Borlak Jürgen, Niehof Monika
Fraunhofer Institute of Toxicology and Experimental Medicine, Medical School of Hannover, Hannover, Germany.
PLoS One. 2009;4(3):e4662. doi: 10.1371/journal.pone.0004662. Epub 2009 Mar 2.
Posttransplantation diabetes mellitus (PTDM) is a frequent complication in immunosuppressive therapy. To better understand the molecular events associated with PTDM we investigated the effect of cyclosporine on expression and activity of hepatic nuclear factor (HNF)1alpha and 4alpha and on genes coding for glucose metabolism in cultures of the rat insulinoma cell line INS-1E, the human epithelial cell line Caco-2 and with Zucker diabetic fatty (ZDF) rats. In the pancreas of untreated but diabetic animals expression of HNF4alpha, insulin1, insulin2 and of phosphoenolpyruvate carboxykinase was significantly repressed. Furthermore, cyclosporine treatment of the insulinoma-1E cell line resulted in remarkable reduction in HNF4alpha protein and INS1 as well as INS2 gene expression, while transcript expression of HNF4alpha, apolipoprotein C2, glycerolkinase, pyruvatekinase and aldolase B was repressed in treated Caco-2 cells. Furthermore, with nuclear extracts of cyclosporine treated cell lines protein expression and DNA binding activity of hepatic nuclear factors was significantly repressed. As cyclosporine inhibits the calcineurin dependent dephosphorylation of nuclear factor of activated T-cells (NFAT) we also searched for binding sites for NFAT in the pancreas specific P2 promoter of HNF4alpha. Notably, we observed repressed NFAT binding to a novel DNA binding site in the P2 promoter of HNF4alpha. Thus, cyclosporine caused inhibition of DNA binding of two important regulators for insulin signaling, i.e. NFAT and HNF4alpha. We further investigated HNF4alpha transcript expression and observed >200-fold differences in abundance in n = 14 patients. Such variability in expression might help to identify individuals at risk for developing PTDM. We propose cyclosporine to repress HNF4alpha gene and protein expression, DNA-binding to targeted promoters and subsequent regulation of genes coding for glucose metabolism and of pancreatic beta-cell function.
移植后糖尿病（PTDM）是免疫抑制治疗中常见的并发症。为了更好地理解与PTDM相关的分子事件，我们研究了环孢素对大鼠胰岛素瘤细胞系INS-1E、人上皮细胞系Caco-2以及Zucker糖尿病脂肪（ZDF）大鼠培养物中肝细胞核因子（HNF）1α和4α的表达与活性以及葡萄糖代谢相关基因的影响。在未经治疗但患有糖尿病的动物胰腺中，HNF4α、胰岛素1、胰岛素2以及磷酸烯醇式丙酮酸羧激酶的表达明显受到抑制。此外，用环孢素处理胰岛素瘤-1E细胞系导致HNF4α蛋白以及INS1和INS2基因表达显著降低，而在处理过的Caco-2细胞中，HNF4α、载脂蛋白C2、甘油激酶、丙酮酸激酶和醛缩酶B的转录本表达受到抑制。此外，用环孢素处理的细胞系的核提取物中，肝细胞核因子的蛋白表达和DNA结合活性明显受到抑制。由于环孢素抑制活化T细胞核因子（NFAT）的钙调神经磷酸酶依赖性去磷酸化，我们还在HNF4α的胰腺特异性P2启动子中寻找NFAT的结合位点。值得注意的是，我们观察到NFAT与HNF4α的P2启动子中的一个新的DNA结合位点的结合受到抑制。因此，环孢素导致胰岛素信号传导的两个重要调节因子，即NFAT和HNF4α的DNA结合受到抑制。我们进一步研究了HNF4α转录本表达，在14名患者中观察到丰度存在200倍以上的差异。这种表达的变异性可能有助于识别有发生PTDM风险的个体。我们提出环孢素会抑制HNF4α基因和蛋白表达、与靶向启动子的DNA结合以及随后对葡萄糖代谢相关基因和胰腺β细胞功能的调节。
