Zhang Y, Akhtar R A
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912, USA.
Curr Eye Res. 1998 Mar;17(3):294-300. doi: 10.1076/ceyr.17.3.294.5223.
Activation of phospholipase D (PLD) is believed to be an important signaling pathway involved in cell growth and differentiation in several tissues, in response to a variety of mitogens. The aim of the present study was to investigate the effect of epidermal growth factor (EGF) on PLD activity in rabbit corneal epithelial cells (RCEC). We have also examined whether the EGF effect is dependent on concurrent activation of phospholipase C (PLC), protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI-3-kinase) in these cells.
RCEC, immortalized with adenovirus SV-40, were cultured until they became confluent. The cells were labeled with [3H]myristic acid and incubated with or without EGF or other agents for specified time intervals. PLD activity was measured by quantifying [3H]phosphatidylethanol in cells incubated in the presence of ethanol. PLC activity was determined by measuring the radioactivity in inositol trisphosphate in myo[3H]inositol-labeled RCEC. PI 3-kinase activity was assessed by measuring the production of PIP3 in 32P-labeled cells.
Addition of EGF to RCEC stimulated PLD activity in a time- and dose-dependent manner. The maximal effect was observed with 150 ng/ml EGF and at 10 min of incubation. The PLD activity was also stimulated when phorbol myristate acetate (PMA) was added to the cells. Treatment of the cells with EGF stimulated PLC activity which was inhibited by U73122, a PLC inhibitor. Under the same experimental conditions, the inhibitor had no effect on EGF-stimulated PLD activity. Down-regulation of PKC or treatment of the cells with RO31-8220, a PKC inhibitor, inhibited the PMA- but not EGF-stimulated PLD activity. Incubation of the cells with wortmannin, a PI 3-kinase inhibitor, abolished the EGF-stimulated PI 3-kinase activity, but potentiated the EGF-stimulated PLD activity. The EGF effect was inhibited by treatment of the cells with tyrphostin B42, a receptor tyrosine kinase inhibitor.
These results indicate that EGF stimulates PLD activity in RCEC by a mechanism that involves tyrosine phosphorylation of a protein(s) in the cascade of biochemical reactions initiated by EGF-receptor interaction, and it is not dependent on concurrent activation of PKC, PLC, or PI 3-kinase in these cells.
磷脂酶D（PLD）的激活被认为是一条重要的信号通路，在多种组织中，响应各种促分裂原，参与细胞生长和分化过程。本研究的目的是调查表皮生长因子（EGF）对兔角膜上皮细胞（RCEC）中PLD活性的影响。我们还研究了EGF的作用是否依赖于这些细胞中磷脂酶C（PLC）、蛋白激酶C（PKC）或磷脂酰肌醇3激酶（PI-3激酶）的同时激活。
用腺病毒SV-40永生化的RCEC培养至汇合。细胞用[3H]肉豆蔻酸标记，并在有或无EGF或其他试剂的情况下孵育特定时间间隔。通过定量在乙醇存在下孵育的细胞中的[3H]磷脂酰乙醇来测量PLD活性。通过测量用肌醇[3H]肌醇标记的RCEC中肌醇三磷酸的放射性来测定PLC活性。通过测量32P标记细胞中PIP3的产生来评估PI 3激酶活性。
向RCEC中添加EGF以时间和剂量依赖性方式刺激PLD活性。在150 ng/ml EGF和孵育10分钟时观察到最大效应。当向细胞中添加佛波醇肉豆蔻酸酯乙酸酯（PMA）时，PLD活性也受到刺激。用EGF处理细胞刺激了PLC活性，该活性被PLC抑制剂U73122抑制。在相同的实验条件下，该抑制剂对EGF刺激的PLD活性没有影响。PKC的下调或用PKC抑制剂RO31-8220处理细胞抑制了PMA刺激的PLD活性，但不抑制EGF刺激的PLD活性。用PI 3激酶抑制剂渥曼青霉素孵育细胞消除了EGF刺激的PI 3激酶活性，但增强了EGF刺激的PLD活性。用受体酪氨酸激酶抑制剂 tyrphostin B42处理细胞抑制了EGF的作用。
这些结果表明，EGF通过一种机制刺激RCEC中的PLD活性，该机制涉及在由EGF受体相互作用引发的生化反应级联中蛋白质的酪氨酸磷酸化，并且它不依赖于这些细胞中PKC、PLC或PI 3激酶的同时激活。
