Kelly J L, Sánchez A, Brown G S, Chesterman C N, Sleigh M J
CSIRO Division of Biomolecular Engineering, North Ryde, Sydney, Australia.
J Cell Biol. 1993 Jun;121(5):1153-63. doi: 10.1083/jcb.121.5.1153.
PDGF is a powerful mitogen initially identified within platelets, but also shown to be produced by a wide variety of cell types. PDGF is encoded on two separate genes. These give rise to three polypeptides, PDGF B and two forms of PDGF A (SA and LA), resulting from alternative splicing of the PDGF A gene primary transcript. We report that in CHO cells transfected with PDGF gene constructs and producing moderate levels of PDGF homodimers, much of the PDGF LA and B produced, but little if any SA, is found in the matrix laid down beneath the cells. Immunoreactive PDGF in cells, and in matrix below expressing cells, was visualized by laser confocal microscopy. Western blotting of protein in matrix extracts, cell extracts, and secreted into the growth medium was used to demonstrate that the range of PDGF A polypeptides seen in the matrix was overlapping with those reported previously to be cell associated in cell types such as NIH3T3 and COS 7. This suggests that attachment to matrix or cell surface may be alternative fates for these polypeptides, with fate dependent on the characteristics of the producing cells. Immunoreactive PDGF A and B could be partially released by incubation of matrix material with heparin but not with other glycosaminoglycans. Digestion of matrix with chondroitin ABC lyase but not heparitinase or collagenase displaced some PDGF from its attachment sites. The results indicate attachment of PDGF to matrix proteoglycans, at least partly through the glycosaminoglycan moieties, and perhaps to additional components. The significance of matrix deposition for PDGF action is discussed.
血小板衍生生长因子（PDGF）是一种强大的促有丝分裂原，最初在血小板中被鉴定出来，但也被证明可由多种细胞类型产生。PDGF由两个独立的基因编码。这些基因产生三种多肽，即PDGF B和两种形式的PDGF A（SA和LA），这是由PDGF A基因初级转录本的可变剪接产生的。我们报告称，在转染了PDGF基因构建体并产生中等水平PDGF同型二聚体的CHO细胞中，所产生的大部分PDGF LA和B，但几乎没有SA（如果有的话），存在于细胞下方沉积的基质中。通过激光共聚焦显微镜观察细胞内以及表达细胞下方基质中的免疫反应性PDGF。对基质提取物、细胞提取物以及分泌到生长培养基中的蛋白质进行蛋白质印迹分析，以证明在基质中看到的PDGF A多肽范围与先前报道的在诸如NIH3T3和COS 7等细胞类型中与细胞相关的多肽范围重叠。这表明这些多肽附着于基质或细胞表面可能是不同的归宿，其归宿取决于产生细胞的特性。免疫反应性PDGF A和B可通过将基质材料与肝素一起孵育而部分释放，但与其他糖胺聚糖一起孵育则不能。用软骨素ABC裂解酶而不是肝素酶或胶原酶消化基质会使一些PDGF从其附着位点上移位。结果表明PDGF至少部分通过糖胺聚糖部分附着于基质蛋白聚糖，也许还附着于其他成分。文中讨论了基质沉积对PDGF作用的意义。
