Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic β-cell apoptosis
Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic β-cell apoptosis
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DOI：
10.1074/jbc.m703612200
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发表时间：
2008-01-25
影响因子：
4.8
通讯作者：
Johnson, James D.
中科院分区：
文献类型：
--
作者：
Alejandro, Emilyn U.;Johnson, James D.
丝氨酸苏氨酸激酶 Raf-1 在许多细胞类型中发挥保护作用，但其在胰腺 β 细胞中的功能尚未阐明。在本研究中，我们检查了原代 β 细胞是否具有 Raf-1，并检验了 Raf-1 对于 β 细胞存活至关重要的假设。使用逆转录酶 PCR、蛋白质印迹和免疫荧光，我们在人胰岛、小鼠胰岛和 MIN6 β 细胞系中鉴定了 Raf-1。使用特定的 Raf-1 抑制剂或显性失活 Raf-1 突变体阻断 Raf-1 活性会导致细胞死亡呈时间和剂量依赖性增加，通过碘化丙啶掺入的实时成像、TUNEL、PCR 增强的 DNA 梯带和 Caspase-3 切割进行评估。尽管细胞凋亡的快速增加与 Erk 磷酸化降低有关，但对两种 Mek 抑制剂的研究表明，经典的 Erk 依赖性途径只能解释抑制 Raf-1 后观察到的部分细胞死亡。 Raf-1 激酶下游另一种独立于 Erk 的通路涉及促凋亡蛋白 Bad，最近在其他组织中得到了表征。抑制 β 细胞中的 Raf-1 会导致丝氨酸 112 处的 Bad 磷酸化显着丧失，并且 Bad 和 Bax 的蛋白质水平增加。总之，我们的数据强烈表明 Raf-1 信号通过 Erk 依赖性和 Bad 依赖性机制在调节 β 细胞存活中发挥重要作用。相反，急性抑制磷脂酰肌醇 3-激酶 Akt 对 β 细胞死亡的影响较小。这些研究将 Raf-1 确定为胰腺 β 细胞中关键的抗凋亡激酶，有助于我们了解这种细胞类型中的生存信号传导。
The serine threonine kinase Raf-1 plays a protective role in many cell types, but its function in pancreatic beta-cells has not been elucidated. In the present study, we examined whether primary beta-cells possess Raf-1 and tested the hypothesis that Raf-1 is critical for beta-cell survival. Using reverse transcriptase-PCR, Western blot, and immunofluorescence, we identified Raf-1 in human islets, mouse islets, and in the MIN6 beta-cell line. Blocking Raf-1 activity using a specific Raf-1 inhibitor or dominant-negative Raf-1 mutants led to a time-and dose-dependent increase in cell death, assessed by real-time imaging of propidium iodide incorporation, TUNEL, PCR-enhanced DNA laddering, and Caspase-3 cleavage. Although the rapid increase in apoptotic cell death was associated with decreased Erk phosphorylation, studies with two Mek inhibitors suggested that the classical Erk-dependent pathway could explain only part of the cell death observed after inhibition of Raf-1. An alternative Erk-independent pathway downstream of Raf-1 kinase involving the pro-apoptotic protein Bad has recently been characterized in other tissues. Inhibiting Raf-1 in beta-cells led to a striking loss of Bad phosphorylation at serine 112 and an increase in the protein levels of both Bad and Bax. Together, our data strongly suggest that Raf-1 signaling plays an important role regulating beta-cell survival, via both Erk-dependent and Bad-dependent mechanisms. Conversely, acutely inhibiting phosphatidylinositol 3-kinase Akt had more modest effects on beta-cell death. These studies identify Raf-1 as a critical anti-apoptotic kinase in pancreatic beta-cells and contribute to our understanding of survival signaling in this cell type.