Wang D J, Huang N N, Gonzalez F A, Heppel L A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
J Cell Physiol. 1991 Mar;146(3):473-82. doi: 10.1002/jcp.1041460319.
We recently reported that extracellular ATP was mitogenic for Swiss 3T3, 3T6, and A431 cells (Huang et al.: Proc. Natl. Acad. Sci. USA, 86:7904-7908, 1989). Here we examined the possible involvement of activation of the protein kinase C (PKC) signal transduction pathway in the mechanism of action of extracellular ATP. A potent synergistic stimulation of DNA synthesis in quiescent cultures of 3T3 and 3T6 cells was observed when ATP was presented in combination with growth factors that activate PKC, such as bombesin, vasopressin, or tumor-promoting phorbol esters. This finding suggests that ATP and these mitogens do not act through a common mechanism. In contrast, ATP was unable to show synergism with phorbol esters in A431 cells. We discovered striking differences when we examined the kinetics of formation of diacylglycerol (DAG) stimulated by ATP among these cell lines. Thus, ATP stimulated a sustained biphasic increase of DAG in A431 cells, but only a rapid transient increase of DAG formation was observed in 3T3 and 3T6 cells. The breakdown of phosphatidylcholine was stimulated by ATP in A431 cells; however, a significantly reduced effect was displayed in 3T6 cells. Furthermore, we found that the diacylglycerol-kinase inhibitor, 1-monooleoylglycerol, greatly potentiated ATP-stimulated DNA synthesis in A431 cells. Finally, down-regulation of PKC by long-term exposure to phorbol dibutyrate (PDBu) prevented stimulation of DNA synthesis induced by bombesin, vasopressin, or phorbol esters in 3T3 or 3T6 cells, while it had no such effect on ATP-stimulated mitogenesis in the presence of insulin or epidermal growth factor. On the other hand, PDBu-mediated down-regulation of PKC partially inhibited [3H [thymidine incorporation stimulated by ATP in A431 cells. Taken together, we conclude that a protein kinase C-dependent pathway is partially involved in ATP-stimulated DNA synthesis in A431 cells, but a protein kinase C-independent pathway exists in 3T3 and 3T6 cells. Pertussis toxin (PTX) inhibited the sustained phase of DAG formation and the breakdown of phosphatidylcholine stimulated by ATP in A431 cells. This suggests involvement of a PTX-sensitive G protein.
我们最近报道，细胞外ATP对瑞士3T3、3T6和A431细胞具有促有丝分裂作用（Huang等人：《美国国家科学院院刊》，86:7904 - 7908，1989）。在此，我们研究了蛋白激酶C（PKC）信号转导途径的激活在细胞外ATP作用机制中可能的参与情况。当ATP与激活PKC的生长因子（如蛙皮素、加压素或促肿瘤佛波酯）联合呈现时，在3T3和3T6细胞的静止培养物中观察到对DNA合成的强效协同刺激。这一发现表明ATP和这些有丝分裂原并非通过共同机制起作用。相比之下，ATP在A431细胞中无法与佛波酯表现出协同作用。当我们研究这些细胞系中由ATP刺激的二酰基甘油（DAG）形成动力学时，发现了显著差异。因此，ATP刺激A431细胞中DAG持续双相增加，但在3T3和3T6细胞中仅观察到DAG形成快速短暂增加。A431细胞中ATP刺激了磷脂酰胆碱的分解；然而，在3T6细胞中其作用显著降低。此外，我们发现二酰基甘油激酶抑制剂1 - 单油酰甘油极大地增强了A431细胞中ATP刺激的DNA合成。最后，长期暴露于佛波二丁酸酯（PDBu）导致PKC下调，阻止了蛙皮素、加压素或佛波酯在3T3或3T6细胞中诱导的DNA合成刺激，而在胰岛素或表皮生长因子存在的情况下，对ATP刺激的有丝分裂没有这种作用。另一方面，PDBu介导的PKC下调部分抑制了A431细胞中ATP刺激的[³H]胸苷掺入。综上所述，我们得出结论，蛋白激酶C依赖性途径部分参与A431细胞中ATP刺激的DNA合成，但3T3和3T6细胞中存在蛋白激酶C非依赖性途径。百日咳毒素（PTX）抑制了A431细胞中ATP刺激的DAG形成的持续阶段以及磷脂酰胆碱的分解。这表明涉及一种对PTX敏感的G蛋白。
