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PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.

Finlay DK, Rosenzweig E, Sinclair LV, Feijoo-Carnero C, Hukelmann JL, Rolf J, Panteleyev AA, Okkenhaug K, Cantrell DA - J. Exp. Med. (2012)

Bottom Line: We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex.This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking.These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. finlayd@tcd.ie

ABSTRACT
mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However, PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

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mTORC1 and HIF1 do not regulate Akt activity or Foxo phosphorylation. (A) Immunoblot analysis of phosphorylated AKT and Foxos in P14-LCMV CTLs treated with and without Akti1/2 or rapamycin for 24 h. (B) P14-LCMV CTLs were treated with and without rapamycin or Akti1/2 for 24 h and subjected to nuclear/cytoplasmic fractionation before immunoblot analysis for Foxo1 and Foxo3a expression. Purity of cytoplasmic and nuclear fractions was confirmed by IκBα and Smc1 expression. (C) Immunoblot analysis of WT or HIF1−/− CTLs for Akt–Foxo and mTORC1 signaling. HIF1−/− CTLs were treated with rapamycin as a negative control for mTORC1 activity. For all panels, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons. Dotted lines indicate that intervening lanes have been spliced out.
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fig6: mTORC1 and HIF1 do not regulate Akt activity or Foxo phosphorylation. (A) Immunoblot analysis of phosphorylated AKT and Foxos in P14-LCMV CTLs treated with and without Akti1/2 or rapamycin for 24 h. (B) P14-LCMV CTLs were treated with and without rapamycin or Akti1/2 for 24 h and subjected to nuclear/cytoplasmic fractionation before immunoblot analysis for Foxo1 and Foxo3a expression. Purity of cytoplasmic and nuclear fractions was confirmed by IκBα and Smc1 expression. (C) Immunoblot analysis of WT or HIF1−/− CTLs for Akt–Foxo and mTORC1 signaling. HIF1−/− CTLs were treated with rapamycin as a negative control for mTORC1 activity. For all panels, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons. Dotted lines indicate that intervening lanes have been spliced out.

Mentions: To explore the links between PI3K, Akt, mTORC1, and HIF1, we addressed two questions. Do mTORC1 and HIF1 regulate Akt activity and Foxo phosphorylation? Is PI3K and Akt activity required for mTORC1 activation and HIF1 expression? In the context of the first question, long-term inhibition of mTORC1 can destabilize mTORC2 complexes in some cell systems and suppress Akt function (Sarbassov et al., 2006). Indeed, rapamycin treatment of CD4 T cells activated with CD3 and CD28 antibodies does diminish Akt S473 phosphorylation, although the impact of this on Akt activity has not been fully assessed (Lee et al., 2010; Delgoffe et al., 2011). If this were true in CTLs, then long-term treatment of T cells with rapamycin would result in the loss of Foxo phosphorylation and cause cells to regain Foxo transcriptional activity and thus CD62L and CCR7 expression. We therefore examined the impact of long-term inhibition of mTORC1 with rapamycin on Foxo phosphorylation and localization in CTLs. We also assessed Akt–Foxo and mTORC1 signaling in IL-2–maintained HIF1- T cells to assess whether loss of HIF1 complexes compromised Foxo phosphorylation/inactivation. Fig. 6 (A and B) shows that inhibition of Akt decreased Foxo phosphorylation (Fig. 6 A) and restored nuclear localization of Foxos in CTLs (Fig. 6 B). In contrast, Akt remained active, i.e., phosphorylated on T308 and S473, and Foxos remained highly phosphorylated and excluded from the nucleus of rapamycin-treated CTLs (Fig. 6, A and B). Additionally, Akt phosphorylation (T308) and the phosphorylation of Foxo transcription factors on Akt substrate sites (T24/32) were unaffected in HIF1β- CTLs, demonstrating that Akt signaling is not altered by HIF1 deletion (Fig. 6 C). It was also evident that the phosphorylation of mTORC1 substrates S6K1 (p70 S6-kinase 1) and 4EBP1 (eIF4E-binding protein 1), on T389 and S65, respectively, and downstream signaling to S6 ribosomal protein (an S6K1 substrate) were normal in HIF1β- CD8+ T cells (Fig. 6 C). Therefore, mTORC1 and HIF1 regulate the expression of CD62L and CCR7 and T cell trafficking but do not control Akt–Foxo phosphorylation and localization.


PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.

Finlay DK, Rosenzweig E, Sinclair LV, Feijoo-Carnero C, Hukelmann JL, Rolf J, Panteleyev AA, Okkenhaug K, Cantrell DA - J. Exp. Med. (2012)

mTORC1 and HIF1 do not regulate Akt activity or Foxo phosphorylation. (A) Immunoblot analysis of phosphorylated AKT and Foxos in P14-LCMV CTLs treated with and without Akti1/2 or rapamycin for 24 h. (B) P14-LCMV CTLs were treated with and without rapamycin or Akti1/2 for 24 h and subjected to nuclear/cytoplasmic fractionation before immunoblot analysis for Foxo1 and Foxo3a expression. Purity of cytoplasmic and nuclear fractions was confirmed by IκBα and Smc1 expression. (C) Immunoblot analysis of WT or HIF1−/− CTLs for Akt–Foxo and mTORC1 signaling. HIF1−/− CTLs were treated with rapamycin as a negative control for mTORC1 activity. For all panels, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons. Dotted lines indicate that intervening lanes have been spliced out.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3526360&req=5

fig6: mTORC1 and HIF1 do not regulate Akt activity or Foxo phosphorylation. (A) Immunoblot analysis of phosphorylated AKT and Foxos in P14-LCMV CTLs treated with and without Akti1/2 or rapamycin for 24 h. (B) P14-LCMV CTLs were treated with and without rapamycin or Akti1/2 for 24 h and subjected to nuclear/cytoplasmic fractionation before immunoblot analysis for Foxo1 and Foxo3a expression. Purity of cytoplasmic and nuclear fractions was confirmed by IκBα and Smc1 expression. (C) Immunoblot analysis of WT or HIF1−/− CTLs for Akt–Foxo and mTORC1 signaling. HIF1−/− CTLs were treated with rapamycin as a negative control for mTORC1 activity. For all panels, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons. Dotted lines indicate that intervening lanes have been spliced out.
Mentions: To explore the links between PI3K, Akt, mTORC1, and HIF1, we addressed two questions. Do mTORC1 and HIF1 regulate Akt activity and Foxo phosphorylation? Is PI3K and Akt activity required for mTORC1 activation and HIF1 expression? In the context of the first question, long-term inhibition of mTORC1 can destabilize mTORC2 complexes in some cell systems and suppress Akt function (Sarbassov et al., 2006). Indeed, rapamycin treatment of CD4 T cells activated with CD3 and CD28 antibodies does diminish Akt S473 phosphorylation, although the impact of this on Akt activity has not been fully assessed (Lee et al., 2010; Delgoffe et al., 2011). If this were true in CTLs, then long-term treatment of T cells with rapamycin would result in the loss of Foxo phosphorylation and cause cells to regain Foxo transcriptional activity and thus CD62L and CCR7 expression. We therefore examined the impact of long-term inhibition of mTORC1 with rapamycin on Foxo phosphorylation and localization in CTLs. We also assessed Akt–Foxo and mTORC1 signaling in IL-2–maintained HIF1- T cells to assess whether loss of HIF1 complexes compromised Foxo phosphorylation/inactivation. Fig. 6 (A and B) shows that inhibition of Akt decreased Foxo phosphorylation (Fig. 6 A) and restored nuclear localization of Foxos in CTLs (Fig. 6 B). In contrast, Akt remained active, i.e., phosphorylated on T308 and S473, and Foxos remained highly phosphorylated and excluded from the nucleus of rapamycin-treated CTLs (Fig. 6, A and B). Additionally, Akt phosphorylation (T308) and the phosphorylation of Foxo transcription factors on Akt substrate sites (T24/32) were unaffected in HIF1β- CTLs, demonstrating that Akt signaling is not altered by HIF1 deletion (Fig. 6 C). It was also evident that the phosphorylation of mTORC1 substrates S6K1 (p70 S6-kinase 1) and 4EBP1 (eIF4E-binding protein 1), on T389 and S65, respectively, and downstream signaling to S6 ribosomal protein (an S6K1 substrate) were normal in HIF1β- CD8+ T cells (Fig. 6 C). Therefore, mTORC1 and HIF1 regulate the expression of CD62L and CCR7 and T cell trafficking but do not control Akt–Foxo phosphorylation and localization.

Bottom Line: We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex.This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking.These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. finlayd@tcd.ie

ABSTRACT
mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However, PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

Show MeSH
Related in: MedlinePlus