Bonner J C, Badgett A, Hoffman M, Lindroos P M
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1995 Mar 17;270(11):6389-95. doi: 10.1074/jbc.270.11.6389.
alpha 2-Macroglobulin (alpha 2M) is a potentially important regulator of platelet-derived growth factor-BB (PDGF-BB)-stimulated cell growth due to our previous observation that PDGF-BB binds to alpha 2M noncovalently (Bonner, J. C., Goodell, A. L., Lasky, J. A., and Hoffman, M. R. (1992) J. Biol. Chem. 267, 12837-12844). We examined the in vitro effect of native and plasmin-activated (receptor-recognized) alpha 2M on the PDGF-BB-induced proliferation of mouse Swiss 3T3 and rat lung fibroblasts. Nondenaturing polyacrylamide gel electrophoresis showed that plasmin converted alpha 2M to its electrophoretically "fast" form at a 2:1 molar ratio and that 125I-PDGF-BB bound both alpha 2M and alpha 2M-plasmin. PDGF-BB-induced growth was not affected by native alpha 2M (0.3 microM) or plasmin (0.6 microM). The combination of plasmin and alpha 2M (2:1 molar ratio) inhibited PDGF-BB-induced cell proliferation 80-90%. Complexes of PDGF-BB.alpha 2M purified by gel filtration chromatography retained growth promoting activity, but the PDGF-BB.alpha 2M-plasmin complex did not. Preincubation of fibroblasts (37 degrees C for 24 h) with alpha 2M-plasmin did not change 125I-PDGF-BB binding or affect gene expression of the 6.5-kilobase PDGF-alpha receptor or 5.2-kilobase PDGF-beta receptor mRNA. However, preincubation with alpha 2M-plasmin (0-4 degrees C for 4 h) increased 125I-PDGF-BB binding 2-fold, and this increase was blocked by a receptor-associated protein antagonist of the alpha 2M-receptor/low density lipoprotein receptor-related protein. The receptor-associated protein antagonist blocked 125I-alpha 2M-methylamine binding, inhibited PDGF-BB-alpha 2M-plasmin uptake from fibroblast-cultured supernatants, and abolished the inhibitory effect of alpha 2M-plasmin on PDGF-stimulated growth. These data suggest that inhibition of PDGF-stimulated proliferation by alpha 2M-plasmin is mediated in part by clearance of PDGF-BB-alpha 2M-plasmin through the lipoprotein receptor-related protein.
α2-巨球蛋白（α2M）可能是血小板衍生生长因子-BB（PDGF-BB）刺激细胞生长的重要调节因子，因为我们之前观察到PDGF-BB与α2M非共价结合（Bonner, J. C., Goodell, A. L., Lasky, J. A., and Hoffman, M. R. (1992) J. Biol. Chem. 267, 12837 - 12844）。我们研究了天然型和纤溶酶激活型（受体可识别型）α2M对PDGF-BB诱导的小鼠瑞士3T3细胞和大鼠肺成纤维细胞增殖的体外作用。非变性聚丙烯酰胺凝胶电泳显示，纤溶酶以2:1的摩尔比将α2M转化为其电泳“快”形式，并且125I-PDGF-BB能与α2M和α2M-纤溶酶结合。PDGF-BB诱导的生长不受天然α2M（0.3微摩尔）或纤溶酶（0.6微摩尔）的影响。纤溶酶和α2M（2:1摩尔比）的组合可抑制PDGF-BB诱导的细胞增殖80 - 90%。通过凝胶过滤色谱法纯化的PDGF-BB·α2M复合物保留了生长促进活性，但PDGF-BB·α2M-纤溶酶复合物则没有。将成纤维细胞在37℃预孵育24小时与α2M-纤溶酶一起，不会改变125I-PDGF-BB的结合，也不影响6.5千碱基的PDGF-α受体或5.2千碱基的PDGF-β受体mRNA的基因表达。然而，在0 - 4℃下与α2M-纤溶酶预孵育4小时会使125I-PDGF-BB的结合增加2倍，并且这种增加被α2M受体/低密度脂蛋白受体相关蛋白的受体相关蛋白拮抗剂所阻断。该受体相关蛋白拮抗剂阻断125I-α2M-甲胺的结合，抑制从成纤维细胞培养上清液中摄取PDGF-BB-α2M-纤溶酶，并消除α2M-纤溶酶对PDGF刺激生长的抑制作用。这些数据表明，α2M-纤溶酶对PDGF刺激的增殖的抑制作用部分是通过脂蛋白受体相关蛋白清除PDGF-BB-α2M-纤溶酶来介导的。
