Study of novel Ras signaling pathway using ras knockout mice

我们建立了H-ras、N-ras和K-ras缺陷小鼠和多个ras突变小鼠，发现H-ras和N-ras突变小鼠以及H-ras N-ras双突变小鼠明显发育正常，但K-ras突变小鼠具有胚胎致死性。我们将人H-ras转基因引入单个和多个ras突变小鼠中，发现H-ras转基因可以挽救这些ras突变小鼠在发育过程中表现出的所有异常，这表明ras基因在胚胎和新生儿发育中的功能是多余的。我们发现，经过20周的TPA治疗后，H-ras缺失突变小鼠的乳头状瘤发生率大约是野生型小鼠的7倍。在H-ras缺失突变小鼠的21个乳头状瘤中，有13个(62%)存在K-ras基因的激活突变。在H-ras缺失突变的海马区，ε受体NR2a(ε1)和NR2b(NR2b)亚基的酪氨酸磷酸化水平增加，相应地NMDA型突触反应选择性增强。此外，长时程增强(LTP)在突变小鼠中显著增强，这很可能是由于NMDA突触反应的选择性增强。因此，尽管Ras蛋白参与了细胞的增殖和分化，但通过下调NMDA受体的磷酸化来调节成年动物的活性依赖性突触可塑性可能是H-Ras蛋白的另一个主要和关键的作用。为了探讨Ras信号通路与NMDA受体的关系，我们用NMDA刺激培养的海马神经细胞，发现NMDA受体发生去磷酸化，ERK和p38MAPK被激活，NMDA刺激激活ERK需要RAS的激活。

We generated H-ras, N-ras and K-ras deficient mice and multiple ras mutant mice, and found that H-ras and N-ras mutant mice as well as H-ras N-ras double mutant mice apparently developed normally, but K-ras mutant mice were embryonic lethal. We introduced human H-ras transgene into a single and multiple ras mutant mice and found that H-ras transgene rescue all abnormality exhibited in the development of these ras mutant mice, suggesting that the function of ras genes is redundant in embryonic and neonatal development.We showed that H-ras null mutant mice develop approximately seven times less papillomas compared with wild-type littermates do after 20 weeks of TPA treatment. 13 (62%) out of 21 papillomas in H-ras null mutant mice have activated mutations of K-ras gene. This suggests that activation of K-ras gene can replace the H-ras activation in the initiation step of skin tumor development.In the H-ras null mutant hippocampus, the tyrosine phosphorylation of NR2A (ε1) and NR2B (ε2) subunits of NMDA receptors is increased and correspondingly NMDA synaptic responses are selectively enhanced. In addition, long-term potentiation (LTP) is markedly enhanced in mutant mice most likely due to a selective enhancement of NMDA synaptic responses. Therefore, although Ras proteins have been implicated in cell proliferation and differentiation, the regulation of activity-dependent synaptic plasticity in the adult animals by downregulation of the phosphorylation of the NMDA receptor may be another major and pivotal role for H-Ras protein. To investigate the relationship between Ras signaling pathway and NMDA receptor, we stimulate hippocampal culture cell by NMDA.We found that dephosphorylation of NMDA receptor occured as well as activation of ERK and p38 MAPK.We found that the activation of ERK by NMDA stimulation requires Ras activity.