Ulrich R G, Cramer C T, Adams L A, Kletzien R F
Investigative Toxicology Unit, Pharmacia & Upjohn, Inc., Kalamazoo, MI 49007, USA.
Cell Biochem Funct. 1998 Jun;16(2):77-85. doi: 10.1002/(SICI)1099-0844(199806)16:2<77::AID-CBF769>3.0.CO;2-U.
Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0.5-1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2.5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor beta subunit; she phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK.
许多肝细胞活动可能受到包括丝裂原活化蛋白激酶（MAPK）在内的细胞内信号蛋白的近端调节。在本研究中，对原代培养的人及大鼠肝细胞中表皮生长因子（EGF）和胰岛素的信号转导事件进行了检测。通过蛋白质免疫印迹法发现，大鼠和人肝细胞在暴露于EGF 0.5 - 1分钟后，EGF受体和MAPK会迅速发生酪氨酸磷酸化。暴露于EGF 2.5分钟后，观察到p42和p44 MAPK的磷酸化。胰岛素处理导致胰岛素受体β亚基发生磷酸化；未观察到其磷酸化。MAPK磷酸化与分子量的变化及激酶活性的增加相对应。仅在人肝细胞中明确观察到胰岛素依赖性的MAPK激活，尽管在大鼠中检测到轻微激活。胰岛素和EGF共同处理导致MAPK磷酸化以及分子量完全电泳迁移，在大鼠而非人肝细胞中酶活性呈相加或协同增加；人肝细胞中的MAPK仅由EGF单独最大程度地刺激。胰高血糖素预处理可阻断胰岛素诱导的MAPK磷酸化、凝胶迁移率改变及激酶活性，但仅部分阻断人肝细胞中EGF诱导的MAPK激活。胰高血糖素还降低了大鼠肝细胞中EGF对MAPK的激活。用福斯可林或环磷酸腺苷类似物预处理可减少大鼠肝细胞中胰岛素、EGF及胰岛素加EGF依赖性的MAPK激活，而不影响受体或MAPK的磷酸化。这些结果表明尽管EGF和胰岛素可能都通过MAPK/ras/raf/MAPK途径发出信号，但这些配体之间以及不同物种之间对MAPK的反应有所不同。此外，在大鼠和人中，胰高血糖素均通过环磷酸腺苷依赖性机制在MAPK下游但在MAPK近端的胰岛素和EGF信号转导途径水平发挥作用。胰高血糖素对人肝细胞中EGF诱导的MAPK磷酸化的部分抑制为MAPK激活的raf - 1非依赖性途径提供了进一步证据。
