Choi M E, Ballermann B J
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 1995 Sep 8;270(36):21144-50. doi: 10.1074/jbc.270.36.21144.
Transforming growth factor-beta 1 (TGF-beta 1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-beta receptors. We explored the possibility of inhibiting TGF-beta-mediated events in glomerular capillary endothelial cells using a TGF-beta type II receptor (T beta R-II) transdominant negative mutant. A mutant TGF-beta type II receptor (T beta R-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat T beta R-II cDNA as template. Since T beta R-II and TGF-beta type I receptor (T beta R-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type T beta R-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with T beta R-IIM, and four independent clones were expanded. That the T beta R-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface T beta R-IIM protein was shown by affinity cross-linking with 125I-TGF-beta 1. In wild-type endothelial cells, TGF-beta 1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 +/- 10% of control (n = 4). In transfected endothelial cells carrying T beta R-IIM, TGF-beta 1 stimulated [3H]thymidine incorporation to 131 +/- 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-beta 1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying T beta R-IIM. This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with T beta R-II signaling inhibits this process in glomerular capillary endothelial cells.
转化生长因子-β1（TGF-β1）在体内可诱导血管生成，在体外可诱导毛细血管形态发生。已鉴定出两种受体丝氨酸/苏氨酸激酶（I型和II型）为信号转导TGF-β受体。我们利用TGF-βII型受体（TβR-II）的显性负突变体，探讨了抑制肾小球毛细血管内皮细胞中TGF-β介导事件的可能性。以大鼠TβR-II cDNA为模板，通过聚合酶链反应制备了一种缺乏胞质丝氨酸/苏氨酸激酶结构域的突变型TGF-βII型受体（TβR-IIM）。由于TβR-II和TGF-βI型受体（TβR-I）会形成异二聚体进行信号转导，因此突变型受体可竞争与野生型TβR-I结合，从而以显性负性方式发挥作用。用TβR-IIM稳定转染肾小球毛细血管内皮细胞，并扩增出四个独立的克隆。通过逆转录-聚合酶链反应、核糖核酸酶保护试验和Northern分析证实了TβR-IIM mRNA的表达。通过与125I-TGF-β1进行亲和交联，显示了细胞表面TβR-IIM蛋白的存在。在野生型内皮细胞中，TGF-β1（2 ng/ml）可将[3H]胸苷掺入量显著抑制至对照的63±10%（n = 4）。在携带TβR-IIM的转染内皮细胞中，TGF-β1可将[3H]胸苷掺入量刺激至对照的131±9%（n = 4，p < 0.005）。此外，在野生型内皮细胞中，内源性和外源性TGF-β1均可诱导细胞凋亡及相关的毛细血管形成。在携带TβR-IIM的转染内皮细胞中，细胞凋亡和毛细血管形成均完全不存在。这首次证明了体外毛细血管形态发生与细胞凋亡相关，并且干扰TβR-II信号传导可抑制肾小球毛细血管内皮细胞中的这一过程。
