Paterson A J, Kudlow J E
Department of Medicine, University of Alabama, Birmingham 35294, USA.
Endocrinology. 1995 Jul;136(7):2809-16. doi: 10.1210/endo.136.7.7789306.
In preparation for the cellular proliferation stimulated by growth factors, the rate of macromolecular synthesis must be increased to allow for the enlargement of the cell that proceeds mitosis. The increased glycoprotein synthesis that follows growth factor stimulation would consume the hexosamines required for protein modification. Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme controlling the synthesis of the hexosamines used in these biosynthetic pathways. We tested the idea that growth factors might activate the transcription of the GFAT gene to increase the cellular content of this rate-limiting enzyme in hexosamine synthesis. We employed a human breast cancer cell line, MDA468 cells, which express high numbers of epidermal growth factor (EGF) receptors, to determine whether EGF could stimulate transcription of the GFAT gene. Our experiments showed that EGF stimulated the accumulation of GFAT messenger RNA (mRNA) to a level 4-fold higher than that in unstimulated cells. This accumulation could be largely accounted for by an increase in transcription, as assessed by nuclear run-on experiments. Furthermore, the GFAT mRNA was highly stable and not further stabilized by EGF. This effect of EGF on GFAT gene transcription required stimulation for 12-16 h with EGF. Interestingly, when cells were exposed to 25 mM glucose instead of 5 mM glucose, this effect of EGF was blocked. Glucose had no effect on the stability of the GFAT mRNA, implying that the effect of glucose was to antagonize the transcriptional effect of EGF on the GFAT gene. Glucosamine had an effect opposite that of glucose, in that it stimulated GFAT mRNA accumulation and had an additive effect with EGF on the accumulation of this mRNA. These results demonstrate that the GFAT gene undergoes a late transcriptional response to EGF and that the provision of high glucose concentrations to the cells blocks this EGF activation. This effect of glucose does not appear to result from its metabolism through GFAT to glucosamine.
为了准备由生长因子刺激引发的细胞增殖，必须提高大分子合成的速率，以允许细胞在进行有丝分裂之前得以增大。生长因子刺激后糖蛋白合成的增加会消耗蛋白质修饰所需的己糖胺。谷氨酰胺：6-磷酸果糖酰胺转移酶（GFAT）是控制这些生物合成途径中所用己糖胺合成的限速酶。我们测试了生长因子可能激活GFAT基因转录以增加该己糖胺合成限速酶细胞含量的想法。我们使用了一种人乳腺癌细胞系MDA468细胞，该细胞系表达大量表皮生长因子（EGF）受体，以确定EGF是否能刺激GFAT基因的转录。我们的实验表明，EGF刺激GFAT信使核糖核酸（mRNA）的积累，使其水平比未刺激细胞高4倍。通过核转录实验评估，这种积累很大程度上可归因于转录的增加。此外，GFAT mRNA高度稳定，EGF不会进一步使其稳定。EGF对GFAT基因转录的这种作用需要用EGF刺激12 - 16小时。有趣的是，当细胞暴露于25 mM葡萄糖而非5 mM葡萄糖时，EGF的这种作用被阻断。葡萄糖对GFAT mRNA的稳定性没有影响，这意味着葡萄糖的作用是拮抗EGF对GFAT基因的转录作用。氨基葡萄糖的作用与葡萄糖相反，它刺激GFAT mRNA的积累，并且与EGF对该mRNA的积累具有相加作用。这些结果表明，GFAT基因对EGF经历晚期转录反应，并且向细胞提供高葡萄糖浓度会阻断这种EGF激活。葡萄糖的这种作用似乎不是由于其通过GFAT代谢为氨基葡萄糖所致。
