ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 6, pp. 910-922 © Pleiades Publishing, Ltd., 2026.
910
Role of AP-1 Proteins in Glucocorticoid
Regulation of Tyrosine Hydroxylase Expression
during Early Ontogeny
Tatyana S. Kalinina
1,2,a
*, Ekaterina V. Sukhareva
1
, Veta V. Bulygina
1
,
Dmitriy A. Lanshakov
1,2
, and Nikolay N. Dygalo
1#
1
Federal Research Center Institute of Cytology and Genetics,
Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia
2
Novosibirsk State University, 630090 Novosibirsk, Russia
a
e-mail: kalin@bionet.nsc.ru
Received January 15, 2026
Revised May 29, 2026
Accepted June 4, 2026
Abstract— Elevated glucocorticoid levels during the sensitive period of early ontogenesis cause long-term
changes in the functioning of neurotransmitter systems and the functions they regulate. One such system
is the noradrenergic system, whose activity depends on the effects of stress or hormonal therapy during
perinatal ontogenesis, altering stress response and psycho-emotional reactions in adult animals. Tyrosine
hydroxylase (TH), a key enzyme in norepinephrine synthesis, is induced by glucocorticoids in the brain
of fetal rats but remains unaffected by hormone administration on the 8th day of life. To evaluate the
involvement of AP-1 transcriptional complex proteins in the age-dependent regulation of TH, we examined
the relationship between the expression levels of the genes and proteins of the Jun and Fos families during
and outside of hormonal induction of the enzyme gene. Hormonal induction of the TH gene and protein
expression was found to occur during the periods of elevated Jun family gene expression (JunB, c-Jun, JunD)
relative to Fos family genes (c-Fos, FosB) in the brainstem of 20-day-old fetuses and 3-day-old rat pups,
and was absent when this ratio decreased on the day 8 of life. Chromatin immunoprecipitation followed
by qPCR (ChIP-qPCR) demonstrated that, following dexamethasone administration on postnatal day 3, the
number of the JunB protein-bound AP-1 sites on the Th gene promoter was significantly higher compared
to the day 8. Consequently, the period of hormonal induction is accompanied by the relative predominance
of the Jun/Jun homodimeric complexes on the Th gene promoter, which activate transcription of the regu-
lated genes. This is in contrast to the day 8 of life, when the balance of Jun/Fos complexes shifts toward
formation of heterodimers that do not alter transcription. The established dynamics of the ratio of AP-1
complex proteins may underlie the age-dependent manifestation of glucocorticoid induction of TH expres-
sion in vivo in the perinatal brain.
DOI: 10.1134/S0006297926600067
Keywords: tyrosine hydroxylase, dexamethasone, AP-1 proteins, Jun/Fos, ChIP-qPCR, brain, perinatal ontogenesis
* To whom correspondence should be addressed.
# Deceased.
INTRODUCTION
Psycho-emotional, metabolic, and immune status
of an adult organism is largely determined by the
early developmental conditions [1]. Exposure to ex-
ternal, typically adverse, factors during the sensitive
period of ontogenesis could permanently alter func-
tioning of neurotransmitter systems and the functions
they regulate [2-9]. It is known that the elevated glu-
cocorticoid levels resulting from stress or hormonal
therapy modify activity of noradrenergic system, al-
tering the stress response and the manifestation of
anxiety and depressive-like behaviors in the adoles-
cent and adult animals [10-12].
ROLE OF AP-1 PROTEINS IN GLUCOCORTICOID REGULATION 911
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
Tyrosine hydroxylase (TH: EC 1.14.16.2), a key
enzyme in norepinephrine synthesis, is an indicator
of the activity of neurotransmitter system [13, 14].
Therefore, studying the effects of stress hormones,
glucocorticoids, on the expression of the enzyme
gene and protein during the critical period of early
development is a necessary step in understanding the
still unclear mechanism of the programming action
of hormones. Expression of the Th gene is regulated
by glucocorticoids in vitro in pheochromocytoma cell
cultures [15-18]. Despite this established fact, depen-
dence of Th gene expression on the stress hormone
levels in vivo is far from clear, and manifestation of
their inducing effects exhibits age-specific features
[19]. For example, administration of glucocorticoids
at the end of embryogenesis increases the levels of
the Th mRNA, enzyme activity, and norepinephrine
concentration in the brainstem of the 20-21-day-old
fetuses within 6-72 h but does not have a similar ef-
fect on the 8th day of life.
The classical mechanism of the glucocorticoid ac-
tion involves interaction of the hormone-activated re-
ceptors with specific glucocorticoid response elements
(GRE) in the promoters of the regulated genes [20, 21].
For many years, the attempts to detect a functionally
active GRE in the promoter of the Th gene have been
unsuccessful [11]. In addition to the canonical mech-
anism, glucocorticoids could alter gene expression
through protein-protein interactions of the receptor
proteins with other transcription factors, particularly
activator protein 1 (AP-1) [20, 22, 23]. A peculiarity
of the hormonal regulation via AP-1 is that enhance-
ment or, conversely, suppression of gene expression
depends on the specific set of proteins forming this
complex. In the case of formation of the Jun/Jun ho-
modimer, gene transcription increases, while forma-
tion of the Jun/Fos heterodimer decreases it [24-26].
The promoter of the rat Th gene contains two AP-1
binding sites: a proximal site (-205/-195bp) and a dis-
tal site (-5728/-5734 bp) [11, 17, 27]. The distal AP-1
binding site is involved in regulation of the gluco-
corticoid-mediated transcription of the Th gene in
rat [17] and human [18] pheochromocytoma cell cul-
tures. Verification of the functioning of non-canonical
mechanism of glucocorticoid action in vivo, mediated
by interaction with the AP-1 transcriptional complex
proteins during the sensitive period of perinatal de-
velopment, was the aim of our study.
MATERIALS AND METHODS
Animals. The study was conducted with Wistar
rats housed at the Shared Research Facility “Vivar-
ium of Conventional Animals of the ICiG SB RAS.”
All manipulations with animals were performed in
accordance with the Directive 2010/63/EU of the Eu-
ropean Parliament and the Council of the EU, Order
no. 199n “On Approval of Good Laboratory Practice
Rules” of the Ministry of Health of the Russian Fed-
eration, and with approval of the Bioethics Commis-
sion of the ICiG SB RAS (Protocol no. 19 dated No-
vember 26, 2013). Animals were maintained under a
controlled 14/10 h light/dark cycle, with free access
to water and food, and at a temperature of 22-24°C.
For mating, one male was placed with four females.
The day of sperm detection in the vaginal smear was
considered the first day of pregnancy, and the day of
birth was considered the first day of life. Pregnant
females were separated into individual cages on days
16-17 of pregnancy. On the second day of life, litters
were adjusted to eight males.
Administration of drugs. Synthetic glucocorti-
coid and glucocorticoid receptor agonist dexametha-
sone (0.2 mg/kg in 20 µL of saline, KRKA, Slovenia)
was administered subcutaneously on the 3rd (P3) and
8th (P8) days of life. Control groups included intact
animals and animals receiving an equivalent volume
of saline. To minimize maternal care bias, each litter
contained pups from all experimental groups. Pups
were marked with a single application of Castellani
paint on the P2 (for animals to be used on P3) and
on P8 (for animals to be used on P8). For each age
group, animals from at least six different litters were
used. After administration, pups were returned to the
maternal cage.
Basal expression levels of AP-1 complex genes
and proteins were studied in brainstem samples from
animals at three ages: embryonic day 20 (E20), P3,
and P8. Fetuses were extracted by cesarean section
after rapid decapitation of pregnant females, and 3
fetuses from 5 different mothers were analyzed.
On P3 and P8, brain samples from intact pups from
litters consisting of all experimental groups were
analyzed.
Sample collection. Animals were euthanized by
rapid decapitation. On day P3, brain tissue samples
were collected at 1, 2, 4, 6, 8, 10, and 24 h after in-
jection of dexamethasone or saline, and on day P8,
samples were collected 6 h after injection of dexa-
methasone or saline.
For analysis, the brainstem region with main
accumulation of noradrenergic neurons in the locus
coeruleus was isolated. In the E20 sample, the brain-
stem without the cerebellum, including the hindbrain
and midbrain, as well as suprahypothalamic region
of the diencephalon were isolated. The boundary
was the plane of the section from the pineal gland
to the optic chiasm. In the P3 and P8 pups, a brain-
stem fragment containing the medulla oblongata and
pons, bounded caudally by the posterior colliculi
and rostrally by the foramen magnum, without the
KALININA et al.912
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
cerebellum, was isolated according to the atlas [28].
Samples were frozen in liquid nitrogen and stored
at −80°C until RNA extraction or ChIP analysis.
Gene expression analysis. Total RNA was isolat-
ed using a one-step method with guanidine isothiocy-
anate [29]. Samples with an A260/280 ratio between
1.8 and 2.0 were used for further analysis.
cDNA was synthesized from 3 µg of total RNA
in a 20 µL reaction mixture containing 0.5 µg oli-
go(dT)15 primer, 1× reverse transcription buffer,
1 mM dNTP, and 50 U of MuLV reverse transcrip-
tase (SibEnzyme, Russia). Incubation was carried out
at 42°C for 90 min followed by heat inactivation of
the enzyme, as described previously [12, 19].
Real-time PCR for the genes Th (NCBI: Th), c-Fos
(NCBI: Fos), FosB (NCBI: Fosb), JunB (NCBI: Junb),
c-Jun (NCBI: Jun), and JunD (NCBI: Jund) was per-
formed using TaqMan® Gene Expression Assays
(Thermo Fisher Scientific, USA): Th (Rn00562500_
m1), Fos (Rn02396759_m1), Fosb (Rn00564121_m1),
Jun (Rn00572991_s1), Junb (Rn00572994_s1), Jund
(Rn00824678_s1). The Actb (Rn00667869_m1) gene
was used as a reference gene. A 20 µL reaction mix-
ture contained 1× qPCRmix-HS LowROX buffer (Evro-
gen, Russia), 1× TaqMan assays, and 2.5 µL of cDNA
(diluted 1 : 25). Amplification was performed with an
ABI VIIA 7 thermocycler (Thermo Fisher Scientific,
USA) using the following program: first cycle: 95°C
for 10 min; followed by 45 cycles: 95°C for 15 s, 60°C
for 1 min. Each sample was analyzed in three tech-
nical replicates [30]. Negative controls were includ-
ed for each PCR reaction: no template control (NTC)
and no reverse transcription control (RT-). Threshold
cycle (Ct) was determined automatically using the
instrument’s software. Amplification efficiency was
verified using the serial dilution of the total cDNA
pool; for all studied genes, efficiency was 97-102%.
Normalization was performed relative to the gene
Actb, whose expression stability in brainstem sam-
ples from animals of the studied ages was confirmed
in preliminary experiments. Average threshold cycle
(Ct) value for Actb at E20 was 19.842 ± 0.193 (28);
at P3: 19.999 ± 0.205 (28); and at P8: 19.847 ± 0.208
(28), respectively, which was not statistically differ-
ent (F
2, 81
= 0.242; p > 0.1). mRNA levels of the target
genes were calculated using the ΔΔCt method, with
the a priori assumption of negligible differences in
the pre-exponential part of the amplification pro-
cess. The ratios of AP-1 complex gene expression
were determined as the ratio of relative mRNA con-
centrations, i.e., the ratio of RQ Jun/RQ Fos, where
RQ = 2
–ΔΔCt
for each sample. The level of Th expres-
sion at each time point after dexamethasone ad-
ministration was expressed as the fold change rel-
ative to the control group (without administration),
RQ = 2
–ΔΔCt
.
Immunohistochemical determination of pro-
tein expression. Transcardial perfusion of P3 and P8
rat pups under deep hypothermic anesthesia was per-
formed with a chilled (4°C) 0.02 M phosphate buffer
(PBS, 20-30 mL), followed by 4% paraformaldehyde in
0.02 M PBS (20-30 mL). The extracted brain was post-
fixed in the same fixative for 4 h at 4°C. After three
washes with 0.02 M PBS, the samples were placed in
a 30% sucrose solution (4°C) for at least 24 h until
fully precipitated, and after that were frozen in iso-
pentane cooled to −40 to −42°C.
Coronal sections of 18 µm thickness were pre-
pared with a HM 550 cryotome (Carl Zeiss), mounted
on SuperFrost Plus slides (Menzel-Gläser), and air-
dried at room temperature for at least 1 h. Anatomical
boundaries were determined using a stereotaxic atlas
of the rat brain [31]. Sections from each animal were
collected sequentially in 5 parallel series. Accordingly,
every 6th section (with an interval of 108 µm) was
placed on each slide, ensuring uniform coverage of
the caudally-rostral extent of the locus coeruleus (LC).
Six sections were mounted on one slide. During sec-
tioning, the presence of the LC region was visually
controlled using a light microscope (objective ×5) to
confirm the correct anatomical level.
Sections were washed in PBS and incubated for
1 h at room temperature in a blocking solution: 1.5%
normal donkey serum (Jackson ImmunoResearch,
#017-000-121) in PBS containing 0.1% Triton X-100
(PBST). Primary antibodies were diluted in the same
solution and sections were incubated overnight at4°C.
The following antibodies were used: rabbit anti-c-Fos
(Cell Signaling, #2250, 1 : 100), mouse anti-JunB (Santa
Cruz, sc-8051, 1 : 100), and sheep anti-TH (Millipore,
AB1542, 1 : 100). TH staining was performed on sep-
arate slides using serial sections alternating with the
sections for c-Fos/JunB detection.
After washing with PBST, sections were incubat-
ed for 2 h at room temperature with secondary anti-
bodies. For double staining of c-Fos/JunB, a mixture
of donkey anti-rabbit Alexa 594 (Jackson ImmunoRe-
search, 711-585-152, 1 : 200) and donkey anti-mouse
Alexa 488 (Jackson ImmunoResearch, 715-546-151,
1 : 200) was used. For TH, donkey anti-sheep Alexa
488 (Jackson ImmunoResearch, 713-546-147, 1 : 200)
was used. Specificity of staining was controlled by
omitting primary antibodies on some sections (nega-
tive control). Sections were mounted in a fluorescent
microscopy medium (Mowiol) containing the interca-
lating fluorophore DAPI for nuclear staining.
Images were obtained using a LSM 780 la-
ser scanning microscope (Carl Zeiss) with a ×20/0.8
Plan-Apochromat objective at resolution of 1024×1024
pixels, using 2× frame averaging to improve image
quality. For each fluorescent label, laser and de-
tector settings were adjusted so that the signal did
ROLE OF AP-1 PROTEINS IN GLUCOCORTICOID REGULATION 913
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
notexceed the dynamic range and were kept constant
for all sections of the experiment. To avoid signal
overlap, imaging was performed in sequential scan-
ning mode: excitation of dyes was carried out with
lasers at wavelengths of 488 nm and 561 nm in sep-
arate tracks. For each fluorophore, laser parameters,
detector gain, and confocal aperture were adjusted to
avoid signal saturation, after which they were kept
constant for all samples within the experiment.
Semi-quantitative analysis of the fluorescent
signal was performed in LC neurons using the ZEN
2012 SP2 software (Carl Zeiss). For each animal, data
from at least 50 cells (4-5 fields of view with well-ex-
pressed LC regions on sections from one slide) were
averaged. In each field of view, average fluorescence
intensity in the cell body region was measured.
Background signal, measured in adjacent tissue ar-
eas without cells, was subtracted from the obtained
values [32]. Each experimental group consisted of 4-6
animals. Statistical analysis was performed on aver-
aged values for each animal (n = number of animals
in the group). Results are presented as the mean flu-
orescence intensity (in arbitrary units).
Chromatin immunoprecipitation with quan-
titative real-time PCR (ChIP-qPCR). Pooled brain-
stem samples from 3-4 P3 and 3-4 P8 rat pups were
homogenized in a phosphate-buffered saline (PBS)
supplemented with a protease inhibitor cocktail:
2 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/mL
leupeptin, pepstatin, and aprotinin. Proteins were
cross-linked to chromatin using 1% formaldehyde
for 10 min at room temperature; the cross-linking
reaction was stopped by adding glycine buffer. Cells
were lysed by pipetting in a sucrose buffer followed
by centrifugation (1100g, 3 min, 4°C). Nuclei were re-
suspended in an SDS-containing buffer and incubated
for 10 min at 4°C.
Lysates were sonicated using a Covaris S2 ultra-
sonicator (Shared Research Facility “Genomics” SB
RAS) according to the manufacturer’s protocol to ob-
tain 100-500 bp DNA fragments (fragmentation con-
trol was performed using agarose gel electrophoresis).
After centrifugation, the supernatant was transferred
to clean tubes and stored at −20°C. For one immunopre-
cipitation reaction, 10-30 µg of chromatin were used.
Magnetic beads (Novex® Dynabeads® Protein G,
Life Technologies™) were prepared according to the
manufacturer’s instructions and incubated for 1 h at
room temperature with antibodies: anti-c-Fos (rab-
bit, Cell Signaling, #2250, dilution 1 : 50), anti-JunB
(mouse, Santa Cruz, sc-8051, 1 : 50), anti-GR (rabbit,
Santa Cruz, H-300, 1 : 50), or control IgG (rabbit,
Jackson ImmunoResearch, 011-000-003, 1 : 1000).
Chromatin samples were then added to the anti-
body-magnetic bead complex and incubated over-
night at 4°C with constant mixing.
After incubation, beads were washed using a mag-
netic bead separation rack with buffer solutions con-
taining increasing NaCl concentrations (150-500 mM),
followed by a lithium salt-based buffer (0.25 M LiCl).
Elution of bound complexes was performed twice
with an elution buffer (1% SDS, 100 mM NaHCO
3
).
Reverse cross-linking was performed by adding 5 M
NaCl and incubating overnight at 65°C. Non-specific
binding was assessed using IgG samples; material loss
was controlled by an aliquot of chromatin (Input)
taken before immunoprecipitation.
All samples were treated with RNase A (10 mg/mL,
30 min, 37°C) and proteinase K (20 mg/mL, 2 h, 65°C).
DNA was extracted with phenol-chloroform mixture,
precipitated with isopropanol in the presence of 3 M
sodium acetate overnight at −20°C, and centrifuged
(14,000 rpm, 20 min, 4°C). The pellet was washed
twice with 75% ethanol, then with 96% ethanol,
dried, and dissolved in 15 µL of water. DNA concen-
tration was determined using a NanoDrop 2000 spec-
trophotometer.
For real-time PCR, 200 ng of DNA in 25 µL of a
qPCR master mix with SYBR Green I (Sintol, Russia),
2% DMSO, and primers specific to the distal AP-1
element (-5772/-5638) of the rat Th gene promoter
[17]: forward 5′-CATGATTGCTGTCACATCACC, reverse
5′-CAACCTGTGCACCAGTGAGT were used. Amplifica-
tion conditions: 95°C for 10 min; followed by 45 cy-
cles: 95°C for 15 s and 60°C for 60 s. Product speci-
ficity was verified by melting curve analysis in the
range of 60-95°C after PCR completion.
Results were normalized to the IgG control,
and enrichment was calculated based on Ct values
according to the manufacturer’s protocol (Thermo
Fisher Scientific; https://www.thermofisher.com/ru/ru/
home/life-science/epigenetics-noncoding-rna-research/
chromatin-remodeling/chromatin-immunoprecipita-
tion-chip/chip-analysis.html).
Statistical analysis. Statistical processing of data
was performed using the Statistica 12 software pack-
age (StatSoft, USA). Normality of the data distribution
of quantitative traits was checked using the Shapiro–
Wilk criterion. Homogeneity of variances was as-
sessed using Levene’s criterion.
For parametric data, the following tests were
used: Student’s t-test for independent samples for
comparing two groups; one-way ANOVA for compar-
ing multiple groups within one age (factor “hormone
administration”) with subsequent post-hoc compar-
ison using Fisher’s least significant difference (LSD)
test; two-way ANOVA for analyzing influence of the
factors “administration” (saline/dexamethasone) and
“time” (hours after injection) on Th gene expression
with intergroup comparisons using Tukey’s test. Since
independent groups of animals were used for each
time point, a model with fixed factors was applied.
KALININA et al.914
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
In the case of significant interaction of factors, an
analysis of simple effects was performed.
In the cases where the assumption of normal-
ity was violated or variances were heterogeneous,
non-parametric methods were used: Kruskal–Wallis
test for multiple comparisons; Mann–Whitney U-test
with Bonferroni correction for subsequent pairwise
comparisons.
Results are presented as box-and-whisker plots
(Tukey’s method). Boundaries of the box correspond
to the 25th and 75th percentiles (interquartile range,
IQR); the line inside is the median. The arithmetic
mean is indicated by an empty square, and individ-
ual values are indicated by black square dots. Whis-
kers are limited to 1.5×IQR. Points beyond the whis-
kers are identified as potential outliers. Analysis was
performed using OriginPro®2015. Differences were
considered statistically significant at p < 0.05.
RESULTS
mRNA levels of Jun and Fos genes in the
brainstem during early ontogenesis. Analysis of the
expression levels of the genes forming the AP-1 tran-
scriptional complex, the Jun family genes (JunB, c-Jun,
JunD), and FosB in the brainstem did not reveal signif-
icant age-related differences: JunB H(2, N=37) = 3.625,
p < 0.163; c-Jun H(2, N=38) = 4.015, p < 0.134; JunD
H(2, N=38) = 1.176, p < 0.556; FosB H(2, N=32) = 3.938,
p < 0.140 (Fig.1). Expression of the c-Fos gene signifi-
cantly increased by the 8th day of postnatal ontogen-
esis (H(2, N=37) = 18.122, p < 0.001) (Fig. 1).
Since direction of the changes in transcription
of the genes regulated through the AP-1 complex is
determined not by the absolute level of the proteins
forming it, but by their ratio to each other [24-26],
we assessed the ratio of basal expression levels of
the Jun family genes relative to the Fos genes in the
brainstem of 20-day-old fetuses and neonatal rats.
Ratio of Jun to Fos expression in the brain-
stem during early ontogenesis. Evaluation of the
ratio between the mRNA levels of Jun family genes
(JunB, c-Jun, JunD) relative to c-Fos gene revealed a
pronounced age dependence in the perinatal brain-
stem. Age influence: JunB/c-Fos H(2, N=37) = 20.945,
p < 0.001; c-Jun/c-Fos H(2, N=38) = 22.194, p < 0.001;
JunD/c-Fos H(2, N=37) = 22.666, p < 0.001. The great-
est shift toward predominance of the Jun transcripts
compared to c-Fos was observed in the brainstem
of E20 and P3, which were not statistically different
from each other in the intergroup comparison with
Bonferroni correction (adjusted p > 0.999) for any
of the presented ratios (Fig. 2a). By P8, the ratios of
JunB, c-Jun, JunD to c-Fos sharply decreased, showing
significant differences compared to both the E20 and
P3 groups (p < 0.001 for each).
Similar patterns in the ratio of mRNA levels of
the Jun family genes (JunB, c-Jun, JunD) were also
found relative to another member of the Fos fam-
ily – FosB (Fig. 2b). In both the brainstem of E20
fetuses and at P3 pups, the Jun/Fos gene expression
ratios, which did not differ statistically between these
age groups (adjusted p > 0.999), significantly exceed-
ed the basal Jun/FosB gene expression ratio observed
in the P8 group. The influence of age was also sig-
nificant: JunB/FosB H(2, N=32) = 11.104, p < 0.004;
c-Jun/FosB H(2, N=32) = 9.753, p < 0.008; JunD/FosB
H(2, N=32) = 10.693, p < 0.005.
The detected shift in the ratio of AP-1 complex
protein transcripts toward excess of Jun over Fos levels
in the brain of E20 fetuses and P3 rats may underlie
the previously established age dependence of the man-
ifestation of hormonal induction of the Th gene [19].
Fig. 1. mRNA levels of Jun family genes (JunB, c-Jun, JunD) and Fos family genes (c-Fos, FosB) in the brainstem of rat fe-
tuses (E20) and pups (P3, P8). * p < 0.05 compared to the animals of other age groups for each gene. Statistical analysis was
performed using the non-parametric Kruskal–Wallis test with subsequent pairwise comparisons using the Mann–Whitney
U-test with Bonferroni correction. Each age group consisted of 12-15 animals.
ROLE OF AP-1 PROTEINS IN GLUCOCORTICOID REGULATION 915
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
Fig. 2. Ratio of mRNA levels of the Jun family genes (JunB, c-Jun, JunD) to c-Fos (a) and to FosB mRNA level (b) in the
brainstem of rat fetuses (E20) and neonatal rats (P3, P8). * p < 0.05 compared to the animals of other age groups for each
ratio. Statistical analysis was performed using the non-parametric Kruskal–Wallis test with subsequent pairwise comparisons
using the Mann–Whitney U-test with Bonferroni correction. Each age group consisted of 12-15 animals.
Fig. 3. Expression of JunB and c-Fos proteins in the locus coeruleus of the brainstem in P3 and P8 rats. a) Representa-
tive microphotographs of immunohistochemical staining: JunB – green (Alexa Fluor 488), c-Fos – red (Alexa Fluor 594).
Scale bar: 50 µm. Arrows indicate nuclear localization of target proteins. b) Ratio of JunB to c-Fos protein levels based on
densitometric analysis. * p < 0.05 compared to P3 pups (Student’s t-test for independent samples). Each age group: n = 4-6.
Profile of the ratio of AP-1 complex protein tran-
scripts during perinatal ontogenesis corresponded to
the ratio of expression levels of immunohistochemical-
ly determined JunB and c-Fos proteins in the locus coe-
ruleus of the brainstem in P3 and P8 rat pups (Fig. 3).
These proteins were chosen for analysis due to
the preferential involvement of the JunB and c-Fos
proteins in the glucocorticoid-mediated regulation of
Th gene expression in the pheochromocytoma cell
culture [17, 18]. The ratio of JunB to c-Fos expression
was significantly higher at P3 than at P8 during post-
natal development (t
8
= 6.1285, p < 0.001).
The identified patterns in the ratio of AP-1 com-
plex transcripts and proteins in the brainstem during
the perinatal period may determine the previously
established age-dependent regulation of the Th gene
expression [19] due to the possible activation of the
non-canonical protein-protein interaction mechanism
KALININA et al.916
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
Fig. 4. Effect of dexamethasone on expression of the tyrosine hydroxylase gene and protein in the brainstem of P3 rats.
a) mRNA level of the Th gene at 1, 2, 4, 6, 8, 10, and 24 h after administration of saline or dexamethasone (0.2 mg/kg).
The “0 h” group consists of intact animals without injections. Each time point: n ≥ 7. b) Representative microphotographs
of immunohistochemical staining of TH protein (green signal – Alexa Fluor 488 fluorophore) in the locus coeruleus of
the brainstem of intact rats and 6 h after administration of saline or dexamethasone. Scale bar: 50 µm. c) Quantitative
assessment of TH fluorescence intensity by densitometric analysis. n = 4-5 per group. Statistical processing: For panel (a),
two-way ANOVA (factors: “administration,” “time”). For panel (c), one-way ANOVA was used. Normality of distribution was
confirmed by the Shapiro–Wilk test (p > 0.05), and homogeneity of variances was confirmed by Levene’s test (p > 0.05).
Post-hoc comparisons of each dexamethasone group with the corresponding control (saline) were performed with Bonfer-
roni correction. The corrected significance threshold for 7 time points: p < 0.0071. Asterisks (*) indicate differences that
reached the corrected significance level.
of glucocorticoid-mediated regulation of the gene ex-
pression. Induction of the enzyme gene in the brain-
stem of E20 and E21 fetuses was observed precisely
during the period of Jun protein predominance iden-
tified in this study, while absence of the hormonal in-
duction of Th in the brain of P8 animals correspond-
ed to the period of minimal values of the Jun to Fos
expression ratio. Therefore, glucocorticoids effects on
Th gene expression and protein in P3 rats, a stage at
which Jun/Fos ratios at both mRNA and protein levels
resemble those found in the fetal brain, would pro-
vide a direct test of our hypothesis that AP-1 complex
proteins mediate hormonal induction of the Th gene
in the neonatal brain.
Effect of glucocorticoids on tyrosine hydrox-
ylase gene and protein expression in the brain-
stem of P3 rats. Dexamethasone administration in-
duced Th gene expression in the brainstem of P3
pups (Fig. 4a). Increase in the transcript expression
was observed 6 h after administration and persist-
ed for 24 h after exposure to the hormone (Hor-
mone administration: F
1,130
= 21.0177, p < 0.001; time:
F
7,130
= 4.5976, p < 0.001; interaction: F
7,130
= 3.1810,
p < 0.003).
Th gene induction was accompanied by a signifi-
cant increase in TH protein levels in the locus coeru-
leus of P3 rats at 6 h after dexamethasone adminis-
tration, compared to intact and saline-treated controls
(F
2,12
= 7.77; p < 0.007) (Fig. 4b, c).
Chromatin immunoprecipitation with quan-
titative real-time PCR (ChIP-qPCR). In the pooled
brainstem samples from the intact and saline-treated
neonatal animals, interaction of the glucocorticoid
receptor with the AP-1 transcriptional complex of
ROLE OF AP-1 PROTEINS IN GLUCOCORTICOID REGULATION 917
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
Fig. 5. Interaction of JunB, c-Fos, and glucocorticoid receptor (GR) proteins with the AP-1 element of the Thgene promoter
(from -5728 to -5734 bp) in the brainstem of P3 and P8 rats 6 h after dexamethasone administration (0.2 mg/kg). Dashed
line represents the signal level during immunoprecipitation with the control antibodies (IgG). * p < 0.05 compared to P3
(Student’s t-test for independent samples). Each determination was performed on a pool of samples from 4 animals in each
age group (n = 4).
the distal promoter of the Th gene using chromatin
immunoprecipitation was not detected with real-time
PCR (ChIP-qPCR) and was statistically indistinguish-
able from the IgG levels (p > 0.1). A significant sig-
nal appeared only after dexamethasone administra-
tion (Fig. 5).
At 6 h after dexamethasone administration,
age-dependent patterns of interaction between AP-1
complex protein, the glucocorticoid receptor (GR),
and the distal AP-1 element of the Th gene promoter
(-5728/-5734 bp) were revealed in pooled brainstem
samples from P3 and P8 pups (Fig. 5).
Binding of antibodies to the glucocorticoid re-
ceptor (GR) with the distal AP-1 element of the Th
gene promoter did not differ between P3 and P8
(t
6
= 0.729; p = 0.541793), same as interaction with
the c-Fos protein, which shifted toward an increase
but did not reach statistical significance (t
6
= 1.943;
p = 0.079568). However, binding of the antibodies to
JunB at the Th promoter region containing the distal
AP-1 element in P3 was significantly higher than in
P8 (t
6
= 6.563; p < 0.022441) (Fig. 5).
Thus, the period of glucocorticoid-mediated in-
duction of the Th gene corresponds to an increase
in the interaction of the JunB protein with the distal
AP-1 element of the enzyme gene promoter. This find-
ing, together with the detected predominance of Jun
family proteins over Fos in the brainstem of fetus-
es and 3-day-old rats, may serve as confirmation of
the involvement of the non-canonical protein-protein
interaction mechanism of hormonal regulation of Th
gene expression in vivo during a specific period of
perinatal ontogenesis.
DISCUSSION
In our study, we established the determining role
of AP-1 transcriptional complex proteins in the regu-
lation of the key enzyme in catecholamine synthesis,
tyrosine hydroxylase, by glucocorticoids in vivo during
critical period of perinatal ontogenesis. The identified
patterns in the ratios of transcripts and proteins in
the AP-1 complex, as well as features of their interac-
tion with the distal AP-1 element of the enzyme gene
promoter, may be a primary factor for the age-depen-
dent manifestation of hormonal induction we previ-
ously discovered [19]. Shift in the ratio of proteins
forming AP-1 toward predominance of the Jun family
proteins over Fos in the brainstem of the 20-day-old
fetuses and 3-day-old rats correlates with the ability
of glucocorticoids to induce a key enzyme of catechol-
amine synthesis at this age. Conversely, decrease in
the Jun to Fos ratio does not allow the stimulating ef-
fect of the hormone to manifest on the 8th day of life.
Existence of canonical and non-canonical mech-
anisms of glucocorticoid hormone action is current-
ly beyond doubt. While the canonical mechanism is
based on the interaction of glucocorticoid receptors
with GRE in the promoter regions of the regulated
genes, the non-canonical mechanism is based on the
interaction of hormone receptors with other tran-
scription factors, such as NF-kB, AP-1, CREB, STAT,
IRF3, GATA-3, Oct1, Ets1, etc. [20, 24, 26, 33]. The
ability to influence transcription through variants
of protein-protein composition of the transcription-
al complexes significantly expands the possibilities
of gene expression regulation by glucocorticoids.
KALININA et al.918
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
Number of the composite dexamethasone binding sites
in the PC-12 culture is about 80%, half of which are
located in the genes that do not have classical GREs
[15], and for which the question of the possibility of
glucocorticoid regulation is of particular importance.
The Th gene also belongs to the genes that do
not have a functionally active GRE site, although that
its expression is induced by glucocorticoids in pheo-
chromocytoma cell cultures [15-18]. To obtain signif-
icant amounts of cDNA for determining nucleotide
sequence of the gene, pheochromocytoma PC-12 cul-
ture treatment with dexamethasone was previously
used [34]. The sequences similar to GRE in the region
of -454 bp and -2421 bp from the transcription start
of the conservatively organized Th promoter [35] gave
false-positive results or were artifacts upon detailed
analysis [17, 34, 36]. The proven involvement of the
distal AP-1 element in the dexamethasone-mediated
regulation of Th gene expression in the rat [17] and
human [18] pheochromocytoma cultures allowed us
to resolve the existing contradiction between the in-
duction of the enzyme gene by glucocorticoids and
absence of a hormone-responsive element in the Th
promoter. The results of our study confirmed involve-
ment of protein-protein interaction in the hormonal
regulation of Th expression in vivo during the peri-
natal period.
Regulation of gene transcription by glucocorti-
coids via AP-1 proteins is a distinctive feature in the
mechanism of hormone action due to the possibility
of bidirectional changes in the expression depending
on the component composition of the proteins form-
ing the complex. In addition to being the most wide-
ly represented partners of glucocorticoid receptors
[37-39], AP-1 proteins enhance transcription of regu-
lated genes through Jun/Jun homodimers and weaken
it through Jun/Fos heterodimers interacting with AP-1
elements of gene promoters [24, 40]. This mechanism
functions not only in the genes of the immune system
during inflammation suppression [41] and osteoblast
differentiation [42] but also participates in the regu-
lation of neuronal genes [17, 18], which is confirmed
by our results. Higher ratio of genes and proteins of
the Jun family relative to Fos in the brainstem was
observed precisely during the period of manifestation
of hormonal induction of TH in the brain of fetuses
and 3-day-old rats. Moreover, as has been established
in our previous study, dexamethasone administration
shifts the ratio of Jun/Fos transcripts in the brainstem
on the 3rd day of life toward predominance of the
Jun transcripts 2 h after hormone administration [43].
It should be noted that all quantitative assess-
ments of relative mRNA abundance obtained in
this work using the ΔΔCt method are based on the
assumption that differences in the kinetics of the
pre-exponential amplification phase are negligible for
the compared genes. Although the use of validated
TaqMan systems, strict design, and high amplification
efficiency make this assumption justified, we cannot
completely exclude its influence on the measured Ct
values. Absolute quantitative assessment (dPCR) could
be the subject of further research for direct verifica-
tion of the copy number. However, the key conclu-
sion of this work concerns not so much the absolute
quantities of the individual mRNAs as relative bal-
ance (ratio) of the Jun and Fos transcripts, which is
an independent and biologically significant parame-
ter reflecting potential for dimerization in the AP-1
complex.
Induction of the Th gene and enzyme protein 6 h
after dexamethasone administration in the region of
the main accumulation of noradrenergic neuron peri-
karyons – brainstem of the 3-day-old animals – agrees
with our hypothesis about the relationship between
the high levels of Jun transcripts and proteins and
the ability of glucocorticoids to regulate TH expres-
sion, and serves as its confirmation. Preservation of
the elevated levels of gene expression for 24 h after
hormone administration was also noted when treat-
ing the pheochromocytoma cell cultures with the
hormone [17]. Furthermore, detection of glucocorti-
coid-mediated TH induction in the brain of 3-day-old
rats expands the hormone-dependent period of regu-
lation of this key neurogene, which determines neu-
rotransmitter system activity [13, 14]. This regulation
occurs not only during the known late intrauterine
development period [10] but also during early post-
natal ontogenesis.
It should be emphasized that, despite the known
cross-activation of mineralocorticoid receptors in the
in vitro experiments [44], the dose of dexamethasone
used in our study is insufficient for activation of
non-glucocorticoid receptors. However, verification of
this possibility requires additional research.
The ChIP-qPCR method showed that on the 3rd
day of life, the number of AP-1 sites bound to the
JunB protein on the Th gene promoter was signifi-
cantly higher than on the 8th day. The obtained data
are consistent with the model according to which,
during the period of hormonal induction (3rd day),
the Jun/Jun homodimeric complexes predominate on
the Th gene promoter, contributing to the transcrip-
tion activation. Thus, for the first time, direct evi-
dence was obtained of the involvement of the AP-1
complex in mediating regulation of the TH gene by
glucocorticoids in the brain in vivo. The absence of
induction on the 8th day is likely due to the signif-
icant decrease in the binding of the JunB protein to
the distal AP-1 element of the Th promoter, which
probably shifts the balance toward formation of the
Jun/Fos heterodimeric complexes that do not activate
gene transcription (Fig. 6). Overall, the obtained data
ROLE OF AP-1 PROTEINS IN GLUCOCORTICOID REGULATION 919
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
Fig. 6. Proposed scheme of glucocorticoid regulation of Th gene expression in the brainstem on the postnatal days3 and8.
indicate priority functioning of the non-canonical
mechanism of interaction of glucocorticoid receptors
with AP-1 but do not exclude contribution of the in-
direct regulatory cascades as a result of, for exam-
ple, activation of the hypothetical post-transcriptional
mechanisms [45].
Resistance of the Th expression to elevated dexa-
methasone levels on the 8th day of postnatal ontogen-
esis may be physiologically justified, as from the days
4 to 14 after birth, the rats exhibit a hyporesponsive
period characterized by the weakened response of the
hypothalamic-pituitary-adrenal system [46, 47]. Unlike
on the 8th day of life, increase in the stress hormone
levels at the end of the prenatal and beginning of the
postnatal ontogenesis causes long-term changes in the
function of the brain’s noradrenergic system [10, 48].
The basis of these changes, as our data indicate, is
hormonal induction of TH expression as a result of
activation of the protein-protein interaction between
glucocorticoid receptors and AP-1 transcriptional
complex proteins. This explains age-dependence of
the hormonal effect and supplements the variety of
the levels of neurogene expression regulation by the
environmental factors. The glucocorticoid-induced
changes in the Th expression may represent one of
the mechanisms linking the stress-induced activation
of the HPA axis with metabolic adaptations. This is
especially relevant in the context of early develop-
ment, when stable patterns of metabolic response to
stress are formed, predetermining the risk of devel-
oping metabolic disorders in adulthood [3, 49].
CONCLUSION
Based on the obtained data, we can conclude that:
1. Age-dependent induction of the tyrosine hydrox-
ylase (TH) gene and protein by glucocorticoids
during perinatal ontogenesis is manifested ex-
clusively during the periods of elevated expres-
sion levels of the Jun family genes and proteins
relative to Fos – in the brainstem of 20-day-old
fetuses and 3-day-old rats – and is not manifest-
ed during the periods of decreased ratio of basal
expression of AP-1 transcriptional complex genes
and proteins – on the 8th day of life.
KALININA et al.920
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
2. The molecular basis of age-dependent regulation
of TH may lie in changes in composition of the
AP-1 transcriptional complexes on the enzyme
gene promoter. The period of hormonal induc-
tion on the 3rd day of life is characterized by
the increase in the number of AP-1 sites bound
to the JunB protein, which may enhance the for-
mation of the Jun/Jun homodimeric complexes
that activate transcription. The obtained data are
consistent with the model according to which,
during the period of activation of the Th gene
transcription by dexamethasone, it is mediated
by the Jun/Jun homodimeric complexes.
Abbreviations
AP-1 activator protein 1
ChIP-qPCR chromatin immunoprecipitation
followed by quantitative PCR
GR glucocorticoid receptor
GRE glucocorticoid response element
TH tyrosine hydroxylase
Th tyrosine hydroxylase gene
Acknowledgments
The authors express their gratitude to the Shared Re-
search Facility for Microscopic Analysis of Biological
Objects of the ICiG SB RAS.
Contributions
N. N. Dygalo– concept; T. S. Kalinina, E. V. Sukhareva,
V. V. Bulygina, and D. A. Lanshakov – conducting ex-
periments and statistical processing of the results;
N. N. Dygalo, T. S. Kalinina, and E. V. Sukhareva –
discussion of the results; V. V. Bulygina – manuscript
correction; T. S. Kalinina – writing the text.
Funding
The work was supported by the federal budget within
the framework of the state assignment of the Feder-
al Research Center Institute of Cytology and Genetics,
SB RAS (No. FWNR-2026-0028).
Ethics approval and consent to participate
All experimental procedures were performed in ac-
cordance with international guidelines for the care
and use of animals and were approved by the Bioeth-
ics Commission of the Institute, Protocol no. 19 dated
November26, 2013. The number of animals was min-
imized to obtain statistically significant results. This
article does not contain any studies involving humans
as research subjects.
Conflict of interest
The authors of this work declare that they have no
conflicts of interest.
REFERENCES
1. Anti, N. A. O., Gheorghe, C. P., Deming, D. D., Adeoye, O. O., Zhang, L., and Mata-Greenwood, E. (2025)
Perinatal glucocorticoid sensitivity in the preterm newborn: molecular mechanisms, endogenous determi-
nants, and clinical implications, Front. Endocrinol. (Lausanne), 16, 1587891, https://doi.org/10.3389/fendo.2025.
1587891.
2. Harris, A., and Seckl, J. (2011) Glucocorticoids, prenatal stress and the programming of disease, Horm.
Behav., 59, 279-289, https://doi.org/10.1016/j.yhbeh.2010.06.007.
3. Reynolds, R. M. (2013) Programming effects of glucocorticoids, Clin. Obstet. Gynecol., 56, 602-609, https://
doi.org/10.1097/GRF.0b013e31829939f7.
4. Pallarés, M. E., and Antonelli, M. C. (2017) Prenatal stress and neurodevelopmental plasticity: relevance to psy-
chopathology, Adv. Exp. Med. Biol., 1015, 117-129, https://doi.org/10.1007/978-3-319-62817-2_7.
5. McGowan, P. O., and Matthews, S. G. (2018) Prenatal stress, glucocorticoids, and developmental programming
of the stress response, Endocrinology, 159, 69-82, https://doi.org/10.1210/en.2017-00896.
6. Krontira, A. C., Cruceanu, C., and Binder, E. B. (2020) Glucocorticoids as mediators of adverse outcomes of pre-
natal stress, Trends Neurosci., 43, 394-405, https://doi.org/10.1016/j.tins.2020.03.008.
7. Kalinina, T., Sukhareva, E., Bulygina, V., Lanshakov, D., and Dygalo, N. (2021) Neonatal dexamethasone prevents
an increase of pro-inflammatory cytokines expression after stress in the brains of adolescent and young adult
rats, Eur. Neuropsychopharmacol., 53, S303-S304, https://doi.org/10.1016/j.euroneuro.2021.10.391.
8. Rim, C., Park, H. S., You, M. J., Yang, B., Kim, H. J., Sung, S., and Kwon, M. S. (2022) Microglia involvement in
sex-dependent behaviors and schizophrenia occurrence in offspring with maternal dexamethasone exposure,
Schizophrenia (Heidelb), 8, 71, https://doi.org/10.1038/s41537-022-00280-6.
9. Briceño-Pérez, C., Briceño-Sanabria, L., Briceño-Sanabria, C., and Reyna-Villasmil, E. (2024) Early life corticoste-
roid overexposure: epigenetic and fetal origins of adult diseases, Int. J. Gynaecol. Obstet., 164, 40-46, https://
doi.org/10.1002/ijgo.14914.
10. Kalinina, T. S., and Dygalo, N. N. (2013) Development of the noradrenergic system in the rat brain af-
ter prenatal exposure to corticosterone [in Russian], Izv. Akad. Nauk Ser. Biol., 4, 447-452, https://doi.org/
10.7868/S0002332913040048.
ROLE OF AP-1 PROTEINS IN GLUCOCORTICOID REGULATION 921
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
11. Sukhareva, E. V., Kalinina, T. S., Bulygina, V. V., and Dygalo, N. N. (2016) Tyrosine hydroxylase in the brain
and its regulation by glucocorticoids [in Russian], Vavilov Zhurn. Genet. Selekt., 20, 212-219, https://doi.org/
10.18699/VJ16.156.
12. Kalinina, T., Sukhareva, E., Lanshakov, D., Bulygina, V., and Dygalo, N. (2019) TpH2 expression after neonatal
dexamethasone treatment and acute or chronic stress in adulthood, Eur. Neuropsychopharmacol., 29, S222,
https://doi.org/10.1016/j.euroneuro.2018.11.361.
13. Kvetnansky, R., Sabban, E. L., and Palkovits, M. (2009) Catecholaminergic systems in stress: structural and mo-
lecular genetic approaches, Physiol. Rev., 89, 535-606, https://doi.org/10.1152/physrev.00042.2006.
14. Tekin, I., Roskoski, R., Jr., Carkaci-Salli, N., and Vrana, K. E. (2014) Complex molecular regulation of tyrosine
hydroxylase, J. Neural. Transm. (Vienna), 121, 1451-1481, https://doi.org/10.1007/s00702-014-1238-7.
15. Polman, J. A., Welten, J. E., Bosch, D. S., de Jonge, R. T., Balog, J., van der Maarel, S. M., de Kloet, E. R., and
Datson, N. A. (2012) A genome-wide signature of glucocorticoid receptor binding in neuronal PC12 cells, BMC
Neurosci., 13, 118, https://doi.org/10.1186/1471-2202-13-118.
16. Tank, A. W., Curella, P., and Ham, L. (1986) Induction of mRNA for tyrosine hydroxylase by cyclic AMP and
glucocorticoids in a rat pheochromocytoma cell line: evidence for the regulation of tyrosine hydroxylase syn-
thesis by multiple mechanisms in cells exposed to elevated levels of both inducing agents, Mol. Pharmacol., 30,
497-503, https://doi.org/10.1016/S0026-895X(25)10392-1.
17. Rani, C. S., Elango, N., Wang, S. S., Kobayashi, K., and Strong, R. (2009) Identification of an activator protein-
1-like sequence as the glucocorticoid response element in the rat tyrosine hydroxylase gene, Mol. Pharmacol.,
75, 589-598, https://doi.org/10.1124/mol.108.051219.
18. Rani, C. S., Soto-Pina, A., Iacovitti, L., and Strong, R. (2013) Evolutionary conservation of an atypical glucocor-
ticoid-responsive element in the human tyrosine hydroxylase gene, J. Neurochem., 126, 19-28, https://doi.org/
10.1111/jnc.12294.
19. Kalinina, T. S., Shishkina, G. T., and Dygalo, N. N. (2012) Induction of tyrosine hydroxylase gene expression
by glucocorticoids in the perinatal rat brain is age-dependent, Neurochem. Res., 37, 811-818, https://doi.org/
10.1007/s11064-011-0676-y.
20. Kalinina, T. S., Sukhareva, E. V., and Dygalo, N. N. (2016) Canonical and non-canonical mechanisms of action
of glucocorticoid stress hormones, Adv. Physiol. Sci., 47, 59-69.
21. Oakley, R. H., and Cidlowski, J. A. (2013) The biology of the glucocorticoid receptor: new signaling mechanisms
in health and disease, J. Allergy Clin. Immunol., 132, 1033-1044, https://doi.org/10.1016/j.jaci.2013.09.007.
22. Teurich, S., and Angel,P. (1995) The glucocorticoid receptor synergizes with Jun homodimers to activate AP-1-reg-
ulated promoters lacking GR binding sites, Chem. Senses, 20, 251-255, https://doi.org/10.1093/chemse/20.2.251.
23. Ratman, D., Vanden Berghe, W., Dejager, L., Libert, C., Tavernier, J., Beck, I. M., and De Bosscher, K. (2013) How
glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering, Mol. Cell
Endocrinol., 380, 41-54, https://doi.org/10.1016/j.mce.2012.12.014.
24. Sapolsky, R. M., Romero, L. M., and Munck, A. U. (2000) How do glucocorticoids influence stress respons-
es? Integrating permissive, suppressive, stimulatory, and preparative actions, Endocr. Rev., 21, 55-89, https://
doi.org/10.1210/edrv.21.1.0389.
25. Cain, D. W., and Cidlowski, J. A. (2017) Immune regulation by glucocorticoids, Nat. Rev. Immunol., 17, 233-247,
https://doi.org/10.1038/nri.2017.1.
26. Kassel, O., and Herrlich, P. (2007) Crosstalk between the glucocorticoid receptor and other transcription factors:
molecular aspects, Mol. Cell. Endocrinol., 275, 13-29, https://doi.org/10.1016/j.mce.2007.07.003.
27. Lenartowski, R., and Goc, A. (2011) Epigenetic, transcriptional and posttranscriptional regulation of the tyrosine
hydroxylase gene, Int. J. Dev. Neurosci., 8, 873-883, https://doi.org/10.1016/j.ijdevneu.2011.07.006.
28. Khazipov, R., Zaynutdinova, D., Ogievetsky, E., Valeeva, G., Mitrukhina, O., Manent, J. B., and Represa, A. (2015)
Atlas of the postnatal rat brain in stereotaxic coordinates, Front Neuroanat., 9, 161, https://doi.org/10.3389/
fnana.2015.00161.
29. Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phe-
nol-chloroform extraction, Anal. Biochem., 162, 156-159, https://doi.org/10.1006/abio.1987.9999.
30. Shishkina, G. T., Kalinina, T. S., and Dygalo, N. N. (2007) Up-regulation of tryptophan hydroxylase-2 mRNA in
the rat brain by chronic fluoxetine treatment correlates with its antidepressant effect, Neuroscience, 150, 404-
412, https://doi.org/10.1016/j.neuroscience.2007.09.017.
31. Paxinos, G., and Watson, C. (2007) The Rat Brain in Stereotaxic Coordinates, 7th Edn. Academic Press, Elsevier,
https://doi.org/10.1016/B978-0-12-547620-1.50006-0.
32. Shihan, M. H., Novo, S. G., Le Marchand, S. J., Wang, Y., and Duncan, M. K. (2021) A simple method for quanti-
tating confocal fluorescent images, Biochem. Biophys. Rep, 25, 100916, https://doi.org/10.1016/j.bbrep.2021.100916.
KALININA et al.922
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026
33. Timmermans, S., Vandewalle, J., and Libert, C. (2022) Dimerization of the glucocorticoid receptor and its im-
portance in (patho)physiology: a primer, Cells, 11, 683, https://doi.org/10.3390/cells11040683.
34. Lewis, E. J., Tank, A. W., Weiner, N., and Chikaraishi, D. M. (1983) Regulation of tyrosine hydroxylase mRNA
by glucocorticoid and cyclic AMP in a rat pheochromocytoma cell line. Isolation of a cDNA clone for tyrosine
hydroxylase mRNA, J.Biol. Chem., 258, 14632-14637, https://doi.org/10.1016/S0021-9258(17)43909-3.
35. Wang, M., Fones, L., and Cave, J. W. (2018) Conserved upstream regulatory regions in mammalian tyrosine
hydroxylase, Mol. Neurobiol., 55, 7340-7351, https://doi.org/10.1007/s12035-018-0936-9.
36. Hagerty, T., Morgan, W. W., Elango, N., and Strong, R. (2001) Identification of a glucocorticoid-responsive el-
ement in the promoter region of the mouse tyrosine hydroxylase gene, J. Neurochem., 76, 825-834, https://
doi.org/10.1046/j.1471-4159.2001.00072.x.
37. Yang-Yen, H. F., Chambard, J. C., Sun, Y. L., Smeal, T., Schmidt, T. J., Drouin, J., and Karin, M. (1990) Transcrip-
tional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to
direct protein-protein interaction, Cell, 62, 1205-1215, https://doi.org/10.1016/0092-8674(90)90396-V.
38. Biddie, S. C., John, S., Sabo, P. J., Thurman, R. E., Johnson, T. A., Schiltz, R. L., Miranda, T. B., Sung, M. H.,
Trump, S., Lightman, S. L., Vinson, C., Stamatoyannopoulos, J. A., and Hager, G. L. (2011) Transcription fac-
tor AP1 potentiates chromatin accessibility and glucocorticoid receptor binding, Mol. Cell, 43, 145-155, https://
doi.org/10.1016/j.molcel.2011.06.016.
39. Karin, M., and Chang, L. (2001) AP-1-glucocorticoid receptor crosstalk taken to a higher level, J. Endocrinol.,
169, 447-451, https://doi.org/10.1677/joe.0.1690447.
40. Seo, J., Koçak, D. D., Bartelt, L. C., Williams, C. A., Barrera, A., Gersbach, C. A., and Reddy, T. E. (2021) AP-1
subunits converge promiscuously at enhancers to potentiate transcription, Genome Res., 31, 538-550, https://
doi.org/10.1101/gr.267898.120.
41. Weikum, E. R., de Vera, I. M. S., Nwachukwu, J. C., Hudson, W. H., Nettles, K. W., Kojetin, D.J., and Ortlund, E. A.
(2017) Tethering not required: the glucocorticoid receptor binds directly to activator protein-1 recognition motifs
to repress inflammatory genes, Nucleic Acids Res., 45, 8596-8608, https://doi.org/10.1093/nar/gkx509.
42. Yamauchi, M., Ogata, Y., Kim, R. H., Li, J. J., Freedman, L. P., and Sodek, J. (1996) AP-1 regulation of the rat bone
sialoprotein gene transcription is mediated through a TPA response element within a glucocorticoid response
unit in the gene promoter, Matrix Biol., 15, 119-130, https://doi.org/10.1016/s0945-053x(96)90153-5.
43. Sukhareva, E. V., Dygalo, N. N., and Kalinina, T. S. (2016) Effect of dexamethasone on the expression of im-
mediate early genes c-fos and c-jun in different regions of the neonatal brain, Mol. Biol., 50, 230-235, https://
doi.org/10.1134/S0026893316020254.
44. Wilkinson, J. M., Hayes, S., Thompson, D., Whitney, P., and Bi, K. (2008) Compound profiling using a panel of ste-
roid hormone receptor cell-based assays, J. Biomol. Screen., 13, 755-765, https://doi.org/10.1177/1087057108322155.
45. Fossom, L. H., Sterling, C. R., and Tank, A. W. (1992) Regulation of tyrosine hydroxylase gene transcription rate
and tyrosine hydroxylase mRNA stability by cyclic AMP and glucocorticoid, Mol. Pharmacol., 42, 898-908, https://
doi.org/10.1016/s0026-895x(25)09194-1.
46. Korte, S. M. (2001) Corticosteroids in relation to fear, anxiety and psychopathology, Neurosci. Biobehav. Rev., 25,
117-142, https://doi.org/10.1016/s0149-7634(01)00002-1.
47. Schmidt, M., Enthoven, L., van Woezik, J. H., Levine, S., de Kloet, E. R., and Oitzl, M. S. (2004) The dynamics
of the hypothalamic-pituitary-adrenal axis during maternal deprivation, J. Neuroendocrinol., 16, 52-57, https://
doi.org/10.1111/j.1365-2826.2004.01123.x.
48. Lanshakov, D. A., Sukhareva, E. V., Bulygina, V. V., Bannova, A. V., Shaburova, E. V., and Kalinina, T. S. (2021)
Single neonatal dexamethasone administration has long-lasting outcome on depressive-like behaviour, Bdnf, Nt-3,
P75ngfr and sorting receptors (SorCS1-3) stress reactive expression, Sci. Rep., 11, 8092, https://doi.org/10.1038/
s41598-021-87652-7.
49. Entringer, S., Buss, C., and Wadhwa, P. D. (2015) Prenatal stress, development, health and disease risk: a psy-
chobiological perspective-2015 Curt Richter Award Paper, Psychoneuroendocrinology, 62, 366-375, https://
doi.org/10.1016/j.psyneuen.2015.08.019.
Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations. AI tools may have been used in the translation or editing of this article.
