低分子量肝素可预防高糖和佛波酯诱导的转化生长因子-β1基因激活。

Low-molecular-weight heparin prevents high glucose- and phorbol ester-induced TGF-beta 1 gene activation.

作者信息

Weigert C, Brodbeck K, Häring H U, Gambaro G, Schleicher E D

机构信息

Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobio-chemistry, University of Tübingen, Tübingen, Germany.

出版信息

Kidney Int. 2001 Sep;60(3):935-43. doi: 10.1046/j.1523-1755.2001.060003935.x.

DOI:10.1046/j.1523-1755.2001.060003935.x

PMID:11532088

Abstract

BACKGROUND

Hyperglycemia-induced overexpression of prosclerotic transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of diabetic nephropathy. Since previous in vivo studies demonstrated a renoprotective effect of low-molecular-weight (LMW) heparin in experimental animals, and recent in vitro data showed an interaction of this drug with the overactivated TGF-beta 1 cascade in high glucose- and phorbol ester-stimulated mesangial cells, we studied the molecular mechanism of these effects on TGF-beta 1 gene expression.

METHODS

Mesangial cells were stimulated with 30 mmol/L glucose or with 0.5 micromol/L phorbol ester [phorbol myristate acetate (PMA)] in the absence or presence of LMW heparin. TGF-beta 1 promoter activity was determined in promoter-reporter luciferase assays. The effect of LMW heparin on the binding of nuclear proteins to a regulatory activator protein-1 (AP-1) site, which mediates the high glucose and PMA responsiveness of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays.

RESULTS

The presence of LMW heparin completely prevented TGF-beta 1 gene activation in both high glucose- and PMA-stimulated cells. Preincubation of the cells with LMW heparin and subsequent stimulation of the cells with both stimuli yielded the same result. Furthermore, treatment with LMW heparin prevented the enhanced binding of nuclear proteins to the regulatory AP-1 site, while binding to a Sp1 site was unaffected. Basal promoter activity and basal AP-1 binding also was reduced by LMW heparin. The LMW heparin effect on basal promoter activity was abolished by mutation of the regulatory AP-1 box B and by deletion of this AP-1 binding site.

CONCLUSIONS

LMW heparin prevents high glucose- and PMA-mediated TGF-beta 1 expression by inhibiting the activation of the TGF-beta 1 promoter and by preventing the enhanced binding of nuclear proteins to the regulatory AP-1 site.

摘要

背景

高血糖诱导的促硬化转化生长因子-β1（TGF-β1）过表达与糖尿病肾病的发病机制有关。由于先前的体内研究证明低分子量（LMW）肝素对实验动物具有肾脏保护作用，且最近的体外数据显示该药物与高糖和佛波酯刺激的系膜细胞中过度激活的TGF-β1级联反应存在相互作用，我们研究了这些作用对TGF-β1基因表达的分子机制。

方法

在不存在或存在LMW肝素的情况下，用30 mmol/L葡萄糖或0.5 μmol/L佛波酯[佛波醇肉豆蔻酸酯乙酸酯（PMA）]刺激系膜细胞。在启动子报告荧光素酶测定中测定TGF-β1启动子活性。通过电泳迁移率变动分析研究LMW肝素对核蛋白与调节激活蛋白-1（AP-1）位点结合的影响，该位点介导TGF-β1启动子的高糖和PMA反应性。

结果

LMW肝素的存在完全阻止了高糖和PMA刺激的细胞中TGF-β1基因的激活。用LMW肝素预孵育细胞并随后用两种刺激物刺激细胞产生了相同的结果。此外，LMW肝素处理可防止核蛋白与调节性AP-1位点的结合增强，而与Sp1位点结合不受影响。LMW肝素也降低了基础启动子活性和基础AP-1结合。通过调节性AP-1框B的突变和该AP-1结合位点的缺失，消除了LMW肝素对基础启动子活性的影响。