Glucose regulates the promoter activity of aldolase A and pyruvate kinase M2 via dephosphorylation of Sp1.

Proliferating cells and tumour cells maintain a high glycolytic rate even under aerobic conditions. FTO2B cells, a rat hepatoma cell line, show high activities of glycolytic enzymes. Within a culture period of 48 h the cell number increases 5-fold. Replacement of glucose by pyruvate in the culture medium lowers glycolytic enzyme activity and prevents proliferation. Transfection assays revealed that glucose deprivation dramatically decreases the transcriptional activities of the Sp1-dependent aldolase and pyruvate kinase promoters leading to reduced reporter gene expression. Sp1 binding activity is also inhibited by ocadaic acid, an inhibitor of protein phosphatase 1. Western blot analyses with nuclear extracts from FTO2B cells cultured in the presence or absence of glucose revealed differences in the phosphorylation state of Sp1. From these results we conclude that glucose increases the amount of the dephosphorylated form of Sp1 which has a higher DNA binding activity. As a consequence gene expression of the glycolytic enzymes is increased which is a prerequisite for cell proliferation.

Redox-regulated expression of glycolytic enzymes in resting and proliferating rat thymocytes.

Resting rat thymocytes partially degrade glucose aerobically to CO2 and H2O and produce reactive peroxide anions. In contrast proliferating cells, due to enhanced induction of glycolytic enzymes, degrade glucose almost completely to lactate thus minimizing the production of reactive oxygen species. In this paper we show that under conditions of oxidative stress the induction of the glycolytic enzymes in cultured rat thymocytes is markedly reduced. Furthermore, transfection assays with a rat hepatoma cell line and Drosophila Schneider cells revealed that reactive oxygen intermediates dramatically decrease the transcriptional activities of the Sp1-dependent aldolase A and pyruvate kinase M2 promoters leading to reduced reporter gene expression. These results indicate that cellular redox changes can regulate gene expression by reversible oxidative inactivation of Sp1 binding.

Differences in DNA-binding efficiency of Sp1 to aldolase and pyruvate kinase promoter correlate with altered redox states in resting and proliferating rat thymocytes.

Thymocytes induce their glycolytic enzymes as they undergo transition from the resting to the proliferating state. Corresponding increases in mRNA levels point to a transcriptional regulation. Electrophoretic mobility shift assays revealed that the DNA-binding efficiency of Sp1 is increased when nuclear extracts from proliferating compared to resting rat thymocytes were used. Here we demonstrate that hydrogen peroxide, added to nuclear extract from proliferating cells, decreases the Sp1 DNA-binding activity, whereas in nuclear extracts from resting cells dithioerythritol fully restores DNA-binding efficiency. Moreover we show that in contrast to resting thymocytes, production of reactive peroxide anions upon priming with phorbol 12-myristate 13-acetate is nearly abolished in the proliferating cells. From these results we propose that reactive oxygen intermediates affect the interaction of the Sp1 transcription factor with its consensus sequence and subsequently regulate glycolytic gene expression.

Down regulation of S-adenosyl-L-methionine decarboxylase activity of Trypanosoma brucei during transition from long slender to short stumpy-like forms in axenic culture.

Long slender trypanosomes, isolated from infected mouse blood or from cryopreserved stabilates, respectively, were unable to grow in conditioned media (cMEM), prepared from the declining phase of axenic bloodstream form cultures. Additionally, mixtures of fresh medium and cMEM led to decreased growth rates and, in accordance to the amount of cMEM used, to a decreased S-adenosyl-L-methionine decarboxylase (Ado-MetDC; E.C. 4.1.1.50) activity. Since addition of polyamines could not overcome the process of transition from dividing to non-dividing cells and the intracellular S-adenosyl-L-methionine (AdoMet), ornithine and putrescine concentrations seemed unaltered during the course of cultivation, we questioned if polyamine metabolism is involved in this transition process. Activities of two key enzymes of polyamine metabolism, AdoMetDC and ornithine decarboxylase (ODC; E.C. 4.1.1.17) were therefore monitored during different growth stages. Our results revealed a specific activity of 44 pmol min-1 mg protein-1 for AdoMetDC and a KM of 10 mu M for AdoMet. Methylglyoxal bis(guanylhydrazone) showed a Ki of 6 mu M. The constant activity of the enzyme during a 7 h time-course in the presence of cycloheximide indicates a t1/2 of more than 7 h for the trypanosomal enzyme. Enzyme activity in trypanosomes isolated from infected laboratory animals and from logarithmic phase bloodstream or procyclic form cultures was high according to a high dividing rate, whereas enzyme activity in parasites isolated from the stationary phase of bloodstream from culture was negligible. In these cultures, AdoMetDC activity decreased with a t1/2 of 7 h during transition from long slender to short stumpy-like forms as soon as the stationary phase was reached. ODC activity was high (approximately 300 pmol min-1 mg protein-1) in dividing trypanosomes isolated from infected animals as well as from logarithmic phase bloodstream or procyclic form cultures and decreased also during transition with a t1/2 of 10 h.