Small molecule inhibition of 6-phosphofructo-2-kinase suppresses t cell activation

Journal of Translational Medicine

Jason Chesney¹  Richard Bucala⁴  John O Trent²  Amy L Clem²  Alden C Klarer²  Brian F Clem²  Sucheta Telang³ 

[1] James Graham Brown Cancer Center, University of Louisville, 505 South Hancock Street, #424, Louisville, KY, 40202, USA;James Graham Brown Cancer Center, Division of Medical Oncology and Hematology, Department of Medicine, University of Louisville, Louisville, KY, USA;University of Louisville, 505 South Hancock Street, #423, Louisville, KY, 40202, USA;Division of Rheumatology, Department of Medicine, Yale University School of Medicine, New Haven, CT, USA

关键词: T cell;    6-bisphosphate;    Fructose-2;    6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase;    Glycolysis;   

Others  :  1205986
DOI  :  10.1186/1479-5876-10-95

 received in 2012-01-26, accepted in 2012-05-16,  发布年份 2012

【 摘 要 】

Background

T cell activation is associated with a rapid increase in intracellular fructose-2,6-bisphosphate (F2,6BP), an allosteric activator of the glycolytic enzyme, 6-phosphofructo-1-kinase. The steady state concentration of F2,6BP in T cells is dependent on the expression of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and the fructose-2,6-bisphosphatase, TIGAR. Of the PFKFB family of enzymes, PFKFB3 has the highest kinase:bisphosphatase ratio and has been demonstrated to be required for T cell proliferation. A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), recently has been shown to reduce F2,6BP synthesis, glucose uptake and proliferation in transformed cells. We hypothesized that the induction of PFKFB3 expression may be required for the stimulation of glycolysis in T cells and that exposure to the PFKFB3 antagonist, 3PO, would suppress T cell activation.

Methods

We examined PFKFB1-4 and TIGAR expression and F2,6BP concentration in purified CD3⁺ T cells stimulated with microbead-conjugated agonist antibodies specific for CD3 and the co-stimulatory receptor, CD28. We then determined the effect of 3PO on anti-CD3/anti-CD28-induced T cell activation, F2,6BP synthesis, 2-[1-¹⁴C]-deoxy-D-glucose uptake, lactate secretion, TNF-α secretion and proliferation. Finally, we examined the effect of 3PO administration on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice.

Results

We found that purified human CD3⁺ T cells express PFKFB2, PFKFB3, PFKFB4 and TIGAR, and that anti-CD3/anti-CD28 conjugated microbeads stimulated a >20-fold increase in F2,6BP with a coincident increase in protein expression of the PFKFB3 family member and a decrease in TIGAR protein expression. We then found that exposure to the PFKFB3 small molecule antagonist, 3PO (1–10 μM), markedly attenuated the stimulation of F2,6BP synthesis, 2-[1-¹⁴C]-deoxy-D-glucose uptake, lactate secretion, TNF-α secretion and T cell aggregation and proliferation. We examined the in vivo effect of 3PO on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice and found that 3PO suppressed the development of both T cell-dependent models of immunity in vivo.

Conclusions

Our data demonstrate that inhibition of the PFKFB3 kinase activity attenuates the activation of T cells in vitro and suppresses T cell dependent immunity in vivo and indicate that small molecule antagonists of PFKFB3 may prove effective as T cell immunosuppressive agents.

【 授权许可】

   
2012 Telang et al.; licensee BioMed Central Ltd.

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【 参考文献 】

• [1]Cooper EH, Barkhan P, Hale AJ: Observations on the proliferation of human leucocytes cultured with phytohaemagglutinin. Br J Haematol 1963, 9:101-111.
• [2]Culvenor JG, Weidemann MJ: Phytohaemagglutinin stimulation of rat thymus lymphocytes glycolysis. Biochim Biophys Acta 1976, 437:354-363.
• [3]Roos D, Loos JA: Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. II. Relative importance of glycolysis and oxidative phosphorylation on phytohaemagglutinin stimulation. Exp Cell Res 1973, 77:127-135.
• [4]Wang T, Marquardt C, Foker J: Aerobic glycolysis during lymphocyte proliferation. Nature 1976, 261:702-705.
• [5]Jones RG, Thompson CB: Revving the engine: signal transduction fuels T cell activation. Immunity 2007, 27:173-178.
• [6]Maciver NJ, Jacobs SR, Wieman HL, Wofford JA, Coloff JL, Rathmell JC: Glucose metabolism in lymphocytes is a regulated process with significant effects on immune cell function and survival. J Leukoc Biol 2008, 84:949-957.
• [7]Bosca L, Mojena M, Diaz-Guerra JM, Marquez C: Phorbol 12,13-dibutyrate and mitogens increase fructose 2,6-bisphosphate in lymphocytes. Comparison of lymphocyte and rat-liver 6-phosphofructo-2-kinase. Eur J Biochem 1988, 175:317-323.
• [8]Moreno-Aurioles VR, Sobrino F: Glucocorticoids inhibit fructose 2,6-bisphosphate synthesis in rat thymocytes. Opposite effect of cycloheximide. Biochim Biophys Acta 1991, 1091:96-100.
• [9]Okar DA, Manzano A, Navarro-Sabate A, Riera L, Bartrons R, Lange AJ: PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate. Trends Biochem Sci 2001, 26:30-35.
• [10]Okar DA, Wu C, Lange AJ: Regulation of the regulatory enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Adv Enzyme Regul 2004, 44:123-154.
• [11]Yalcin A, Telang S, Clem B, Chesney J: Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. Exp Mol Pathol 2009, 86:174-179.
• [12]Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R, Gottlieb E, Vousden KH: TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell 2006, 126:107-120.
• [13]Rider MH, Bertrand L, Vertommen D, Michels PA, Rousseau GG, Hue L: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis. Biochem J 2004, 381:561-579.
• [14]Okar DA, Lange AJ: Fructose-2,6-bisphosphate and control of carbohydrate metabolism in eukaryotes. Biofactors 1999, 10:1-14.
• [15]Chesney J: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and tumor cell glycolysis. Curr Opin Clin Nutr Metab Care 2006, 9:535-539.
• [16]Telang S, Yalcin A, Clem AL, Bucala R, Lane AN, Eaton JW, Chesney J: Ras transformation requires metabolic control by 6-phosphofructo-2-kinase. Oncogene 2006, 25:7225-7234.
• [17]Wu C, Okar DA, Newgard CB, Lange AJ: Increasing fructose 2,6-bisphosphate overcomes hepatic insulin resistance of type 2 diabetes. Am J Physiol Endocrinol Metab 2002, 282:E38-E45.
• [18]Wu C, Okar DA, Stoeckman AK, Peng LJ, Herrera AH, Herrera JE, Towle HC, Lange AJ: A potential role for fructose-2,6-bisphosphate in the stimulation of hepatic glucokinase gene expression. Endocrinology 2004, 145:650-658.
• [19]Chesney J, Mitchell R, Benigni F, Bacher M, Spiegel L, Al-Abed Y, Han JH, Metz C, Bucala R: An inducible gene product for 6-phosphofructo-2-kinase with an AU-rich instability element: role in tumor cell glycolysis and the Warburg effect. Proc Natl Acad Sci U S A 1999, 96:3047-3052.
• [20]Chesney J, Telang S, Yalcin A, Clem A, Wallis N, Bucala R: Targeted disruption of inducible 6-phosphofructo-2-kinase results in embryonic lethality. Biochem Biophys Res Commun 2005, 331:139-146.
• [21]Clem B, Telang S, Clem A, Yalcin A, Meier J, Simmons A, Rasku MA, Arumugam S, Dean WL, Eaton J, et al.: Small-molecule inhibition of 6-phosphofructo-2-kinase activity suppresses glycolytic flux and tumor growth. Mol Cancer Ther 2008, 7:110-120.
• [22]Van Schaftingen E, Lederer B, Bartrons R, Hers HG: A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Application to a microassay of fructose 2,6-bisphosphate. Eur J Biochem 1982, 129:191-195.
• [23]Ishizaki M, Akimoto T, Muromoto R, Yokoyama M, Ohshiro Y, Sekine Y, Maeda H, Shimoda K, Oritani K, Matsuda T: Involvement of tyrosine kinase-2 in both the IL-12/Th1 and IL-23/Th17 axes in vivo. J Immunol 2011, 187:181-189.
• [24]Chatzidakis I, Mamalaki C: T cells as sources and targets of TNF: implications for immunity and autoimmunity. Curr Dir Autoimmun 2010, 11:105-118.
• [25]Ghilardi N, Kljavin N, Chen Q, Lucas S, Gurney AL, De Sauvage FJ: Compromised humoral and delayed-type hypersensitivity responses in IL-23-deficient mice. J Immunol 2004, 172:2827-2833.
• [26]Nestle FO, Kaplan DH, Barker J: Psoriasis. N Engl J Med 2009, 361:496-509.
• [27]van der Fits L, Mourits S, Voerman JS, Kant M, Boon L, Laman JD, Cornelissen F, Mus AM, Florencia E, Prens EP, Lubberts E: Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J Immunol 2009, 182:5836-5845.