Heldman A W, Kandzari D E, Tucker R W, Crawford L E, Fearon E R, Koblan K S, Goldschmidt-Clermont P J
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md, USA.
Circ Res. 1996 Feb;78(2):312-21. doi: 10.1161/01.res.78.2.312.
Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21ras (EJ-Ras) produces morphogenic changes characterized by decreased attachment to the substratum, with retraction and rounding of the cell body. Transformed fibroblasts lose their "stressed" conformation and adopt a "relaxed" morphology. The specific molecular mechanisms responsible for these changes remain uncharacterized. We found that EJ-Ras transformation of NIH 3T3 cells decreased the cellular content of polymerized actin, particularly at the expense of actin stress fibers, but induced the accumulation of actin filaments in peripheral ruffling membranes. Polymerization of actin could be induced in EJ-Ras-transformed cells by exposure to platelet-derived growth factor (PDGF)-BB to an extent similar to that observed in wild-type NIH 3T3 cells. In EJ-Ras cells, actin polymerization was independent of phospholipase C gamma 1 (PLC gamma 1) activity, because inositol tris-phosphate (IP3) production observed in control NIH 3T3 cells in response to PDGF-BB was absent. Although PDGF-BB did stimulate tyrosine phosphorylation of PLC gamma 1, the phospholipase was strongly inhibited by an inhibitory factor present in the cytoplasm of EJ-Ras-transformed cells. In addition, cytoplasmic extracts of EJ-Ras, but not of control cells, inhibited phosphatidylinositol 4,5-diphosphate (PIP2) hydrolysis catalyzed by a recombinant PLC gamma 1 in vitro. Although PIP2 hydrolysis could not contribute to the reorganization of the actin cytoskeleton induced by PDGF-BB in EJ-Ras-transformed cells, phosphatidylinositol 3-kinase (PI3-K) was necessary for actin polymerization. Wortmannin, a specific PI3-K inhibitor, not only blocked actin polymerization in both control and EJ-Ras-transformed cells but actually led to rapid actin depolymerization when these cells were exposed to PDGF-BB. Thus, in EJ-Ras-transformed cells, cell morphogenic changes in response to PDGF-BB rely importantly on PI3-K and can occur in the complete absence of IP3 production despite tyrosine phosphorylation of PLC gamma 1.
持续激活的与GTP结合的p21ras同工型（EJ-Ras）对成纤维样细胞（NIH 3T3）的转化会产生形态发生变化，其特征是细胞与基质的附着减少，细胞体回缩并变圆。转化后的成纤维细胞失去其“应激”构象，呈现“松弛”形态。导致这些变化的具体分子机制仍不清楚。我们发现，EJ-Ras对NIH 3T3细胞的转化降低了聚合肌动蛋白的细胞含量，尤其是以肌动蛋白应力纤维为代价，但诱导了外周褶皱膜中肌动蛋白丝的积累。通过暴露于血小板衍生生长因子（PDGF）-BB，可在EJ-Ras转化细胞中诱导肌动蛋白聚合，其程度与野生型NIH 3T3细胞中观察到的相似。在EJ-Ras细胞中，肌动蛋白聚合与磷脂酶Cγ1（PLCγ1）活性无关，因为在对照NIH 3T3细胞中响应PDGF-BB观察到的肌醇三磷酸（IP3）生成不存在。尽管PDGF-BB确实刺激了PLCγ1的酪氨酸磷酸化，但该磷脂酶受到EJ-Ras转化细胞细胞质中存在的一种抑制因子的强烈抑制。此外，EJ-Ras的细胞质提取物而非对照细胞的提取物在体外抑制了重组PLCγ1催化的磷脂酰肌醇4,5-二磷酸（PIP2）水解。尽管PIP2水解对PDGF-BB在EJ-Ras转化细胞中诱导的肌动蛋白细胞骨架重组没有作用，但磷脂酰肌醇3-激酶（PI3-K）对肌动蛋白聚合是必需的。渥曼青霉素是一种特异性PI3-K抑制剂，它不仅阻断了对照细胞和EJ-Ras转化细胞中的肌动蛋白聚合，而且当这些细胞暴露于PDGF-BB时实际上导致了肌动蛋白的快速解聚。因此，在EJ-Ras转化细胞中，对PDGF-BB的细胞形态发生变化重要地依赖于PI3-K，并且尽管PLCγ1发生酪氨酸磷酸化，但在完全没有IP3生成的情况下也能发生。
