Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells
Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells
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尿激酶型纤溶酶原激活剂及其 1 型抑制剂在培养的人成纤维细胞和肉瘤细胞中的独特定位
作者：
J. Pöllänen;O. Saksela;E. Salonen;P. Andreasen;L. Nielsen;K. Danø;A. Vaheri
我们研究了尿激酶型纤溶酶原激活物（u-PA）和1型纤溶酶原激活物抑制剂（派-1）在人成纤维细胞和肉瘤细胞的免疫细胞化学定位，使用多克隆和单克隆抗体。发现u-PA位于离散的细胞-基质接触部位，并且也位于细胞-细胞接触区域，而派-1分布为均匀的地毯，不包括细胞下基质上的条纹状区域。为了确认u-PA和派-1的细胞外定位，我们在固定前在0 ℃下对细胞进行活体染色。双标实验显示u-PA和派-1在细胞下的分布不同，尤其是在细胞周围。u-PA和派- 1的染色模式抵抗用0.2%皂苷处理，然后机械去除细胞，这是先前报道的分离成纤维细胞的焦点接触膜的方法。我们进一步证明了通过酶谱法将u-PA沉积到通过皂苷处理获得的细胞的接触区域，并且通过代谢标记、反向酶谱法、免疫印迹和免疫沉淀来证实派-1的沉积。纤连蛋白也存在于制剂中。用10（-6）M地塞米松处理肉瘤细胞后，派-1和纤连蛋白的沉积均增强。限制u-PA离散的接触点和派-1的细胞基质上的更均匀的分布可以解释细胞产生大量的酶抑制剂可以产生PA介导的局灶性蛋白水解。
We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell- cell contacts, whereas PAI-1 was distributed as a homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI- 1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u- PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10(-6) M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PA-mediated focal proteolysis.
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DOI：
10.1016/s0021-9258(17)42691-3
发表时间：
1984-12
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J. V. van Mourik;D. Lawrence;D. Loskutoff
通讯作者：
J. V. van Mourik;D. Lawrence;D. Loskutoff
成纤维细胞与含纤连蛋白的基质的紧密接触和局部接触粘附。
DOI：
--
发表时间：
1985
期刊：
Federation proceedings
影响因子：
--
作者：
Lark,MW;Laterra,J;Culp,LA
通讯作者：
Culp,LA
DOI：
10.1016/s0021-9258(18)88883-4
发表时间：
1985-06
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Randy W. Scott;Barbara L. Bergman;Ani 1 Bajpai;R. Hersh;Henry Rodriguez;Barry N. Jonesd;Carlos Barredad;S. Watts;Joffre B. Bakera
通讯作者：
Randy W. Scott;Barbara L. Bergman;Ani 1 Bajpai;R. Hersh;Henry Rodriguez;Barry N. Jonesd;Carlos Barredad;S. Watts;Joffre B. Bakera
DOI：
--
发表时间：
1984-05
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
D. Eaton;R. W. Scott;J. Baker
通讯作者：
D. Eaton;R. W. Scott;J. Baker