Cao Y, Pettersson R F
Ludwig Institute for Cancer Research, Stockholm, Sweden.
Growth Factors. 1993;8(4):277-90. doi: 10.3109/08977199308991573.
Acidic fibroblast growth factor (aFGF) lacks a classical signal sequence for secretion via the exocytic pathway but yet has to be released from cells in order to interact with high affinity receptors on the cell surface. To study the release process, we have expressed human aFGF in HeLa cells using a T7 RNA polymerase-driven vaccinia virus system. The high level of expression in combination with an efficient antibody allowed us to analyze the release of aFGF by pulse-chase experiments, and to immunolocalize the protein in transfected cells. In the absence of heparin, only negligible amounts of aFGF were detected in the medium during a 15 hr chase period. However, if heparin was present during the chase, readily detectable amounts (about 10-20% of total) of aFGF were found in the medium during the 15 hr chase. Extracellular aFGF was first detected at 8 hr and increased during the chase. Concomitantly, only small amounts of lactate dehydrogenase activity, used as a cytoplasmic marker, was released from the cells. Further analyses indicated that heparin both stabilized the protein from degradation and prevented the binding of released aFGF to extracellular heparan-sulfate proteoglycans. Thus, both factors contributed to the increased recovery of aFGF in the presence of heparin. The slow and inefficient release of aFGF is consistent with our previous results obtained in insect cells expressing aFGF to a very high level, as well as with those obtained by others in cultured cells producing FGF. Immunolocalization using an affinity purified antibody made against native aFGF, showed strong fluorescence in the nuclei in most cells, while staining in the cytoplasm was usually weaker and varied between cells. The nuclear localization was confirmed by subcellular fractionation and immunoblot analysis. At an early time point following transfection (4 hr), aFGF was preferentially localized to the nuclei, while the distribution of the protein between cytoplasm and nuclei was about equal at later time points (12 hr). Thus, we conclude that aFGF is capable of efficiently entering the nucleus and apparently becoming trapped there.
酸性成纤维细胞生长因子（aFGF）缺乏通过胞吐途径分泌的经典信号序列，但仍必须从细胞中释放出来，以便与细胞表面的高亲和力受体相互作用。为了研究释放过程，我们使用T7 RNA聚合酶驱动的痘苗病毒系统在HeLa细胞中表达了人aFGF。高水平的表达与高效抗体相结合，使我们能够通过脉冲追踪实验分析aFGF的释放，并在转染细胞中对该蛋白进行免疫定位。在没有肝素的情况下，在15小时的追踪期内，培养基中仅检测到极少量的aFGF。然而，如果在追踪期间存在肝素，则在15小时的追踪期内，培养基中可检测到大量（约占总量的10 - 20%）的aFGF。细胞外aFGF在8小时首次被检测到，并在追踪过程中增加。与此同时，仅少量用作细胞质标记物的乳酸脱氢酶活性从细胞中释放出来。进一步分析表明，肝素既能稳定蛋白质使其不被降解，又能阻止释放的aFGF与细胞外硫酸乙酰肝素蛋白聚糖结合。因此，这两个因素都导致了在肝素存在下aFGF回收率的增加。aFGF缓慢且低效的释放与我们之前在高表达aFGF的昆虫细胞中获得的结果一致，也与其他人在产生FGF的培养细胞中获得的结果一致。使用针对天然aFGF制备的亲和纯化抗体进行免疫定位，在大多数细胞的细胞核中显示出强烈的荧光，而细胞质中的染色通常较弱且细胞之间存在差异。通过亚细胞分级分离和免疫印迹分析证实了核定位。在转染后的早期时间点（4小时），aFGF优先定位于细胞核，而在后期时间点（12小时），蛋白质在细胞质和细胞核之间的分布大致相等。因此，我们得出结论，aFGF能够有效地进入细胞核并显然被困在那里。
