Protein Kinase C Delta (PKCδ) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1

BackgroundHigh fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. Methodology and Principal FindingsUsing genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21Cip1/WAF1. This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21Cip1/WAF1 at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21Cip1/WAF1 was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21Cip1/WAF1 with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. Conclusions and SignificanceThese observations disclose PKCδ as negative regulator of p21Cip1/WAF1, which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.

研究背景：高脂饮食诱导的高血糖与棕榈酸刺激的细胞凋亡，可通过β细胞中蛋白激酶Cδ（protein kinase C delta, PKCδ）的特异性抑制得以阻断。为更深入阐释PKCδ的生物学功能，本研究在无应激条件下，分析了PKCδ活性变化对胰岛素分泌细胞增殖与存活的影响。研究方法与主要结果：通过遗传与药理学手段，本研究检测了PKCδ活性上调与下调对胰岛素分泌细胞增殖、凋亡及细胞周期调控的作用。采用蛋白质印迹法（Western blotting）与共聚焦激光扫描显微镜对蛋白样本进行分析。野生型PKCδ（PKCδWT）过表达可显著促进INS-1E细胞的增殖，并伴随细胞周期抑制剂p21Cip1/WAF1的表达下调及其从细胞核向细胞质的转位。该核外排过程由PKCδ介导的p21Cip1/WAF1在Ser146位点的磷酸化所调控。在激酶失活型PKCδ（PKCδKN）过表达的细胞中，以及经罗素亭（rottlerin）抑制内源性PKCδ活性或通过RNA干扰敲低PKCδ后，p21Cip1/WAF1的磷酸化水平均显著降低，这促进了该抑制剂的核积累并诱导INS-1E细胞发生凋亡。人与小鼠胰岛细胞表达p21Cip1/WAF1且呈现显著的核积累，而在PKCδWT转基因小鼠的胰岛细胞中，该抑制剂则定位于细胞质。研究结论与意义：上述结果揭示PKCδ可作为p21Cip1/WAF1的负调控因子，在无应激条件下促进胰岛素分泌细胞的增殖；同时提示，额外的应激诱导变化可使PKCδ转向其已知的促凋亡功能。