Goldkorn T, Ding T
Yale School of Medicine, New Haven, CT 06510, USA.
Adv Exp Med Biol. 1997;400A:461-72. doi: 10.1007/978-1-4615-5325-0_62.
Transforming growth factor beta 1 (TGF beta 1) increases the phosphorylation of the epidermal growth factor (EGF) receptor and inhibits the growth of A431 cells, but the mechanism of TGF beta 1 signaling is unknown. Recent studies from this and other laboratories suggest a novel sphingomyelin signal transduction pathway (1-4). Ceramide, which is generated by sphingomyelinase action, can be deacylated to sphingoid bases, which are potential inhibitors of protein kinase C (PKC). Ceramide appears to have bioeffector properties. Cell-permeable ceramide analogs stimulate monocytic differentiation of human leukemia (HL60) cells (1), as well as the phosphorylation of the EGF receptor at Thr669 in A431 human epidermoid carcinoma cells (2). Further studies (3,4) demonstrate the existence of a ceramide-activated protein kinase (CAPK) that may mediate some of these aspects. The present studies aim to investigate the mechanism of TGF beta 1 signaling and to explore whether TGF beta 1's pathway involves activation of PKC by 1,2-Diacylglycerol (DAG) and/or stimulation of a CAPK by ceramide. Ceramide and DAG levels of A431 cells are determined by thin layer chromatography (TLC) after treatment with either TGF beta 1 or with EGF. 100 pM TGF beta 1 treatment for 1 hr increases the cellular contents of DAG 2-fold. 20 nM EGF treatment for 15 min decreases it 0.5-fold. Ceramide levels are reduced 2-fold by TGF beta 1 and almost unaffected by EGF. To evaluate the involvement of other components of signal transduction, the effects of TGF beta 1 and EGF on PKC activity are studied. 20 nM EGF decreases membrane PKC activity to 0.5-fold of controls, whereas 100 pM TGF beta 1 treatment of A431 cells increases this activity 4-fold. Modulation of PKC activity is paralled by translocation of the enzyme between the cytosol and the membrane as determined by Western immunoblot analysis. These studies suggest that TGF beta 1 and EGF may have regulatory effects on both sphingolipid and phospholipid metabolisms which could transmodulate both the CAPK and the PKC mediated signal tranduction pathways.
转化生长因子β1（TGFβ1）可增加表皮生长因子（EGF）受体的磷酸化水平，并抑制A431细胞的生长，但其信号传导机制尚不清楚。本实验室及其他实验室最近的研究提示了一种新的鞘磷脂信号转导途径（1-4）。由鞘磷脂酶作用产生的神经酰胺可脱酰基生成鞘氨醇碱，后者可能是蛋白激酶C（PKC）的潜在抑制剂。神经酰胺似乎具有生物效应特性。可透过细胞的神经酰胺类似物可刺激人白血病（HL60）细胞的单核细胞分化（1），以及A431人表皮样癌细胞中EGF受体在苏氨酸669位点的磷酸化（2）。进一步的研究（3,4）证明存在一种神经酰胺激活的蛋白激酶（CAPK），它可能介导其中的某些作用。本研究旨在探讨TGFβ1信号传导的机制，并探索TGFβ1的信号途径是否涉及1,2-二酰基甘油（DAG）对PKC的激活和/或神经酰胺对CAPK的刺激。在用TGFβ1或EGF处理后，通过薄层层析（TLC）测定A431细胞中的神经酰胺和DAG水平。用100 pM TGFβ1处理1小时可使细胞内DAG含量增加2倍。用20 nM EGF处理15分钟可使其降低0.5倍。TGFβ1可使神经酰胺水平降低2倍，而EGF对其几乎无影响。为了评估信号转导其他成分的参与情况，研究了TGFβ1和EGF对PKC活性的影响。20 nM EGF可使膜PKC活性降至对照的0.5倍，而用100 pM TGFβ1处理A431细胞可使该活性增加4倍。如通过蛋白质免疫印迹分析所确定的，PKC活性的调节与该酶在胞浆和膜之间的转位同时发生。这些研究提示，TGFβ1和EGF可能对鞘脂和磷脂代谢均具有调节作用，这可能会对CAPK和PKC介导的信号转导途径产生相互调节作用。
