Rapamycin inhibits human renal epithelial cell proliferation: Effect on cyclin D3 mRNA expression and stability
Rapamycin inhibits human renal epithelial cell proliferation: Effect on cyclin D3 mRNA expression and stability
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DOI：
10.1111/j.1523-1755.2005.00350.x
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发表时间：
2005-06-01
影响因子：
19.6
通讯作者：
Anglicheau, D
中科院分区：
文献类型：
--
作者：
Pallet, N;Thervet, E;Anglicheau, D
背景资料。最近的数据表明，移植后早期使用雷帕霉素与移植延迟功能(DGF)延长有关。由于雷帕霉素在其他细胞类型中的作用已知，我们推测这一作用可能继发于人肾上皮细胞(HRECs)对增殖的抑制。原代培养的HRECs与不同浓度的雷帕霉素孵育。细胞毒实验检测细胞增殖情况。流式细胞仪分析细胞周期。用Western印迹法检测蛋白质表达水平。用实时定量聚合酶链式反应(PCR)检测细胞周期蛋白D3的表达水平。瞬时转染法检测细胞周期蛋白D3基因的转录活性。雷帕霉素通过抑制细胞从G(1)向S的转变，对生长的人脐静脉内皮细胞有明显的抑制增殖作用，且呈浓度依赖性。导致细胞周期进程的p70(S6)激酶通路被发现是活跃的，低浓度的雷帕霉素显著降低了p70(S6)激酶的磷酸化。雷帕霉素可完全抑制胎牛血清诱导的细胞周期蛋白D3蛋白表达增加和mRNA积聚，但不影响细胞周期蛋白E和CDK抑制因子的表达水平。这种对细胞周期蛋白D3蛋白表达的调节主要是由于其mRNA的不稳定。雷帕霉素使其半衰期减少26%(4.81.3h vs.6.5h，P<；0.001)。雷帕霉素抑制HRECs对有丝分裂刺激的增殖反应，并导致细胞周期停滞于G1期早期，不仅通过抑制p70(S6K)途径的非特异性过程，而且通过直接影响细胞周期蛋白D3mRNA的稳定性。
Background. Recent data have suggested that rapamycin use during the initial period after transplantation is associated with prolonged delayed graft function (DGF). Because of the known effects of rapamycin in other cell types, we speculated that this action may be secondary to human renal epithelial cells (HRECs) inhibition of proliferation.Methods. Primary cultures of HRECs were incubated with various concentrations of rapamycin. Cell proliferation was evaluated by cytotoxicity assays. The cell cycle was analyzed by flow cytometry. Protein expression levels were assessed by Western blot. Cyclin D3 mRNA levels were measured by quantitative real-time polymerase chain reaction (PCR). The transcriptional activity of the cyclin D3 gene was evaluated using transient transfection.Results. Rapamycin exerted a significant concentration-dependent antiproliferative effect on growing HRECs by inhibiting the G(1) to S transition. The p70(S6) kinase pathway leading to cell cycle progression was found to be active, and low concentrations of rapamycin dramatically reduced p70(S6) kinase phosphorylation. Rapamycin completely inhibited the increase in cyclin D3 protein expression and mRNA accumulation induced by fetal calf serum, but did not affect cyclin E or cdk-inhibitor expression levels. This regulation of cyclin D3 protein expression is mainly due to a destabilization of its mRNA. Rapamycin reduced the mRNA half-life by 26% (4.8 +/- 1.3 hours vs. 6.5 +/- 1.0 hours, P < 0.001).Conclusion. Rapamycin inhibits the proliferative response of HRECs to mitogenic stimuli, and causes cell cycle arrest in the early G 1 phase, not only by a nonspecific process due to inhibition of the p70(S6k) pathway, but also by a direct effect on cyclin D3 mRNA stability.