Klein S B, Fisher G J, Jensen T C, Mendelsohn J, Voorhees J J, Elder J T
Department of Dermatology, University of Michigan Medical School, Ann Arbor, 48109-0672.
J Cell Physiol. 1992 May;151(2):326-36. doi: 10.1002/jcp.1041510214.
Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.
转化生长因子-α（TGF-α）是表皮角质形成细胞的一种自分泌生长因子，可诱导其自身表达（自诱导）。由于这一过程的调控对于表皮生长的控制可能很重要，我们研究了表皮生长因子（EGF）受体酪氨酸激酶和蛋白激酶C（PKC）在培养的人角质形成细胞TGF-α自诱导中的作用。抗磷酸酪氨酸免疫印迹分析表明，EGF和TGF-α能快速且显著地刺激生长因子缺乏的角质形成细胞中一种170 kDa蛋白的酪氨酸磷酸化。使用抗EGF受体单克隆抗体进行免疫沉淀，该蛋白被鉴定为EGF受体。针对EGF受体的单克隆抗体以及EGF受体酪氨酸激酶抑制剂RG50864均抑制了EGF和TGF-α诱导的酪氨酸磷酸化以及TGF-α mRNA积累，表明受体的酪氨酸激酶活性参与了TGF-α自诱导。该单克隆抗体以相似效力（IC50约为0.1 μg/ml）抑制角质形成细胞生长和TGF-α自诱导。TGF-α和PKC激活剂十四酰佛波醇12-肉豆蔻酸酯13-乙酸酯（TPA）对TGF-α稳态mRNA水平有相似影响，提示PKC激活可能是TGF-α自诱导的下游介质。然而，苔藓抑素预处理使角质形成细胞PKC活性下调超过90%，消除了TPA诱导的TGF-α mRNA，但不影响自诱导反应或EGF刺激的酪氨酸磷酸化。这些结果表明，EGF受体和PKC通过不同途径刺激TGF-α基因表达，提示该系统中TGF-α自诱导不需要PKC。此外，PKC下调后EGF刺激的酪氨酸磷酸化和TGF-α自诱导未增强，这一事实表明PKC对正常人角质形成细胞中的EGF受体酪氨酸激酶活性没有持续性抑制作用。
