Analysis of the Regulatory Mechanism and Function of a Novel Class of Phospholipase C, PLCε

1、我们构建了磷脂酶C_ε(PLC_ε)基因敲除小鼠，结果表明，这些小鼠的心脏发生了心室扩张，这是由于明显的返流和半月瓣(主动脉和肺动脉)轻度狭窄而导致的容量超负荷所致。我们分析了导致这些表型的先天性半月瓣膜发育缺陷的机制，得出结论：PLC_ε基因敲除小鼠的半月瓣叶异常增厚和形态异常是由于半月瓣膜发育后期瓣膜重塑过程中细胞的异常增殖所致。通过与携带减弱的表皮生长因子受体的小鼠或肝素结合的表皮生长因子缺乏的小鼠的表型相似，我们推测PLC_ε作为表皮生长因子受体下游的RAS效应器，在抑制瓣膜细胞增殖方面发挥了关键作用。事实上，我们观察到在PLC_ε基因敲除小鼠中，诱导BMP受体下游瓣膜细胞增殖的Smad1/5/8的磷酸化水平增加。2.通过将以二甲基丁酸为启动子和佛波酯TPA为启动子的两阶段皮肤化学致癌方案应用于PLC_ε基因敲除小鼠，我们证明了PLC_ε在ras癌基因诱导的良性肿瘤的发生和恶性进展中起着关键作用。进一步分析了PLC_ε的作用机理。PLC_ε基因敲除小鼠在TPA诱导的基底层细胞增殖和表皮增生中表现出明显的抑制作用，提示PLC_ε在TPA下游信号传导中起着关键作用。3、我们在模型动物秀丽线虫中干扰了PLC_ε基因，并观察到了由于精囊括约肌异常收缩而导致的不育表型。4通过使用表达PDGFR突变体的BaF3细胞，我们通过建立依赖于PDGF的PLC_γ激活诱导的Ca^&lt；2+&gt；增加的检测系统，展示了PH和RA结构域的重要性。

1, We constructed phospholipase C_ε(PLC_ε) knockout mice and showed that the hearts of these mice develop ventricular dilation, which is caused by a volume overload resulting from marked regurgitation and mild stenosis of the semilunar (aortic and pulmonic) valves. We analysed the mechanism of the congenital semilunar valvulogenesis defect causing these phenotypes and concluded that the abnormal thickening and morphology of the semilunar valve leaflets in PLC_ε knockout mice were caused by aberrant cellular proliferation in valve remodeling at the late stages of semilunar valvulogenesis. By analogy with the phenotypes of mice carrying an attenuated epidermal growth factor (EGF) receptor or deficient in heparin-binding EGF, we speculated a crucial role of PLC_ε, as a Ras effector downstream of the EGF receptor, in inhibition of valvular cell proliferation. In fact, we observed increased phosphorylation of Smad1/5/8, which induce valvular cell proliferation downstream of the BMP receptor, in PLC_ε knockout mice.2, By applying the two stage skin chemical carcinogenesis protocol using DMBA as an initiator and a phorbor ester TPA as a promoter to PLC_ε knockout mice, we showed that PLC_ε plays a crucial role in ras oncogene-induced development of benign tumors as well as in their malignant progression. We further analysed the mechanism of action of PLC_ε. PLC_ε knockout mice showed marked reduction in proliferation of basal layer cells and epidermal hyperplasia induced by TPA, suggesting a crucial role of PLC_ε in downstream signaling from TPA.3, We disrupted the PLC_ε gene in a model organism Caenorhabditis elegans and observed a sterile phenotype, caused by abnormal contraction of sphincter muscles of the spermatheca.4, By using BaF3 cells expressing a PDGF receptor mutant lacking the PLC_γ-binding sites, we showed the importance of PH and RA domains by establishing an assay system for Ca^<2+> increase induced by PDGF-dependent PLC_ε activation.