Search

Background: Pulmonary artery hypertension (PAH) is a severe proliferative disease characterized by the remodeling of small pulmonary arteries leading to a progressive increase in pulmonary vascular resistance and ultimately to right ventricular failure and death. Many studies have indicated that endothelial dysfunction is a key element in the pathogenesis of this disease. In this study, we investigated whether endothelial glycolysis plays a critical role in the development of hypoxia-induced PAH by knocking down endothelial PFKFB3 (encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3), a critical regulator of glycolysis. Methods and Results: Immunostaining results showed that pulmonary artery endothelial PFKFB3 was increased in the lung of PAH patients. Mice deficient in endothelial Pfkfb3 showed decreased right ventricular systolic pressure, attenuated right ventricular hypertrophy and normal morphology of distal pulmonary arteries after four weeks of hypoxia compared with control mice. Furthermore, PFKFB3-deficient pulmonary arterial ECs secreted less growth factors, which ameliorated the proliferation of pulmonary artery smooth muscle cells. Additionally, the levels of endothelial derived inflammatory cytokines were also decreased. Besides, the inhibitory effect of endothelial Pfkfb3 supression on PAH formation were demonstrated in Sugen 5416/hypoxia rat pulmonary hypertension model with 3PO, a specific PFKFB3 inhibitor. Mechanistically, genetic deletion of PFKFB3 or 3PO treatment decreased the TNF-α induced NLRP3 inflammasome activation in pulmonary arterial ECs. Conclusions: Endothelial PFKFB3 inhibition is able to ameliorate the development of hypoxia-induced pulmonary hypertension.

6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is a key regulatory enzyme of glucose metabolism. This bifunctional enzyme controls glycolysis and gluconeogenesis as well as glucose phosphorylation and metabolism. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) plays significant role in the glycolysis regulation in the heart as well as in some other organs (kidney, brain and lung). Results of this investigation clearly demonstrated that in the glioma cells as well as in some mouse organs are presented several new unique alternative splice variants of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2. These PFKFB-2 splice variants are a result of deletions or/and inserts in catalytic regions of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase as well as in С-terminal regulatory region. Alternative splice variants of PFKFB-2 from glioma and mouse cells contain different inserts after 2nd exon but mouse splice variant has also deletion in 6-phosphofructo-2-kinase region and insert in C-terminus. Another splice variants of mouse PFKFB-2 have deletions in 6-phosphofructo-2-kinase or fructose-2,6-bisphosphatase regions, since one splice variant has deletion of all catalytic domains. In conclusion, this study provides evidences that alternative splicing of PFKFB-2 mRNA possibly has a significant role in glycolysis regulation via creation of monofunctional isoforms of this enzyme.

A bifunctional PFKFB enzyme is a key regulator of glycolysis. Four different genes encode the synthesis of multiple PFKFB isoforms. We analyzed data concerning structural organization of PFKFB genes and alternative splice variants of PFKFB mRNA, as well as expression and molecular mechanisms of regulation of different PFKFB isoforms in malignant tumors and at hypoxia, and mechanisms of hormonal regulation. Hypoxia inducible factor (HIF)-dependent mechanisms of regulation of the expression of different PFKFB variants are presented and discussed.

Methyl tertial butyl ether, an oxygenated compound of gasoline, is a toxic and ecolo­gically dangerous chemical compound with an ability to introduce a variety of neurotoxic, allergic and respiratory diseases, as well as cancer and leukemia. We used RT/PCR or quan­titative PCR to study the effect of methyl tertial butyl ether (everyday injection for one month) on the expression of mRNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) and vascular endothelial growth factor (VEGF), which are important for tumor growth and metastasis in the lung and liver. The level of PFKFB-3 and VEGF mRNA expression was increased in the liver in methyl tertial butyl ether treated rats with respect to the control animals. However, methyl tertial butyl ether had opposite ef­fect on the expression of PFKFB-3 and VEGF mRNA in the lung: with strong induction of PFKFB-3 and suppression of VEGF mRNA expression. Moreover, alternative splicing of PFKFB-3 mRNA was changed in methyl tertial butyl ether treated rats in organ specific manner. Thus, methyl tertial butyl ether induces alternative splicing of PFKFB-3 mRNA accompanied by appearing of variants with longer C-terminus in the liver, and with shorter C-terminus in the lung. The results of this investigation clearly demonstrate that methyl tertial butyl ether affects in organ-specific manner the expression and alternative spli­cing of mRNA coding for PFKFB-3 and VEGF, key regulatory factors of glycolysis, and angiogenesis.

The expression of the F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA was studied during liver regeneration by three independent assays: Northern blot analysis, reverse transcription-polymerase chain reaction and ribonuclease protection. We demonstrate the presence of F-type mRNA in foetal and adult rat livers and a transient increase in its levels with a maximum at 12 h after partial liver resection. The time course of F-type mRNA induction differs from that reported for the L-type isoform, suggesting differences in the regulation of the expression of F- and L-type isoforms of the bifunctional enzyme during liver regeneration.

This work was supported by grants 9110229 and 9210267 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Spain.

A chicken liver cDNA for 6-phosphofructo-2-kinase/fructose-2,6. bisphosphatase was isolated from a Lambda ZAP2 phage library. The chicken liver cDNA codes for a protein that has 89.1, 88.4 and 88.0% amino acid identity with the human, rat and bovine liver isoforms, respectively. The kinetic properties of the rat and chicken liver enzymes, purified to homogeneity after expression in E. coli , were different including negative cooperativity for ATP binding and inhibition by Mg 2+ for the chicken liver 6-phosphofructo-2-kinase but not for the rat liver kinase. Differences in the β-loop ATP signature sequences in the chicken and rat liver kinase domains may explain the kinetic differences and represent the major divergence in the evolution of the enzyme from birds to mammals.

Lys-356 has been implicated as a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Li, L., Lin, K., Correia, J., and Pilkis, S. J. (1992) J. Biol. Chem. 267, 16669-16675). To ascertain whether the three other basic residues (Arg-352, Arg-358, and Arg-360), which are located in a surface loop (residues 331-362) which contains Lys-356, are important in substrate binding, these arginyl residues were mutated to Ala, and each arginyl mutant was expressed in Escherichia coli and purified to homogeneity. The far UV circular dichroism spectra of the mutants were identical to that of the wild-type enzyme. The kinetic parameters of 6-phosphofructo-2-kinase of the mutants revealed only small changes. However, the Km for fructose 2,6-bisphosphate, Ki for fructose 6-phosphate, and Ka for inorganic phosphate of fructose-2,6-bisphosphatase for Arg352Ala were, respectively, 2,800-, 4,500-, and 1,500-fold higher than those for the wild-type enzyme, whereas there was no change in the maximal velocity or the Ki for inorganic phosphate. The Km for fructose 2,6-bisphosphate and Ki for inorganic phosphate of Arg360Ala were 10- and 12-fold higher, respectively, than those of the wild-type enzyme, whereas the maximal velocity and Ki for fructose 6-phosphate were unchanged. In addition, substrate inhibition was not observed with Arg352Ala and greatly reduced with Arg360Ala. The properties of the Arg358Ala mutant were identical to those of the wild-type enzyme. The results demonstrate that in addition to Lys-356, Arg-352 is another critical residue in fructose-2,6-bisphosphatase for binding the C-6 phospho group of fructose 2,6-bisphosphate and that Arg-360 binds the C-2 phospho group of fructose 2,6-bisphosphate in the phosphoenzyme.fructose 2,6-bisphosphate complex. The results also provide support for Arg-352, Lys-356, and Arg-360 constituting a specificity pocket for fructose-2,6-bisphosphatase.

Transgenic carnations were produced with a modified mammalian bifunctional enzyme cDNA coding 6-phosphofructo-2- kinaseffructose 2,6-bisphosphatase. Relative activity of this enzyme determines the fructose 2,6-bisphosphate (fru 2,6-P2) cytosolic concentration. This metabolite — as a signal molecule — is one of the carbohydrate metabolism regulators. The regenerated Dianthus chinensis and Dianthus caryophyllus shoots were selected on MS basal medium containing 150 mg/1 kanamycin. Transgene integration was proven by PCR analysis with cDNA specific primers followed by Southern hybridization of DNA isolated from selected green shoots, which survived on kanamycin containing medium, so 3 D. chinensis and 20 D. caryophyllus transgenic plants were produced. Transgene expression were examined by RT-PCR. Transformed and control plants were potted in glasshouse to evaluate the effect of modified fru 2,6-P2 on development, growth and carbohydrate metabolism.

Neoplastic cells metabolize abundant glucose relative to normal cells in order to satisfy the increased energetic and anabolic needs of the transformed state. This review will summarize the requirement of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases for the regulation of glycolysis in cancer cells and their potential utility as targets for the development of antineoplastic agents.The steady-state concentration of fructose-2,6-bisphosphate controls the overall rate of glycolysis by allosterically activating a rate-limiting enzyme, 6-phosphofructo-1-kinase. The intracellular concentration of fructose-2,6-bisphosphate is controlled by a family of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases that are encoded by four independent genes (PFKFB1-4). The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase encoded by the PFKFB3 gene has the highest kinase:phosphatase activity ratio of the four enzymes and thus contributes significantly to the synthesis of fructose-2,6-bisphosphate. PFKFB3 is activated by mitogenic, inflammatory and hypoxic stimuli, and was recently found to be constitutively expressed by several human leukemias and solid tumor cells. By setting the intracellular fructose-2,6-bisphosphate concentration, PFKFB3 controls glycolytic flux to lactate and the nonoxidative pentose shunt, and is selectively required for the tumorigenic growth of ras-transformed cells.These findings demonstrate a key role for the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in neoplastic transformation and provide rationale for the development of agents that selectively inhibit the PFKFB3 enzyme as antineoplastic agents.

The Fru-6-P/Fru-2,6-P2 exchange reaction of rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was almost entirely dependent on the presence of Pi. This exchange was not due to a reversal of the bisphosphatase nor to trace amounts of adenine nucleotide in the enzyme. Exchange activity was maximal at pH 7, activated by ADP, and equal to 10-15 percent of the kinase Vmax. The ADP/ATP exchange reaction was more resistant to various protein modifying agents than the kinase. These studies confirm the existence of both exchange reactions but do not prove they are related to the kinase reaction.

Stationary states of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle were investigated in relation to the input concentration of fructose 6-phosphate. Below a critical input concentration of fructose 6-phosphate very low levels of fructose 2,6-bisphosphate were obtained. Above this point the fructose 2,6-bisphosphate changes in direct proportion to the input of fructose 6-phosphate. Phosphorylation of the enzyme causes an increase of the critical input concentration of fructose 6-phosphate. The control coefficients for fructose 2,6-bisphosphate have their maximum at the critical input concentration of fructose 6-phosphate.