Growth factors and corneal endothelial cells: II. Characterization of epidermal growth factor receptor from bovine corneal endothelial cells.
Growth factors and corneal endothelial cells: II. Characterization of epidermal growth factor receptor from bovine corneal endothelial cells.
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生长因子和角膜内皮细胞：II。
DOI：
10.1097/00003226-199201000-00002
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发表时间：
1992
期刊：
影响因子：
2.8
通讯作者：
Schultz,GS
中科院分区：
文献类型：
--
作者：
Woost,PG;Jumblatt,MM;Eiferman,RA;Schultz,GS
表皮生长因子（EGF）是一种有效的角膜内皮细胞丝裂原，可能在角膜内皮伤口愈合中起作用。为了进一步表征EGF与内皮细胞的相互作用，我们测量了125I-EGF与培养的牛角膜内皮细胞（BCEC）结合的生化参数，确定了EGF诱导的蛋白磷酸化模式，并研究了视黄酸（RA）和转化生长因子（TGF-[β]）对EGF诱导的DNA合成和受体水平的影响。125I-EGF与BCEC的结合依赖于时间，在37[度]C下约2小时后达到平台，对EGF具有特异性，并且具有高亲和力（Kd= 100 pM），每个细胞约有21,000个受体。EGF受体激酶的细胞底物可能作为EGF的初始第二信使，通过十二烷基硫酸钠聚丙烯酰胺凝胶32p标记的BCEC蛋白的放射自显影检测。EGF刺激了170、37、21和20 kda蛋白的磷酸化。在无血清培养基中培养24 h的BCEC中加入1 nM、100 nM和10[微]M RA，与对照培养相比，逐渐抑制DNA合成高达80%。然而，当与5 nM的EGF联合添加时，1 nM和100 nM的RA协同刺激DNA合成，比EGF刺激水平高80%，而不会改变EGF受体水平或结合亲和力。因此，BCEC短期暴露于RA增强了egf刺激的DNA合成，很可能是通过受体后步骤起作用。在无血清培养基中培养24 h的BCEC中加入10 nM TGF-[β]，刺激低水平的DNA合成，并没有显著改变egf诱导的DNA合成，也没有改变egf诱导的蛋白质磷酸化模式。这些结果表明，BCEC中的EGF受体具有与其他哺乳动物细胞中的EGF受体相似的生化特征，包括刺激几种细胞蛋白的磷酸化，这些蛋白可能介导BCEC中EGF的生物作用，并且EGF可能与其他影响角膜内皮细胞有丝分裂的药物协同作用。
Epidermal growth factor (EGF) is a potent mitogen for corneal endothelial cells and may play a role in endothelial wound healing. To further characterize the interaction of EGF with endothelial cells, we measured biochemical parameters of 125I-EGF binding to cultured bovine corneal endothelial cells (BCEC), determined the pattern of EGF-induced protein phosphorylation, and investigated the influence of retinoic acid (RA) and transforming growth factor [beta](TGF-[beta]) on EGF-induced DNA synthesis and receptor levels. Binding of 125I-EGF to BCEC was dependent on time, reaching a plateau after~ 2 h at 37 [degrees] C, was specific for EGF, and had high affinity (Kd= 100 pM) with approximately 21,000 receptors per cell. Cellular substrates for the EGF receptor kinase, which may function as initial second messengers for EGF, were detected by autoradiography of sodium dodecyl sulfate polyacrylamide gels of 32P-labeled BCEC proteins. EGF stimulated phosphorylation of 170, 37, 21 and 20-kDa proteins. Addition of 1 nM 100 nM and 10 [micro], M RA to BCEC cultured in serum-free medium for 24 h progressively inhibited DNA synthesis by up to 80% compared with control cultures. However, when added in combination with 5 nM EGF, 1 nM and 100 nM RA synergistically stimulated DNA synthesis by up to 80% above the level of EGF stimulation without altering EGF receptor levels or binding affinity. Thus, short-term exposure of BCEC to RA potentiated EGF-stimulated DNA synthesis, most likely by acting at a postreceptor step. Addition of 10 nM TGF-[beta] to BCEC cultured in serum-free medium for 24 h stimulated low levels of DNA synthesis and did not significantly alter EGF-induced DNA synthesis nor alter EGF-induced protein phosphorylation patterns. These results demonstrate that EGF receptor in BCEC has biochemical characteristics that are similar to EGF receptors in other mammalian cells including stimulation of phosphorylation of several cellular proteins that may mediate the biological action of EGF in BCEC, and that EGF may interact synergistically with other agents that influence corneal endothelial cell mitosis.