Department of Surgery, James A. Haley Veterans Affairs Medical Center, University of South Florida Health Sciences Center, Tampa, FL, USA.
Surgery. 2011 Jan;149(1):135-42. doi: 10.1016/j.surg.2010.04.017. Epub 2010 Jun 8.
We have demonstrated that activated Kupffer cells undergo accelerated apoptosis via Toll-like receptor (TLR)-4 and protein kinase C (PKC)-ζ-dependent nuclear factor (NF)-κB activation. Because PKC-ζ plays a pivotal role in cell signaling, we sought to determine the signaling pathway of PKC-ζ in Kupffer cell apoptosis.
Mouse Kupffer cell line (MKCL3-2) were transfected with PKC-ζ small interfering RNA (siRNA) and then treated with elastase alone or elastase along with the extracellular signal-regulated kinase (ERK) inhibitor U0126. Cell extracts were assayed for PKC-ζ (protein and activity), TLR-4, NF-κB nuclear translocation, phosphorylated ERK-1/2, activated caspase-3, and DNA fragmentation. All n ≥3; data are expressed as mean values ± standard deviations; means were compared using the t test; P < .05 was considered significant.
Elastase upregulated TLR-4, PKC-ζ, NF-κB, ERK-1/2, caspase-3, and DNA fragmentation (all P < .01 versus control). Transfection with PKC-ζ siRNA attenuated the elastase-induced upregulation of PKC-ζ activity, NF-κB, ERK-1/2, caspase-3, and DNA fragmentation (all P < .01 versus control). The interaction of PKC-ζ with ERK-1/2 was increased by elastase and was attenuated by PKC-ζ siRNA as confirmed by co-immunoprecipitation and immunofluorescent staining.
Activation of Kupffer cells upregulates PKC-ζ activity, increases apoptosis, and induces nuclear translocation of NF-κB via ERK-1/2-dependent pathways. Inhibiting the activity of PKC-ζ significantly attenuates Kupffer cell apoptosis, NF-κB, and ERK-1/2 activation. The interaction of PKC-ζ and ERK-1/2 warrants further investigation.
我们已经证明，通过 Toll 样受体 (TLR)-4 和蛋白激酶 C (PKC)-ζ 依赖性核因子 (NF)-κB 激活，激活的枯否细胞(Kupffer cell)经历加速凋亡。由于 PKC-ζ 在细胞信号转导中发挥关键作用，我们试图确定 PKC-ζ 在枯否细胞凋亡中的信号通路。
用 PKC-ζ 小干扰 RNA(siRNA)转染小鼠枯否细胞系 (MKCL3-2)，然后用弹性蛋白酶单独或弹性蛋白酶加细胞外信号调节激酶 (ERK)抑制剂 U0126 处理。细胞提取物用于检测 PKC-ζ(蛋白和活性)、TLR-4、NF-κB 核易位、磷酸化 ERK-1/2、活化的 caspase-3 和 DNA 片段化。所有 n ≥3；数据表示为平均值 ± 标准差；使用 t 检验比较均值；P <.05 被认为有统计学意义。
弹性蛋白酶上调 TLR-4、PKC-ζ、NF-κB、ERK-1/2、caspase-3 和 DNA 片段化(均 P <.01 与对照相比)。PKC-ζ siRNA 转染减弱了弹性蛋白酶诱导的 PKC-ζ 活性、NF-κB、ERK-1/2、caspase-3 和 DNA 片段化的上调(均 P <.01 与对照相比)。弹性蛋白酶增加了 PKC-ζ 与 ERK-1/2 的相互作用，PKC-ζ siRNA 减弱了这种相互作用，这通过共免疫沉淀和免疫荧光染色得到证实。
激活的枯否细胞通过 ERK-1/2 依赖性途径上调 PKC-ζ 活性，增加细胞凋亡，并诱导 NF-κB 核易位。抑制 PKC-ζ 的活性显著减弱枯否细胞凋亡、NF-κB 和 ERK-1/2 的激活。PKC-ζ 和 ERK-1/2 的相互作用值得进一步研究。
