Date post: | 02-Jan-2017 |
Category: |
Documents |
Upload: | truongphuc |
View: | 227 times |
Download: | 0 times |
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 1
Electrophilic Prostaglandins and Lipid Aldehydes Repress Redox Sensitive
Transcription Factors p53 and HIF by Impairing the Selenoprotein
Thioredoxin Reductase
Philip J. Moos1, Kornelia Edes, Pamela Cassidy2, Edmund Massuda, F.A.
Fitzpatrick1,2
Huntsman Cancer Institute, Department of Oncological Science1 & Medicinal
Chemistry2
2000 Circle of Hope
University of Utah
Salt Lake City, UT 84112-5550
Correspond with:
F.A. Fitzpatrick, Ph.D.
Dee Glenn and Ida W. Smith Chair of Cancer Research
Huntsman Cancer Institute
2000 Circle of Hope
University of Utah
Salt Lake City, Utah 84112-5550
Telephone: 801-581-6204
Fax: 801-585-0011
E.mail [email protected]
Running title: Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase
1Abreviations used: PG, prostaglandins; 4-HNE, 4-hydroxy-2-nonenal; TrxR,
thioredoxin reductase; IKK, I-kappa-β kinase; APB, amidopentyl biotin; MDA,
malodialdehyde.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 2
SUMMARY
Tumor suppressor p53 exhibits an enigmatic phenotype in cells exposed to
electrophilic, cyclopentenone prostaglandins of the A- and J-series. Namely, cells
harboring a wild type p53 gene accumulate p53 protein that is conformationally
and functionally impaired. This occurs via an unknown molecular mechanism. We
report that electrophilic cyclopentenone prostaglandins covalently modify and
inhibit thioredoxin reductase, a selenoprotein that governs p53 and other redox
sensitive transcription factors. This mechanism accounts fully for the unusual p53
phenotype in cells exposed to electrophilic prostaglandins. Based on this
mechanism we derived, tested, and affirmed several predictions regarding the
kinetics of p53 inactivation; the protective effects of selenium; the structure-activity
relationships for inhibition of thioredoxin reductase and impairment of p53 by
electrophilic lipids; the susceptibility of hypoxia-inducible factor to inactivation by
electrophilic lipids; and the equivalence of chemical inactivation of p53 to deletion
of a p53 allele. Chemical precepts dictate that other electrophilic agents should
also inhibit thioredoxin reductase and impair its governance of redox sensitive
proteins. Our results provide a novel framework to understand how endogenous
and exogenous electrophiles might participate in carcinogenesis; how
selenoproteins and selenium might confer protection against cancer; how certain
tumors might acquire their paradoxical p53 phenotype, and how chronic
inflammation might heighten the risk for cancer.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 3
INTRODUCTION
Cyclopentenone prostaglandins (PG1), of the A- and J- series impair the
conformation and function of tumor suppressor p53 by a novel, but unknown,
mechanism of action (1,2). Cyclopentenone PG penetrate cells and accumulate in
the cytosol and nucleus (3) where they can react covalently with other molecules
via their electrophilic β carbon (4). Few of the proximal molecular targets of these
PG are established (5-7). There are two formal hypotheses to explain how
electrophilic PG might inactivate p53. First, they might act directly, via covalent
reaction with p53 itself. Second, they might act indirectly, via covalent reaction
with regulatory proteins that govern p53 conformation and function. A direct
mechanism of action is incompatible with our observation that PGA1 and A2
antagonize only the apoptosis mediated by p53 (1), but not the cell-cycle arrest.
These PG should antagonize all functions mediated by p53 if their molecular
mechanism of action involved its modification directly. Accordingly, we sought
candidate proteins consistent with an indirect molecular mechanism of action.
Thioredoxin reductase (TrxR) is notable from biological and chemical perspectives.
Biologically, TrxR-Trx cycling modulates sulfhydryl-disulfide isomerization
reactions that govern the conformation and function of p53, as well as several other
redox sensitive transcription factors, like NFκB and hypoxia-inducible factor (HIF)
(8-10). Trr1, the yeast ortholog of TrxR, is essential for transcription by p53
expressed ectopically in yeast (11,12). Chemically, TrxR is a selenoprotein.
Selenocysteine residues are typically more nucleophilic than cysteine under
comparable conditions. Chemical precepts dictate that electrophilic agents, like
cyclopentenone PG, should react readily with selenoproteins under conditions
encountered in cells.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 4
Herein we report that: i) a prototypical PGA analog forms a covalent adduct
with TrxR; ii) analogous to the cyclopentenone PG, several representative, naturally
occurring or synthetic aldehydes and ketones with electrophilic β carbons (15-keto-
PG, 4-hydroxy-2-nonenal, ethacrynic acid) impair p53 conformation and function,
indirectly, via inhibition of TrxR; iii) other redox-sensitive transcription factors
governed by TrxR-Trx cycling, e.g. HIF, are also susceptible to inactivation by lipid
aldehydes and ketones with electrophilic β carbons; iv) supplementation of cell
culture medium with inorganic Se spares p53 from inactivation by lipid
electrophiles; v) impairment of p53 by lipid electrophiles is comparable in severity
to loss of one allele of the p53 gene. Our results provide a novel framework to
understand how these agents and numerous chemically related, endogenous and
exogenous electrophiles might participate in carcinogenesis; how selenoproteins
and dietary selenium may confer protection against cancer (13); how cells might
acquire an unusual and unexplained p53 phenotype observed in some tumors (14-
17); and how chronic inflammation might heighten the risk for cancer (18).
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 5
EXPERIMENTAL PROCEDURES
Materials. We used DMEM and McCoy’s 5A medium and supplements
(GIBCO/BRL); prostaglandins (Cayman Chemicals); Auranofin (ICN Biomedicals);
cobalt chloride and etoposide (Sigma); malondialdehyde (Fluka); 4-hydroxy-2-
nonenal (Oxis International, Inc.); protease inhibitor mixture and FuGene-6
transfection reagent (Roche Molecular Biochemicals); enhanced chemiluminescence
reagents (Amersham Pharmacia); luciferase reporter lysis buffer and reporter
detection reagents (Promega); monoclonal antibodies directed against p53 (Pab240
– Santa Cruz; Pab1620 (Ab5) – Oncogene Sciences); polyclonal antibodies against
TrxR (Upstate Biotechnology) and p53 (FL-393-G, Santa Cruz); horseradish
peroxidase-conjugated secondary antibodies; protein A/G PLUS-Agarose (Santa
Cruz Biotechnology); Neutravidin-conjugated beads (Pierce); Ac-DEVD-MCA
(Peptides International); luciferase reporter constructs for p53 (p53-Luc,
Stratagene) and HIF-1 (p2.1, ref 19; gift of G.L. Semenza, Johns Hopkins
University, Baltimore); and a β-galactosidase expression vector (pCMVβ;
CLONTECH).
Cell Culture. We maintained HCT 116 p53+/+, p53+/- and p53-/- (ref. 20 gift of
B. Vogelstein) cells in McCoy’s 5A medium and RKO cells (gift of M. Meuth,
Institute for Cancer Studies, University of Sheffield, Sheffield, U.K.) in DMEM at
37°C in a humidified incubator with 5% CO2. We supplemented media with 2 mM
L-glutamine, 1 mM sodium pyruvate, 50 units/ml penicillin and streptomycin, and
10% (vol/vol) FBS. In certain experiments, cells were metabolically labeled with
100 µCi [75Se], obtained from the University of Missouri Research Reactor, for 48
hrs to ensure that selenoproteins were labeled to steady state.
Isolation of Proteins Labeled by PGA1-ABP: Neutravidin Sequestration. We
treated RKO and HCT 116 cells with 60 µM PGA1-APB for 4 hrs, or as described in
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 6
the text. We lysed cells in 250 mM sucrose 50 mM Tris, pH 7.4/25 mM KCl/5 mM
MgCl2/1 mM EDTA/1× complete protease inhibitor/2 mM NaF/2 mM sodium
orthovanadate. We sonicated the lysate twice for 5 s at 4°C. After centrifugation at
13,000 × g, we incubated samples containing 200 µg of total cell lysate for 16 h at
4°C with 100 µl of Neutravidin beads in 1 ml of PBS with 0.4% Tween 20. We
centrifuged the samples at 500×g, 5 min to isolate the Neutravidin–biotin
complexes. We washed the beads 5 × 1 ml of PBS/0.4% Tween 20. We fractionated
samples by SDS/PAGE and detected the biotinylated proteins with Streptavidin-
HRP. We also reprobed the membrane with antibodies directed against Trx, TrxR,
p53, p50/p105 of NFκB, and IKKα to determine if they adhered to the Neutravidin
beads.
Immunoprecipitation of p53. We lysed cells and incubated 200 µg of total cell
lysate for 16 h at 4°C with 1 µg of either Pab240 or Pab1620, antibodies that
specifically recognize p53 in its mutant or wild-type conformation, respectively,
(21). We added 20 µl of protein A/G PLUS-Agarose in 1 ml of PBS with 0.4% Tween
20. We centrifuged the samples at 500× g, for 5 min to isolate the antigen–antibody
immune complexes. We washed the immunoprecipitate 2 × 1 ml of PBS/0.4%
Tween 20. We fractionated samples by SDS/PAGE and measured the amount of
conformationally mutant or wild type p53 in the immunoprecipitate by
hybridization with a separate anti-p53 polyclonal antibody (FL-393).
p53 and HIF-1 Transcriptional Activity. We transfected 105 RKO cells per well
with 1 µg of p53-Luc or p2.1 and 50 ng of pCMVβ in 3 µl of FuGene-6. After 48 h,
we incubated cells for 6 h with vehicle (DMSO), 0–60 µM of various electrophiles,
plus 50 µM etoposide or 100 µM CoCl2, respectively. We aspirated media, washed
cells with PBS (pH 7.4) at 4°C, and then lysed cells at 4°C in 100 µl of Reporter
Lysis Buffer. We centrifuged the lysate at 20,000×g for 15 min at 4°C and
quantified luciferase and β-galactosidase activity in the supernatant fractions.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 7
Thioredoxin reductase activity. We lysed RKO cells in 50 mM Tris, pH
7.4/0.1M NaCl/2 mM EDTA/1% SDS/1% deoxycholate/1 mM NaF/1 mM sodium
orthovanadate/1× complete protease inhibitors. We added 20 µl of protein lysate
(3-4 µg/µl) to 100 µl of 20 µM Tris, pH 7.4, containing 70 µM insulin, 66.6 µM E.
coli thioredoxin, and 120 nM NADPH and 5.5 mM EDTA. We monitored the
oxidation of NADPH at 340 nm for 0-5 min at 25oC, or longer to ensure we
measured the linear portion of the progress curve.
Caspase-3 Activity. We treated 106 HCT116 cells for 48 hrs with 20 µM
amethopterin plus 0, 6, or 20 µM PGA2. We measured caspase-3 activity as an
index of apoptosis (22). We verified that PGA2 did not interfere with caspase-3.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 8
RESULTS
To determine whether electrophilic PGs react with TrxR or other proteins we
used PGA1 amidopentyl biotin (PGA1-APB). PGA1-APB retains the α, β unsaturated
ketone substituent and the electrophilic β carbon of PGA1. Its C1 biotin amide,
instead of a C1 carboxyl group, facilitates the detection of any covalent adducts it
might form with proteins (6). [Figure 1]
To calibrate the utility of PGA1-ABP we examined its interaction with IKKα, a
protein putatively modified by cyclopentenone PG (7). We incubated intact RKO
and HCT 116 colon cancer cells with PGA1-ABP (60 µM) for 4 hr; sequestered any
proteins with biotin epitopes on neutravidin beads; fractionated these same
biotinylated proteins by SDS-PAGE; and identified them immunochemically. Cells
contained approximately 15 proteins labelled de novo with PGA1-APB under these
conditions - two of these proteins were IKKα [Figure 2] and the p50/p105 subunit
of NFκB [data not shown]. Our data are the first direct evidence that
cyclopentenone PG do covalently modify cellular IKK, as hypothesized (7), and they
suggest that PGA1-ABP is useful for isolating and identifying proteins that react
with cyclopentenone PG.
Accordingly, we determined if PGA1-APB reacted with TrxR, the strongest
candidate molecule for an indirect mechanism of action that leads to impairment of
p53. RKO cells and two HCT 116 cell lines contained a protein modified covalently
by PGA1-ABP and identified as TrxR immunochemically with anti-TrxR antibody
[Figure 2A, lanes 3]. The fact that HCT 116 p53+/+ and HCT 116 p53-/- cells each
contained this ∼ 56-kDA protein excludes the possibility that it is a p53-PGA1-ABP
adduct that migrates more slowly than p53 during fractionation by SDS-PAGE
[Figure 2A, lanes 3]. Incubation of cells with PGA1 alone, or aminopentyl biotin
alone, did not generate proteins with biotin epitopes inserted de novo [Figure 2A,
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 9
lanes 2 & 4]. Thus, the biotin epitope on the 56-kDa TrxR protein and other
cellular proteins originates from their covalent reaction with PGA1-ABP, not its
hydrolysis products in cells. We exploited the fact that TrxR is a selenoprotein to
strengthen our conclusion that it forms an adduct with PGA1-ABP. We
metabolically labelled RKO and HCT 116 cells with 75Se to incorporate it into their
selenoproteins; repeated the previous experiment; and found a 75Se-labeled, 56 kDa
protein recognized by anti-TrxR antibody among the proteins sequestered on
neutravidin beads [Figure 2B].
RKO cells and HCT 116 p53+/+ cells that are haplosufficient in p53 contained
barely detectible amounts of p53 covalently modified by PGA1-APB when we
conducted experiments analogous to those described above. In corresponding
experiments where we immunoprecipated p53 with anti-p53 antibodies;
fractionated the precipitate by SDS-PAGE; and probed with neutravidin-HRP we
found no detectable biotinylated p53 adduct [data not shown]. Thus, PGA1-ABP
does not react to an appreciable extent with p53, itself, in intact cells. As expected,
the HCT 116 p53-/- cells that lack p53 did not contain any modified protein
corresponding to p53.
In addition to TrxR, we examined thioredoxin (Trx), the substrate of TrxR.
RKO cells contained a 12-kDa species labeled by PGA1-ABP and identified as Trx
with anti-Trx antibody [data not shown]. The 12-kDa species also
immunoprecipitated with anti-Trx antibody and reacted positively with
streptavidin-HRP, consistent with its annotation as a Trx:PGA1-ABP adduct.
Several representative electrophilic lipids also inhibited p53 transactivation,
p53 conformation, TrxR activity and HIF transactivation with a similar rank-order
of potency [Figure 3]. 4-Hydroxy-2-nonenal (4-HNE), a decomposition product of
linoleic or arachidonic acid hydroperoxides was the most potent inhibitor followed
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 10
by J- and A-series cyclopentenone PG; ethacryinic acid, and 15-keto PGF2α, the
main pulmonary metabolite of PGF2α. Under our experimental conditions MDA and
PGB1 were inactive, as expected, because exogenously added MDA does not
penetrate cell membranes readily and PGB1 has an inert β-carbon.
We investigated the kinetic features of p53 inactivation by PGA1 to assess
their compatibility with inhibition of TrxR. We exposed RKO cells to 50 µM
etoposide for 6 hr to initiate genomic damage and time-dependent accumulation of
wild type p53. We added PGA1 simultaneously with etoposide at t = 0 hr or at 1, 2
and 4 hr after the addition of etoposide. PGA1 impaired p53 transcription
maximally when present throughout the entire 6 hr duration of the experiment.
When added at intervals after etoposide, PGA1 impaired p53 transactivation
proportionately to exposure time [Figure 4A]. For example, when present only for
the final two hr, t = 4 - 6 hr, PGA1 did not impair p53 transactivation or
conformation significantly (95% versus 100%).
TrxR is a selenoprotein whose cellular steady-state level depends on selenium
availability (23,24). Most tissue culture media is partially deficient in selenium
[∼ 0.1 µM]. Tumor suppressor p53 was less vulnerable to impairment by PGA2 when
cells were grown in media supplemented with 1µM inorganic Se. Half-maximal
impairment of p53 required exposure to ∼ 3-fold more PGA2 [IC50 = 60 µM PGA2] in
cells grown with supplemental selenium versus cells with no supplementation
[IC50= 20 µM PGA2] [Figure 4B].
Auranofin is chemically unrelated to cyclopentenone PG or other electrophilic
lipids, but it is an inhibitor of TrxR activity (23,25). Auranofin impaired the
conformation of p53 and transcription by p53 and HIF [Figure 4C], analogously to
the electrophilic lipids depicted in Figure 3.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 11
Bunz and colleagues recently established that p53 haplosufficiency is
proportional to p53-mediated apoptosis in vitro using a panel of isogenic HCT 116
cells whose p53 alleles were disrupted experimentally (20). We used these same
cell lines to compare the functional consequences of p53 inactivation by
electrophilic agents versus the functional consequences of genetic deletion of p53
alleles. Consistent with previous results (20) amethopterin induced apoptosis
proportional to p53 haplosufficiency (caspase-3 activity in HCT 116 p53+/+ > HCT
116 p53+/- > HCT 116 p53-/-) [Figure 5 bars to right]. When we incubated the HCT
116 p53+/+ cells with amethopterin plus PGA2, the PGA2 antagonized apoptosis in a
concentration-dependent manner [Figure 5 bars to left]. Apoptosis in HCT 116
p53+/+ cells treated with 6 to 20 µM PGA1 plus amethopterin corresponded
approximately to apoptosis in the HCT 116 p53+/- cells treated with amethopterin
alone. In other words, chemical impairment of p53 approximates the loss of at least
one allele of p53.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 12
DISCUSSION
We recently discovered that electrophilic cyclopentenone PG impair the p53
tumor suppressor by a novel mechanism that is distinct from mutation of the p53
gene or functional inactivation of p53 by oncoproteins like mdm-2. Since our
initial report (1) we have sought the precise molecular mechanism responsible for
this effect. Here we report that cyclopentenone PG act by covalently modifying and
inhibiting TrxR. This indirect mechanism can account fully for the p53 phenotype
in cells exposed to cyclopentenone PG, as well as other representative electrophilic
lipids.
To develop our mechanism of action hypothesis we integrated three
independent observations: i) PGA1 and A2 impair the conformation, transactivation
and function of p53 (1); ii) PGA1-ABP reacts covalently with TrxR and Trx [Figure 2];
and iii) TrxR-Trx coupling maintains p53 and several other several redox-sensitive
proteins and transcription factors in an active state (8-10). Collectively, these data
suggested a model with several, testable predictions. First, our model predicts that
many other chemical agents with an α,β unsaturated carbonyl and accessible,
electrophilic β carbons should impair p53 conformation and function. Second,
these chemical agents should impair p53 transcription, p53 conformation and
inhibit TrxR activity with the same rank-order of potency, if they act indirectly via
TrxR. Third, these agents should impair transactivation by other redox-sensitive
transcription factors, e.g. hypoxia-inducible factor (10,26) with the same rank-
order of potency that they impair TrxR and p53. Our results affirm each of these
predictions. Our model also predicts that PGA1 will impair p53 only as it
accumulates during the initial, early stage of the cellular response to DNA damage;
but not later, after it has assumed a transcriptionally active, wild type
conformation. By inhibiting the disulfide reductase activity of TrxR-Trx, PGA1
should prevent assembly of p53 into a mature conformation, but it should not
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 13
convert p53 from an active to an inactive conformation. Kinetic experiments
affirmed this prediction. Lastly, the molecular mechanism of action we propose can
resolve a paradox. Namely, A-series PG antagonise p53-dependent apoptosis, but
not cell cycle arrest. These effects are fully compatible with inhibition of TrxR.
Inhibition of TrxR-Trx cycling deranges the assembly of p53 into a transcriptionally
competent form; this manifests as antagonism of p53 mediated processes, like
apoptosis. Inhibition of TrxR-Trx cycling can derange the redox status and
catalytic competence of ribonucleotide reductase; this manifests as cell cycle arrest
in G1 because ribonucleotide reductase is the rate-limiting enzyme in DNA
synthesis.
Our mechanistic framework is supported, but not necessarily proven, by our
data. Notably, our framework aligns well with yeast genetic experiments indicating
that TrxR is essential for transcription by p53 (11,12). In addition to its effects on
TrxR, we observed that PGA1-APB binds covalently to thioredoxin, the substrate of
TrxR. TrxR and Trx are vital components of a regulatory cycle and they act
coordinately to maintain p53 conformation and function. Biologically, the
reductase activity of TrxR maintains Trx in a reduced state so it is competent to
function as a sulfhydryl-disulfide isomerase (8). It is possible that covalent binding
to, and direct inactivation of Trx is also important for p53 inactivation. In other
words, electrophilic lipids inhibit Trx directly, via irreversible, covalent binding and
indirectly via their effects on TrxR.
Certain α,β unsaturated carbonyl compounds with electrophilic carbons are
known risk factors for cancer, e.g. acrolein, MDA, 4-hydroxy-2-nonenal. Their
carcinogenic mechanism is best understood in terms of their direct interaction with
DNA (27-29). Our data and model provide a new molecular basis for appreciating
their carcinogenic effects. Aside from these well-established carcinogens many
chemically complex α,β unsaturated ketones and α,β unsaturated aldehydes are
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 14
considered safer because they have low rates of reaction with DNA and reduced
glutathione (GSH) (30). Dipple and colleagues have termed these agents "stealth
carcinogens" (31) because the unusual elements of this phenotype occur in tumors.
Our data and model provide a new molecular basis for appreciating, and re-
assessing the safety of such compounds. The cyclopentenone PG provoke an
unusual and distinctive phenotype typified by accumulation of p53 protein, in an
abnormal conformation that cannot support DNA binding and transcription.
We have tested a set of representative electrophilic chemicals that include:
agents derived from the cyclooxygenase pathway (malondialdehyde, metabolites of
prostaglandins, e.g. 15-keto-PGF2 α, and dehydration products, e.g. PGA2 or ∆12-
PGJ2) and; agents derived from the lipoxygenase (LOX) pathways (e.g. 4-HNE
generated by decomposition of hydroperoxy-octadecadienoic acids. Based on
chemical precepts we anticipate that electrophilic carbons on numerous other
compounds would confer a similar ability to inhibit TrxR. Cells can encounter
electrophilic chemicals via environmental exposure, dietary exposure, or normal
metabolic processes. We draw special attention to inflammation as one of these
processes. As part of their normal host-defence function inflammation inevitably
exposes proximal epithelial and stromal cells to substances with mutagenic
potential in vitro (18). Individual eicosanoids with α,β unsaturated ketone
substituents like PGA1 or PGA2 may not occur in µM concentrations at a site of
inflammation. However, inflammatory exudate contains a blend of electrophiles
typified by α,β unsaturated aldehydes derived from eicosanoid biosynthesis or lipid
peroxidation (4-HNE); α,β unsaturated ketones derived from eicosanoid metabolism
(15-keto-PGF2α, 15-keto-PGE2, 5-, 12-, and 15-oxo-ETE); and α,β unsaturated
ketones derived from albumin dehydrating PGE2 to PGA2 and, PGD2 to ∆12- PGJ2
and 15-deoxy-∆12-PGJ2 (32-35). Thus, inflammation likely exposes cells to a
mixture of electrophiles in quantities sufficient to impair TrxR. Note that 4-HNE, a
common product from lipid peroxidation potently inactivates the selenoprotein
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 15
TrxR. The level of endogenous 4-HNE in tissues ranges from 0.1-3.0 µM and
increases to ∼ 10 µM in conditions of oxidative stress (36).
Although purely speculative at this point, our mechanistic framework aligns
well with lipid mediator class switching hypothesis recently proposed by Serhan
and colleagues (37). It has been suggested that J-series cyclopentenone PGs are
present during resolution phases of inflammation and attenuate the inflammatory
response (33). Electrophilic lipoxins would also be present and perhaps act
through a common mechanism involving TrxR inhibition.
The same electrophilic cyclopentenone PG and lipid aldehydes that impair
conformation and repress transcription by p53 can also repress transcription and
antagonize the effects of NF-κB (7,38-40). This may also occur via an irreversible,
covalent modification of proteins. For instance, cyclopentenone PG of the A- and J-
series, or 4-HNE can form adducts with IKK (7,40) or NF-κB (39), in situ.
Investigators have inferred that repression of NF-κB in cells derives from this
mechanism. However, they have demonstrated adduct formation only with isolated
NFκB and IκB proteins in situ, not in cells (7,38-40). With the PGA1-APB analog,
we have now demonstrated direct alkylation of IKK in cells. Therefore, electrophilic
lipids may resolve inflammation through direct and indirect mechanisms to
attenuate cellular signalling pathways. The fact that several of these types of
chemicals can impair the p53 tumor suppressor and that the functional
consequences of its impairment rival the loss of a p53 allele may have broad
implications for cancer progression.
The fact that electrophilic lipids act indirectly, via a selenoprotein may have
implications for cancer prevention. For instance, our model predicts that selenium
supplementation of culture media should maintain TrxR activity, and thereby spare
p53 from inactivation by electrophilic lipids. We affirmed this prediction. Clark and
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 16
colleagues have reported that supplementation of dietary selenium lowers the risk
of prostate, colon and certain other cancers (41). The molecular basis for this
phenomenon, especially the role of selenoproteins, is uncertain. Our observation
that selenium spares an important tumor suppressor, p53, provides an explicit
mechanistic framework to understand how dietary selenium confers protection
against cancer. Approximately 50-60% of cancer patients have tumors harboring
mutations or deletions of p53. These patients typically have a poorer prognosis
than patients with tumors harboring wild type p53 (20,42). Likewise, not all
mutations are equally pernicious - certain classes may be worse than others. In
particular, mutations associated with an altered conformation of p53 protein
correlate with significantly shorter survival and poorer prognosis in patients with
colorectal cancer (43,44). We draw attention to reports about colon, breast and
neuroblastoma tumors with a wild type p53 gene that paradoxically express a
dysfunctional p53 protein with a mutant conformation (14,15,17,45-48). The
latency model of p53 function (49), in its current form, does not account for the
peculiar p53 phenotype described in these reports. We can recapitulate this
unusual and unexplained p53 phenotype in cells by impairing their TrxR-Trx
activity with electrophilic lipids, suggesting a potential role for TrxR-Trx, or related
disulfide reductases, in its emergence.
In summary, various electrophilic lipids have the capacity to repress
transactivation by several redox-responsive transcription factors by covalently
modifying regulatory proteins in the pathways - IKK in the case of the NFκB
pathways and TrxR-Trx in the case of p53 and HIF.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 17
ACKOWLEDGEMENTS:
Dr. B. Vogelstein genereously provided HCT116 cell lines and p53 expression
construct, pC53-SN3. Dr. G. L. Semanski generously provided the HIF-1 luciferase
reporter construct. We thank the Huntsman Cancer Foundation and R01 AI26730
for support of this work. F.A.F. holds the Dee Glenn and Ida W. Smith Chair for
Cancer Research.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 18
References
1. Moos, P. J., Edes, K., and Fitzpatrick, F. A. (2000) Proc Natl Acad Sci U S A
97(16), 9215-20.
2. Mullally, J. E., Moos, P. J., Edes, K., and Fitzpatrick, F. A. (2001) J Biol
Chem 276(32), 30366-73.
3. Narumiya, S., and Fukushima, M. (1986) J Pharmacol Exp Ther 239(2), 500-
5.
4. Honn, K. V., and Marnett, L. J. (1985) Biochem Biophys Res Commun 129(1),
34-40
5. Narumiya, S., Ohno, K., Fukushima, M., and Fujiwara, M. (1987) J
Pharmacol Exp Ther 242(1), 306-11.
6. Parker, J. (1995) Prostaglandins 50(5-6), 359-75.
7. Rossi, A., Kapahi, P., Natoli, G., Takahashi, T., Chen, Y., Karin, M., and
Santoro, M. G. (2000) Nature 403(6765), 103-8.
8. Mustacich, D., and Powis, G. (2000) Biochem J 346 Pt 1, 1-8.
9. Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T.
(1992) Nucleic Acids Res 20(15), 3821-30.
10. Huang, L. E., Arany, Z., Livingston, D. M., and Bunn, H. F. (1996) J Biol
Chem 271(50), 32253-9.
11. Pearson, G. D., and Merrill, G. F. (1998) J Biol Chem 273(10), 5431-4.
12. Casso, D., and Beach, D. (1996) Mol Gen Genet 252(5), 518-29.
13. Ganther, H. E. (1999) Carcinogenesis 20, 1657-66
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 19
14. Moll, U. M., Riou, G., and Levine, A. J. (1992) Proc Natl Acad Sci U S A
89(15), 7262-6.
15. Moll, U. M., LaQuaglia, M., Benard, J., and Riou, G. (1995) Proc Natl Acad
Sci U S A 92(10), 4407-11.
16. Schlamp, C. L., Poulsen, G. L., Nork, T. M., and Nickells, R. W. (1997) J Natl
Cancer Inst 89(20), 1530-6.
17. Bosari, S., Viale, G., Roncalli, M., Graziani, D., Borsani, G., Lee, A. K., and
Coggi, G. (1995) Am J Pathol 147(3), 790-8.
18. Fitzpatrick, F. A. (2001) Int Immunopharmacol 1(9-10), 1651-67.
19. Semenza, G. L., Jiang, B. H., Leung, S. W., Passantino, R., Concordet, J. P.,
Maire, P., and Giallongo, A. (1996) J Biol Chem 271(51), 32529-37.
20. Bunz, F., Hwang, P. M., Torrance, C., Waldman, T., Zhang, Y., Dillehay, L.,
Williams, J., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1999) J Clin
Invest 104(3), 263-9.
21. Legros, Y., Meyer, A., Ory, K., and Soussi, T. (1994) Oncogene 9(12), 3689-
94.
22. Moos, P. J., and Fitzpatrick, F. A. (1998) Cell Growth Differ 9(8), 687-97
23. Berggren, M., Gallegos, A., Gasdaska, J., and Powis, G. (1997) Anticancer
Res 17(5A), 3377-80.
24. Gallegos, A., Berggren, M., Gasdaska, J. R., and Powis, G. (1997) Cancer Res
57(21), 4965-70.
25. Gromer, S., Arscott, L. D., Williams, C. H., Jr., Schirmer, R. H., and Becker,
K. (1998) J Biol Chem 273(32), 20096-101.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 20
26. Ema, M., Hirota, K., Mimura, J., Abe, H., Yodoi, J., Sogawa, K., Poellinger,
L., and Fujii-Kuriyama, Y. (1999) Embo J 18(7), 1905-14.
27. Basu, A. K., O'Hara, S. M., Valladier, P., Stone, K., Mols, O., and Marnett, L.
J. (1988) Chem Res Toxicol 1(1), 53-9.
28. Basu, A. K., and Marnett, L. J. (1984) Cancer Res 44(7), 2848-54.
29. Basu, A. K., and Marnett, L. J. (1983) Carcinogenesis 4(3), 331-3
30. Parker, J., and Ankel, H. (1992) Biochem Pharmacol 43(5), 1053-60.
31. Khan, Q. A., Vousden, K. H., and Dipple, A. (1997) Carcinogenesis 18(12),
2313-8.
32. Shibata, T., Kondo, M., Osawa, T., Shibata, N., Kobayashi, M., and Uchida,
K. (2002) J Biol Chem 277(12), 10459-66
33. Gilroy, D. W., Colville-Nash, P. R., Willis, D., Chivers, J., Paul-Clark, M. J.,
and Willoughby, D. A. (1999) Nat Med 5(6), 698-701.
34. Fitzpatrick, F. A., and Waynalda, M. A. (1981) Biochemistry 20(21), 6129-34.
35. Fitzpatrick, F. A., and Wynalda, M. A. (1983) J Biol Chem 258(19), 11713-8.
36. Esterbauer, H., Eckl, P., and Ortner, A. (1990) Mutat Res 238(3), 223-33.
37. Levy, B. D., Clish, C. B., Schmidt, B., Gronert, K., and Serhan, C. N. (2001)
Nat Immunol 2(7), 612-9
38. Straus, D. S., Pascual, G., Li, M., Welch, J. S., Ricote, M., Hsiang, C. H.,
Sengchanthalangsy, L. L., Ghosh, G., and Glass, C. K. (2000) Proc Natl Acad
Sci U S A 97(9), 4844-9.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 21
39. Cernuda-Morollon, E., Pineda-Molina, E., Canada, F. J., and Perez-Sala, D.
(2001) J Biol Chem 276(38), 35530-6.
40. Ji, C., Kozak, K. R., and Marnett, L. J. (2001) J Biol Chem 276(21), 18223-8.
41. Clark, L. C., Combs, G. F., Jr., Turnbull, B. W., Slate, E. H., Chalker, D. K.,
Chow, J., Davis, L. S., Glover, R. A., Graham, G. F., Gross, E. G., Krongrad,
A., Lesher, J. L., Jr., Park, H. K., Sanders, B. B., Jr., Smith, C. L., and
Taylor, J. R. (1996) Jama 276(24), 1957-63.
42. Goh, H. S., Yao, J., and Smith, D. R. (1995) Cancer Res 55(22), 5217-21.
43. Borresen-Dale, A. L., Lothe, R. A., Meling, G. I., Hainaut, P., Rognum, T. O.,
and Skovlund, E. (1998) Clin Cancer Res 4(1), 203-10.
44. Webley, K. M., Shorthouse, A. J., and Royds, J. A. (2000) J Pathol 191(4),
361-7.
45. Sun, X. F., Carstensen, J. M., Zhang, H., Stal, O., Wingren, S., Hatschek, T.,
and Nordenskjold, B. (1992) Lancet 340(8832), 1369-73.
46. Sun, X. F., Carstensen, J. M., Zhang, H., Arbman, G., and Nordenskjold, B.
(1996) Eur J Cancer 32A(11), 1963-7.
47. Lou, M. A., Tseng, S. L., Chang, S. F., Yue, C. T., Chang, B. L., Chou, C. H.,
Yang, S. L., Teh, B. H., Wu, C. W., and Shen, C. Y. (1997) Br J Cancer 75(5),
746-51
48. Stenmark-Askmalm, M., Stal, O., Sullivan, S., Ferraud, L., Sun, X. F.,
Carstensen, J., and Nordenskjold, B. (1994) Eur J Cancer 2, 175-80
49. Kubbutat, M. H., and Vousden, K. H. (1998) Mol Med Today 4(6), 250-6.
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 22
Figure 1. Structure of PGA1 and PGA1-ABP. The electrophilic β-carbon is indicated
with a star.
Figure 2. A) Neutravidin sequestration of PGA1-ABP-labeled proteins in RKO and
HCT 116 cells. The upper panel shows the proteins identified with anti-IKKα, anti-
TrxR and anti-p53 antibodies formed de novo in cells incubated with PGA1-ABP.
The lower panel shows the immunochemical identification of TrxR and p53 in the
whole cell lysates. IKKα and TrxR occurs as a PGA1-ABP conjugate in all three cell
lines and a minimal amount of p53 occurs as a PGA1-ABP conjugate in the HCT116
p53+/+ cells. Each cell line was incubated with lane 1) vehicle control (DMSO), lane
2) 60 µM PGA1, lane 3) 60 µM PGA1-ABP, and lane 4) 180 µM biotin pentylamine
linker. B) The identification of selenoprotein TrxR was further supported by
metabolically labelling RKO and HCT 116 cells with 75Se, and repeating the
experiment using PGA1-ABP and anti-TrxR antibodies: lane 5) RKO cells incubated
with PGA1, lane 6) RKO cells incubated with PGA1-ABP, lane 7) HCT 116 p53-/-
cells incubated with PGA1-ABP, and lane 8) HCT 116 p53+/+ cells incubated with
PGA1-ABP.
Figure 3. Electrophilic lipids attenuate A) p53 activity as measured by luciferase
reporter constructs in RKO cells in a dose-dependent manner. Electrophiles used
include: 4-HNE (filled square), ∆12-PGJ2 (filled up-triangle), 15-dideoxy-∆12, ∆14-
PGJ2 (filled down-triangle), PGA2 (filled diamond), 15-keto-PGF2α (filled circle), 15-
keto-PGE2 (open down-triangle), PGB1 (open up-triangle), and malondiadehyde (open
circle). B) These lipids also result in a change in conformation of p53 to the ‘mutant’
conformation. C) The lipids attenuate HIF-1 transcriptional with a similar rank
order of potency to p53. D) Electrophilic lipids attenuate TrxR activity as measured
by Trx-dependent NADPH oxidase activity in RKO cell lysates.
Figure 4. A) Selenium supplementation of RKO cells spares p53 from impairment
by PGA2. Cells were either incubated in media supplemented with 1 µM sodium
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 23
selenite (filled squares) for 48 hr or maintained in standard media (filled circles) and
then stimulated with etoposide in the presence of PGA2. B) PGA2 attenuates
transcriptionally active p53 in a time-dependent manner. Luciferase activity was
monitored (right panel) from a p53_luc reporter construct in RKO cells that were
stimulated with 50 µM etoposide (dark bars, left panel) and 60 µM PGA2 was added
after 0, 1, 2 or 4 hr (light gray bars, left panel). PGA2 must be present during the
initial 1-2 hrs of the p53 response to attenuate p53 transcription. Delayed addition
of PGA2 , 4 hrs after the initiation of the p53 has no effect on p53 transcription. C)
Incubation of RKO cells with 10 µM auranofin for 6 hr deranges the conformation
(left panel) of p53 as determined by immunoprecipitations with conformation
sensitive antibodies. The content of p53 recognized by Pab240 (‘mutant’
conformation) increased; and the content of p53 recognized by Pab1620 (wild type
conformation) decreased. Auranofin also attenuated p53 and HIF-1 transcription
in RKO cells (right panel) as measured by luciferase reporter assays.
Figure 5. Electrophilic antagonism of apoptosis corresponds to the loss of a p53
allele. Caspase acitivity was measured as a determinant of apoptosis in HCT 116
p53+/+, p53+/-, and p53-/- cells incubated with 20 µM amethopterin for 48 hr (right
panel). PGA2 functionally impaired apoptosis in p53+/+ cells at a level comparable
to the p53+/-, haploinsufficient, cells (left panel).
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 24
Figure 1:
SCOO
OH
NH
NH
CO
NH NH
O
O
OH
COOH
PGA1
PGA1-amidopentylbiotin
SCOO
OH
NH
NH
CO
NH NH
O
O
OH
COOH
PGA1
PGA1-amidopentylbiotin
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 25
Figure 2:
NeutrAvidin Pull-down
1 2 4321432143
∝ -TrxR1
∝ -p53
TrxR
p53
RKO HCT 116p53+/+
HCT 116p53-/-
Whole Cell Lysate
∝ -IKKα
A
5 6 87
Whole Cell Lysate
NeutrAvidin Pull-down
5 6 87
TrxR
TrxR
∝ -TrxR1
[75Se] metabolic
label
B
NeutrAvidin Pull-down
1 2 4321432143
∝ -TrxR1
∝ -p53
TrxR
p53p53
RKO HCT 116p53+/+
HCT 116p53-/-
Whole Cell LysateWhole Cell Lysate
∝ -IKKα
A
5 6 87
Whole Cell Lysate
NeutrAvidin Pull-down
5 6 87
TrxR
TrxR
∝ -TrxR1
[75Se] metabolic
label
B
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 26
Figure 3:
C
D
0 10 1000
20
40
60
80
100
120
Nor
mal
ized
HIF
-1 R
epor
ter A
ctiv
ity(6
hr C
obal
t Chl
orid
e, 1
00 µ
M)
Concentration (µM)
0 1000
20
40
60
80
100
Concentration (µM)
Nor
mal
ized
NAD
PHO
xida
se A
ctiv
ity
C
D
0 10 1000
20
40
60
80
100
120
Nor
mal
ized
HIF
-1 R
epor
ter A
ctiv
ity(6
hr C
obal
t Chl
orid
e, 1
00 µ
M)
Concentration (µM)
0 1000
20
40
60
80
100
Concentration (µM)
Nor
mal
ized
NAD
PHO
xida
se A
ctiv
ity
∆12
-PG
J 2
PGA 2
PGA 1
DMSO
blan
k
15-d
ideo
xy∆∆
PGJ 2
15-k
eto-
PGF 2
α
4-hy
drox
ynon
enal
Mal
ondi
alde
hyde
A
B
IP: 240
0 10 1000
20
40
60
80
100
Concentration
Nor
mal
ized
p53
Rep
orte
r Act
ivity
(6 h
r Eto
posi
de, 5
0 µM
)
∆12
-PG
J 2
PGA 2
PGA 1
DMSO
blan
k
15-d
ideo
xy∆∆
PGJ 2
15-k
eto-
PGF 2
α
4-hy
drox
ynon
enal
Mal
ondi
alde
hyde
A
B
IP: 240
∆12
-PG
J 2
PGA 2
PGA 1
DMSO
blan
k
15-d
ideo
xy∆∆
PGJ 2
15-k
eto-
PGF 2
α
4-hy
drox
ynon
enal
Mal
ondi
alde
hyde
A
B
IP: 240
0 10 1000
20
40
60
80
100
Concentration
Nor
mal
ized
p53
Rep
orte
r Act
ivity
(6 h
r Eto
posi
de, 5
0 µM
)
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 27
Figure 4:
Aura
nofin
EtO
H
IP: 240
IP: 1620
Lysate
0
20
40
60
80
100
120
CoCl2+Auranofin
EtOH CoCl2Etop Etop+Auranofin
EtOH
Nor
mal
ized
p53
Rep
orte
r Act
ivity
(% M
axim
um)
0
20
40
60
80
100
120
Nor
mal
ized
HIF
-1 R
epor
ter A
citiv
ity(%
Max
imum
)
B
C
20 40 60 80 100
Normalized p53 Reporter Activity(% Maximum)
PG
A 2 E
top
Trea
tmen
t
Treatment Time (hr)0 1 2 3 4 5 6
A
0 10 1000
20
40
60
80
100
PGA2 Concentration (µM)
B
Nor
mal
ized
p53
Rep
orte
r Act
ivity
(6 h
r Eto
posi
de, 5
0 µM
)Au
rano
fin
EtO
H
IP: 240
IP: 1620
Lysate
0
20
40
60
80
100
120
CoCl2+Auranofin
EtOH CoCl2Etop Etop+Auranofin
EtOH
Nor
mal
ized
p53
Rep
orte
r Act
ivity
(% M
axim
um)
0
20
40
60
80
100
120
Nor
mal
ized
HIF
-1 R
epor
ter A
citiv
ity(%
Max
imum
)
Aura
nofin
EtO
H
IP: 240
IP: 1620
Lysate
Aura
nofin
EtO
H
IP: 240
IP: 1620
Lysate
0
20
40
60
80
100
120
CoCl2+Auranofin
EtOH CoCl2Etop Etop+Auranofin
EtOH
Nor
mal
ized
p53
Rep
orte
r Act
ivity
(% M
axim
um)
0
20
40
60
80
100
120
Nor
mal
ized
HIF
-1 R
epor
ter A
citiv
ity(%
Max
imum
)
B
C
20 40 60 80 100
Normalized p53 Reporter Activity(% Maximum)
PG
A 2 E
top
Trea
tmen
t
Treatment Time (hr)0 1 2 3 4 5 6
A
0 10 1000
20
40
60
80
100
PGA2 Concentration (µM)
B
Nor
mal
ized
p53
Rep
orte
r Act
ivity
(6 h
r Eto
posi
de, 5
0 µM
)
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 28
Figure 5:
0 6 200
50
100
150
200
250
300
350
400
450HCT116 p53+/+
Amethopterin DMSO
Cap
ase
Activ
ity (p
mol
e/m
in/m
g pr
otei
n)(D
EVD
sub
stra
te)
PGA2 Concentration (µM)
0
50
100
150
200
250
300
350
400
450
+/++/--/-
HCT116 p53 status
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from
FitzpatrickPhilip J. Moos, Kornelia Edes, Pamela Cassidy, Edmund Massuda and Frank A.
reductasetranscription factors p53 and HIF by impairing the selenoprotein thioredoxin
Electrophilic prostaglandins and lipid aldehydes repress redox sensitive
published online November 6, 2002J. Biol. Chem.
10.1074/jbc.M211134200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on February 13, 2018http://w
ww
.jbc.org/D
ownloaded from