Synergistic effect of hypoxia and TNF-alpha on production of PAI-1 in human proximal renal tubular cells.
Synergistic effect of hypoxia and TNF-alpha on production of PAI-1 in human proximal renal tubular cells.
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DOI：
10.1111/j.1523-1755.2005.00435.x
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发表时间：
2005-08
影响因子：
19.6
通讯作者：
Xuan Li;H. Kimura;K. Hirota;K. Kasuno;K. Torii;T. Okada;H. Kurooka;Y. Yokota;H. Yoshida
中科院分区：
文献类型：
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作者：
Xuan Li;H. Kimura;K. Hirota;K. Kasuno;K. Torii;T. Okada;H. Kurooka;Y. Yokota;H. Yoshida
慢性缺氧被认为是各种慢性炎症性肾脏疾病进展中小管间质纤维化的共同机制，其中纤溶酶原激活物抑制剂-1 （PAI-1）通过抑制纤溶酶依赖的ECM降解在细胞外基质（ECM）的积累中起重要作用。在本研究中，我们通过肾活检样本的免疫染色研究了PAI-1在肾小管细胞中的存在。我们还仔细研究了缺氧和肿瘤坏死因子- α (tnf - α)对培养的人近端肾小管细胞（HPTECs）中PAI-1表达的影响。方法将在Dulbecco改良Eagle培养基（DMEM）中生长停滞24小时的融合细胞暴露于缺氧（1% O(2)）和/或tnf - α (10 ng/mL)中长达48小时。采用酶联免疫吸附法（ELISA）、TaqMan定量聚合酶链反应（PCR）或cDNA阵列分析分别测定刺激后细胞PAI-1蛋白和mRNA的含量，并与对照组（18% O(2)不含tnf - α）培养的细胞进行比较。免疫印迹和免疫荧光分析证实了缺氧诱导因子-1 α (hif -1 α)的存在。用荧光素酶报告基因法测定人PAI-1启动子活性。结果新月形肾小球肾炎中，近端小管簇特异性染色PAI-1。cDNA阵列分析发现PAI-1是缺氧诱导的主要基因。低氧处理24小时，tnf - α及其联合处理诱导PAI-1蛋白分泌增加2.8倍、1.8倍和4.6倍，PAI-1 mRNA水平分别增加3.6倍、3.3倍和12.1倍。免疫印迹分析和免疫细胞化学显示，缺氧诱导因子-1 α (hif -1 α)在暴露于缺氧16小时后，在细胞裂解物中明显积累，并专门转移到细胞核中，而tnf - α则没有。荧光素酶报告基因检测显示，缺氧、tnf - α及其联合作用使PAI-1的转录活性分别提高1.8倍、1.4倍和2.2倍。hif -1 α的显性阴性形式显著抑制缺氧诱导的PAI-1转录活性。抑制核因子- kappab （NF-kappaB）导致缺氧条件下PAI-1的产生适度降低。结论缺氧诱导PAI-1表达的途径是hif -1 α的核积累，部分途径是NF-kappaB的激活。tnf - α可协同增强缺氧诱导的PAI-1表达。
BACKGROUND Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where plasminogen activator inhibitor-1 (PAI-1) plays an important role in the accumulation of extracellular matrix (ECM) through inhibition of plasmin-dependent ECM degradation. In the present study, we investigated the presence of PAI-1 in renal tubular cells by immunostaining renal biopsy samples. We also closely examined the effects of hypoxia and tumor necrosis factor-alpha (TNF-alpha) on PAI-1 expression in cultured human proximal renal tubular cells (HPTECs). METHODS Confluent cells growth-arrested in Dulbecco's modified Eagle's medium (DMEM) for 24 hours were exposed to hypoxia (1% O(2)) and/or TNF-alpha at 10 ng/mL for up to 48 hours. Amounts of PAI-1 protein and mRNA after stimulation were measured by enzyme-linked immunosorbent assay (ELISA) and TaqMan quantitative polymerase chain reaction (PCR) or cDNA array analysis, respectively, and compared to those in cells incubated under control conditions (18% O(2) without TNF-alpha). Hypoxia-inducible factor-1alpha (HIF-1alpha) was demonstrated by immunoblot and immunofluorescence analyses. Human PAI-1 promoter activity was estimated by luciferase reporter gene assay. RESULTS In crescentic glomerulonephritis, clusters of proximal tubules were specifically stained for PAI-1. cDNA array analysis identified PAI-1 as a major gene highly induced by hypoxia in HPTECs. Treatment of 24 hours with hypoxia, TNF-alpha, and their combination induced a 2.8-fold, a 1.8-fold, and a 4.6-fold increase in PAI-1 protein secretion, and produced a 3.6-fold, a 3.3-fold, and a 12.1-fold increase at the PAI-1 mRNA level, respectively. Immunoblot analysis and immunocytochemistry revealed that hypoxia-inducible factor-1alpha (HIF-1alpha) was markedly accumulated in the cell lysates and exclusively translocated to nuclei after 16 hours' exposure of HPTECs to hypoxia but not to TNF-alpha. Luciferase reporter gene assay showed that hypoxia, TNF-alpha, and their combination increased PAI-1 transcription activity by 1.8-fold, 1.4-fold, and 2.2-fold, respectively. A dominant-negative form of HIF-1alpha significantly suppressed PAI-1 transcription activity induced by hypoxia. Inhibition of nuclear factor-kappaB (NF-kappaB) caused a moderate decrease in PAI-1 production under hypoxia. CONCLUSION Hypoxia induces PAI-1 expression via remarkable nuclear accumulation of HIF-1alpha and partially via NF-kappaB activation in HPTECs. TNF-alpha can synergistically enhance this hypoxia-induced PAI-1 expression.