PHORBOL ESTER INDUCES THE RAPID PROCESSING OF CELL-SURFACE HEPARIN-BINDING EGF-LIKE GROWTH-FACTOR - CONVERSION FROM JUXTACRINE TO PARACRINE GROWTH-FACTOR ACTIVITY
PHORBOL ESTER INDUCES THE RAPID PROCESSING OF CELL-SURFACE HEPARIN-BINDING EGF-LIKE GROWTH-FACTOR - CONVERSION FROM JUXTACRINE TO PARACRINE GROWTH-FACTOR ACTIVITY
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DOI：
10.1091/mbc.6.8.967
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发表时间：
1995-08-01
影响因子：
3.3
通讯作者：
TANIGUCHI, N
中科院分区：
文献类型：
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作者：
GOISHI, K;HIGASHIYAMA, S;TANIGUCHI, N
Vero细胞肝素结合表皮生长因子样生长因子（HB-EGF）是一种20至30 kDa的膜锚定HB-EGF前体（proHB-EGF）。在过度表达重组HB-EGF的Vero细胞（Vero H细胞）中检测了组成型和12-O-十四烷酰佛波醇13-乙酸酯（TPA）诱导型proHB-EGF的定位和加工。流式细胞术和荧光免疫染色结果表明，Vero细胞proHB-EGF与细胞表面结合，定位于细胞与细胞接触的界面。细胞表面生物素化和免疫沉淀检测到一个20至30 kDa的异质性proHB-EGF物种。Vero H细胞表面proHB-EGF组成型翻转，半衰期为1.5h。一些20- 30-kDa的细胞表面相关的proHB-EGF的处理和14-kDa的生物活性HB-EGF的物种被缓慢释放，但大多数的proHB-EGF被内化，显示扩散的免疫荧光染色模式和积累的proHB-EGF的内涵体。加入TPA可诱导proHB-EGF在pro（148）-瓦尔（149）位点的快速加工，半衰期为7 min。TPA的作用可被蛋白激酶C抑制剂staurosporine和H7所消除。动力学分析表明，加入TPA后30 min，细胞表面proHB-EGF的损失最大，proHB-EGF重新合成，并在12-24 h内恢复初始细胞表面水平。细胞表面的proHB-EGF的损失是伴随着出现的14-和19-kDa的可溶性HB-EGF物质的条件培养基。Vero H细胞相关的proHB-EGF是共培养中EP 170.7细胞的促生长因子。proHB-EGF的处理导致了HB-EGF旁分泌活性的丧失和可溶性HB-EGF旁分泌促有丝分裂活性的同时增加。得出的结论是，加工调节HB-EGF的生物活性，将其从细胞表面的旁分泌生长因子转化为加工，释放的可溶性旁分泌生长因子。
Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a pro(148)-Val(149) site with a half-life of 7 min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.