヒビタ-; フォルボ-ル・エステル; サイクリックAMP; プロスタグランディンE1; ジアシルグリセロ-ル; 相乗効果

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

1990年至1992年，在日本文部科学省科学研究补助金（C）的资助下，我对纤溶酶原激活物抑制剂2（派-2）的结构和功能进行了研究。PL-21是产生派-2的早幼粒细胞白血病细胞系。通过向PL-21细胞培养基中加入细胞分化促进剂如佛波醇肉豆蔻酸酯乙酸酯（PMA）、维甲酸（RA）、地塞米松（Dex）和重组细胞因子包括肿瘤坏死因子（TNF）、转化生长因子（TGF）、粒细胞集落刺激因子（G-CSF）和白细胞介素-6（IL-6）来研究派- 2的分化相关表达。派活性在培养基和细胞裂解液中增加约70倍后，暴露于PMA。地塞米松也增加细胞内派活性约6倍，与派-2抗原平行。AS与PMA、TNF和IL-6诱导PL-21细胞的情况相同 ...更多信息 巨噬细胞样细胞，但不影响派活性。其他细胞因子检查没有增加派活性。Dex对纤溶系统有不同的影响，Dex可使人纤维肉瘤细胞派-2 mRNA表达减少，本文研究了Dex和PMA对人前B细胞淋巴瘤细胞株RC-K8产生尿激酶（u-PA）的调节作用。PMA可上调u-PA的分泌，但不诱导PAI的产生; Dex可通过抑制u-PA的表达而下调u-PA的分泌，但不诱导PAI的产生。提高细胞内cAMP的药物单独使用时几乎不增加派-2的产生，但与PMA显示出协同作用。为了阐明其机制，采用从人胎盘cDNA文库中克隆的派-2 cDNA探针，通过北方印迹杂交技术研究了PMA和/或cAMP诱导的派-2基因表达。派-2 mRNA的稳态水平在PMA刺激时显著升高，在9 h达到最高。用放线菌酮（CHX）抑制蛋白质合成可抑制诱导。派-2 mRNA水平在cAMP刺激过程中略有增加，但与PMA的情况相反，增加仍持续24小时后。此外，CHX并未抑制这种增加，而是增强了这种增加。细胞核Run-on分析显示，PMA处理的细胞派-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诱导派-2 mRNA的表观半衰期分别约为9 h和3 h。CHX稳定PMA或cAMP诱导的派-2 mRNA。这些数据表明，PMA诱导的派-2 mRNA表达需要从头蛋白质合成，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的弱激活，其中不需要从头蛋白质合成。少

项目成果

Kenji NIIYA, et al: "Dexamethasone Down-regulates the Urokinase Secretion in a Human Lymphoma Cell Line RC-K8 without Inducing the Plasminogen Activator Inhibitors" Thrombosis Research. 63. 311-321 (1992)

Kenji NIIYA 等人：“地塞米松下调人淋巴瘤细胞系 RC-K8 中的尿激酶分泌，而不诱导纤溶酶原激活剂抑制剂”血栓形成研究。

T HAYASHI,K NIIYA,et al: "Synergistic Stimulating Effect between Cyclic AMP and phorbol Ester on Plasminogen Activator Inhibitor Type 2 Production in human Promyelocytic Leukemia Cell Line..." Biochimica et Biophtsica Acta. 1134. 273-277 (1992)

T HAYASHI、K NIIYA 等人：“环 AMP 和佛波醇酯对人早幼粒细胞白血病细胞系中纤溶酶原激活剂抑制剂 2 型产生的协同刺激作用……”Biochimica et Biophtsica Acta。

Kenji Niiya: "The regulation of plasminogen activator inhibitor-2 production in a leukemia cell line (in Japanese)." Jpn J of Clinical Hematology. 32. 490-496 (1991)

Kenji Niiya：“白血病细胞系中纤溶酶原激活剂抑制剂 2 产生的调节（日语）。”

Kenji Niiya and Nobuo Sakuragawa: "Effects of cAMP and phorbol ester on the productions of urinary type plasminogen activator and its inhibitor in human lymphoma and leukemia cell lines (in Japanese)." Nippon Rinshyou. 50. 325-329 (1992)

Kenji Niiya 和 Nobuo Sakurakawa：“cAMP 和佛波酯对人淋巴瘤和白血病细胞系中尿型纤溶酶原激活剂及其抑制剂产生的影响（日语）。”

Kenji Niiya.et al: "Dexamethasone Downーregulates the Urokinase Secretion in a Human Lymphoma Cell Line RCーK8 without Inducing the Plasminogen Activator Inhibitors" Thrombosis Research. 63. 311-321 (1992)

Kenji Niiya.等人：“地塞米松下调人淋巴瘤细胞系 RC-K8 中的尿激酶分泌，而不诱导纤溶酶原激活剂抑制剂”血栓形成研究 63. 311-321 (1992)。