Perianayagam M C, Madias N E, Pereira B J G, Jaber B L
Department of Medicine, Tufts University School of Medicine, and Department of Medicine, Caritas St. Elizabeth's Medical Center, Boston, MA 02135, USA.
Eur J Clin Invest. 2006 May;36(5):353-61. doi: 10.1111/j.1365-2362.2006.01637.x.
Complement fragment C5a and neutrophils have been implicated in the pathogenesis of renal disease and C5a has also been shown to delay apoptosis of human neutrophils via a transcription-independent pathway. However, transcription-dependent pathways have not been well described. The present study examined whether activation of HL-60-derived neutrophils by C5a modulates the transcription of two members of the Bcl2 family, Bax (pro-apoptotic) and Bcl2 (anti-apoptotic) molecules, and whether the cAMP-response element-binding protein (CREB) transcription factor mediates these effects through the phosphatidylinositol 3-kinase (PI3K)/Akt and extra-cellular signal-regulated kinase (ERK) signalling pathways.
The human promyelocytic leukaemia HL-60 cell line was differentiated into neutrophils using 1.25% DMSO. Differentiated cells were incubated with recombinant human C5a for 30-120 min with, or without, pretreatment with wortmannin or PD98059. The cells were lysed and quantified for gene-specific Bax and Bcl2 mRNA. In separate experiments, cells were incubated with C5a for 5-30 min with, or without, pretreatment with wortmannin, PD98059, or alkaline phosphatase. Cells were then lysed and immunoblotted using antihuman phospho-CREB (Ser133) antibody. Apoptosis was assessed by measuring active caspase-3 in differentiated HL-60 cells.
C5a inhibited caspase-3 activation in HL-60-derived neutrophils (P=0.003). C5a significantly increased the expression of Bcl2 mRNA (P=0.028), which was time-dependent, peaking at 30 min, and was abrogated in the presence of either wortmannin or PD98059 (both P=0.028). The C5a had no impact on Bax mRNA expression. The Bax : Bcl2 mRNA ratio markedly decreased at 30 min (P=0.028). Time-dependent effect of C5a on CREB phosphorylation was demonstrable and rapid, peaking at 5 min, and was abrogated by either wortmannin or PD98059 (both P=0.028). Phosphorylation of CREB, but not of Akt and ERK, was inhibited by alkaline phosphatase (P=0.028). The effect of C5a on Bcl2 mRNA expression was abrogated by alkaline phosphatase (P=0.028). The Bax : Bcl2 mRNA ratio markedly increased in the presence of alkaline phosphatase (P=0.046).
This study demonstrates that C5a induces Bcl2 mRNA transcription in HL-60-derived neutrophils, which is mediated in part by CREB through the convergence of the PI3K/Akt and ERK-signalling pathways.
补体片段C5a和中性粒细胞与肾脏疾病的发病机制有关，并且C5a还被证明可通过转录非依赖途径延迟人中性粒细胞的凋亡。然而，转录依赖途径尚未得到充分描述。本研究检测了C5a对HL-60来源的中性粒细胞的激活是否会调节Bcl2家族的两个成员，即促凋亡分子Bax和抗凋亡分子Bcl2的转录，以及环磷酸腺苷反应元件结合蛋白（CREB）转录因子是否通过磷脂酰肌醇3激酶（PI3K）/Akt和细胞外信号调节激酶（ERK）信号通路介导这些效应。
使用1.25%二甲基亚砜（DMSO）将人早幼粒细胞白血病HL-60细胞系分化为中性粒细胞。将分化后的细胞与重组人C5a孵育30 - 120分钟，有无渥曼青霉素或PD98059预处理。细胞裂解后对基因特异性的Bax和Bcl2 mRNA进行定量。在单独的实验中，细胞与C5a孵育5 - 30分钟，有无渥曼青霉素、PD98059或碱性磷酸酶预处理。然后细胞裂解并使用抗人磷酸化CREB（Ser133）抗体进行免疫印迹。通过测量分化的HL-60细胞中的活性半胱天冬酶-3来评估细胞凋亡。
C5a抑制HL-60来源的中性粒细胞中半胱天冬酶-3的激活（P = 0.003）。C5a显著增加Bcl2 mRNA的表达（P = 0.028），这是时间依赖性的，在30分钟时达到峰值，并且在渥曼青霉素或PD98059存在的情况下被消除（两者P = 0.028）。C5a对Bax mRNA表达没有影响。在30分钟时，Bax : Bcl2 mRNA比值显著降低（P = 0.028）。C5a对CREB磷酸化的时间依赖性效应是可证明的且迅速，在5分钟时达到峰值，并且被渥曼青霉素或PD98059消除（两者P = 0.028）。碱性磷酸酶抑制CREB的磷酸化，但不抑制Akt和ERK的磷酸化（P = 0.028）。碱性磷酸酶消除了C5a对Bcl2 mRNA表达的影响（P = 0.028）。在碱性磷酸酶存在的情况下，Bax : Bcl2 mRNA比值显著增加（P = 0.046）。
本研究表明，C5a诱导HL-60来源的中性粒细胞中Bcl2 mRNA转录，这部分是由CREB通过PI3K/Akt和ERK信号通路的汇聚介导的。
