Transcript akt model

The Transcription Factor Myc Controls
Metabolic Reprogramming Upon T
Lymphocyte Activation
Zeba Shahnaz
09-arid-1567
PhD Zoology
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The Myc family genes i.e., c-Myc, MYCN and L-Myc genes
encode transcription factors that play essential roles in cell
proliferation, cell growth, differentiation, and apoptosis
The Myc protein consists of two regions
1- N-terminal transactivation domain (TAD)
(required for transcriptional activation)
2- C-terminal domain (CTD)
(critical for DNA binding and protein Interactions)
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Additionally, Myc has four conserved regions known as Myc
boxes (MB) that are essential for different functions.
MBI
MBII
MBIII
MBIV
The CTD encompasses a basic region required for binding to
the consensus CACGTG E-box and a helix-loop-helix leucine
zipper (HLHZip) domain, necessary for dimerization with
Max.
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The function of c-Myc is modulated by the availability of its
heterodimeric binding partner Max and the concentrations of
competing
Max/Max
homodimers
and
Max/Mad
heterodimers, all of which bind to a hexameric DNA sequence
termed the E box.
Myc/Max heterodimers bound to DNA recruit protein
complexes (TRRAP) associated with histone acetylase activity
that modify chromatin and activate transcription, whereas
Max/Mad heterodimers recruit inhibitory complexes (Sin3/NCoR) associated with histone deacetylase activity.
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Proliferating animal cells consume considerable amounts of
energy and require de novo synthesis of macromolecules to
support their growth and proliferation
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One of the fundamental problems in animal cells is
coordination of the metabolic program with cell growth- and
proliferation-associated bioenergetic demand.
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Prokaryotes and unicellular eukaryotes rely primarily on
homeostatic regulation of cell metabolism, while metabolic
programs in multicellular eukaryotes have evolved to be
actively controlled by extracellular signals (Deberardinis et al.,
2008).
Antigenic stimulation
T- cell
T-cell expansion
+differentiation
Immune response
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Accumulation of cell biomass during the initial growth and
rapid proliferation during the expansion phase is associated
with dramatically increased bioenergetic and biosynthetic
demand.
So, T cell activation provides a unique physiological cellular
model to help us understand how animal cells coordinate their
metabolic program with cell growth and proliferation in
response to extracellular fate-decisive signals
Polyclonal mitogen
(concanavalin A
&
phytohaemagglutinin )
↑glycolysis
&
Glutamine oxidation
(In thymocyte &
Mature lymphocytes)
The uptake of glutamine and glucose and the consumption of the
latter, mainly through glycolysis, are substantially up regulated
upon stimulation with anti-CD3 and anti-CD28
Signaling via ERK
and AKT pathways
are used in the
uptake
of
glutamine
and
glucose,
respectively
mammalian target of
rapamycin (mTOR), an
essential component of PI3KAkt pathway, used in
regulating fatty acid
metabolism in memory T cells
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However, the robustness of metabolic changes after T cell
activation suggests the presence of additional regulatory steps
in the T cell metabolic program, and the molecular
mechanisms behind these remain to be explored.
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The metabolic changes associated with T cell activation are
reminiscent of the characteristic metabolic activities of tumor
cells and may represent a general metabolic reprogramming
during cell growth and proliferation .
Murine Tlymphocytes
IL-7
Resting
Anti-CD4 &
anti-CD28
Active
Cell
proliferation
↑ metabolites that are involved
in anabolic pathway s of lipids,
amino acids & nucleotides
↑ glutamine & glucose
consumption
↓carnitne
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Activation of T cells dramatically up regulated glycolysis
In contrast, both mitochondria-dependent fatty acid βoxidation (FAO) and pyruvate oxidation through the
tricarboxylic acid cycle (TCA cycle) were markedly down
regulated upon activation.
Glutamine and glucose consumption through oxidative
catabolism and pentose phosphate pathway (PPP) were
significantly elevated in activated T cells.
Therefore, activation rapidly switches T cell metabolism
from FAO and pyruvate oxidation via the TCA cycle to
aerobic glycolysis, PPP, and glutamine oxidation.
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To explore the impact of metabolic reprogramming on T cell
growth and proliferation, T cells were stimulated in either
complete or nutrient-free media.
Deprivation of glutamine but not glucose led to an impairment
of activation-induced cell growth associated with a reduction
of lipid and protein biosynthesis .
However, both conditions resulted in impaired activationinduced cell proliferation
2DG (I)
HK
DHEA(I)
G6PD
Blocked T-cell proliferation
DON (I)
GLs
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In order to test the dependency of T cell proliferation on
glucose and glutamine catabolism in vivo, 2DG and DON
were applied in an adoptive transfer model by using
ovalbumin (OVA)-specific, TCR-transgenic OT-II T cells.
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Both 2DG and DON partially inhibited the proliferation of
antigen-specific active T cells after OVA immunization in
vivo .
These results show that both glucose and glutamine catabolic
pathways are required for activation-induced T cell
proliferation in vitro and in vivo.
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Two transcription factors, hypoxia inducible factor 1, alpha
subunit (HIF1α), and myelocytomatosis oncogene (Myc) were
used to know the regulation of metabolic reprogramming in
activated T cells .
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Both HIF1 α and Myc are recently suggested to regulate cell
metabolism in transformed cells , and were highly induced at
both the transcription and protein levels upon T cell activation
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The induction of these two transcription factors caused the
up regulation of glycolysis and glutaminolysis
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A mouse model was generated carrying a conditional HIF1a allele
(Hif1aflox/flox) and a tamoxifen-inducible Cre recombinase
(CreERT2) transgene to delete HIF1a flox alleles in an acute manner.
T cells
Cultured With 4Hydroxytamoxif
en (4OH)
activated with
anti-CD3 and
antiCD28 (active)
Deletion of
HIF1 α
IL-7
Cultured
Without 4Hydroxytamoxif
en (4OH)
No/less
effect on
glycolysis,
glutaminolysi
s & FAO
activated with
anti-CD3 and
antiCD28 (active)
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Another mouse model was generated carrying a conditional Myc
allele (Myc flox/flox) and a tamoxifen-inducible Cre recombinase
(CreERTam) to delete Myc flox alleles in an acute manner.
T cells
Cultured With 4Hydroxytamoxif
en (4OH)
activated with
anti-CD3 and
antiCD28 (active)
Deletion of
Myc
IL-7
Cultured
Without 4Hydroxytamoxif
en (4OH)
cell
proliferation
stopped,Low
accumulation
of lipid, a.a,
nucleotides
activated with
anti-CD3 and
antiCD28 (active)
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The role of Myc in regulating T cell proliferation and growth
in vivo was observed by using the staphylococcal enterotoxin
B (SEB) which acts as an antigen and activate the naive Vb8+
T cells.
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The Myc deletion blocked SEB-induced Vb8+ T cell
proliferation and growth ( consistent with in vitro results)
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Deletion of Myc significantly impaired activation-induced
glycolytic flux at both early and late time points.
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Myc deficiency led to a reduction of HK2 and pyruvate kinase
muscle isoform 2 (PKM2) at the protein level.
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In vivo studies ( effects of deletion of Myc in regulating T cell
metabolism in response to SEB) also showed that Myc is
required for the induction of enhanced glycolytic activity and
metabolic gene expression in SEB-responsive T cells in in
vivo.
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As glycolysis interconnects with other metabolic pathways,
such as the PPP, the TCA cycle, and FAO, the metabolic flux
through these pathways was investigated.
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So, Acute deletion of Myc moderately inhibited activationinduced upregulation of metabolic activity through the PPP
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The induction of two key metabolic enzymes in the PPP, Tkt
and G6PDx, was compromised in Myc-deficient T cells upon
activation.
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There are a large number of Myc target genes that are
activated by an increase in the level of Myc.
Many of these Myc targets have an E-box element containing
the 5′-CACGTG-3′ sequence.
In quiescent G0 or differentiated cells, the many Myc target
genes are repressed by Max/Mad or Max/Mnt dimers, together
with the SIN3/HDAC transcriptional co-repressor complex.
The HDACs deacetylate chromatin, making it inaccessible to
transcription.
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Myc activates transcription by replacing the Mad/Mnt, which
removes the repressor complex.
The Myc/Max dimer bind to the E-box and provides a
complex to bring in various co activators.
The histone acetyltransferases (HATs), such as CBP/p300,
CGN5 and TIP60, acetylate histones to open up the chromatin
to make it accessible so that the Myc target genes can be
transcribed.
These target genes contribute to cell growth and activation of
the cell cycle.
Module 4: Figure Myc as a gene activator
Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2012
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Myc drives the expression of Gfpt1, Cad, Ppat, and Oat ,
which not only are required for glutaminolysis but also control
critical steps in the hexosamine, pyrimidine, purine, and
polyamine biosynthetic pathways .
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Difluoromethylornithine (DFMO), a potent inhibitor of
ornithine decarboxylase (ODC) and polyamine biosynthesis,
inhibited activation-induced T cell proliferation in vitro and in
vivo , and the addition of exogenous polyamines completely
restored proliferation in the presence of DFMO in vitro
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Addition of either hypoxanthine and thymidine (HT) or
polyamines (PAs) alone was insufficient to permit cell
proliferation in the absence of glutamine.
Because a-KG is required to maintain cell viability and
minimal cell proliferation in the absence of glutamine,
addition of a-KG alone was sufficient to drive only one cell
division in the absence of glutamine.
However, the combination of a-KG with either HT or with PAs
promoted additional cell divisions and growth .
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While in Myc deficient T cells even the addition of a-KG with
HT and PAs failed to promote cell proliferation in such Mycdeficient T cells.
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This indicates that Myc-driven glutaminolysis coordinates
multiple biosynthetic pathways to support activation-induced
cell proliferation and growth.
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Although ornithine is produced largely from arginine via the
action of arginase as part of the urea cycle , urea cycle
enzymes including arginase were not induced upon activation
nor were any defects in proliferation observed in T cells
lacking arginase I, the dominant isoform in the thymus, these
results indicate that arginine is not the only precursor for
ornthine in T cells.
Previous studies in bovine endothelial and intestinal epithelial
cells suggested the possible presence of a noncanonical
metabolic pathway, which generates ornithine from glutamine
.
Intriguingly, the metabolic enzymes Aldh18A1, Prodh, and
OAT that link glutamine and proline to ornithine synthesis
were induced in a Myc dependent manner upon T cell
activation.
 Therefore, U-13C-isotope labeled glutamine
(U-13C-glutamine) flux were followed and the incorporation
of 13C into all five carbons of ornithine (U-13C-ornithine)
were observed, which accounted for up to 30% of total
ornithine in active T cells but only 1% in resting T cells.
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Finally, deletion of Myc partially abolished the incorporation
of glutamine-derived carbons into both ornithine and
putrescine .
These results indicate that a Myc-dependent noncanonical
ornithine biosynthetic pathway is coupling glutaminolysis to
ornithine and polyamine biosynthesis in activated T cells .
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To fulfill the bioenergetic and biosynthetic demand of proliferation, T cells
reprogram their metabolic pathways from fatty acid b-oxidation and
pyruvate oxidation via the TCA cycle to the glycolytic, pentose-phosphate,
and glutaminolytic pathways.
HIF1a and Myc, transcription factors were induced upon T cell activation,
but only the acute deletion of Myc markedly inhibited activation induced
glycolysis and glutaminolysis in T cells.
Glutamine as an important source for biosynthetic precursors in active T
cells.
A Myc-dependent metabolic pathway link glutaminolysis to the
biosynthesis of polyamines. Therefore, drives metabolic reprogramming in
activated, primary T lymphocytes.
This may represent a general mechanism for metabolic reprogramming
under patho-physiological conditions.
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Dang CV. 2011. Therapeutic targeting of Myc-reprogrammed cancer cell
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DeBerardinis, R.J., Lum, J.J., Hatzivassiliou, G., and Thompson, C.B.
(2008).The biology of cancer: metabolic reprogramming fuels cell growth
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