REGULATION OF GLUTAMINE-FRUCTOSE-6-PHOSPHATE AMIDOTRANSFERASE GENE-TRANSCRIPTION BY EPIDERMAL GROWTH-FACTOR AND GLUCOSE

In preparation for the cellular proliferation stimulated by growth fac tors, the rate of macromolecular synthesis must be increased to allow for the enlargement of the cell that proceeds mitosis. The increased g lycoprotein synthesis that follows growth factor stimulation would con sume the hexosamines required for protein modification. Glutamine:fruc tose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme c ontrolling the synthesis of the hexosamines used in these biosynthetic pathways. We tested the idea that growth factors might activate the t ranscription of the GFAT gene to increase the cellular content of this rate-limiting enzyme in hexosamine synthesis. We employed a human bre ast cancer cell line, MDA468 cells, which express high numbers of epid ermal growth factor (EGF) receptors, to determine whether EGF could st imulate transcription of the GFAT gene. Our experiments showed that EG F 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 g ene transcription required stimulation for 12-16 h with EGF. Interesti ngly, 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 stabili ty of the GFAT mRNA, implying that the effect of glucose was to antago nize the transcriptional effect of EGF on the GFAT gene. Glucosamine h ad 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 g lucose concentrations to the cells blocks this EGF activation. This ef fect of glucose does not appear to result from its metabolism through GFAT to glucosamine.