Biochemical Pharmacology 177 (2020) 113936

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Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

The super-cooling compound icilin stimulates c-Fos and Egr-1 expression T

and activity involving TRPM8 channel activation, Ca2+ ion influx and
activation of the ternary complex factor Elk-1
⁎
Myriam Ulricha, Ulrich Wissenbachb, Gerald Thiela,
a
Department of Medical Biochemistry and Molecular Biology, Germany
b
Experimental and Clinical Pharmacology, Saarland University Medical Faculty, D-66421 Homburg, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: The TRPM8 cation channel can be activated by the cooling compound icilin. Recently, we showed that stimu-
Ca2+ channel lation of TRPM8 channels induces a signaling cascade leading to the activation of the transcription factor AP-1.
Egr-1 Additionally, expression of the AP-1 constituent c-Fos has been shown to be induced following TRPM8 stimu-
Elk-1 lation. c-Fos is frequently used as a marker for neuronal activity. Here, we have analyzed the mechanism
c-Fos
connecting TRPM8 stimulation and c-Fos expression. Furthermore, we analyzed the expression of the neuronal
TRPM8
Transient receptor potential
activity-responsive transcription factor Egr-1 following TRPM8 activation. The results show that icilin-induced
stimulation of TRPM8 channels increased c-Fos promoter activity and induced c-Fos expression. Moreover, icilin
Chemical compounds: stimulation increased Egr-1 promoter activity and induced the expression of Egr-1. Pharmacological inhibition of
icilin, PubChem CID: 161930 TRPM8 blocked the icilin-induced expression of Egr-1 and c-Fos. An influx of Ca2+ ions into the cells via TRPM8
RQ-00203078, PubChem CID: 49783953 was necessary to stimulate Egr-1 and c-Fos expression following icilin treatment. Genetic experiments revealed
that serum response elements within the Egr-1 and c-Fos promoters are crucial to couple TRPM8 stimulation
with enhanced transcription of both the Egr-1 and c-Fos genes. These data were corroborated by experiments
showing that TRPM8 stimulation increased the transcriptional activation potential of Elk-1, a SRE binding
protein. c-Fos is important for neuronal excitability and survival. Egr-1 plays an important role in synaptic
plasticity, consolidation and reconsolidation of long-term memory. Elk-1 may preserve neurons against toxic
insults but may also induce depressive behaviour. The fact that TRPM8 stimulation activates the transcription
factors c-Fos, Egr-1, and Elk-1 connects TRPM8 signaling with maintaining important brain functions.

1. Introduction Recently, we showed that stimulation of TRPM8 channels with icilin

Transient receptor potential melastatin-8 (TRPM8) channels are
non-selective cation channels that are expressed in peripheral sensory
neurons and in several non-neuronal tissues, including the prostate,
testis, lung, and uterus [1–3]. Stimulation of TRPM8 channels permits
the influx of Ca2+ and Na+ ions into the cells. TRPM8 belongs to the
temperature sensing TRP channels (thermo-TRPs) that can be activated
by cold temperature and natural and synthetic “cooling” agents such as
menthol, eucalyptol, and icilin [3–5]. The analysis of transgenic
TRPM8-deficient mice confirmed the role of TRPM8 channels in ther-
mosensation [6–8]. In trigeminal and dorsal root ganglion neurons
TRPM8 channels function as cold nociceptors [7,8]. Pharmacological
TRPM8 inhibitors have been discussed for their use in the treatment of
cold hypersensitivity following nerve injury and inflammation [3].
leads to the activation of the transcription factor AP-1 [9,10], indicating
that stimulation of TRPM8 triggers changes of the cellular expression
pattern. It is also known that stimulation of TRPM8 channels activates
expression of c-Fos, a basic region leucine zipper transcription factor
that dimerizes with other bZIP proteins to form the AP-1 transcription
factor complex. c-Fos is widely used as a marker for neuronal activity
and this activity has been used to monitor TRPM8 activation in neurons
[11–15]. Therefore, we asked how TRPM8 stimulation induces c-Fos
expression.
The biosynthesis of the transcription factor Egr-1 is also induced by
neuronal activity. Egr-1 is a zinc finger protein that is synthesized as a
result of cellular stimulation with neurotransmitters, hormones, and
growth factors [16–19]. We recently showed that stimulation of TRPM8
induces the activation of extracellular signal-regulated protein kinase

Abbreviations: SRE, serum response element; SRF, serum response factor; TRP, transient receptor potential
⁎
Corresponding author. Department of Medical Biochemistry and Molecular Biology, Saarland University, Building 44, D-66421 Homburg, Germany.
E-mail address: gerald.thiel@uks.eu (G. Thiel).

https://doi.org/10.1016/j.bcp.2020.113936
Received 20 February 2020; Accepted 23 March 2020
Available online 26 March 2020
0006-2952/ © 2020 Elsevier Inc. All rights reserved.
M. Ulrich, et al. Biochemical Pharmacology 177 (2020) 113936

ERK1/2 [10]. This kinase is known to be a strong activator of Egr-1. We

therefore asked whether TRPM8 stimulation induces the biosynthesis of
biologically active Egr-1.
The results of this study show that icilin-mediated stimulation of
TRPM8 channels induces the biosynthesis of Egr-1 and c-Fos. The
analyses of the icilin-induced signaling cascade leading to the upregu-
lation of Egr-1 and c-Fos expression revealed that an influx of Ca2+ ions
is required to couple TRPM8 stimulation with increased Egr-1 and c-Fos
concentrations. On the transcriptional level, we identified Elk-1, a key
transcriptional regulator of serum response element (SRE)-driven gene
transcription, as an important transcription factor for both Egr-1 and c-
Fos expression following stimulation of TRPM8 channels with icilin.
The fact that stimulation of TRPM8 increased the transcriptional acti-
vation potential of Elk-1 supports the view that TRPM8 stimulation
regulates gene transcription via Elk-1.
c-Fos has been shown to be important for neuronal excitability and
survival. Egr-1 activity has been correlated with synaptic plasticity,
consolidation and reconsolidation of long-term memory. Finally, Elk-1
has been shown to preserve neurons against toxic insults but may also
induce depressive behaviour. Together, our data connect TRPM8 sig-
naling with the activation of three transcription factors, c-Fos, Egr-1,
and Elk-1 that play important roles in brain function.

2. Materials and methods

2.1. Cell culture and reagents

HEK293 cells expressing TRPM8 channels (HEK293-M8 cells) have

been described elsewhere [9,10,20]. The cells were incubated for 24 h
in DMEM containing 0.05% fetal bovine serum before stimulation.
Stimulation with icilin (1 μM, Santa Cruz Biotechnology, Heidelberg,
Germany, # sc-201557) was performed for 24 h in medium containing
0.05% fetal bovine serum. Cells were preincubated for 3 h with the
compound RQ-00203078 (1 μM, dissolved in DMSO, a kind gift of
Alomone’s laboratories, Jerusalem, Israel) before stimulation. Stimu-
lation with icilin was performed in the presence of RQ-00203078.

2.2. Lentiviral gene transfer

The lentiviral transfer vectors pFUW-REST-Elk-1ΔC, pFUW-GAL4-

Elk-1, and pFUW-GAL4-Sp1 have been described elsewhere [21–23].
Viral particles were produced as previously described [24,25].

2.3. Reporter assays

The lentiviral transfer vectors pFWEgr-1.2.luc, pFWEgr-1.1.luc,

pFWEgr-1SRE.luc, pFWEBS24.luc, pFWmc-Fos.luc, pmc-Fos.lucΔEts,
pFWmc-Fos.lucΔCArG, and pFWUAS5Sp12.luc have been described
elsewhere [23,26–28]. Infected cells were maintained in medium con-
taining 0.05% fetal bovine serum for 24 h and then stimulated with
icilin (1 μM) for 24 h. Cell extracts were prepared using reporter lysis
buffer (Promega, Mannheim, Germany) and analyzed for luciferase
activities. Luciferase activity was normalized to the protein con-
centration.
2.4. Western blots
Nuclear extracts were prepared as described [29]. Thirty μg of nu-
clear proteins were separated by SDS-PAGE and the blots were in-
cubated with antibodies directed against Egr-1 (Cell Signaling Tech-
nology, Frankfurt am Main, Germany, # 4154), c-Fos (Cell Signaling
Technology, Frankfurt am Main, Germany, # 2250) or HDAC1 (Santa
Cruz Biotechnology, Heidelberg, Germany, # sc-81598). Im-
munoreactive bands were detected via enhanced chemiluminescence as
described [30,31].
Fig. 1. Icilin stimulation induces Egr-1 expression and activity in HEK293-M8
cells. (A) HEK293-M8 cells were cultured for 24 h in medium containing 0.05%
serum and then stimulated with icilin (1 μM) as indicated. Nuclear extracts
were prepared and subjected to Western blot analysis using an antibody di-
rected against Egr-1. The antibody directed against histone deacetylase-1
(HDAC1) was used as a loading control. (B, C) Icilin stimulation increases Egr-1
activity in HEK293-M8 cells. (B) Schematic depiction of an integrated provirus
encoding the Egr-1-responsive reporter gene EBS24.luc. The chromatin-em-
bedded transcription unit contained the luciferase reporter gene, a minimal
promoter consisting of four binding sites for Egr-1, a TATA box and an initiator
element. The U3 region of the 5’-LTR of the transfer vector was deleted. The
location of the woodchuck hepatitis virus posttranscriptional regulatory ele-
ment (WPRE) and the HIV flap element are shown. (C) HEK293-M8 cells were
infected with a recombinant lentivirus containing the Egr-1-responsive reporter
gene EBS24.luc. The cells were serum-starved for 24 h and then stimulated with
icilin (1 μM) for 24 h. Cell extracts were prepared and analyzed for luciferase
activities. Luciferase activity was normalized to the protein concentration. Data
shown are means +/− SD of six experiments performed in quadruplicate

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M. Ulrich, et al.
2.5. Statistics
The two-tailed Studentś t-test was used for the statistical analyses.
Statistical probability is expressed as ***P < 0.001; **P < 0.01, and
*P < 0.05. We considered values significant when P < 0.05.
3. Results
3.1. Treatment of HEK293-M8 cells with icilin stimulates the biosynthesis of
the transcription factor Egr-1 and activates Egr-1-controlled gene
transcription
Stimulation of HEK293-M8 cells, HEK293 cells expressing TRPM8
channels, with icilin leads to the activation of AP-1, a stimulus-re-
sponsive transcription factor [9,10]. Icilin stimulation triggers a sig-
naling cascade that includes the activation of extracellular signal-
regulated protein kinase ERK1/2 [10]. This protein kinase is known to
strongly activate the biosynthesis of the zinc finger protein Egr-1 [32].
We therefore asked whether TRPM8 activation leads to the expression
of Egr-1. HEK293-M8 cells were serum-starved for 24 h and then sti-
mulated with icilin for 24 h. Egr-1 immunoreactivity was low in the
absence of stimulation. In contrast, icilin treatment strikingly increased
the expression of Egr-1 (Fig. 1A). Stimulation of the cells with icilin
induced a transient expression of Egr-1 with a peak expression occur-
ring 1 h following stimulation.
Next, we tested whether the newly synthesized Egr-1 protein was
biologically active. We used an Egr-1-responsive luciferase reporter
gene (EBS24.luc) that was embedded into the chromatin of HEK293-M8
cells via lentiviral gene transfer (Fig. 1B). By this means we ensured
that the reporter gene had the same nucleosomal architecture as the
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Biochemical Pharmacology 177 (2020) 113936
Fig. 2. Stimulation of HEK293-M8 cells with icilin
activates transcription of Egr-1 promoter/luciferase
reporter genes. (A) Schematic representation of the
chromatin-integrated Egr-1 promoter/luciferase re-
porter genes Egr-1.2.luc and Egr-1.1.luc containing
sequences from −490 to +235 or from −239 to
+235 derived from the human Egr-1 gene. The co-
pies of the serum response element (SRE) are de-
picted. The SRE consists of the serum response factor
binding site encompassing the consensus sequence
CC[A/T]6GG (CArG box), and the ternary complex
factor binding site (Ets) with the consensus core se-
quence GGAA/T. (B) HEK293-M8 cells were infected
with one of the recombinant lentiviruses encoding an
Egr-1 promoter/luciferase reporter gene. The in-
fected cells were treated with icilin (1 μM) for 24 h as
indicated. Cell extracts were prepared and analyzed
for luciferase activities. Luciferase activity was nor-
malized to the protein concentration. Data shown are
means +/− SD of 3 experiments performed in
quadruplicate (***P < 0.001). (C) Schematic re-
presentation of the chromatin-embedded Egr-
1.SRE.luc reporter gene that contains the two prox-
imal SREs # 1 and 2 of the Egr-1 promoter upstream
of a minimal promoter. (D) HEK293-M8 cells were
infected with a recombinant lentivirus containing the
Egr-1.SRE.luc reporter gene. The infected cells were
treated with icilin (1 μM) for 24 h as indicated. Cell
extracts were prepared and analyzed for luciferase
activities. Luciferase activity was normalized to the
protein concentration. Data shown are means +/−
SD of 3 experiments performed in quadruplicate
(***P < 0.001).
other human genes. The cells were stimulated with icilin for 24 h using
a concentration of 1 μM. The results show that icilin strongly increased
transcription of the Egr-1-responsive reporter gene (Fig. 1C). Thus,
stimulation of HEK293-M8 cells with icilin resulted in the expression of
biologically active Egr-1. These experiments identified the GC-rich Egr-
1 DNA binding site as an icilin-responsive element.
3.2. Treatment of HEK293-M8 cells with icilin induces transcription of Egr-
1 promoter- controlled reporter genes
To identify the genetic element within the Egr-1 gene that functions
as an icilin-responsive element, we analyzed Egr-1 promoter-controlled
luciferase reporter genes Egr-1.2.luc and Egr-1.1.luc, as depicted in
Fig. 2A. The most important genetic elements of the Egr-1 promoter are
five copies of a serum response element (SRE), binding sites for the
serum response factor (SRF) and for ternary complex factors (TCF). The
TCF proteins contact DNA and bind to a SRF dimer. Egr-1 promoter/
luciferase reporter genes were inserted into the chromatin of HEK293-
M8 cells via lentiviral gene transfer. The reporter genes contained ei-
ther 490 nucleotides (Egr1.2.luc) or 239 nucleotides (Egr1.1.luc) of the
human Egr-1 gene 5’-upstream region together with 235 nucleotides of
the 5’-nontranslated region. Stimulation of HEK293-M8 cells with icilin
induced reporter gene transcription controlled by 490 nucleotides of
the human Egr-1 promoter (reporter gene Egr1.2.luc) (Fig. 2B, left
panels). Transcription of the reporter gene controlled by 239 nucleo-
tides of the Egr-1 regulatory region (reporter gene Egr1.1.luc) was also
induced by icilin, but to a lesser extent (Fig. 2B, right panels). These
results suggest that transcription of the reporter gene depends on the
number of SREs. To directly test the icilin-responsiveness of the SRE, we
analyzed the Egr-1.SRE.luc reporter gene that contained the two
M. Ulrich, et al. Biochemical Pharmacology 177 (2020) 113936

Fig. 3. Stimulation of TRPM8 channels

with icilin upregulates c-Fos promoter
activity and stimulates c-Fos expres-
sion. (A) HEK293-M8 cells were cul-
tured for 24 h in medium containing
0.05% serum and then stimulated with
icilin (1 μM) as indicated. Nuclear ex-
tracts were prepared and subjected to
Western blot analysis using an antibody
directed against c-Fos and an antibody
directed against HDAC1. (B) Schematic
representation of an integrated pro-
virus containing a c-Fos promoter/lu-
ciferase reporter gene (mc-Fos.luc). The
cyclic AMP response element (CRE),
the serum response element (Ets, CArG
box), the c-sis-inducible element (SIE),
and the AP-1 sites are depicted. In ad-
dition, the sequence of the SRE and
point mutantions within the promoter
mutants ΔEts and ΔCArG are depicted.
(C, D) HEK293-M8 cells were infected
with one of the recombinant lenti-
viruses containing the luciferase gene
under control of the wild-type c-Fos
promoter or under control of either the
c-Fos promoter mutant ΔEts (C) or
ΔCArG (D). The infected cells were
treated with icilin (1 μM) for 24 h. Cell
extracts were prepared and analyzed
for luciferase activities. Luciferase ac-
tivity was normalized to the protein
concentration. Data shown are means
+/− SD of 3 experiments performed
in quadruplicate (***P < 0.001).

proximal SREs # 1 and 2 of the Egr-1 promoter upstream of a minimal
promoter (Fig. 2C). HEK293-M8 cells were infected with a recombinant
lentivirus containing the Egr-1.SRE.luc reporter gene and stimulated
with icilin. The results, depicted in Fig. 2D, show that transcription of
this reporter gene was significantly induced following stimulation of
the cells with icilin. These data identified the SRE as an icilin-re-
sponsive element.
3.3. Treatment of HEK293-M8 cells with icilin stimulates c-Fos promoter
activity and expression of c-Fos
Stimulation of TRPM8 channels induces the expression of c-Fos, a
transcription factor that is often used as a marker for neuronal activity
[11–15]. While the Egr-1 gene is mainly controlled by multiple SREs,
the c-Fos gene contains only one very well characterized SRE, [33,34],
but does have binding sites for the transcription factors CREB, STAT,
and AP-1. We asked whether the expression of c-Fos is induced by icilin
in HEK293-M8 cells. Cells were serum-starved for 24 h and then sti-
mulated with icilin for 24 h. Fig. 3A shows that treatment of the
HEK293-M8 cells with icilin increased the expression of c-Fos. In-
creased c-Fos expression was detectable 2 h after stimulation. Next, we
measured the activity of the c-Fos promoter. A schematic depiction of
the integrated provirus encoding the luciferase reporter gene under the
control of the regulatory region of the c-Fos gene is shown in Fig. 3B.
The location of the SRE is depicted as well as binding sites for the
transcription factors CREB (CRE), STAT (SIE), and AP-1. Fig. 3C (left
panels) shows that stimulation of TRPM8 channels with icilin strongly
induced transcription controlled by the c-Fos promoter. To elucidate
the importance of the SRE of the c-Fos promoter for the icilin-induced
activation of c-Fos expression, we analyzed mutants of the c-Fos
promoter that contained base pair mutations of the binding sites for the
ternary complex factor (Ets), and the serum response factor (CArG box),
respectively, as depicted in Fig. 3B. The results show that mutations of
both binding sites for ternary complex factors (Fig. 3C) and SRF
(Fig. 3D) significantly impaired icilin-induced upregulation of c-Fos
promoter/luciferase gene transcription.
3.4. Pharmacological inhibition of TRPM8 channels attenuates expression
and activation of Egr-1 and c-Fos in icilin-stimulated HEK293-M8 cells
To verify that icilin functions via TRPM8 activation, we used a
pharmacological compound. The compound RQ-00203078 (Fig. 4A)
has been shown to inhibit menthol-induced calcium influx and men-
thol-induced whole-cell current via TRPM8 channels [35,36]. We re-
cently showed that treatment of HEK293-M8 cells with RQ-00203078
completely blocked icilin- and menthol-induced activation of the tran-
scription factor AP-1 [10]. HEK293-M8 cells were serum-starved for
24 h, preincubated with RQ-00203078 (1 μM) and then stimulated with
icilin in the presence of the inhibitor. Fig. 4B shows that the upregu-
lation of Egr-1 expression in icilin-treated HEK293-M8 cells was com-
pletely blocked in the presence of the TRPM8 inhibitor RQ-00203078.
Next, we measured the cellular Egr-1 activity. HEK293-M8 cells con-
taining the Egr-1-responsive reporter gene EBS24.luc were preincubated
with RQ-00203078 and then stimulated with icilin in the presence of
the inhibitor. Fig. 4C shows that incubation of the cells with RQ-
00203078 abolished the activation of Egr-1 in icilin-treated HEK293-
M8 cells. Next, we analyzed the expression of c-Fos and the activity of
the c-Fos promoter in icilin-stimulated HEK293-M8 cells, either in the
presence or absence of RQ-00203078. Fig. 4D shows that icilin-induced
expression of c-Fos was blocked when TRPM8 channels were inhibited

4
M. Ulrich, et al.
(1 μM) or solvent. Cells were stimulated with icilin for 24 h in the presence or absence of RQ-00203078. Cell extracts were prepared and analyzed for luciferase
activities. Luciferase activity was normalized to the protein concentration. Data shown are means +/− SD of 8 experiments performed in quadruplicate
(***P < 0.001, n.s., not significant).
with the compound RQ-00203078. Likewise, no activation of the c-Fos
promoter could be measured in icilin-stimulated HEK293-M8 cells in
the presence of RQ-00203078 (Fig. 4E).
3.5. Activation of Egr-1 and c-Fos expression in icilin-stimulated HEK293-
M8 cells requires in influx of Ca2+ ions into the cells
TRPM8 channels function as Ca2+ permeable cation channels. We
assessed the role of extracellular Ca2+ ions in icilin-induced activation
of Egr-1 and c-Fos. Fig. 5A shows that the icilin-induced biosynthesis of
Egr-1 was attenuated when the cells were cultured in Ca2+-free
medium. In addition, no increase in Egr-1 activity was observed in
icilin-stimulated HEK293-M8 cells under these conditions (Fig. 5B).
Western blot experiments showed that icilin stimulation only margin-
ally activated c-Fos expression in HEK293-M8 cells, cultured in Ca2+-
free medium (Fig. 5C), and no elevation of the c-Fos promoter activity
was observed (Fig. 5D). Hence, an influx of Ca2+ ions into the cells via
TRPM8 and the subsequent elevation of [Ca2+]i is essential for the
activation of Egr-1 and c-Fos expression following stimulation of
TRPM8 channels with icilin.
3.6. Suppression of ternary complex factor activity blocks the biosynthesis
and activation of Egr-1 and c-Fos in icilin-stimulated HEK293-M8 cells
The experiments involving Egr-1 promoter-controlled reporter
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Biochemical Pharmacology 177 (2020) 113936
Fig. 4. The TRPM8 inhibitor RQ-00203078
blocks the expression of Egr-1 and c-Fos
mediated by stimulation of TRPM8 channels
with icilin. (A) RQ-00203078. (B) HEK293-
M8 cells were cultured for 24 h in medium
containing 0.05% serum. Cells were pre-
incubated with RQ-00203078 for 24 h and
then stimulated with icilin (1 μM) for 1 h in
the presence of the TRPM8 blocker. Nuclear
extracts were prepared and subjected to
Western blot analysis using an antibody di-
rected against Egr-1 and an antibody di-
rected against HDAC1. (C) HEK293-M8 cells
expressing the Egr-1-responsive EBS24.luc
reporter gene were serum-starved for 24 h,
preincubated for 3 h with RQ-00203078
(1 μM), and then stimulated with icilin
(1 μM) for 24 h in the presence of the in-
hibitor. Cell extracts were prepared and
analyzed for luciferase activities. Luciferase
activity was normalized to the protein con-
centration. Data shown are means +/− SD
of 4 independent experiments performed in
quadruplicate (***P < 0.001, n.s., not sig-
nificant). (D) HEK293-M8 cells were cultured
for 24 h in medium containing 0.05% serum,
preincubated for 24 h with either RQ-
00203078 (1 μM) or solvent, and then sti-
mulated with icilin (1 μM) for 5 h in the
presence or absence of the compound RQ-
00203078. Nuclear extracts were prepared
and subjected to Western blot analysis using
an antibody directed against c-Fos and an
antibody directed against HDAC1. (E)
HEK293-M8 cells were infected with a re-
combinant lentivirus containing the c-Fos
promoter/luciferase reporter gene. The in-
fected cells were cultured for 24 h in medium
containing 0.05% serum and then pre-
incubated for 3 h with either RQ-00203078
genes showed that the SREs are important for the coupling of TRPM8
channels with the transcription of the Egr-1 gene. Likewise, an analysis
of c-Fos promoter mutants underlined the importance of the SRE of the
c-Fos promoter for the icilin-induced expression of c-Fos. An important
component of SRE-regulated gene transcription is the ternary complex
factor which couples signaling with gene transcription via phosphor-
ylation. We therefore tested the involvment of the ternary complex
factor Elk-1 in the TRPM8-induced activation of Egr-1 using the
dominant-negative mutant of Elk-1 termed REST/Elk-1ΔC. The mutant
contains the DNA binding and serum response factor (SRF) interaction
domains of Elk-1, but lacks its C-terminal activation domain, which
contains crucial phosphorylation sites. REST/Elk-1ΔC additionally
contains the N-terminal repression domain of the transcriptional re-
pressor REST, which recruits histone deacetylases to the Elk-1 regulated
transcription unit. The modular structure of Elk-1 and REST/Elk-1ΔC is
depicted in Fig. 6A. Expression of REST/Elk-1ΔC significantly reduced
the biosynthesis of Egr-1 in icilin-stimulated HEK293-M8 cells (Fig. 6B)
and completely blocked the icilin-induced increase in Egr-1 activity
(Fig. 6C). These data indicate that Elk-1 connects the transcription of
the Egr-1 gene and the activation of Egr-1 with the icilin-induced in-
tracellular signaling cascade. Moreover, icilin-induced expression of c-
Fos was impaired in HEK293-M8 cells expressing the Elk-1 mutant
REST/Elk-1ΔC and no stimulation of c-Fos promoter activity was ob-
served under these conditions. Thus, Elk-1 connects the transcription of
the c-Fos gene with the icilin-induced intracellular signaling cascade.
M. Ulrich, et al.
Fig. 5. Activation of Egr-1 and c-Fos in icilin-stimulated HEK293-M8 cells re-
quires an influx of Ca2+ ions into the cells. (A) HEK293-M8 cells were serum-
starved for 48 h in either DMEM or Ca2+-free medium. Cells were stimulated
with icilin (1 μM) for 1 h. Nuclear extracts were prepared and subjected to
Western blot analysis using an antibody directed against Egr-1 and an antibody
directed against HDAC1. (B) HEK293-M8 cells were infected with a re-
combinant lentivirus containing the Egr-1-responsive EBS24.luc promoter/lu-
ciferase reporter gene. The cells were serum-starved for 24 h in either DMEM or
Ca2+-free medium. Cells were stimulated with icilin (1 μM) for 24 h. Cell ex-
tracts were prepared and analyzed for luciferase activities. Luciferase activity
was normalized to the protein concentration. Data shown are means +/− SD
of 5 independent experiments performed in quadruplicate (***P < 0.001). (C)
HEK293-M8 cells were serum-starved for 48 h in either DMEM or Ca2+-free
medium. Cells were stimulated with icilin (1 μM) for 5 h. Nuclear extracts were
prepared and subjected to Western blot analysis using an antibody directed
against c-Fos and an antibody directed against HDAC1. (D) HEK293-M8 cells
were infected with a recombinant lentivirus containing a c-Fos promoter pro-
moter/luciferase reporter gene. The cells were serum-starved for 24 h in either
6
Biochemical Pharmacology 177 (2020) 113936
DMEM or Ca2+-free medium. Cells were stimulated with icilin (1 μM) for 24 h.
Cell extracts were prepared and analyzed for luciferase activities. Luciferase
activity was normalized to the protein concentration. Data shown are means
+/− SD of 6 independent experiments performed in quadruplicate
(***P < 0.001).
3.7. Stimulation of TRPM8 channels with icilin increases the transcriptional
activation potential of Elk-1
To confirm the association of Elk-1 with the icilin/TRPM8-induced
signaling cascade, we analyzed whether TRPM8 stimulation leads di-
rectly to an activation of Elk-1. As a sensor for the biological activity of
Elk-1, we used a GAL4-Elk-1 fusion protein consisting of the DNA
binding domain of GAL4 and the phosphorylation-regulated activation
domain of Elk-1 (Fig. 7A). As a control, we measured the activity of a
GAL4-Sp1 fusion protein, consisting of the DNA binding domain of
GAL4 and the glutamine-rich activation domain of the transcription
factor Sp1 (Fig. 7B). To detect the transcriptional activation of these
GAL4 fusion proteins we implanted a reporter gene into the chromatin
of HEK293-M8 cells that contained binding sites for GAL4 (termed UAS,
upstream activating sequence) (Fig. 7C). The results show that stimu-
lation of TRPM8 channels with icilin strongly increased the transcrip-
tional activation potential of Elk-1. The transcriptional activation po-
tential of Sp1 was slightly but significantly increased in icilin-
stimulated HEK293-M8 cells (Fig. 7D).
4. Discussion
Stimulation of TRP channels induces an intracellular signaling cas-
cade that results in changes in the gene expression pattern of the cells,
mediated by the activation of stimulus-responsive transcription factors.
These proteins stimulate transcription of delayed response genes. The
proteins encoded by these delayed response genes may be responsible
for biochemical and physiological changes following TRP channel ac-
tivation [19]. This scenario has nicely been documented for the sti-
mulus-induced activation of TRPV1 channels. In sensory neurons,
TRPV1 stimulation leads to the activation of the transcription factor
CREB that binds to the promoter region of the calcitonin gene-related
peptide gene, triggering expression of the 47 amino acid peptide CGRP
[37]. CGRP in turn promotes neurogenic inflammation. Stimulation of
the TRP channels TRPC6 and TRPM3 has also been connected with the
activation of stimulus-responsive transcription factors [19,38].
The objective of this study was to investigate the transcriptional
changes following stimulation of TRPM8 cation channels. Recently, we
showed that TRPM8 stimulation leads to the activation of the tran-
scription factor AP-1 [9,10]. In addition, increased expression of c-Fos
in neurons expressing activated TRPM8 channels has been shown. The
biosynthesis of c-Fos is induced by neuronal activity and the detection
of c-Fos expression is widely used to detect activated neurons. Ac-
cordingly, c-Fos immunoreactivity was used as a neuronal marker to
monitor activated TRPM8 channels [11–15]. In this study, we in-
vestigated how TRPM8 stimulation induces c-Fos expression. The zinc
finger transcription factor Egr-1 is also activated by neuronal activity
and other stimuli, including ligands for G protein-coupled receptors or
receptor tyrosine kinases. Recently, we showed that TRPM8 stimulation
triggers the activation of the protein kinase ERK1/2, a protein kinase
that functions as a very potent inducer of Egr-1 expression
[22,31,32,39,40]. We therefore hypothesized that Egr-1 expression may
be induced as a result of TRPM8 channel stimulation.
The data show that TRPM8 stimulation with icilin rapidly triggered
an elevation of Egr-1 and c-Fos promoter activities and Egr-1 and c-Fos
expression. Using an Egr-1-responsive reporter gene we could directly
show that Egr-1 activity is increased in cells following the stimulation
of TRPM8 channels with icilin. The importance of TRPM8 within the
signaling cascade was verified pharmacologically by using a specific
M. Ulrich, et al.
TRPM8 inhibitor. The expression of c-Fos and Egr-1 was significantly
attenuated in icilin-stimulated cells in the presence of the inhibitor.
Furthermore, our data revealed that icilin-induced activation of tran-
scription requires the influx of Ca2+ ions into the cells.
To elucidate the TRPM8-induced signaling cascade leading to c-Fos
and Egr-1 expression, we used promoter mutagenesis and genetic tools.
The results show that the SREs within the Egr-1 and c-Fos promoters are
crucial to connect TRPM8 stimulation with Egr-1 and c-Fos expression.
The SRE is composed of the CArG box, the binding site for a dimer of
the serum response factor, and the Ets site, the binding site for ternary
complex factors (TCFs) such as Elk-1. TCFs not only contact DNA but
also bind to a dimer of the serum response factor (SRF) with its B do-
main, thus generating a ternary complex. Elk-1 is a major nuclear
substrate for the MAP kinases extracellular signal-regulated protein
kinase (ERK1/2), p38, and c-Jun N-terminal protein kinase (JNK)
[41,42]. Phosphorylation of Elk-1 by MAP kinases connects stimulus-
induced MAP kinase activation with transcriptional activation of serum
response elements (SRE)-responsive genes [34,43]. The importance of
Elk-1 in orchestrating gene transcription following TRPM8 stimulation
was supported by the observation that the transcriptional activation
7
Biochemical Pharmacology 177 (2020) 113936
Fig. 6. Icilin-induced stimulation of Egr-1
and c-Fos expression requires the ternary
complex factor Elk-1. (A) Modular structure
of Elk-1 and the dominant-negative mutant
REST/Elk-1ΔC. (B) HEK293-M8 cells were
infected with a lentivirus encoding either β-
galactosidase (mock) or REST/Elk-1ΔC. The
infected cells were maintained in serum-re-
duced medium for 48 h and then stimulated
with icilin (1 μM) in serum-reduced medium
for 1 h. Nuclear extracts were prepared and
subjected to Western blot analysis using an
antibody directed against Egr-1 and an an-
tibody directed against HDAC1. (C)
HEK293-M8 cells were infected with a len-
tivirus containing the Egr-1-responsive
EBS24.luc reporter gene. In addition, cells
were either mock-infected or infected with a
recombinant lentivirus encoding REST/Elk-
1ΔC. Stimulation with icilin (1 μM) was
performed for 24 h in serum-reduced
medium. Cell extracts were prepared and
analyzed for luciferase activities. Luciferase
activity was normalized to the protein con-
centration. Data shown are means +/− SD
of six independent experiments performed
in quadruplicate (***P < 0.001). (D)
HEK293-M8 cells were infected with a len-
tivirus encoding either β-galactosidase
(mock) or REST/Elk-1ΔC. The infected cells
were stimulated with icilin (1 μM) in serum-
reduced medium for 5 h. Nuclear extracts
were prepared and subjected to Western blot
analysis using an antibody directed against
c-Fos and an antibody directed against
HDAC1. (E) HEK293-M8 cells were infected
with a lentivirus containing a c-Fos pro-
moter/luciferase reporter gene. In addition,
cells were either mock-infected or infected
with a recombinant lentivirus encoding
REST/Elk-1ΔC. Stimulation with icilin
(1 μM) was performed for 24 h in serum-
reduced medium. Cell extracts were pre-
pared and analyzed for luciferase activities.
Luciferase activity was normalized to the
protein concentration. Data shown are
means +/− SD of 6 independent experi-
ments performed in quadruplicate
(***P < 0.001).
potential of Elk-1 increased as a result of TRPM8 stimulation. We
conclude that Elk-1 activation is a key step in connecting TRPM8 sti-
mulation with enhanced Egr-1 and c-Fos expression and function.
Moreover, Elk-1 has been identified as regulator of AP-1 via controlling
c-Fos expression [40,44–46], indicating that TRPM8-induced signaling
cascades converge to Elk-1, and thus connecting TRPM8 activation with
gene transcription. Elk-1 is strongly expressed in neurons and can be
viewed as a master regulator of neuronal activity-controlled genes en-
coding the transcription factors Egr-1 and c-Fos.
c-Fos is important for neuronal excitability and survival [47]. Egr-1
orchestrates changes in gene transcription in neurons responsible for
synaptic plasticity, stabilization of long-term memory and memory re-
consolidation [48–51]. Elk-1 has been proposed to support neuronal
survival, to counteract excitotoxic neuronal death in a model of striatal
damage and to show beneficial effects in in vitro and in vivo models of
Huntington’s disease [52–54]. Overexpression of Elk-1 in the brain may
produce depressive behaviour [55], suggesting that the expression level
of Elk-1 is important for its neuronal function. The fact that TRPM8
stimulation activates c-Fos, Egr-1, and Elk-1 indicates that activated
TRPM8 channels influence the biological activity of neurons.
M. Ulrich, et al.
Fig. 7. Stimulation of TRPM8 channels with icilin increases the transcriptional
activation potential of Elk-1. (A) Modular structure of Elk-1 and GAL4-Elk-1.
The DNA binding domain of Elk-1 is localized at the N-terminus. The phos-
phorylation-responsive transcriptional activation domain is localized on the C-
terminus, with the key phosphoacceptor sites S383 and S389. A truncated Elk-1
is expressed as a fusion protein together with the N-terminal DNA-binding
domain of GAL4. (B) Modular structure of Sp1 and GAL4-Sp1. The GAL4-Sp1
fusion protein contains the N-terminal activation domain of Sp1 (amino acids
88 to 620), fused to the GAL4 DNA binding domain. (C) Provirus depicting the
GAL4-responsive reporter gene UAS5Sp12.luc. (D) HEK293-M8 cells were in-
fected with a lentivirus containing the GAL4-responsive luciferase reporter
gene. In addition, cells were infected with a lentivirus encoding either the
GAL4-Elk-1 fusion protein or the GAL4-Sp1 fusion protein as indicated. The
infected cells were treated with icilin (1 μM) for 24 h. Cell extracts were pre-
pared and analyzed for luciferase activities. Luciferase activity was normalized
to the protein concentration. Data shown are means +/− SD of three experi-
ments performed in quadruplicate (***P < 0.001).
8
Biochemical Pharmacology 177 (2020) 113936
Acknowledgements
We thank Alomone’s laboratories for their kind gifts of the com-
pound RQ-00203078 and Libby Guethlein for critical reading of the
manuscript. This study was supported by the Saarland University,
Germany (LOM-T201000492).
Authorship contributions
GT conceived and coordinated the study, analyzed the data and
wrote the paper. MU performed the experiments and analyzed the data.
UW contributed indispensible reagents. All authors reviewed the re-
sults, corrected the manuscript, and approved the final version of the
manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
References
[1] Y. Liu, N. Qin, TRPM8 in health and disease: Cold sensing and beyond, Adv. Exp.
Med. Biol. 704 (2011) 185–208.
[2] Y. Yudin, T. Rohacs, Regulation of TRPM8 channel activity, Mol. Cell. Endocrinol.
353 (2012) 68–74.
[3] L. Almaraz, J.-A. Manenschijn, E. de la Peña, F. Viana, TRPM8, In: Mammalian
transient receptor potential (TRP) cation channels (Eds., B. Nilius, V. Flockerzi),
Handbook of Experiment. Pharmacol. 222 (2014) 547-579.
[4] D.D. McKemy, W.M. Neuhausser, D. Julius, Identification of a cold receptor reveals
a general role of TRP channels in thermosensation, Nature 416 (2002) 52–58.
[5] A.M. Peier, A. Moqrich, A.C. Hergarden, A.J. Reeve, D.A. Andersson, G.M. Story,
T.J. Earley, I. Dragoni, P. McIntyre, S. Bevan, A. Patapoutian, A TRP channel that
senses cold and menthol, Cell 108 (2002) 705–715.
[6] D.M. Bautista, J. Siemens, J.M. Glazer, P.R. Tsuruda, A.I. Basbaum, C.L. Stuck,
S.E. Jordt, D. Julius, The menthol receptor TRPM8 is the principal detector of en-
vironmental cold, Nature 448 (2007) 204–208.
[7] R.W. Colburn, M.L. Lubin, D.J. Stone Jr, Y. Wang, D. Lawrence, M.R. D́ Andrea,
M.R. Brandt, Y. Liu, C.M. Flores, N. Qin, Attenuated cold sensitivity in TRPM8 null
mice, Neuron 54 (2007) 379–386.
[8] A. Dhaka, A.N. Murray, J. Mathur, T.J. Earley, M.J. Petrus, A. Patapoutian, TRPM8
is required for cold sensation in mice, Neuron 54 (2007) 371–378.
[9] G. Thiel, J. Welck, U. Wissenbach, O.G. Rössler, Dihydrotestosterone activates AP-1
in prostate cancer cells, Int. J. Biochem. Cell Biol. 110 (2019) 9–20.
[10] G. Thiel, T.M. Backes, J. Welck, S. Steinhausen, A.-L. Fischer, D.S. Langfermann,
M. Ulrich, U. Wissenbach, O.G. Rössler, Pharmacological inhibition of TRPM8-in-
duced gene transcription, Biochem. Pharmacol. 170 (2019) 113678, , https://doi.
org/10.1016/j.bcp.2019.113678.
[11] W.M. Knowlton, A. Fisher, D.M. Bautista, D.D. McKemy, TRPM8, but not TRPA1, is
required for neural and behavioral responses to acute noxious cold temperatures
and cold-mimetics in vivo, Pain 150 (2010) 340–350.
[12] M.C. Almeida, T. Hew-Butler, R.N. Soriano, S. Rao, W. Wang, J. Wang, N. Tamayo,
D.L. Oliveira, T.B. Nucci, P. Aryal, A. Garami, D. Bautista, N.R. Gavva,
A.A. Romanovsky, Pharmacological blockade of the cold receptor TRPM8 attenu-
ates autonomic and behavioral cold defenses and decreases deep body temperature,
J. Neurosci. 32 (2012) 2086–2099.
[13] P. Beukema, K.L. Cecil, E. Peterson, V.R. Mann, M. Matsushita, Y. Takashima,
S. Navlakha, A.L. Barth, TrpM8-mediated somatosensation in mouse neocortex, J.
Comp. Neurol. 526 (2018) 1444–1456.
[14] K. Tamura, S. Sugita, T. Tokunaga, Y. Minegishi, N. Ota, TRPM8-mediated cuta-
neous stimulation modulates motor neuron activity during treadmill stepping in
mice, J. Physiol. Sci. 69 (2019) 931–938.
[15] P.R. Lee, J.Y. Lee, H.B. Kim, J.H. Lee, S.B. Oh, TRPM8 mediates hyperosmotic sti-
muli-induced nociception in dental afferents, J. Dental Res. 99 (2020) 107–114.
[16] G. Thiel, G. Cibelli, Regulation of life and death by the zinc finger transcription
factor Egr-1, J. Cell. Physiol. 193 (2002) 287–292.
[17] O.G. Rössler, L. Stefano, I. Bauer, G. Thiel, Stimulus-transcription coupling in the
nervous system – The zinc finger protein Egr-1, in: G. Thiel (Ed.), Transcription
factors in the nervous system – Development, brain function, and disease, Wiley-
VCH, Weinheim, Germany, 2006, pp. 379–395.
[18] G. Thiel, O.G. Rössler, Resveratrol regulates gene transcription via activation of
stimulus-responsive transcription factors, Pharmacol. Res. 117 (2017) 166–176.
[19] G. Thiel, A. Lesch, S. Rubil, T.M. Backes, O.G. Rössler, Regulation of gene tran-
scription following stimulation of transient receptor potential (TRP) channels, Int.
Rev. Cell. Mol. Biol. 335 (2018) 167–189.
[20] M. Bödding, U. Wissenbach, V. Flockerzi, Characterization of TRPM8 as a phar-
macophore receptor, Cell Calcium 42 (2007) 618–628.
[21] L. Stefano, J. Al Sarraj, O.G. Rössler, C. Vinson, G. Thiel, G. Up-regulation of tyr-
osine hydroxylase gene transcription by tetradecanoylphorbol acetate is mediated
by the transcription factors Ets-like protein-1 (Elk-1) and Egr-1, J. Neurochem. 97
M. Ulrich, et al.
(2006) 92-104.
[22] G. Thiel, O.G. Rössler, Immediate-early transcriptional response to angiotensin II in
human adrenocortical cells, Endocrinology 152 (2011) 4211–4223.
[23] M. Ekici, A. Keim, O.G. Rössler, M. Hohl, G. Thiel, Chromatin structure and ex-
pression of the AMPA receptor subunit GluR2 in human glioma cells: major reg-
ulatory of REST and Sp1, J. Cell. Biochem. 133 (2012) 528–543.
[24] A. Keim, I. Müller, G. Thiel, Efficient genetic manipulation of 1321N1 astrocytoma
cells using lentiviral gene transfer, J. Neurosci. Meth. 206 (2012) 138–142.
[25] O.G. Rössler, G. Thiel, Regulation of gene transcription following stimulation of
Gαq-coupled designer receptors, in: G. Thiel (Ed.), Designer receptors exclusively
activated by designer drugs, 108 Springer, Humana Press, New York,
Neuromethods, 2015, pp. 49–60.
[26] O.G. Rössler, I. Henß, G. Thiel, Transcriptional response to muscarinic acetylcholine
receptor stimulation: Regulation of Egr-1 biosynthesis by Elk-1, ERK, MKP-1 and
calcineurin in carbachol stimulated human neuroblastoma cells, Arch. Biochem.
Biophys. 470 (2008) 93–102.
[27] O.G. Rössler, G. Thiel, Thrombin induces Egr-1 expression in fibroblasts involving
elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and ac-
tivation of ternary complex factor, BMC Mol. Biol. 10 (2009) 40.
[28] S. Rubil, O.G. Rössler, G. Thiel, CREB AP-1, ternary complex factors and MAP ki-
nases connect transient receptor potential melastatin-3 (TRPM3) channel stimula-
tion with increased c-Fos expression, Brit. J. Pharmacol. 173 (2016) 305–318.
[29] K. Kaufmann, G. Thiel, Epidermal growth factor and thrombin induced proliferation
of immortalized human keratinocytes is coupled to the synthesis of Egr-1, a zinc
finger transcriptional regulator, J. Cell. Biochem. 85 (2002) 381–391.
[30] D. Spohn, O.G. Rössler, S.E. Philipp, M. Raubuch, S. Kitajima, D. Griesemer,
M. Hoth, G. Thiel, Thapsigargin induces expression of activating transcription factor
3 in human keratinocytes involving Ca2+ Ions and c-Jun N-terminal protein kinase,
Mol. Pharmacol. 78 (2010) 865–876.
[31] S.I. Mayer, I. Müller, S. Mannebach, T. Endo, G. Thiel, Signal transduction of
pregnenolone sulfate in insulinoma cells. Activation of Egr-1 expression involving
TRPM3, voltage-gated calcium channels, ERK, and ternary complex factors, J. Biol.
Chem. 286 (2011) 10084–10096.
[32] K. Kaufmann, K. Bach, G. Thiel, Extracellular signal-regulated protein kinases Erk1/
Erk2 stimulate expression and biological activity of the transcriptional regulator
Egr-1, Biol. Chem. 382 (2001) 1077–1081.
[33] R. Treisman, Journey to the surface of the cell: Fos regulation and the SRE, EMBO J.
14 (1995) 4905–4913.
[34] M.A. Cahill, R. Janknecht, A. Nordheim, Signal uptake by the c-fos serum response
element. In: Inducible gene expression, Vol. 2. (1995), Ed. P.A. Baeuerle,
Birkhäuser, Boston/ Basel/Berlin, pp 39-72.
[35] Y. Okamoto, T. Ohkubo, T. Ikebe, J. Yamazaki, Blockade of TRPM8 activity reduces
the invasion potential of oral squamous carcinoma cell lines, Int. J. Oncol. 40
(2012) 1431–1440.
[36] M. Ohmi, Y. Shishido, T. Inoue, K. Ando, A. Fujiuchi, A. Yamada, S. Watanabe,
K. Kawamura, Identification of a novel 2-pyridyl-benzensulfonamide derivative,
RQ-0020378, as a selective and orally active TRPM8 antagonist, Bioorg. Med.
Chem. Lett. 24 (2014) 5364–5368.
[37] M. Nakanishi, K. Hata, T. Nagayama, T. Sakurai, T. Nishisho, H. Wakabayashi,
T. Hiraga, S. Ebisu, T. Yoneda, Acid activation of Trpv1 leads to an up-regulation of
calcitonin gene-related peptide expression in dorsal root ganglion neurons via the
CamK-CREB cascade: A potential mechanism of inflammatory pain, Mol. Biol. Cell
21 (2010) 2568–2577.
[38] G. Thiel, S. Rubil, A. Lesch, L.A. Guethlein, O.G. Rössler, Transient receptor po-
tential TRPM3 channels: Pharmacology, signaling, and biological dfunctions,
Biochemical Pharmacology 177 (2020) 113936
Pharmacol. Res. 124 (2017) 92–99.
[39] O.G. Rössler, D. Glatzel, G. Thiel, Resveratrol upregulates Egr-1 expression and
activity involving extracellular signal-regulated protein kinase and ternary complex
factors, Exp. Cell Res. 332 (2015) 116–127.
[40] G. Thiel, A. Lesch, A. Keim, Transcriptional response to calcium-sensing receptor
stimulation, Endocrinology 153 (2012) 4716–4728.
[41] A.J. Whitmarsh, S.H. Yang, M.S.S. Su, A.D. Sharrocks, R.J. Davis, Role of p38 and
JNK mitogen-activated protein kinases in the activation of ternary complex factors,
Mol. Cell. Biol. 17 (1997) 2360–2371.
[42] S.H. Yang, A.J. Whitmarsh, R.J. Davis, A.D. Sharrocks, Differential targeting of MAP
kinases to the ETS-domain transcription factor Elk-1, EMBO J. 17 (1998)
1740–1749.
[43] P.E. Shaw, J. Saxton, Ternary complex factors: prime nuclear targets for mitogen-
activated protein kinases, Int. J. Biochem. Cell Biol. 35 (2003) 1210–1226.
[44] G. Thiel, O.G. Rössler, Resveratrol stimulates AP-1-regulated gene transcription,
Mol. Nutr. Food Res. 58 (2014) 1402–1413.
[45] A. Lesch, X. Hui, P. Lipp, G. Thiel, Transient receptor potential melastatin-3
(TRPM3)-induced activation of AP-1 requires Ca2+ ions and the transcription fac-
tors c-Jun, ATF2, and ternary complex factor, Mol. Pharmacol. 87 (2015) 617–628.
[46] I. Müller, T. Endo, G. Thiel, Regulation of AP-1 activity in glucose-stimulated in-
sulinoma cells, J. Cell. Biochem. 110 (2010) 1481–1494.
[47] J. Zhang, D. Zhang, J.S. McQuade, M. Behbehani, J.Z. Tsien, M. Xu, c-fos regulates
neuronal excitability and survival, Nat. Genet. 30 (2002) 416–420.
[48] M.W. Jones, M.L. Errington, P.J. French, A. Fine, T.V.P. Bliss, S. Garel, P. Charnay,
B. Bozon, S. Laroche, S. Davis, A requirement of the immediate early gene Zif268 in
the expression of late LTP and long-term memories, Nature Neurosci. 4 (2001)
289–296.
[49] B. Bozon, S. Davis, S. Laroche, A requirement for the immediate early gene zif268 in
reconsolidation of recognition memory after retrieval, Neuron 40 (2003) 695–701.
[50] J.L.C. Lee, B.J. Everitt, K.L. Thomas, Independent cellular processes for hippo-
campal memory consolidation and reconsolidation, Science 304 (2004) 839–843.
[51] Z. Penke, E. Morice, A. Veyrac, A. Gros, C. Chagneau, P. LeBlanc, N. Samson,
K. Baumgärtl, I.M. Mansuy, S. Davis, S. Laroche, Zif268/Egr1 gain of function fa-
cilitates hippocampal synaptic platicity and long-term spatial recognition memory,
Phil. Trans. R. Soc. B 369 (2014) 20130159.
[52] I. Ferrer, R. Blanco, M. Carmona, Differential expression of active, phosphorylation-
dependent MAP kinases, MAPK/ERK, SAPK/JNK and p38, and specific transcription
factors substrates following quinolinic acid excitotoxicity in the rat, Brain Res. Mol.
Brain Res. 94 (2001) 48–58.
[53] M. Anglada-Huguet, A. Giralt, E. Perez-Navarro, J. Alberch, X. Xifró, Activation of
Elk-1 participates as a neuroprotective compensatory mechanism in models of
Huntingtońs disease, J. Neurochem. 121 (2012) 639–648.
[54] F. Yildirim, C.W. Ng, V. Kappes, T. Ehrenberger, S.K. Rigby, V. Stivanello,
T.A. Gipson, A.R. Soltis, P. Vanhoutte, J. Caboche, D.E. Housman, E. Fraenkel, Early
epigenomic and transcriptional changes reveal Elk-1 transcription factor as a
therapeutic target in Huntingtońs disease, Proc. Nat. Acad. Sci. USA 116 (2019)
24840–24851.
[55] K. Apazoglou, S. Farley, V. Gorgievski, R. Belzeaux, J.P. Lopez, J. Grenier,
E.C. Ibrahim, M.-A. El Khoury, Y.C. Tse, R. Mongredien, A. Barbé, C.E.A. de
Macedo, W. Jaworski, A. Bochereau, A. Orrico, E. Isingrini, C. Guinaudie,
L. Mikasova, F. Louis, S. Gautron, L. Groc, C. Massaad, F. Yildirim, V. Vialou,
S. Dumas, F. Marti, N. Mechawar, E. Morice, T.P. Wong, J. Caboche, G. Turecki,
B. Giros, E.T. Tzavara, Antidepressive effects of targeting ELK-1 signal transduction,
Nature Med. 24 (2018) 591–597.