Biochimica et Biophysica Acta 1853 (2015) 660670

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Biochimica et Biophysica Acta

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Retinoids induce Nur77-dependent apoptosis in mouse thymocytes

Beta Kiss a,1, Katalin Tth a,1, Zsolt Sarang b, va Garabuczi a, Zsuzsa Szondy a,
a
b

Department of Dental Biochemistry, Signaling and Apoptosis Research Group, Research Center of Molecular Medicine, University of Debrecen, Debrecen H-4012, Hungary
Department of Biochemistry and Molecular Biology, Signaling and Apoptosis Research Group, Research Center of Molecular Medicine, University of Debrecen, Debrecen H-4012, Hungary

a r t i c l e

i n f o

Article history:
Received 21 November 2014
Received in revised form 20 December 2014
Accepted 30 December 2014
Available online 6 January 2015
Keywords:
Apoptosis
Nur77
Retinoid
Thymocyte

a b s t r a c t
Nur77 is a transcription factor, which plays a determinant role in mediating T cell receptor-induced cell death of
thymocytes. In addition to regulation of transcription, Nur77 contributes to apoptosis induction by targeting mitochondria, where it can convert Bcl-2, an anti-apoptotic protein into a proapoptotic molecule. Previous studies
have demonstrated that retinoids are actively produced in the mouse thymus and can induce a transcriptiondependent apoptosis in mouse thymocytes. Here we show that retinoic acids induce the expression of Nur77,
and retinoid-induced apoptosis is completely dependent on Nur77, as retinoids were unable to induce apoptosis
in Nur77 null thymocytes. In wild-type thymocytes retinoids induced enhanced expression of the apoptosisrelated genes FasL, TRAIL, NDG-1, Gpr65 and Bid, all of them in a Nur77-dependent manner. The combined action
of these proteins led to Caspase 8-dependent Bid cleavage in the mitochondria. In addition, we could demonstrate the Nur77-dependent induction of STAT1 leading to enhanced Bim expression, and the mitochondrial
translocation of Nur77 leading to the exposure of the Bcl-2/BH3 domain. The retinoid-induced apoptosis was dependent on both Caspase 8 and STAT1. Our data together indicate that retinoids induce a Nur77-dependent cell
death program in thymocytes activating the mitochondrial pathway of apoptosis.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Vitamin A deciency has been known for a long time to be accompanied by immune deciency and susceptibility to a wide range of infectious diseases [1] due to the various effects of vitamin A on the
immune functions [2]. Suggestions have been made that the active metabolites of vitamin A, that mediate its effects on the immune system,
are the retinoic acids (RAs)all-trans RA (ATRA) and 9-cis RA (9cRA)
[3,4]. Among many others, retinoids were shown to be produced in
the thymus [59], and to induce apoptosis in immature thymocytes
[912].
ATRA and 9cRA are suggested natural ligands for the nuclear retinoic
acid receptor family [13]. ATRA and 9cRA are equipotent in activating
retinoic acid receptors (RARs), while activation of retinoid X receptor
(RXR) by ATRA is 50-fold less than by 9cRA [14]. Though ATRA does
not bind to RXR receptors, the latest observation is explained by
Abbreviations: ATRA, all-trans retinoic acid; Bcl-2, B cell lymphoma 2; Bid, BH3
interacting domain death agonist; DP, double positive; 9cRA, 9-cis retinoic acid; FasL, Fas ligand; NDG-1, Nur77 dependent gene-1 coded protein; RALDH, retinaldehyde dehydrogenase; RAR, retinoic acid receptor; RXR, retinoid X receptor; STAT1, Signal Transducers and
Activators of Transcription 1
Corresponding author at: Department of Dental Biochemistry, Signaling and
Apoptosis Research Group, Research Center of Molecular Medicine, University of
Debrecen, H-4012 Debrecen, Nagyerdei krt.98, Hungary. Tel.: +36 52 416432; fax: +36
52 314 989.
E-mail address: szondy@med.unideb.hu (Z. Szondy).
1
Beta Kiss and Katalin Tth contributed equally to the experiments presented in this
work and share rst authorship.

http://dx.doi.org/10.1016/j.bbamcr.2014.12.035
0167-4889/ 2015 Elsevier B.V. All rights reserved.

conversion of the ATRA to 9cRA within the cells reaching an intracellular

concentration sufcient to activate RXR receptors at high extracellular
concentrations of ATRA [15]. In the presence of RAs retinoid receptors
function in the form of RAR/RXR heterodimers or RXR/RXR homodimers
[16], and modulate gene expression either by direct binding to their
cognate response elements or via interaction with other transcription
factors. In addition, RXR can form heterodimers with various other
members of the steroid/thyroid/retinoid receptor family, including
Nur77 [17].
Nur77 also belongs to the steroid/thyroid hormone receptor superfamily, and is an orphan receptor for which no ligand is known [18]. Besides forming a heterodimer with the retinoid X receptor to confer
9cRA-dependent transcription to reporters containing the DR5 regulatory element, it can also bind in monomeric form to promoters containing the Nur77 binding response element [19], as well as a homodimer to
the Nur77 response element carrying ones [20]. Nur77 was shown to be
a determinant transcription factor in mediating T cell receptor-induced
cell death of both thymocytes (negative selection) and peripheral T cells
or T cell lines [11,2124]. It was shown to be induced during T cell
receptor-induced death of T cells, and the binding of it to its response elements correlated well with the onset of apoptosis. In addition, constitutively active Nur77 was found to induce thymocyte apoptosis [25,
26]. To identify globally the targets of Nur77 transcription that regulate
apoptosis in thymocytes, differential gene expression experiments were
conducted with RNA isolated from Nur77 transgenic and wild-type fetal
thymi with cDNA microarrays. These studies identied Fas ligand (FasL)
and TRAIL, two known death receptor ligands, and Nur77 dependent

B. Kiss et al. / Biochimica et Biophysica Acta 1853 (2015) 660670

gene-1 coded protein (NDG-1) that regulates Caspase 8 activity by unknown mechanisms, as mediators of Nur77-dependent apoptosis [27].
Studies done on cancer cell lines revealed rst, that in addition to
regulating transcription, Nur77 is also able to induce apoptosis independent of its transcriptional activity by targeting mitochondria, where it
can convert Bcl-2, an antiapoptotic protein into a proapoptotic molecule. This effect is achieved via its association with the linker region of
Bcl-2 between the BH3 and BH4 domains resulting in the exposure of
the Bcl-2 BH3 domain [28]. In thymocytes during negative selection
Nur77 also translocates into the mitochondria and contributes to the
apoptosis induction [29]. This mitochondrial targeting is regulated by
protein kinase C [30] and requires heterodimerization with RXR,
which carries a putative nuclear export sequence present in its
carboxyl-terminal region [31].
Previous studies in our laboratory have shown that retinoids induce
a transcription-dependent apoptosis in thymocytes via activating RAR
[12]. However, the mechanism of the apoptosis induction was not investigated so far. Our data presented in this paper demonstrate that retinoids induce a Nur77-dependent apoptosis program in thymocytes
involving both transcription-dependent and -independent mechanisms
mediated by Nur77.
2. Materials and methods

661

using the Bio-Rad electrophoresis and transfer system. The free binding
sites of membranes were blocked with 5% non fat dry milk powder in
20 mM Tris, 0.1 M NaCl buffer with 0.1% Tween overnight at 4 C. One
g anti-Nur77 (BD Pharmingen), anti-Bid (R&D Systems), anti-Bim or
anti-STAT1 antibodies (Santa Cruz Biotechnology) were added to the
membranes. To detect Nur77, Bid, Bim or STAT1 signals peroxidaselabeled anti-mouse IgG (1:1000), anti-rat IgG (1:10,000) or anti-rabbit
IgG (1:10,000) were used and the enhanced chemiluminescence was visualized using the ECL System (Amersham). Equal loading of protein
was demonstrated with probing the membranes with anti--tubulin
(Santa Cruz Biotechnology) or -actin (Sigma-Aldrich) antibodies.
2.5. Affymetrix analysis
Mouse Affymetrix 430Av2 arrays carrying probes for 22690 transcripts were used to identify retinoid-regulated apoptosis genes in
mouse thymocytes. Nur77+/+ and/ control (DMSO exposed) and
4 h 9cRA (0.3 M) treated thymocyte samples were run in duplicates.
Hybridization was carried out in Genomics Core Facility in Heidelberg.
Raw intensity data was scaled and normalized in Affymetrix Microarray
suite 5.0. Filtering of changed transcripts was carried out in Microsoft
Access and Excel software. Increased transcripts were judged as
Present and increased + 2 fold in both duplicates. Decreased transcripts had minimum of 1.6 fold change value in both duplicates.

2.1. Reagents
2.6. qRT-PCR for detecting changes in the mRNA expression of various genes
All reagents were obtained from (Sigma-Aldrich, Budapest) except
indicated otherwise.
2.2. Thymocyte suspensions
Thymocyte suspensions were prepared from thymus glands of
4 week-old Nur77+/+ mice and their Nur77 decient [32] littermates
by mincing the glands in RPMI 1640 media supplemented with 10%
charcoal-treated FCS, 2 mM glutamine, and 100 IU penicillin/100 g
streptomycin/ml. In a few experiments thymocytes were isolated from
STAT1/ mice (Jackson Laboratories [33], as well. Thymocytes were
washed three times and diluted to a nal concentration of 5 106
cells/ml before incubation at 37 C in a humidied incubator under an
atmosphere of 5% CO2/95% air. Cell death was measured by trypan
blue uptake. A total of 9598% of cells routinely excluded trypan blue
after the isolation procedures. These studies have been reviewed and
approved by the review committee of the University of Debrecen
(DEMB).
2.3. Determination of the percentage of apoptotic thymocytes
Thymocytes were incubated in 24 well plates in the presence of the
indicated concentrations of retinoids for 12, dexamethasone-acetate
(0.1 M) or Jo2 antibody (1 g/ml) for 6 h. To test the involvement of
caspases thymocytes were preincubated with 40 M z-IETD.fmk (BD
Pharmingen) or 75 M z-LEHD.fmk Calbiochem (San Diego, CA, USA)
for 1 h. For determining the amount of cells containing degraded DNA
characteristic for apoptosis, a rapid technique using propidium iodide
DNA staining was applied. For staining cells were xed with 70% ice
cold ethanol for 5 min, then washed and redissolved in 100 l PBS containing 100 g/ml RNase and incubated for 10 min at room temperature.
Finally 400 l PBS containing 50 g/ml propidium iodide was added. % of
cells carrying decreased amount of DNA due to apoptosis (sub G0G1
cells) was determined on DNA histograms by ow cytouorometry
using FACScan (BD Biosciences).
2.4. Western blot analysis of Nur77, Bid, Bim and STAT1 expression
Whole cell homogenate was used. 40 g protein was run on an polyacrylamide gel, and blotted onto polyvinylidene diuoride membranes

Thymocytes (5 106/ml) were exposed to ATRA or 9cRA for 4 h. At

the end of the incubation total RNA was isolated with TRI reagent from
treated cells. Transcript quantitation was accomplished via quantitative
real-time RT (reverse transcriptase) PCR (polymerase chain reaction)
using Taqman gene expression assay. cDNA was synthesized using
High-Capacity cDNA Archive Kit (Applied Biosystems) according to
manufacturer's instruction. Real-time monitoring was carried out
using an ABI Prism 7900 performing 40 cycles of 94 C for 12 s and
60 C for 1 min with FAM-MGB-labeled specic probes (Applied
Biosystems). Threshold cycle number was determined using SDS 2.0
software. All samples were assayed in triplicates and the gene expression was normalized to cyclophilin.
2.7. Preparation of mitochondria and Western blot to detect mitochondrial
translocation of Nur77/RXR
To obtain mitochondrial fraction of thymocytes, cell homogenates (10 7 cell/sample) were fractionated by the ProteoExtract
Cytosol/Mitochondria Fractionation Kit from Calbiochem according
to the manufacturer's instructions. At the end of the procedure mitochondrial samples were denatured in 5 Laemmli buffer. Nur77 and
RXR were detected on Western blot by using the antibody against
Nur77 (BD Pharmingen) and RXR (Santa Cruz Biotechnology).
The blots were reprobed with antibodies to mitochondrial Hsp60
as loading control or the nuclear Lamin B (Santa Cruz Biotechnology)
to detect potential nuclear contamination.
2.8. Intracellular staining for Bcl-2/BH3
To detect intracellular levels of the Bcl-2 BH3 domain thymocytes
(2 106/sample) were washed twice with PBS, were xed and permeabilized using 250 l of Cytox/Cytoperm solution (BD Biosciences) for
20 min at 4 C. After xation and blocking steps the samples were
washed twice using perm/wash solution (200 l/sample) and stained
with the anti-Bcl-2 BH3 domain antibody (Abgent, San Diego, USA
1:100) for overnight at 4 C. For the detection cells were labeled with
FITC- or Cy3-conjugated anti-rabbit IgG and were analyzed by ow
cytomerty. Data was acquired using FACS Calibur (BD Biosciences) and
analyzed using WinMDI 2.9 software.

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2.9. Detection of TNF alpha content in thymocyte supernatant

5 106/0.5 ml wild type thymocytes were plated onto 24-well plates
and were stimulated with retinoids and the combination of phorbol
dibutyrate and ionomycin for 18 h. The samples were than centrifuged
at 4000 g for 5 min. The cytokine quantity of the supernatant culture
medium was determined by DuoSet Mouse TNF alpha ELISA Kit (R&D
Systems).
2.10. Isolation of CD4 + CD8 + double positive thymocytes by ow
cytometry sorting
Thymocytes isolated freshly from 4 week-old Nur77+/+ mice were
stained in PBS with CD4-PE and CD8-FITC antibodies (Sigma-Aldrich)
at room temperature for 10 min. CD4/CD8 double positive thymocytes
were separated with FACS Aria III instrument (BD Biosciences). Data
analysis was carried out by using BD FACSDiva Version 6.1.3 software.
2.11. Statistical analyses
All the data are representative of at least three independent experiments carried out on three different days. Values are expressed as
mean S.D. P values were calculated by using two-tailed Student's ttest for two samples of unequal variance, when data for the wild-type
and Nur77 null thymocytes were compared, and Student's t-test when
thymocytes from the same mouse were treated in various ways. Statistical signicance is indicated by a single asterisk (P b 0.05).
3. Results
3.1. Nur77 is upregulated and essential in retinoid induced apoptosis of
mouse thymocytes
Previous studies in our laboratory have shown that retinoids induce
a transcription-dependent apoptosis in mouse thymocytes via activating RAR [12]. Subsequent studies have revealed that ligation of RAR
can also induce the expression of Nur77 and its target FasL in a mouse
T cell hybridoma cell line [34]. To test whether retinoids could also induce Nur77 in mouse thymocytes, thymocytes isolated from 4 week
old mice were exposed to ATRA and 9cRA, and Nur77 expression was
detected on both mRNA (Fig. 1A) and protein levels (Fig. 1B). As seen
in Fig. 1, both ATRA and 9cRA were able to induce the expression of
Nur77 at concentrations where they saturate RARs, however, 9cRA
was more effective indicating that ligation of RXR might contribute to
the induction of Nur77. To decide whether Nur77 is required for
retinoid-induced apoptosis, apoptosis induction by retinoids was tested
on thymocytes isolated from both wild-type and Nur77 null mice [32].
As shown in Fig. 1C, retinoids induced a dose dependent cell death in
the wild-type thymocytes. In line with the lower expression of Nur77
in the presence of ATRA, ATRA was much less potent in apoptosis induction than 9cRA at each concentrations tested. However, none of these
compounds could induce apoptosis in Nur77 null thymocytes indicating
an essential role of Nur77 in mediating retinoid-induced apoptosis of
thymocytes. Interestingly, even the background rate of apoptosis was
lower in Nur77 null thymocytes indicating that the spontaneous cell
death involves Nur77-dependent elements. These results were not related to a Nur77-dependent developmental defect of thymocytes, as
we did not observe any change in the age-dependent size or thymocyte
composition of the Nur77 null thymuses (Fig. 1D and E). Our observation is in line with previously published data [32].
3.2. FasL, TRAIL, NDG1, GRP65 and Bid are induced by retinoids to initiate
thymocyte apoptosis
To identify genes mediating retinoid-induced apoptosis, mRNA levels
isolated from control (DMSO-treated) and retinoid-treated thymocytes

were compared by Affymetrix 430Av2 arrays carrying probes for

22,690 transcripts. As an effective retinoid, 0.3 9cRA was selected
for these studies. We conrmed Nur77, as one of the genes, that was signicantly upregulated (Table 1). The Nur77 family consists of Nur77,
Nor-1, and Nurr1 with overlapping biological activity, however, only
Nur77 was upregulated by 9cRA in mouse thymocytes. These data
were also conrmed by qRT-PCR analysis (data not shown). To decide
which apoptosis-related genes are regulated in a Nur77-dependent
manner during retinoid-induced apoptosis, both wild-type and Nur77
null thymocytes were exposed to 9cRA for 4 h and the changes in their
mRNA levels were compared by Affymetrix 430Av2 arrays. We found
ten apoptosis related genes to be induced by retinoids (Table 1). Interestingly most of these genes were induced in a Nur77-dependent manner.
NDG-1 was already a known Nur77-dependent gene [27]. However G
protein-coupled receptor 65 (Gpr65), also known as T-cell deathassociated gene 8, which belongs to a group of acid-sensing receptors
in the G2A G protein-coupled receptor (GPCR) family coupled to the adenylate cyclase [35], and the BH3 interacting domain death agonist (Bid),
which is activated by Caspase 8 cleavage and initiates the mitochondrial
pathway of apoptosis [36], were unexpected to be found among the
Nur77-regulated genes. To conrm the induction of these 3 genes by retinoids, their mRNA expression was also determined by qRT-PCR analysis
following 4 h exposure to ATRA or 9cRA. In addition, we also determined
the expression of FasL and TRAIL, which are known Nur77-regulated apoptosis related genes, but were not detectable by the Affymetrix analysis.
As shown in Fig. 2, all these genes were induced by retinoids in wild-type
thymocytes, but not in Nur77 null thymocytes conrming that these
genes are indeed retinoid-induced Nur77 dependent genes. 9cRA was
found to be more effective in the induction of all the Nur77-dependent
genes than ATRA, in accordance with the higher levels of Nur77 induced
by it.
3.3. Caspase 8 activation contributes to the initiation of
retinoid-induced apoptosis
Since retinoids induced the expression of TRAIL and FasL, two cell
death receptor ligands, and NDG-1, which similar to death receptors,
can also activate Caspase-8 though by unknown mechanisms [27], we
decided to check whether Caspase 8 plays a role in retinoid-induced apoptosis. First we tested whether Caspase 8 is cleaved during retinoid induced apoptosis. As shown in Fig. 3A, cleaved Caspase 8 protein can be
detected in retinoid-induced apoptosis. Since Bid was also induced by
retinoids (Table 1), and Bid is a Caspase 8 substrate [36], to demonstrate
that Caspase 8 is activated during retinoid-induced apoptosis we
checked, whether Bid is also cleaved. As shown in Fig. 3B, we could detect cleavage of Bid in thymocytes treated by 9cRA, but not in thymocytes treated by 9cRA in the presence of z-IETD-fmk, a Caspase 8
inhibitor. To test the role of Caspase 8 activation in retinoid-induced apoptosis, we inhibited its activity. Preincubaton of thymocytes with zIETD-fmk signicantly attenuated retinoid-induced cell death, but not
inhibited it fully (Fig. 3C), similar to the glucocorticoid-induced cell
death, in which Caspase 8 has, but not a determining role [37,38]. At
the same time z-IETDK completely inhibited Fas-mediated death.
These data indicate that Caspase 8 contributes to the initiation of
retinoid-induced apoptosis, but the death is not entirely dependent on
its activity.
3.4. Caspase 9 also plays a determining role in retinoid-induced apoptosis
Thymocytes are considered to be Type I cells, in which cell death receptor activated Caspase 8 generates sufcient amount of Caspase 3 to
drive cell death, without the need for the mitochondrial pathway [39].
Thus Fas mediated apoptosis of thymocytes is not affected by the loss
of Caspase 9 [40]. Though we found signs for Caspase 8 activation during
retinoid-induced apoptosis, we decided to test by inhibiting Caspase 9,
whether retinoid-induced apoptosis requires Caspase 9 activity (thus

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663

Fig. 1. Nur77 is upregulated and essential in retinoid induced apoptosis of mouse thymocytes. (A) Retinoids induce the mRNA expression of Nur77 in mouse thymocytes. Thymocytes were
exposed to the indicated concentrations of ATRA or 9cRA dissolved in DMSO in the presence of charcoal treated FCS. The nal concentration of DMSO was 0.5% in each assay. mRNA expression was determined 4 h later by qRT-PCR analysis using cyclophilin as a reference gene. (B) Retinoids induce the protein expression of Nur77 in mouse thymocytes. Thymocytes were
exposed to the indicated concentrations of ATRA or 9cRA. Protein expression was determined 4 h later by Western blot analysis. -actin was used for loading control. (C) Retinoids fail to
induce apoptosis in Nur77-null thymocytes. Thymocytes isolated from 4 weeks old wild-type mice and their Nur77 null littermates were exposed to the indicated concentrations of ATRA
and 9cRA. The percentage of thymocytes in subG1 population was determined in propidium iodide stained samples 12 h later. (D) Loss of Nur77 does not affect the age-dependent changes
in the thymic weight in mice. (E) Unaltered total thymocyte cell number and double positive thymocyte population size in the thymus of four week old Nur77 null mice. Data represent
mean S.D. of three independent experiments. *Signicantly different from the DMSO-treated control (p b 0.05).

the mitochondrial pathway). Fas-induced apoptosis was used as a control. As seen in Fig. 3C, while the Caspase 9 inhibitor z-LEHD.fmk had
only slight effect on the apoptosis induced by the Jo2 anti-Fas antibody,
it completely prevented retinoid-induced and dexamethasone-induced
apoptosis. These data indicate that though Caspase 8 is activated during
retinoid-induced apoptosis, its apoptosis-inducing action requires the
mitochondrial pathway of apoptosis involving Caspase 9. In line with
this observation we also detected the activation of Caspase 9 (Fig. 3D).
Interestingly, activation of Caspase 8 preceded in time that of Caspase
9 indicating that Caspase 8 plays an initiator role in the retinoidinduced apoptosis.
3.5. Gpr65-mediated signals do not seem to be critical in retinoid-induced
apoptosis of thymocytes
Previous studies have shown that Gpr65 can activate adenylate cyclase [35], and increases in cytosolic cAMP levels might lead to apoptosis
in thymocytes by enhancing the production of TNF- [41] or by inducing Bim [42]. Since 9cRA induced the expression of Gpr65, we decided to

test whether addition of retinoids results in TNF- production by

checking its mRNA expression by qRT-PCR and by detecting its protein
level by ELISA. Exposure of thymocytes to retinoids did not result in detectable TNF- production (data not shown). However, as shown in
Fig. 4A and B, retinoids signicantly induced the expression of Bim at
both mRNA and protein levels, but this increase was not inhibited by
Rp-cAMPS triethylamine or H89, specic membrane-permeable inhibitors of cAMP dependent protein kinase I and II [43]. Addition of PKA inhibitors had no effect on the retinoid-induced apoptosis of the
thymocytes either (data not shown). All together our data indicate
that though the expression of Gpr65 is increased, under our culture conditions there is no signicant acidication which would activate it and
the coupled adenylate cyclase pathway.
3.6. Bim is induced in a STAT1-dependent manner during retinoid-mediated
apoptosis of thymocytes and contributes to cell death induction
Previous studies have shown that STAT1 can also regulate Bim expression in thymocytes [44]. The Affymetrix data suggested STAT1 is

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Table 1
9-cis retinoic acid regulates the expression of Nur77 and several Nur77-dependent and independent apoptosis-related genes in mouse thymocytes.
Gene description

Symbol

Nur77+/+ Nur77/

apoptosis is STAT1 dependent. In addition, loss of STAT1, attenuated the

retinoid-induced apoptosis of thymocytes (Fig. 4D) indicating that
STAT1-induced Bim expression contributes to the initiation of the
retinoid-induced apoptosis program.

Fold change
Nuclear receptor subfamily 4, group A, member 1
Nur77 downstream gene 1
BH3 interacting domain death agonist
G-protein coupled receptor 65
Signal transducer and activator of transcription 1
Transglutaminase 2
Protein tyrosine phosphatase, non-receptor type 6
Myeloid differentiation primary response
gene 116

Nur77
3.1
NDG-1
33.3
Bid
6.5
Gpr65
3.7
STAT1
2.2
TG2
4.8
Ptpn6
2.2
Myd116
2.4

2.7
1.9
1.9
1.1
4.8
1.0
2.4

Wild-type and Nur77 null thymocytes were exposed to 0.3 M 9cRA for 4 h and the changes in their mRNA levels were compared by Affymetrix 430Av2 arrays. Data represent fold
changes as compared to the DMSO-treated controls based on two independent experiments. Bold letters highlight dose genes the expression of which is independent of Nur77.

also induced during retinoid-induced apoptosis in a Nur77-dependent

manner (Table 1), what we could also conrm on protein levels
(Fig. 4D). Thus we decided to test by using STAT1 knock out thymocytes
[33] whether the induction of Bim expression during retinoid-induced
apoptosis is STAT1 dependent. As shown in Fig. 4B and C, our results indicate that enhancement in the Bim expression during retinoid-induced

3.7. Nur77 translocates into the mitochondria during retinoid-induced

apoptosis and leads to the exposure of the Bcl-2 BH3 domain
Previous studies have shown that in addition to its transcription inducing ability, Nur77 can also contribute to apoptosis induction during
negative selection in thymocytes by targeting mitochondria, where it
converts Bcl-2, an antiapoptotic protein into a proapoptotic molecule
after associating with it and leading to the exposure of the Bcl-2/BH3
domain [29]. That is why we tested whether retinoids are capable of inducing the translocation of Nur77 into the mitochondria. As control, we
used the phorbol dibutyrate/ionomycin treatment, which mimics the
signaling of negative selection. As shown in Fig. 5A, treatment with
phorbol dibutyrate/ionomycin resulted in a time dependent translocation of Nur77 into the mitochondria appearing as a wide band due to
the heavy phosphorylation of the protein [22]. 9cRA and, with a much
less efciency, ATRA also induced a time-dependent translocation of
Nur77 with a peak at 2 h for each treatment. However, the protein appeared with a much thinner band that was found in phorbol
dibutyrate/ionomycin-treated cells, indicating a less prominent or lack

Fig. 2. Retinoids induce the expression of FasL, NDG-1, TRAIL, Grp65 and Bid in a Nur77-dependent manner. Effect of retinoids on the mRNA expression of (A) Bid, (B) NDG-1, (C) Gpr65,
(D) FasL and (E) TRAIL in wild type and Nur77 null thymocytes. Thymocytes were exposed to the indicated concentrations of retinoids and mRNA expressions were determined 4 h later by
qRT-PCR analysis using cyclophilin as a reference gene. Data represent mean S.D. of four independent experiments. *Signicantly different from the DMSO-treated control (p b 0.05).

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Fig. 3. Activation of both Caspase 8 and 9 is required for retinoid-induced apoptosis. (A) Retinoids induce the activation of caspase 8 during apoptosis. Thymocytes were exposed to 0.3 M
9cRA or 3 M ATRA, and the cleavage of Caspase 8 was followed in the absence or the presence of 40 M specic Caspase 8 inhibitor, zIETD-fmk by Western blot at the indicated time
points. Tubulin was used as loading control. (B) Bid is cleaved during retinoid-induced apoptosis. Thymocytes were treated with 0.3 M 9cRA in the absence or the presence of 40 M
zIETD-fmk for 2, 4 and 6 h. Bid expression was determined by Western bot from total cell lysate. -actin was used as loading control. (C) Retinoids induce the activation of Caspase 9 during
apoptosis. Thymocytes were exposed to 0.3 M 9cRA, 3 M ATRA or 0.1 M dexamaethasone acetate and the time-dependent cleavage of Caspase 9 was detected by Western blot analysis.
(D) Inhibition of both Caspase 8 and 9 prevents retinoid-induced apoptosis. Thymocytes were exposed to 3 M ATRA, 0.9 M 9cRA for 12 h, to 0.1 M dexamethasone acetate or 1 g/ml Jo2
antibody for 6 h in the presence or absence of 40 M zIETD-fmk, or 75 M Caspase 9 inhibitor Z-LEHD-FMK. The percentage of thymocytes in subG1 population was determined in
propidium iodide stained samples. Data represent mean S.D. of three independent experiments. *Signicantly different from the sample, where no caspase inhibitor was present
(p b 0.05).

of phosphorylation of it during retinoid-induced signaling. Nur77 can

form heterodimers with the retinoid X receptor (RXR), and previous
studies indicated that heterodimer formation is required for the nuclear
export of Nur77 [31]. Interestingly, we detected mitochondrial translocation of RXR in both Nur77 +/+ and / thymocytes. In phorbol
dibutyrate/ionomycin-treated cells the kinetics of the mitochondrial appearance of RXR was different from that of Nur77, and the presence or
absence of Nur77 did not alter it. In retinoid treated cells, however,
the peak of both Nur77 and RXR translocations was detected at 2 h
after treatment. Next we checked whether association of Nur77 with
mitochondria leads to Bcl-2 conformational change and exposure of
its BH3 domain. The Bcl-2 BH3 domain is usually buried within the
folded Bcl-2 protein and is undetectable by the Bcl-2/BH3-specic antibodies [29]. Exposure of this domain correlates with the proapoptotic
activities of Bcl-2 [29] and can be detected by mimicking signals of negative selection by adding phorbol dibutyrate/ionomycin to the thymocytes [30]. That is why we measured Bcl-2/BH3 exposure during
retinoid-induced apoptosis as well, and found a time dependent exposure of the BH3 domain (Fig. 5B). The exposure of the domain was related to Nur77, as the exposure of this domain was not observed in Nur77
null thymocytes exposed to 9cRA.

3.8. Other molecules that might contribute to retinoid-induced apoptosis

of thymocytes
In line with our previously published data [9] we detected the
induction of transglutaminase 2 upon exposure to 9cRA in a Nur77 independent manner. Transglutaminase 2 is a multifunctional protein
crosslinking enzyme, which was shown to be related to the mitochondrial pathway of apoptosis [45,46]. In addition, we found increased expression of two proteins that negatively regulate protein phosphorylation,
Gadd34 and Protein tyrosine phosphatase, non-receptor type 6

(Ptpn6). Gadd34 (also known as MyD116) was originally described as

a growth arrest and DNA damage-inducible gene. It facilitates protein
phosphatase type 1 activity through both direct binding to the protein,
as well as through binding to other proteins that also modulate phosphatase activity. Increased expression of Gadd34 was subsequently found to
correlate with apoptosis, and forced overexpression of the protein leads
to apoptosis [47]. Ptpn6, on the other hand, is known to be required for
receptor-mediated cytotoxic signaling that causes intracellular acidication and apoptosis [48]. While the retinoid-induced induction Ptpn6 was
Nur77 dependent, the induction of Gadd34 was not.

3.9. Retinoids induce both Nur77-dependent transcription and the

appearance of Bcl-2/BH3 domain in the DP thymocytes
Previous studies have shown that retinoids induce apoptosis primarily in double positive (DP) thymocytes [12], but the basal expression of
Nur77 shows a differentiation-dependence [49]. While in DP thymocytes the expression of Nur77 is low, both positive and negative selection induces the expression of Nur77, the expression correlating with
the strength of the TCR signal. As a result, in the thymus of a transgenic
mouse which expressed GFP under the control of the Nur77 promoter,
mostly medullary (positively selected) thymocytes were GFP-positive
[50]. Thus we decided to test whether the Nur77-dependent events
that we observed in non-separated thymocytes are related to DP thymocytes using DP thymocytes received by sorting (Fig. 6A). As shown
in Fig. 6B, DP thymocytes expressed Nur77 and reacted to both 9cRA
and the PdBu/ionomycin treatment by upregulating Nur77. Since we
could generate small amounts of sorted cells we decided to check the
retinoid-induction of three Nur77-regulated genes: Gpr65, NDG-1 and
Bid. As shown in Fig. 6C, retinoids induced the mRNA expression of all
these three genes. To test whether Bcl-2/BH3 exposure can be detected
in DP thymocytes, thymocytes were exposed to retinoids and then

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Fig. 4. Bim is induced in a STAT1-dependent manner during retinoid-mediated apoptosis. (A) Retinoids fail to induce Bim in Nur77 decient thymocytes. Thymocytes were treated with
the indicated concentrations of 9cRA or ATRA for 4 h and mRNA level of Bim was measured by qRT-PCR and normalized to cyclophilin. (B) Retinoids induce the appearance of Bim protein
in a Nur77- and STAT1-dependent, but a protein kinase A-independent manner. Wild type thymocytes were exposed to 0.3 M 9cRA or 3 M ATRA, and Bim protein was detected 6 h later
in the absence or presence of protein kinase A I and II inhibitors (10 M H89 or 50 nM Rp.cAMPS triethylamine). Thymocytes derived from Nur77 or STAT1 null mice were treated with
0.3 M 9cRA or 3 M ATRA and Bim protein was detected 6 h later by Western blot analysis. -actin was used as loading control. (C) Retinoids fail to induce the expression of Bim in STAT1
null thymocytes. Thymocytes were exposed to the indicated retinoid concentrations and Bim expression was determined 6 h later by qRT-PCR analysis using cyclophilin as a reference
gene. (D) Retinoids induce the expression of STAT1 in thymocytes. Thymocytes derived from wild type mice were treated with 0.3 M 9cRA or 3 M ATRA and STAT1 protein was detected
2 or 4 h later by Western blot analysis. -actin was used as loading control. (E) Loss of STAT1 attenuates retinoid induced apoptosis of thymocytes. Wild type and STAT1 null thymocytes
were exposed to 3 M ATRA, 0.9 M 9cRA for 6 h. The percentage of thymocytes in subG1 population was determined in propidium iodide stained samples. Data represent mean S.D. of
three independent experiments. *Signicantly different from the DMSO-treated control (p b 0.05).

labeled with anti-CD8 and anti-Bcl-2/BH3 antibodies. As shown in

Fig. 6D, we could detect the exposure of Bcl-2/BH3 domain in the
CD8+ population. Since the majority of CD8+ thymocytes are DP thymocytes, these data indicate that Nur77 can translocate into the mitochondria in these cells. Finally we tested whether the retinoid-induced
death of the sorted DP thymocytes is dependent on Caspase 8 and Caspase 9. As shown in Fig. 6E, both inhibitors attenuated retinoid-induced
apoptosis similarly as in the unsorted thymocytes.
4. Discussion
Previous studies in our and other laboratories have shown that retinoids applied alone are capable of inducing apoptosis in mouse thymocytes [11,12] and in T cell lines [34,50]. The studies carried out on the
Jurkat T cell line revealed that similar to T cell receptor signaling retinoids can induce the expression of Nur77 [50].
Our data presented in this paper demonstrate that retinoids are capable of inducing the expression of Nur77 also in mouse thymocytes.
Retinoids not only induced the expression of Nur77, but retinoidinduced apoptosis was fully dependent on Nur77. This was a surprise,

since TCR-mediated apoptosis, which is also coupled to an induction

in the expression of Nur77, was not affected by the loss of Nur77 [32].
This observation was explained by the simultaneous induction of Nor1
by TCR signals, which shows overlapping biological activities with
Nur77 [51]. However, unlike during TCR signaling, in retinoid-induced
apoptosis other members of the Nur77 family were not induced.
These data indicate that though Nur77 and Nor1 show overlapping biological activities, they might be regulated by divergent signals.
Our data also demonstrate that 9cRA is more effective in inducing
Nur77 than ATRA indicating that ligation of RXR might contribute to
the Nur77 induction in mouse thymocytes. Our previous studies have already shown that 9cRA is more effective in inducing apoptosis in thymocytes than ATRA is, but the molecular mechanism was not known. Here
we propose that 9cRA is more effective in inducing Nur77, and more
Nur77 leads to a more effective apoptosis induction in thymocytes.
Nur77 is a known transcription factor. In a search for the retinoidinduced apoptosis genes, we found the induction of nine apoptosisrelated genes, the majority of which was induced in a Nur77dependent manner. FasL, TRAIL and NDG-1 were previously known to
be Nur77 targets in thymocytes [27]. In the present study we identied

B. Kiss et al. / Biochimica et Biophysica Acta 1853 (2015) 660670

667

Fig. 5. Nur77 translocates into the mitochondria during retinoid-induced apoptosis and leads to the exposure of the Bcl-2 BH3 domain. (A) Detection of time-dependent Nur77 and RXR
translocation into mitochondria in wild-type and Nur77 null thymocytes following retinoid or 1 M phorbol dibutyrate/2 g/ml ionomycin treatment. Mitochondrial fractions of thymocytes were isolated at the indicated time points following treatment and were blotted with anti-Nur77 and RXR antibodies. For fraction purity the blot was also probed for the nuclear
Lamin B and the mitochondrial Hsp60. (B) Time dependent Bcl-2/BH3 exposure of wild-type thymocytes exposed to retinoids. 1 M phorbol dibutyrate/2 g/ml ionomycin treatment
of wild-type thymocytes was used as positive control, while exposure of Nur77 null cells to the same compounds was used as negative control.

four additional Nur77-dependent genes, Bid, a BH3-only protein, Gpr65,

a pH sensitive receptor, Bim, another BH3 only protein, and Ptpn6, a
protein phosphatase, during retinoid-induced apoptosis. We have demonstrated that Caspase 8 is activated and Bid is cleaved during retinoidinduced apoptosis.
Since FasL, TRAIL and NDG-1 all could contribute to Caspase 8 activation, we tested the role of Caspase 8 in retinoid-induced apoptosis by a
specic Caspase 8 inhibitor, in a concentration at which it only slightly
affected dexamethasone-induced cell death. This concentration of the
inhibitor, however, completely prevented the cleavage of Bid and significantly attenuated the retinoid-induced cell death indicating a central
role of Caspase 8 in retinoid induced apoptosis of thymocytes. Though
thymocytes are considered to be type I cells in the context of Fasinduced cell death [39], our data, which demonstrate that Caspase 9 inhibition also prevents retinoid-induced apoptosis, indicate that during
retinoid-induced apoptosis Caspase 8 drives the mitochondrial pathway
of apoptosis in thymocytes, in which Caspase 8-induced Bid cleavage
and activation might play a mediator event.
The importance of the mitochondrial pathway was also demonstrated by the fact that retinoid-induced STAT1 leads to the upregulation of
Bim, another BH3-only protein, and loss of STAT1 prevented Bim upregulation and signicantly attenuated retinoid-induced apoptosis. Interestingly, retinoid induced STAT1 expression and phosphorylation was
observed in other types of cells as well [52] indicating that STAT1 is

not a unique mediator of retinoid signaling in the thymocytes. In addition, transglutaminase 2, which was previously demonstrated to act
on the mitochondria [45,46], was also upregulated.
Besides acting as transcription factor, Nur77 protein is known to act
directly in the mitochondria to induce apoptosis [28,29]. Here we demonstrated that Nur77 can translocate into the mitochondria also during
retinoid-induced apoptosis of thymocytes. This translocation was detected with a similar time kinetics, but Nur77 found in the mitochondria
seemingly lacked heavy phosphorylation, which characterizes the protein during negative selection [22]. Interestingly, 9cRA was more effective in inducing Nur77 translocation than ATRA, in contrast to a previous
nding which suggested that ligation of RXR might interfere with Nur77
translocation [31]. However, those studies were conducted in the
presence of another apoptosis-inducing signal, which results in a protein kinase C-dependent phosphorylation of Nur77. Thus mitochondrial
translocation of Nur77 might be simultaneously affected by both its
phosphorylation status and the presence of the RXR ligand. The translocation of Nur77 correlated with the appearance of the proapoptotic BH3
domain of Bcl-2.
Though the majority of our studies were conducted on unsorted thymocytes, we repeated some key experiments on sorted DP thymocytes
as well. We could prove that Nur77 is induced, Nur77-dependent genes
are transcribed, the Bcl-2/BH3 domain appears in these cells, and the
death is Caspase 8 and 9 dependent. Based on our ndings we propose

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Fig. 6. Retinoids induce the expression of Nur77-dependent genes as well as the exposure of the Bcl-2/BH3 domain in DP thymocytes. (A) Result of the sorting of DP thymocytes. (B) Nur77
is induced at protein levels by 0.3 M 9cRA or by 1 M phorbol dibutyrate/2 g/ml ionomycin treatments in sorted DP thymocytes. (C) The mRNA levels of Nur77 and three Nur77-dependent genes are induced by retinoids in DP thymocytes. Thymocytes were exposed to 3 M ATRA or 0.3 M 9cRA and mRNA expressions were determined 4 h later by qRT-PCR analysis
using cyclophilin as a reference gene. Data represent mean S.D. of four independent experiments. *Signicantly different from the DMSO-treated control (p b 0.05). (D) The Bcl-2/
BH3 domain is exposed following retinoid treatment in DP thymocytes. Thymocytes were exposed to 3 M ATRA, 0.9 M 9cRA or the vehicle control. 4 h later cells were labeled with
both FITC-labeled anti-CD8 and with Cy3-labeled anti-Bcl-2/BH3 antibodies. The exposure of Bcl-2/BH3 is shown in CD8+ thymocytes, which represent mostly DP cells. (E) Inhibition
of both Caspase 8 and 9 prevents retinoid-induced DP thymocyte apoptosis. Thymocytes were exposed to 3 M ATRA, 0.3 M 9cRA, in the presence or absence of 40 M zIETD-fmk,
or 75 M Caspase 9 inhibitor Z-LEHD-FMK. The percentage of thymocytes in subG1 population was determined in propidium iodide stained samples 12 h later. Data represent
mean S.D. of three independent experiments. *Signicantly different from the sample, where no caspase inhibitor was present (p b 0.05).

that retinoids induce a Nur77-dependent cell death program in mouse DP

thymocytes, which involves already known and new Nur77-dependent
elements, which all together contribute to the induction of the mitochondrial pathway of apoptosis (Fig. 7). Can developing thymocytes be exposed to retinoids under in vivo conditions in the thymus? Data coming
from our laboratory and that of Agace indicate that cells located in the
thymus are capable of producing RAs generating an endogenous RA milieu within the tissue [68]. Thus, we detected an age-dependent mRNA
expression of retinaldehyde dehydrogenases (RALDH1 and 2), cellular
RA binding protein-II and CYP26A1, proteins responsible for the synthesis, nuclear transport and degradation of RA in the postnatally developing
thymus [6]. We could also demonstrate the existence of an age- and
RALDH-dependent production of RAR-activating ligand(s) by measuring
the induction of an RAR-responsive transgene in two independent transgenic mouse strains, in which the expression of the transgene reects
functionally relevant retinoic acid synthesis [6]. We also found that
their production is related to engulng macrophages [9]. Surprisingly,
however, we could not detect the presence of any of the classical retinoids in the mouse thymus [6], instead metabolites of the retinol saturase
pathway seem be produced [53]. Thus, our data indicate that in vivo

novel retinoids might contribute to the regulation of thymocyte apoptosis

which are produced in the thymic cortex during the course of the constant apoptotic cell engulfment.
5. Conclusions
Our data presented in this paper indicate that retinoids induce a
Nur77-dependent apoptosis in immature thymocytes. Data coming
from our laboratory and that of Agace indicate that cells located in the
thymus are capable of producing RAs generating an endogenous RA milieu within the tissue [59], and the source of retinoids in the postnatal
thymus are the engulng macrophages [9]. Besides retinoids, various
other molecules, such as TGF-, carbon monoxide, ATP or adenosine,
are also released by macrophages while they engulf apoptotic cells. Interestingly, all these molecules have been described to induce or promote apoptosis in thymocytes in the absence of TCR signaling. Thus
we propose that thymic macrophages, because they continually engulf
apoptotic cells, might constantly provide these cell death-inducing signals including retinoids and thus contribute to the formation of a thymic
milieu that ensures the effective induction of death by neglect [54].

B. Kiss et al. / Biochimica et Biophysica Acta 1853 (2015) 660670

Fig. 7. Proposed model for retinoid-induced apoptosis in DP mouse thymocytes. Retinoids

induce the expression of Nur77. The transcription factor Nur77 induces the expression of
FasL, TRAIL, NDG-1, Gpr65 and STAT1. FasL, TRAIL and NDG-1 may contribute to Caspase 8
activation. Caspase 8 will cleave Bid, the expression of which is also enhanced during retinoid-induced apoptosis. Retinoid-induced STAT1 leads to the upregulation of Bim. Retinoids also induce the expression of transglutaminase 2. Cleaved Bid, TG2 and Nur77 can
directly act on the mitochondria to initiate the mitochondrial pathway of apoptosis.

Transparency document
The Transparency document associated with this article can be
found, in the online version.
Acknowledgments
The authors thank Dr. J. Milbrandt for providing the nur77 null mice.
The technical assistance of Edit Komczi and Zsolt Hartmann is gratefully
acknowledged. This study was supported by Hungarian grants from the
National Research Fund (OTKA K104228 and K83865) and the TMOP
4.2.2.A-11/1/KONV-2012-0023 VD-ELEM project co-nanced by the
European Social Fund and the European Regional Development Fund.
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