Brasse-Lagnel Carole, Fairand Alain, Lavoinne Alain, Husson Annie
Groupe Appareil Digestif, Environnement et Nutrition, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Université de Rouen, 76183 Rouen cedex, France.
J Biol Chem. 2003 Dec 26;278(52):52504-10. doi: 10.1074/jbc.M306752200. Epub 2003 Oct 21.
Glutamine stimulates the expression of the argininosuccinate synthetase (ASS) gene at both the level of enzyme activity and mRNA in Caco-2 cells. Searching to identify the pathway involved, we observed that (i) the stimulating effect of glutamine was totally mimicked by glucosamine addition, and (ii) its effect but not that of glucosamine was totally blocked by 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of amidotransferases, suggesting that the metabolism of glutamine to glucosamine 6-phosphate was required. Moreover, run-on assays revealed that glucosamine was acting at a transcriptional level. Because three functional GC boxes were identified on the ASS gene promoter (Anderson, G. M., and Freytag, S. O. (1991) Mol. Cell Biol. 11, 1935-1943), the potential involvement of Sp1 family members was studied. Electrophoretic mobility shift assays using either the Sp1 consensus sequence or an appropriate fragment of the ASS promoter sequence as a probe demonstrated that both glutamine and glucosamine increased Sp1 DNA binding. Immunoprecipitation-Western blot experiments demonstrated that both compounds increased O-glycosylation of Sp1 leading to its translocation into nucleus. Again, the effect of glutamine on Sp1 was inhibited by the addition of DON but not of glucosamine. Taken together, the results clearly demonstrate that the metabolism of glutamine through the hexosamine pathway leads to the cytosolic O-glycosylation of Sp1, which, in turn, translocates into nucleus and stimulates the ASS gene transcription. Collectively, the results constitute the first demonstration of a functional relationship between a regulating signal (glutamine), a transcription factor (Sp1), and the transcription of the ASS gene.
谷氨酰胺在酶活性和mRNA水平上均能刺激Caco-2细胞中精氨琥珀酸合成酶（ASS）基因的表达。为了确定其中涉及的途径，我们观察到：（i）添加氨基葡萄糖可完全模拟谷氨酰胺的刺激作用；（ii）氨基转移酶抑制剂6-重氮-5-氧代-L-正亮氨酸（DON）可完全阻断谷氨酰胺的作用，但不影响氨基葡萄糖的作用，这表明谷氨酰胺代谢生成6-磷酸氨基葡萄糖是必需的。此外，核转录分析表明氨基葡萄糖作用于转录水平。由于在ASS基因启动子上鉴定出了三个功能性GC框（Anderson, G. M., and Freytag, S. O. (1991) Mol. Cell Biol. 11, 1935 - 1943），因此研究了Sp1家族成员的潜在参与情况。使用Sp1共有序列或ASS启动子序列的适当片段作为探针进行的电泳迁移率变动分析表明，谷氨酰胺和氨基葡萄糖均增加了Sp1与DNA的结合。免疫沉淀-蛋白质印迹实验表明，这两种化合物均增加了Sp1的O-糖基化，导致其易位至细胞核。同样，添加DON可抑制谷氨酰胺对Sp1的作用，但不影响氨基葡萄糖的作用。综上所述，结果清楚地表明，谷氨酰胺通过己糖胺途径的代谢导致Sp1在细胞质中的O-糖基化，进而易位至细胞核并刺激ASS基因转录。总体而言，这些结果首次证明了调节信号（谷氨酰胺）、转录因子（Sp1）与ASS基因转录之间的功能关系。
