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Prostaglandin D2-Mediated DP2 and AKT Signal Regulate the Activation of Androgen Receptors in Human Dermal Papilla Cells

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Prostaglandin D2 (PGD2) and prostaglandin D2 receptor 2 (DP2) is known to be an important factor in androgenetic alopecia (AGA). However, the effect of PGD2 in human dermal papilla cells (hDPCs) is not fully understood. The function of PGD2-induced expression of the androgen receptor (AR), DP2, and AKT (protein kinase B) signal were examined by using real time-PCR (qRT-PCR), western blot analysis, immunocytochemistry (ICC), and siRNA transfection system. PGD2 stimulated AR expression and AKT signaling through DP2. PGD2 stimulated AR related factors (transforming growth factor beta 1 (TGFβ1), Creb, lymphoid enhancer binding factor 1 (LEF1), and insulin-like growth factor 1, (IGF-1)) and AKT signaling (GSK3β and Creb) on the AR expression in hDPCs. However, these factors were down-regulated by DP2 antagonist (TM30089) and AKT inhibitor (LY294002) as well as DP2 knockdown in hDPCs decreased AR expression and AKT signaling. Finally, we confirmed that PGD2 stimulates the expression of AR related target genes, and that AKT and its downstream substrates are involved in AR expression on hDPCs. Taken together, our data suggest that PGD2 promotes AR and AKT signal via DP2 in hDPCs, thus, PGD2 and DP2 signal plays a critical role in AR expression. These findings support the additional explanation for the development of AGA involving PGD2-DP2 in hDPCs.
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International Journal of
Molecular Sciences
Article
Prostaglandin D2-Mediated DP2 and AKT Signal
Regulate the Activation of Androgen Receptors in
Human Dermal Papilla Cells
Kwan Ho Jeong ID , Ji Hee Jung ID , Jung Eun Kim and Hoon Kang * ID
Department of Dermatology, St. Paul’s Hospital, College of Medicine, The Catholic University of Korea,
Seoul 02259, Korea; jaykh86@gmail.com or kwanho16@nate.com (K.H.J.); kpajjh@naver.com (J.H.J.);
mdkjeun@naver.com (J.E.K.)
*Correspondence: johnkang@catholic.ac.kr; Tel.: +82-2-958-2143; Fax: +82-2-969-8999
Received: 15 January 2018; Accepted: 9 February 2018; Published: 12 February 2018
Abstract:
Prostaglandin D2 (PGD2) and prostaglandin D2 receptor 2 (DP2) is known to be an
important factor in androgenetic alopecia (AGA). However, the effect of PGD2 in human dermal
papilla cells (hDPCs) is not fully understood. The function of PGD2-induced expression of the
androgen receptor (AR), DP2, and AKT (protein kinase B) signal were examined by using real
time-PCR (qRT-PCR), western blot analysis, immunocytochemistry (ICC), and siRNA transfection
system. PGD2 stimulated AR expression and AKT signaling through DP2. PGD2 stimulated AR
related factors (transforming growth factor beta 1 (TGF
β
1), Creb, lymphoid enhancer binding factor
1 (LEF1), and insulin-like growth factor 1, (IGF-1)) and AKT signaling (GSK3
β
and Creb) on the AR
expression in hDPCs. However, these factors were down-regulated by DP2 antagonist (TM30089)
and AKT inhibitor (LY294002) as well as DP2 knockdown in hDPCs decreased AR expression and
AKT signaling. Finally, we confirmed that PGD2 stimulates the expression of AR related target genes,
and that AKT and its downstream substrates are involved in AR expression on hDPCs. Taken together,
our data suggest that PGD2 promotes AR and AKT signal via DP2 in hDPCs, thus, PGD2 and DP2
signal plays a critical role in AR expression. These findings support the additional explanation for
the development of AGA involving PGD2-DP2 in hDPCs.
Keywords: androgen receptor; dermal papilla cell; prostaglandin D2; AKT; CRTH2/DP2
1. Introduction
Androgenetic alopecia (AGA) is the most common hair loss disorder in men. AGA is characterized
by the replacement of thick terminal hair with fine small vellus hair on the genetic predisposition
area of scalp such as frontal and vertex area [
1
]. The major pathologic changes of hair follicles in
AGA are hair cycle dynamics which shows gradually shortening of anagen phase. 5
α
-reductase plays
the key role in dermal papillar cells for the transformation of testosterone (T) to dihydrotestosterone
(DHT). After strong binding of DHT to androgen receptors (AR), following cascade signaling alters hair
growth and affected hair follicles are miniaturized after all [
2
,
3
]. However, the mechanisms underlying
AGA are not fully understood.
Histopathologically, inflammatory cell infiltration around the follicular bulge is commonly found
in AGA hair follicles [
4
]. Mahe et al. hypothesized that the inflammatory process in AGA is triggered
by pro-inflammatory cytokines such as MCP-1, IL-6 and IL-8 [
5
]. These inflammatory reactions have
attracted the interest of many researchers studying the underlying pathogenesis of AGA.
Cotsarelis and colleagues reported that the levels of prostaglandin D2 synthase (PTGDS) and its
catalytic product, prostaglandin D2 (PGD2), were elevated in the balding scalp compared with the
non-balding scalp of patients with AGA, as well as PGD2 inhibits mouse and human hair growth
Int. J. Mol. Sci. 2018,19, 556; doi:10.3390/ijms19020556 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2018,19, 556 2 of 12
through DP2 [
6
]. The biological effects of PGD2 are usually mediated by its two G protein-coupled
receptors (PGD2 receptor; PTGDR): prostaglandin receptor 1 (DP1) and prostaglandin receptor 2 (DP2,
also known as chemoattractant homologous receptor expressed on Th2 cells; CRTH2). The induction
of PGD2 could result from increased androgen levels, since androgens have been shown to stimulate
PTGDS [
7
]. Some evidences suggest that PGD2 promotes the onset of catagen phase and decreased
hair lengthening, leading to an increase in telogen follicles and miniaturization of the hair follicles,
and PGD2 also inhibits hair follicle regeneration involved in wound healing [
6
,
8
,
9
]. These findings have
demonstrated the effect of PGD2 on hair growth and its roles in AGA. However, our understanding
of how the PGD2 pathway functions in DPCs of AGA remains limited. Thus, we focused on the
expression of AR related genes by PGD2-DP2 at cellular level.
2. Results
2.1. Prostaglandin D2 Receptor 2 (DP2) Antagonist Regulates Dihydrotestosterone (DHT)-Induced
Prostaglandin D2 (PGD2) Pathway
To determine whether DHT affects the PGD2 pathway, human dermal papilla cells (hDPCs) were
treated with various doses of DHT for 24 h. Treatment with 100 nM DHT increased cyclooxygenase-2
(COX2), PTGDS and DP2 mRNA expression (Figure 1A–C). Moreover, the protein level of DP2 was
increased by 100 nM of DHT treatment at 3 h (1.4-fold) and 5 h (1.9-fold), respectively (Figure 1D).
In addition, the levels of PGD2 receptor were significantly upregulated upon treatment with high
concentrations of DHT, such as 100 nM and 1000 nM (Figure 1E). We next examined whether a
DP2 antagonist affects AR expression. hDPCs were pretreated with TM30089 (DP2 antagonist) at
20
µ
M for 1 h and then treated with 100 nM DHT for 5 h. Stimulation with TM30089 inhibited the
upregulation of AR and DP2 (Figure 1F,G). Additionally, immunocytochemistry data showed that
DHT-stimulated AR and DP2 expression was detected in the nucleus, and the expression of AR and
DP2 in the TM30089-treated group was weaker than that in the DHT-treated group (Figure S1A,B).
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 12
Cotsarelis and colleagues reported that the levels of prostaglandin D2 synthase (PTGDS) and its
catalytic product, prostaglandin D2 (PGD2), were elevated in the balding scalp compared with the
non-balding scalp of patients with AGA, as well as PGD2 inhibits mouse and human hair growth
through DP2 [6]. The biological effects of PGD2 are usually mediated by its two G protein-coupled
receptors (PGD2 receptor; PTGDR): prostaglandin receptor 1 (DP1) and prostaglandin receptor 2
(DP2, also known as chemoattractant homologous receptor expressed on Th2 cells; CRTH2). The
induction of PGD2 could result from increased androgen levels, since androgens have been shown
to stimulate PTGDS [7]. Some evidences suggest that PGD2 promotes the onset of catagen phase and
decreased hair lengthening, leading to an increase in telogen follicles and miniaturization of the hair
follicles, and PGD2 also inhibits hair follicle regeneration involved in wound healing [6,8,9].
These findings have demonstrated the effect of PGD2 on hair growth and its roles in AGA.
However, our understanding of how the PGD2 pathway functions in DPCs of AGA remains limited.
Thus, we focused on the expression of AR related genes by PGD2-DP2 at cellular level.
2. Results
2.1. Prostaglandin D2 Receptor 2 (DP2) Antagonist Regulates Dihydrotestosterone (DHT)-Induced
Prostaglandin D2 (PGD2) Pathway
To determine whether DHT affects the PGD2 pathway, human dermal papilla cells (hDPCs)
were treated with various doses of DHT for 24 h. Treatment with 100 nM DHT increased
cyclooxygenase-2 (COX2), PTGDS and DP2 mRNA expression (Figure 1A–C). Moreover, the
protein level of DP2 was increased by 100 nM of DHT treatment at 3 h (1.4-fold) and 5 h (1.9-fold),
respectively (Figure 1D). In addition, the levels of PGD2 receptor were significantly upregulated
upon treatment with high concentrations of DHT, such as 100 nM and 1000 nM (Figure 1E). We
next examined whether a DP2 antagonist affects AR expression. hDPCs were pretreated with
TM30089 (DP2 antagonist) at 20 μM for 1 h and then treated with 100 nM DHT for 5 h. Stimulation
with TM30089 inhibited the upregulation of AR and DP2 (Figure 1F,G). Additionally,
immunocytochemistry data showed that DHT-stimulated AR and DP2 expression was detected in
the nucleus, and the expression of AR and DP2 in the TM30089-treated group was weaker than that
in the DHT-treated group (Figure S1A,B).
Figure 1. Cont.
Int. J. Mol. Sci. 2018,19, 556 3 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 12
Figure 1. Prostaglandin D2 receptor (DP2) antagonist (TM30089) decreases dihydrotestosterone
(DHT)-induced androgen receptor (AR) and prostaglandin expression in human dermal papilla cells
(hDPCs). The mRNA expression of cyclooxygenase-2 (COX2), prostaglandin D2 synthase (PTGDS)
and DP2 was examined in hDPCs treated with DHT for 24 h. The mRNA expression of COX2 (A),
PTGDS (B) and DP2 (C) was induced by 100 nM DHT. DP2 protein expression was strongly
induced by 100 nM DHT at 5 h (D). hDPCs were cultured for 24 h with DHT, as indicated. The level
of PGD2 receptor in the supernatant was evaluated in three independent experiments (E). The
relative mRNA levels were normalized to that of GAPDH. hDPCs were pretreated with 20 μM
TM30089 for 1 h and then treated with 100 nM DHT for 5 h. The protein level of AR (F) and DP2 (G)
was measured by western blot. TM30089 decreased the DHT-induced AR and DP2 expression.
β-actin served as a loading control for protein normalization. The results are expressed as the
mean ± SD of three independent experiments: CTL; control. * p < 0.05 compared with the control (0
nM DHT). # p < 0.05 compared with the DHT 100 nM.
2.2. The Effects of PGD2 on AR Expression and hDPCs
To determine whether PGD2 directly regulates AR expression, hDPCs were stimulated with
various concentrations of PGD2 in serum-free medium for 24 h. PGD2 at 50 nM–1000 nM induced
the expression of AR. At 200 nM in particular, PGD2 treatment increased the expression of AR
(2.3-fold) mRNA at 24 h compared with 0 nM group (Figure 2A). The mRNA expression of AR was
increased at 24 h in compare with PGD2 treatment for 5 h group (Figure 2B). On the other hand, the
protein level of AR was increased at 3 h and 5 h (Figure 2C). We examined whether AR related
factors are mediated by PGD2 in hDPCs. We observed that the mRNA expression of AR related
factors (TGFβ1, Creb, LEF1, and IGF-1) was increased by PGD2 treatment (200 nM for 24 h) (Figure
2D). We next examined whether PGD2 is involved in the growth inhibition of hDPCs. hDPCs were
treated with various concentrations of PGD2 (0 nM–1000 nM) for 72 h. PGD2 treatment
dose-dependently inhibited cell viability at 72 h (Figure 2E). Furthermore, the mRNA expression of
apoptosis-related genes, including caspase-1, -3, and -9, was dose-dependently increased by PGD2
treatment for 24 h (Figure 2F). In addition, apoptosis in various concentration of PGD2 treated with
hDPCs detected by TUNEL assay. We found that the number of apoptotic cells dose-dependently
increased in the PGD2-treated groups (Figure S2A). Also, we examined the changes in protein
levels of the Bcl2 and Bax genes, which are known to regulate apoptotic cell death. The Bax/Bcl2
ratio was 3.5-fold higher in the PGD2 (1000 nM) at 24 h compared with 5 h (Figure S2B).
Figure 1.
Prostaglandin D2 receptor (DP2) antagonist (TM30089) decreases dihydrotestosterone
(DHT)-induced androgen receptor (AR) and prostaglandin expression in human dermal papilla cells
(hDPCs). The mRNA expression of cyclooxygenase-2 (COX2), prostaglandin D2 synthase (PTGDS)
and DP2 was examined in hDPCs treated with DHT for 24 h. The mRNA expression of COX2 (
A
),
PTGDS (
B
) and DP2 (
C
) was induced by 100 nM DHT. DP2 protein expression was strongly induced
by 100 nM DHT at 5 h (
D
). hDPCs were cultured for 24 h with DHT, as indicated. The level of PGD2
receptor in the supernatant was evaluated in three independent experiments (
E
). The relative mRNA
levels were normalized to that of GAPDH. hDPCs were pretreated with 20
µ
M TM30089 for 1 h and
then treated with 100 nM DHT for 5 h. The protein level of AR (
F
) and DP2 (
G
) was measured by
western blot. TM30089 decreased the DHT-induced AR and DP2 expression.
β
-actin served as a loading
control for protein normalization. The results are expressed as the mean
±
SD of three independent
experiments: CTL; control. * p< 0.05 compared with the control (0 nM DHT). # p< 0.05 compared with
the DHT 100 nM.
2.2. The Effects of PGD2 on AR Expression and hDPCs
To determine whether PGD2 directly regulates AR expression, hDPCs were stimulated with
various concentrations of PGD2 in serum-free medium for 24 h. PGD2 at 50 nM–1000 nM induced the
expression of AR. At 200 nM in particular, PGD2 treatment increased the expression of AR (2.3-fold)
mRNA at 24 h compared with 0 nM group (Figure 2A). The mRNA expression of AR was increased at
24 h in compare with PGD2 treatment for 5 h group (Figure 2B). On the other hand, the protein level of
AR was increased at 3 h and 5 h (Figure 2C). We examined whether AR related factors are mediated by
PGD2 in hDPCs. We observed that the mRNA expression of AR related factors (TGF
β
1, Creb, LEF1,
and IGF-1) was increased by PGD2 treatment (200 nM for 24 h) (Figure 2D). We next examined whether
PGD2 is involved in the growth inhibition of hDPCs. hDPCs were treated with various concentrations
of PGD2 (0 nM–1000 nM) for 72 h. PGD2 treatment dose-dependently inhibited cell viability at 72 h
(Figure 2E). Furthermore, the mRNA expression of apoptosis-related genes, including caspase-1, -3,
and -9, was dose-dependently increased by PGD2 treatment for 24 h (Figure 2F). In addition, apoptosis
in various concentration of PGD2 treated with hDPCs detected by TUNEL assay. We found that the
number of apoptotic cells dose-dependently increased in the PGD2-treated groups (Figure S2A). Also,
we examined the changes in protein levels of the Bcl2 and Bax genes, which are known to regulate
apoptotic cell death. The Bax/Bcl2 ratio was 3.5-fold higher in the PGD2 (1000 nM) at 24 h compared
with 5 h (Figure S2B).
Int. J. Mol. Sci. 2018,19, 556 4 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 12
Figure 2. PGD2 regulates the AR expression and viability of hDPCs. hDPCs were cultured in
serum-free DMEM for 24 h, and then treated with the indicated concentrations of PGD2 for 24 h (A).
For optimal condition of AR mRNA expression, hDPCs were cultured in serum-free DMEM for 24
h, and then 200 nM PGD2 treated in hDPCs for 5 and 24 h (B). hDPCs were treated with PGD2 (200
nM) for the indicated times and harvested. The AR protein level was determined using western blot
analysis (C). The mRNA expression of transforming growth factor beta 1 (TGFβ1), Creb, lymphoid
enhancer binding factor 1 (LEF1) and insulin-like growth factor 1 (IGF-1) was measured by qRT-PCR
(D). Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium
bromide (MTT) assay after incubation with different concentrations of PGD2 (0, 50, 100, 200, 500,
1000 nM) for 72 h (E). The mRNA expression of caspase-1, -3, and -9 was measured by qRT-PCR (F).
The results are expressed as the mean ± SD of three independent experiments. * p < 0.05, compared
with the control (0 nM PGD2).
2.3. PGD2-Induced AR Expression is Regulated by AKT Signalling
To investigate the association of the AKT and AR signalling pathways in PGD2-induced
hDPCs, hDPCs were treated with PGD2 for different amounts of time, up to 24 h. AKT
phsphorylation was observed at 3 and 5 h (Figure 3A). Second, hDPCs were treated with LY294002
(AKT inhibitor) at 20 μM for 1 h before AR, AKT and GSK3β (AKT/GSK3β) phosphorylation was
analysed using western blot. Stimulation with PGD2 increased AR and AKT/GSK3β
phosphorylation in hDPCs compared with control. Treatment with LY294002 along with PGD2
further decreased AKT/GSK3β phosphorylation compared with PGD2 treated group (Figure 3B).
We also examined the mRNA level of AR and AKT signal related factors LEF1, Creb, and IGF-1. All
mRNA of examined molecules related to the AR were blocked by treatment with LY294002
(Figure 3C).
Figure 2.
PGD2 regulates the AR expression and viability of hDPCs. hDPCs were cultured in serum-free
DMEM for 24 h, and then treated with the indicated concentrations of PGD2 for 24 h (
A
). For optimal
condition of AR mRNA expression, hDPCs were cultured in serum-free DMEM for 24 h, and then
200 nM PGD2 treated in hDPCs for 5 and 24 h (
B
). hDPCs were treated with PGD2 (200 nM) for the
indicated times and harvested. The AR protein level was determined using western blot analysis
(
C
). The mRNA expression of transforming growth factor beta 1 (TGF
β
1), Creb, lymphoid enhancer
binding factor 1 (LEF1) and insulin-like growth factor 1 (IGF-1) was measured by qRT-PCR (
D
).
Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide
(MTT) assay after incubation with different concentrations of PGD2 (0, 50, 100, 200, 500, 1000 nM) for
72 h (
E
). The mRNA expression of caspase-1, -3, and -9 was measured by qRT-PCR (
F
). The results are
expressed as the mean
±
SD of three independent experiments. * p< 0.05, compared with the control
(0 nM PGD2).
2.3. PGD2-Induced AR Expression is Regulated by AKT Signalling
To investigate the association of the AKT and AR signalling pathways in PGD2-induced hDPCs,
hDPCs were treated with PGD2 for different amounts of time, up to 24 h. AKT phsphorylation was
observed at 3 and 5 h (Figure 3A). Second, hDPCs were treated with LY294002 (AKT inhibitor) at 20
µ
M
for 1 h before AR, AKT and GSK3
β
(AKT/GSK3
β
) phosphorylation was analysed using western blot.
Stimulation with PGD2 increased AR and AKT/GSK3
β
phosphorylation in hDPCs compared with
control. Treatment with LY294002 along with PGD2 further decreased AKT/GSK3
β
phosphorylation
compared with PGD2 treated group (Figure 3B). We also examined the mRNA level of AR and AKT
signal related factors LEF1, Creb, and IGF-1. All mRNA of examined molecules related to the AR were
blocked by treatment with LY294002 (Figure 3C).
Int. J. Mol. Sci. 2018,19, 556 5 of 12
Figure 3.
PGD2 regulates the AKT signal. The AKT phosphorylation was determined using western
blot analysis. hDPCs were treated with PGD2 (200 nM) for the indicated times and harvested (
A
).
hDPCs were pretreated with LY294002 for 1 h, and then PGD2 (200 nM) treatment for 5 h. The protein
levels of AR, and AKT/GSK3β/Creb phosphorylation was measured using western blot analysis (B).
The mRNA expression of AR, LEF1, Creb, and IGF-1 was measured using qRT-PCR (
C
).
β
-actin served
as a loading control for protein normalization. GAPDH was used as an internal control for mRNA
normalization. The results are expressed as the mean
±
SD of three independent experiments.
*p< 0.05
,
compared with the control (0 nM PGD2), # p< 0.05 compared with PGD2.
2.4. PGD2-Induced AR Expression and AKT Signalling Are Regulated by a DP2 Antagonist
We confirmed that PGD2-DP2 affects AR expression via AKT and its involved factors (including
LEF1, Creb, and IGF-1). We hypothesized that suppression of DP2 would inactivate AR expression
by inhibiting AR-related factors and AKT signalling. Thus, we examined whether inhibition of DP2
could regulate the activity of AR and its related factors. TM30089 has been known as a highly potent
antagonist on mouse CRTH2/DP2 [
10
]. PGD2-induced AR, DP2, and COX2 mRNA expression was
reduced by TM30089 (Figure 4A–C). We also found that the mRNA expression of TGF
β
1, Creb,
LEF1, and IGF-1, which are related to the activity of AR and AKT signalling, was blocked by TM30089
(Figure 4D–G). In addition, protein levels of AR and phosphorylation of AKT/GSK3
β
was also reduced
by the TM30089. (Figure 4H). PGD2-inhibited cell viability was significantly recovered by 30% upon
treatment with TM30089 compared with the PGD2-treated group (Figure 4I). These results indicated
that AR expression and hDPC viability were regulated by PGD2 through DP2.
Int. J. Mol. Sci. 2018,19, 556 6 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 12
Figure 4. The effect of a DP2 antagonist on PGD2-induced AR related genes and AKT signalling in
hDPCs. hDPCs were pretreated with TM30089 (20 μM) or LY294002 (20 μM) for 1 h and then
stimulated with 200 nM PGD2 for 24 h. The mRNA expression of AR, DP2, COX2, IGF-1, LEF1,
Creb, and TGFβ1 was measured by qRT-PCR (AG). hDPCs in serum-free DMEM were pretreated
with TM30089 (20 μM) for 1 h and then stimulated with 200 nM PGD2 for 5 h. The protein level of
AR, and AKT/GSK3β phosphorylation was measured using western blot analysis. The histogram
shows quantitative representation of the levels of PGD2-induced phosphorylation obtained from a
densitometric analysis of three independent experiments (H). An MTT-based assay was performed
to determine the effects of PGD2 after 72 h of treatment. TM30089 (20 μM) restored the viability of
hDPCs that was inhibited by 200 nM PGD2 (I). β-actin served as a loading control for protein
normalization. GAPDH was used as an internal control for mRNA normalization. The results are
expressed as the mean ± SD of three independent experiments. TM; TM30089, * p < 0.05 compared
with the control (0 nM PGD2), # p < 0.05 compared with PGD2.
2.5. The Functions of DP2 on PGD2-Induced AR Expression
Next, to study the involvement of DP2 in PGD2-induced AR expression, hDPCs were
transfected with DP2-targeting siRNA (20 nM). Transfection with DP2 siRNA significantly knocked
down the protein level of AR, DP2, COX2 and AKT/GSK3β/Creb phosphorylation, whereas the
negative control siRNA (siNC) (20 nM) had no effect (Figure 5A). We also confirmed DP2 gene
silencing at the mRNA level. PGD2-induced the target of AR or AKT genes (including AR, COX2,
DP2, LEF1, and Creb) and cell apoptosis genes such as caspase-3 and caspase-9 were markedly
attenuated by DP2-targeting siRNA transfection (Figure 5B). These data suggest that DP2 is
important for PGD2-mediated AKT signal on AR expression in hDPCs.
Figure 4.
The effect of a DP2 antagonist on PGD2-induced AR related genes and AKT signalling
in hDPCs. hDPCs were pretreated with TM30089 (20
µ
M) or LY294002 (20
µ
M) for 1 h and then
stimulated with 200 nM PGD2 for 24 h. The mRNA expression of AR, DP2, COX2, IGF-1, LEF1,
Creb, and TGF
β
1 was measured by qRT-PCR (
A
G
). hDPCs in serum-free DMEM were pretreated
with TM30089 (20
µ
M) for 1 h and then stimulated with 200 nM PGD2 for 5 h. The protein level of
AR, and AKT/GSK3
β
phosphorylation was measured using western blot analysis. The histogram
shows quantitative representation of the levels of PGD2-induced phosphorylation obtained from a
densitometric analysis of three independent experiments (
H
). An MTT-based assay was performed to
determine the effects of PGD2 after 72 h of treatment. TM30089 (20
µ
M) restored the viability of hDPCs
that was inhibited by 200 nM PGD2 (
I
).
β
-actin served as a loading control for protein normalization.
GAPDH was used as an internal control for mRNA normalization. The results are expressed as the
mean
±
SD of three independent experiments. TM; TM30089, * p< 0.05 compared with the control
(0 nM PGD2), # p< 0.05 compared with PGD2.
2.5. The Functions of DP2 on PGD2-Induced AR Expression
Next, to study the involvement of DP2 in PGD2-induced AR expression, hDPCs were transfected
with DP2-targeting siRNA (20 nM). Transfection with DP2 siRNA significantly knocked down the
protein level of AR, DP2, COX2 and AKT/GSK3
β
/Creb phosphorylation, whereas the negative control
siRNA (siNC) (20 nM) had no effect (Figure 5A). We also confirmed DP2 gene silencing at the mRNA
level. PGD2-induced the target of AR or AKT genes (including AR, COX2, DP2, LEF1, and Creb) and
cell apoptosis genes such as caspase-3 and caspase-9 were markedly attenuated by DP2-targeting
siRNA transfection (Figure 5B). These data suggest that DP2 is important for PGD2-mediated AKT
signal on AR expression in hDPCs.
Int. J. Mol. Sci. 2018,19, 556 7 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 12
Figure 5. Knockdown of DP2 suppress AR related genes and AKT signal. After transfection with
negative control (siNC) or DP2 siRNA, and then treated with PGD2 (200 nM) for 5 h. The protein
levels of AR, DP2, COX2, and AKT/GSK3β/Creb phosphorylation was measured using western blot
analysis (A). After transfection with siNA or DP2 siRNA for 24 h, and then treated with PGD2 (200
nM) for 24 h. The mRNA expression of AR, DP2, COX2, LEF1, Creb, and caspases (-3, and -9) was
measured by qRT-PCR (B). β-actin served as a loading control for protein normalization. GAPDH
was used as an internal control for mRNA normalization. The results are expressed as the mean ±
SD of three independent experiments. * p < 0.05 compared with the siNC (siRNA negative control),
# p < 0.05 compared with PGD2.
3. Discussion
Human dermal papilla cells (hDPCs) play an important role in hair follicle formation and hair
regeneration and growth [11]. In particular, the regulation of growth and apoptosis in hDPCs has
been reported to be necessary for maintaining hair growth [12]. Some studies have suggested that
the factors secreted from hDPCs in response to DHT can induce male hair loss by affecting the
activity of various genes in hair follicles [13,14]. DHT-induced androgens stimulate the secretion of
hair growth inhibitory factors such as transforming growth factor beta 1 and 2 (TGFβ1/2) [15,16].
DHT is involved in several cellular signalling mechanisms. For example, DHT increases cell death
and inhibits the cell cycle [12]. DHT modulates hair growth, hair cycling, and hair loss in
AGA-susceptible hair follicles only [17]. Although definitive evidence has been reported for
pathological mechanisms of AGA, the function of DPCs in AGA remain unclear.
DKK-1 and TGFβ1, which are cell death factors, are produced by DHT to destroy hair follicle
cells and induce them to enter catagen stage, thereby causing hair loss [14,18]. Importantly, in
susceptible individuals, DHT is also thought to precipitate an abbreviated anagen phase, as well as
structural miniaturization in the hair follicle and associated anatomical structures.
Interestingly, DHT simulated prostaglandin D2 signalling through the expression of COX2,
PTGDS, and DP2. Although various stimuli may induce the expression of COX2 in many cells
[19,20], we used DHT, which promoted AR expression by affecting DP2 and COX2. We also
investigated the changes the activity of AR by DP2 antagonist. Our results showed that DP2
antagonist has the potential to suppress AR signal by reducing the protein expression of DP2. These
findings indicated that activation of AR is associated with DHT as well as prostaglandin pathway.
Cyclooxygenase-2 (COX2), a pro-inflammatory inducible enzyme, is a key enzyme in
prostaglandin (PG) biosynthesis that converts arachidonic acid (AA) to PGG2 and subsequently to
PGH2, which is metabolized by various PG synthases to other PGs [21]. PGs are potent biologically
active lipid mediators that are produced from AA by almost every cell type and are known to
regulate immune responses. One of them, PGD2 is involved in wound healing [8], and hair loss [6]
Figure 5.
Knockdown of DP2 suppress AR related genes and AKT signal. After transfection with
negative control (siNC) or DP2 siRNA, and then treated with PGD2 (200 nM) for 5 h. The protein levels
of AR, DP2, COX2, and AKT/GSK3
β
/Creb phosphorylation was measured using western blot analysis
(
A
). After transfection with siNA or DP2 siRNA for 24 h, and then treated with PGD2 (200 nM) for
24 h. The mRNA expression of AR, DP2, COX2, LEF1, Creb, and caspases (-3, and -9) was measured
by qRT-PCR (
B
).
β
-actin served as a loading control for protein normalization. GAPDH was used as
an internal control for mRNA normalization. The results are expressed as the mean
±
SD of three
independent experiments. * p< 0.05 compared with the siNC (siRNA negative control), # p< 0.05
compared with PGD2.
3. Discussion
Human dermal papilla cells (hDPCs) play an important role in hair follicle formation and hair
regeneration and growth [
11
]. In particular, the regulation of growth and apoptosis in hDPCs has been
reported to be necessary for maintaining hair growth [
12
]. Some studies have suggested that the factors
secreted from hDPCs in response to DHT can induce male hair loss by affecting the activity of various
genes in hair follicles [
13
,
14
]. DHT-induced androgens stimulate the secretion of hair growth inhibitory
factors such as transforming growth factor beta 1 and 2 (TGF
β
1/2) [
15
,
16
]. DHT is involved in several
cellular signalling mechanisms. For example, DHT increases cell death and inhibits the cell cycle [
12
].
DHT modulates hair growth, hair cycling, and hair loss in AGA-susceptible hair follicles only [
17
].
Although definitive evidence has been reported for pathological mechanisms of AGA, the function of
DPCs in AGA remain unclear.
DKK-1 and TGF
β
1, which are cell death factors, are produced by DHT to destroy hair follicle cells
and induce them to enter catagen stage, thereby causing hair loss [
14
,
18
]. Importantly, in susceptible
individuals, DHT is also thought to precipitate an abbreviated anagen phase, as well as structural
miniaturization in the hair follicle and associated anatomical structures.
Interestingly, DHT simulated prostaglandin D2 signalling through the expression of COX2,
PTGDS, and DP2. Although various stimuli may induce the expression of COX2 in many cells [
19
,
20
],
we used DHT, which promoted AR expression by affecting DP2 and COX2. We also investigated
the changes the activity of AR by DP2 antagonist. Our results showed that DP2 antagonist has the
potential to suppress AR signal by reducing the protein expression of DP2. These findings indicated
that activation of AR is associated with DHT as well as prostaglandin pathway.
Cyclooxygenase-2 (COX2), a pro-inflammatory inducible enzyme, is a key enzyme in
prostaglandin (PG) biosynthesis that converts arachidonic acid (AA) to PGG2 and subsequently
to PGH2, which is metabolized by various PG synthases to other PGs [
21
]. PGs are potent biologically
Int. J. Mol. Sci. 2018,19, 556 8 of 12
active lipid mediators that are produced from AA by almost every cell type and are known to regulate
immune responses. One of them, PGD2 is involved in wound healing [
8
], and hair loss [
6
] actions are
mediated through DP1 and CRTH2/DP2 [
22
]. We observed that DHT treatment enhanced the target of
PGD2 pathway (COX2, PTGDS, and DP2) in hDPCs.
Based on the above results, we performed
in vitro
analysis to investigate the effect of PGD2 on the
expression of AR in hDPCs. Firstly, we investigated the AR signal pathway involved in the effects of
PGD2 in hDPCs. We focused on the TGF
β
1 and TGF
β
2, which involved in hair growth inhibition and
apoptosis [
14
]. We also investigated the androgen specific transcription factors such as Creb, and LEF1,
which plays an essential role in the regulation of prostate cancer cells; however, these factors have
not been observed in the DPCs of hair follicles in AGA. Although IGF-1 was known as growth factor
in hair development, some study reported that IGF-1 directly stimulated the activity of the 5
α
R and
AR [
23
]. We found that PGD2 treatment enhances the expression of AR, Creb, TGF
β
1, IGF-1, and LEF1
mRNA. Although androgens did not alter the proliferation of hDPCs [
11
], our results showed that
PGD2 did affect the viability of hDPCs. In similar to previous findings about the effect of PGD2 on
cellular viability [
24
,
25
], our results showed that at certain concentration, PGD2 can inhibit cell growth.
Apoptosis is related to the activation of caspases such as caspase-3 and caspase-9. Caspase-9,
an initiator caspase, can directly cleave and activate caspase-3 [
26
]. We found that PGD2 treatment
significantly increased the expression of caspase-1, caspase-3 and caspase-9 at 24 h. TUNEL is
well known for effective method for detecting programmed cell death [
27
]. Our results showed
that treatment with 1000 nM of PGD2 caused a significant in the number of apoptosis hDPCs.
PGD2 increased the expression of Bax (pro-apoptotic gene), while it caused a decrease in the expression
of Bcl2 (anti-apoptotic gene) in hDPCs. These results indicate that cell apoptosis was induced by
treatment with high concentration of PGD2, indicating the direct involvement of the caspase pathway.
Several studies have demonstrated that AKT is involved in signal transduction
pathway downstream of a variety of inflammatory mediators, glycogen metabolism and
proliferation apoptosis [
28
]. Phosphorylated AKT targets glycogen synthase kinase 3 (GSK3),
subsequently phosphorylates GSK3
β
and GSK3
α
. Function of AKT and GSK3
β
were known as a key
regulator of AR activation [
29
]. Activated GSK3
β
promoted the production of inflammatory
molecules such as iNOS and COX2. Creb is regulated by a number of signaling kinase,
including mitogen-activated protein kinase (MAPK) and AKT [
30
]. AKT signal that leads to
induction of the AR in other cell systems [
31
]. Thus, we examined the role of AKT in PGD2-dependent
signal pathway on AR expression in hDPCs. LY294002, a specific inhibitor of AKT, did affect
the PGD2-induced upregulation of the AR, IGF-1, Creb, and LEF1 as well as phosphorylated
AKT/GSK3
β
/Creb signal. We also observed that inhibition of AKT activation blocks the increases
in expression of AR related genes induced by PGD2. Interestingly, according to previous studies,
the knockdown of AKT/GSK3
β
suppressed AR related gene expression [
32
]. These results suggest
that AKT/GSK3
β
phosphorylation is involved in AR expression. Although the role of PGD2-mediated
AKT/GSK3
β
/Creb phosphorylation is uncertain in AR expression on hDPCs, our results indicated
that PGD2-mediated AKT/GSK3β/Creb has been linked as a key factor in AR expression.
Based on the results of this study, PGD2-induced expression of AR might occur as a result of
cell growth inhibition and various genes upregulation because of the activation of DP2. The DP2
antagonist TM30089 is known to regulate the viability of various cell types [
10
]. Thus, we observed that
TM30089 has a reversal effect on the PGD2-induced decrease in cell viability. Besides, TM30089 did
affect the PGD2-induced upregulation of the AR, DP2, and COX2 level as well as AKT/GSK3
β
/Creb
phosphorylation level in hDPCs. In addition, the knockdown of DP2 (DP2 siRNA) on hDPCs did
reduce the PGD2-induced AR, AKT and caspase pathway. These data indicated that AR expression of
hDPCs by PGD2 was partly dependent on the presence of the DP2.
Taken together, we sought to determine which pathway(s) is critical for the induction of DP2 by
PGD2 in hDPCs. Activation of DP2 by PGD2 leads to the AKT signal through G protein-dependent
Int. J. Mol. Sci. 2018,19, 556 9 of 12
pathway, and more recent results show that activation of the DP2 induces apoptosis through the
intrinsic pathway [33,34].
4. Materials and Methods
4.1. Human Dermal Papilla Cell (hDPCs) Culture and Reagents
Human dermal papilla cells (hDPCs, sourced from scalp of a 57-old female) were purchased from
PromoCell (Heidelberg, Germany). hDPCs were cultured in Follicle Dermal Papilla Cell Growth Media
(PromoCell) supplemented with provided mixture reagent at 37
C in a humidified atmosphere of 5%
CO
2
. Dihydrotestosterone (DHT) from Sigma-Aldrich (St. Louis, MO, USA). LY294002 (AKT inhibitor)
were from Cell Signaling Technology (Beverly, MA, USA). TM30089 (DP2 antagonist) was from Caymen
Chemical (Ann Arbor, MI, USA). For treatment, the reagents were dissolved in 100% methanol and
DMSO to a concentration at 10 mM. Three to fourth-passage DPCs were used in each experiment.
4.2. Cell Viability Assay
hDPCs were seeded in a 24-well plate at a density of 1
×
10
4
cells/well. To test whether DP2
antagonist participate in the viability of PGD2, hDPCs were seeded in a 24-well plate at a density of
1×104
cells/well. After 24 h, the medium was replaced with serum-free medium. TM30089 (20
µ
M)
were pretreated with cells for 1 h, and then incubated with or without 200 nM of PGD2 for 72 h. With the
addition of 100
µ
L/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT,
Sigma) to each well, the cells were incubated at 37
C for 4 h. Then, the supernatant was harvested
and then treated with 400
µ
L of dimethyl sulfoxide (DMSO, Sigma). The absorbance was measured at
a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA, VersaMax Microplate,
Thermo Fisher, MA, USA) reader.
4.3. Real Time-PCR (qRT-PCR)
Total RNA from the hDPCs using the TRIzolTM reagent (Invitrogen, Carlsbad, CA, USA) and
cDNA synthesis with QuantiTect Rev. Transcription kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. The cDNA used for real time-PCR, which was carried out with SYBR
Green (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers sequences and PCR conditions
are listed in Supplementary Table S1.
4.4. Western Blotting Analysis
The protocol for western blot analysis was described in a previous report [
35
]. Briefly, the protein
lysates from cultured hDPCs were prepared in RIPA cell lysis buffer containing protease inhibitor
cocktail. The membranes were subsequently incubated with primary antibodies against total (AKT,
Creb) and phosphorylation (AKT, Creb, GSK3
β
) (Cell Signaling Technology, Danvers, MA, USA), AR,
COX2, Bax, Bcl-2 and
β
-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), CRTH2/DP2
(Novus Biologicals LLC, Littleton, CO, USA) overnight at 4 C on a rotary shaker.
4.5. Immunofluoresence of Androgen Receptor and DP2
hDPCs were plated in 8-chamber slides (SPL, Korea) at a density of 2
×
10
3
cells per well
and cultured in serum-free medium in the presence of DHT or vehicle control (methanol) for
5 h. Immunofluorescence staining of AR was performed as previously described [
36
]. Briefly,
proteins were immunolabeled by incubating with anti-AR antibody (1:100, Cell Signaling Technology),
anti-CRTH2/DP2 antibody (1:100, Novus) and anti-rabbit Alex Fluor 488 conjugated antibody (1:200;
Invitrogen, OR, USA). Slides were examined under Axiovert 200 microscope (ZEISS, Germany).
Int. J. Mol. Sci. 2018,19, 556 10 of 12
4.6. ELISA
PGD2 receptor ELISA kit (Abbexa, Cambridge, UK) was used according to the manufacturer’s
protocol. For the measurement of PGD2 receptor in conditioned medium of DHT-induced hDPCs,
cells from passages 3–4 were plated overnight at a density of 2
×
10
5
cells per 6 well culture dish,
washed three times with phosphate-buffered saline (PBS), and then incubated in serum-free medium
for 24 h for the collection of conditioned medium. To examine PGD2 receptor induction in response to
DHT in hDPCs were treated with varying concentrations of DHT in serum-free medium for 5 or 24 h
and concentrations of PGD2 receptor in conditioned medium were measured. Optical density was
measured by an ELISA reader at 450 nm.
4.7. DP2 Gene Silencing Experiments
Small interfering RNA (siRNA) targeted at DP2 (Santa Cruz Biotechnology) was used to knockout
DP2. hDPCs were cultured and incubated at 37
C in a 5% CO
2
incubator until 70–80% confluent.
Thereafter, 2
µ
L DP2 siRNA duplex was diluted into 100
µ
L of siRNA transfection medium (Santa Cruz
Biotechnology). In a separate tube, 2
µ
L of transfection reagent (Santa Cruz biotechnology) was diluted
into 100
µ
L of siRNA transfection medium. The dilutions were mixed gently and incubated for 30 min
at room temperature. Next, cells were incubated in negative control (siNC) or DP2 siRNA transfection
cocktail for 5 h at 37
C. Following transfection, media was changed in all cells to complete media and
incubated for a further 18 h. Effects of PGD2 on AR related genes and AKT signaling in normal and
DP2-silenced hDPCs were then investigated.
4.8. TUNEL Assay
hDPCs seeded in 24-well plates at a density of 2
×
10
4
cell/well were treated with PGD2 at various
concentrations (0, 200, 500, 1000 nM) or DMSO as a control for 72 h. Cell apoptosis was examined
with the in situ cell death detection kit (Roche, Mannheim, Germany) according to the manufacturer’s
instructions. The cells were fixed, permeated with 0.1% Triton X-100 solution, labelled for DNA
breaks with terminal deoxynucleotide transferase (TdT) dUTP fluorescein nick end labeling (TUNEL,
green fluorescence) and observed under Axiovert 200 microscope (ZEISS)
4.9. Statistical Analysis
All data are representative data from three independent experiments. The statistical significance
of the differences among groups was tested using one-way ANOVA (SigmaPlot 12.3 software, San Jose,
CA, USA). All graphs were generated using GraphPad Prism 5 (La Jolla, CA, USA). pvalue < 0.05 was
considered statistically significant.
5. Conclusions
PGD2 directly stimulates the expression of androgen target genes, AKT and its downstream
substrates are involved in mediating these effects. Thus, our data in this study provide that the activity
of AR could be regulated not only DHT but also various signal changes by PGD2 in hDPCs.
Supplementary Materials: The following are available online at www.mdpi.com/1422-0067/19/2/556/s1.
Acknowledgments:
This research was supported by the Ministry of Trade, Industry & Energy (MOTIE),
Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries
of Economic Cooperation Region (R0005754).
Author Contributions:
Kwan Ho Jeong and Ji Hee Jung performed the research, statistical analysed the data
and wrote the manuscript. Jung Eun Kim conducted data collection, analysed and critically reviewed the study.
Hoon Kang supervised the whole study process and wrote the manuscript. All authors contributed to this article.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2018,19, 556 11 of 12
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Supplementary resource (1)

... In addition, the kinetics of Smad2 phosphorylation by PGD 2 was relatively slower than that of TGF-β1 (Fig. 3C). In line with this, many other reports also suggest that prostanoids facilitate the expression of TGF-β in a variety of cells [41][42][43]. Secondly, it has been reported that Smad2 phosphorylation is essential to the functioning of the TGF-β receptor signaling cascade [3,44]. Our results also showed that stimulation of A549 cells with PGD 2 strongly induced the phosphorylation of Smad2 (Fig. 3C, Fig. 4A and Fig. 5C). ...
Article
Background/aims: Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. Methods: A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-β1 and TGFβ type I receptor (TGFβRI), and protein expression was assessed by immunoblotting and immunofluorescence. Results: Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-β1 expression, and silencing of TGF-β1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFβRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. Conclusion: Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.
... The other mechanism of insulin sensitization is the Akt/mTOR pathway, which is promoted by PPAR-γ agonism. Since both Akt (Jeong, Jung, Kim, & Kang, 2018) and mTOR are upregulated in balding scalp (Chew et al., 2016;Stamatas et al., 2017), then it is possible that insulin sensitization is creating a problem. Hence, therapeutic intervention with IGF-1 is not foreseen to be the best solution. ...
Article
Background The success of 5α-reductase inhibitors in the 1990s vindicated the role of androgens and cast doubt on the role of diet in androgenetic alopecia (AGA). However, poor glucose control and high cholesterol are now recognised as comorbidities, which are salient characters of the ‘western diet’. Scope and approach In glucose potentiated hair loss, continuous monosaccharide flux to the liver promotes the polyol pathway, causing fatty liver and attenuating synthesis of sex hormone binding globulin, accommodating the increased ratio of dihydrotestosterone (DHT) to testosterone. The scalp of the balding phenotype is characterised by overactive PPAR-γ receptors, increased fatty acid synthesis, enlarged sebaceous glands and sebum secretions. Sebum feeds lipophilic bacteria, such as Propionibacterium acnes, which augment the expression of prostaglandin-type (PGD2 & 15d-PGJ2) ligands of PPAR-γ and increase local insulin sensitivity via Akt/mTOR pathways. In hyperglycaemic events the androgen dependent polyol pathway depletes glucose and generates purine by-products that antagonise adenosine receptors. Mitochondrial reactive oxygen species accumulate, and ATP levels reduce, slowing gluconeogenesis in the outer root sheath keratinocytes of the hair follicle. Furthermore, the current commentary suggests that an important mineral in hair health is magnesium, which is relevant to both glucose and cholesterol potentiated hair loss. Magnesium deficiency not only reinforces insulin resistance, but in cholesterol potentiated hair loss, local magnesium dependent monooxygenase enzymes that metabolise cholesterol and vitamin D are impaired. Furthermore, magnesium deficient muscles at the occipital and temporal region of the skull create mechanical strain against the galea aponeurotica. Key findings and conclusions Taking all of this into consideration, treatment options for androgenetic alopecia should include a low cholesterol and low glycaemic index diet, improved glucose control, and fortification with magnesium. Furthermore, the current narrative does not endorse severe caloric restriction for obvious health reasons.
... In this pathway, 30 genes were expressed, within which spermine synthase (SMS) was the only gene that presented significant differences (p < 0.05) between Gc and Gs1, showing higher relative expression in Gc than in Gs5 and Gs1. SMS is a polyamine with antioxidant and anti-inflammatory properties that have been reported to significantly inhibit the production of nitric oxide (NO), prostaglandins, and cytosines [139], and reduce intracellular MDA levels. This indicates that the RBCs have a constitutive expression of SMS, speD, and E3.3.1.1, ...
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To understand changes in enzyme activity and gene expression as biomarkers of exposure to methylmercury, we exposed loggerhead turtle erythrocytes (RBCs) to concentrations of 0, 1, and 5 mg L−1 of MeHg and de novo transcriptome were assembled using RNA-seq. The analysis of differentially expressed genes (DEGs) indicated that 79 unique genes were dysregulated (39 upregulated and 44 downregulated genes). The results showed that MeHg altered gene expression patterns as a response to the cellular stress produced, reflected in cell cycle regulation, lysosomal activity, autophagy, calcium regulation, mitochondrial regulation, apoptosis, and regulation of transcription and translation. The analysis of DEGs showed a low response of the antioxidant machinery to MeHg, evidenced by the fact that genes of early response to oxidative stress were not dysregulated. The RBCs maintained a constitutive expression of proteins that represented a good part of the defense against reactive oxygen species (ROS) induced by MeHg.
... 15 PGD2 was found to activate androgen receptors in the human dermal papilla cells (hDPC) via the protein kinase B (AKT) pathways leading to the release of multiple cytokines that induce follicular apoptosis. 16 Moreover, PGD2 through its effects on G proteincoupled receptor 44 (GR44) found in the outer root sheath of hair follicles and dermal papillae was found to inhibit hair growth cycles and lead to miniaturization of hair follicles. 17 Notably, GR44 ...
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Background Androgentic alopecia (AGA) is the most common form of alopecia in men. Cetrizine, a second generation H1 blocker is known for its anti‐inflammatory properties as well as its ability to decrease prostaglandin D2 (PGD2) production. Aim To evaluate the efficacy and tolerability of topical cetirizine in male patients with AGA. Methods Two groups of 30 patients each (healthy males aged between 22 and 55 years) with different grades of AGA classified according to Hamilton Norwood classification were recruited for this study. Group A subjects applied 1 ml of 1% topical cetirizine daily while group B subjects served as controls and were instructed to apply 1 ml of a placebo solution for 6 months. Results Dermoscopic assessment revealed significantly higher hair regrowth among the cetirizine‐treated group (P<0.001). The patients’ satisfaction was significantly higher among the cetirizine‐treated group (p < 0.001). Conclusion The current study highlights a potential role cetirizine might have in treating AGA. It should be noted that studies are lacking in this regard and more randomized and controlled trials are warranted in order to confirm or refute such early findings.
... Furthermore, hair folicles are a source of pro-angiogenic factors, such as VEGF 52 . Few studies have investigated the effects of fatty acids on skin hair growth 53,54 . ...
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Wound healing is an essential process for organism survival. Some fatty acids have been described as modulators of wound healing. However, the role of omega-3 fatty acids is unclear. In the present work, we investigate the effects of oral administration of eicosapentaenoic acid (EPA)-rich oil on wound healing in mice. After 4 weeks of EPA-rich oil supplementation (2 g/kg of body weight), mice had increased serum concentrations of EPA (20:5ω-3) (6-fold) and docosahexaenoic acid (DHA; 22:6ω-3) (33%) in relation to control mice. Omega-3 fatty acids were also incorporated into skin in the EPA fed mice. The wound healing process was delayed at the 3rd and 7th days after wounding in mice that received EPA-rich oil when compared to control mice but there was no effect on the total time required for wound closure. Collagen reorganization, that impacts the quality of the wound tissue, was impaired after EPA-rich oil supplementation. These effects were associated with an increase of M2 macrophages (twice in relation to control animals) and interleukin-10 (IL-10) concentrations in tissue in the initial stages of wound healing. In the absence of IL-10 (IL-10−/− mice), wound closure and organization of collagen were normalized even when EPA was fed, supporting that the deleterious effects of EPA-rich oil supplementation were due to the excessive production of IL-10. In conclusion, oral administration of EPA-rich oil impairs the quality of wound healing without affecting the wound closure time likely due to an elevation of the anti-inflammatory cytokine IL-10.
... In addition, ACM-induced HO-1 expression in microglia was inhibited by DP2 PGD 2 receptor antagonists, CAY10471, but not by a DP1 antagonist, BW A868C (Fig. 5B). Since PGD 2 induces HO-1 expression through AKT activation (Jeong et al., 2018), we further examined the levels of AKT activation in WT ACM-and KO ACM-treated microglia. As expected, DJ-1 KO ACM less increased phosphor-AKT (pAKT) levels than WT ACM (Fig. 5C). ...
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Dysfunctional regulation of inflammation may contribute to the progression of neurodegenerative diseases. The results of this study revealed that DJ-1, a Parkinson's disease (PD) gene, regulated expression of prostaglandin D 2 synthase (PTGDS) and production of prostaglandin D 2 (PGD 2 ), by which DJ-1 enhanced anti-inflammatory function of astrocytes. In injured DJ-1 knockout (KO) brain, expression of tumor necrosis factor-alpha (TNF-α) was more increased, but that of anti-inflammatory heme oxygenase-1 (HO-1) was less increased compared with that in injured wild-type (WT) brain. Similarly, astrocyte-conditioned media (ACM) prepared from DJ-1-KO astrocytes less induced HO-1 expression and less inhibited expression of inflammatory mediators in microglia. With respect to the underlying mechanism, we found that PTGDS that induced expression of HO-1 was lower in DJ-1 KO astrocytes and brains compared with their WT counterparts. In addition, PTGDS levels increased in the injured brain of WT mice, but barely in that of KO mice. We also found that DJ-1 regulated PTGDS expression through Sox9. Thus, Sox9 siRNAs reduced PTGDS expression in WT astrocytes, and Sox9 overexpression rescued PTGDS expression in DJ-1 KO astrocytes. In agreement with these results, ACM from Sox9 siRNA-treated astrocytes and that from Sox9-overexpression astrocytes exerted opposite effects on HO-1 expression and anti-inflammation. These findings suggest that DJ-1 positively regulates anti-inflammatory functions of astrocytes, and that DJ-1 dysfunction contributes to the excessive inflammatory response in PD development.
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Inflammation and apoptosis are regulated by similar factors, including ultraviolet B (UVB) radiation and cannabinoids, which are metabolized by cyclooxygenase-2 (COX-2) into pro-apoptotic prostaglandin derivatives. Thus, the aim of this study was to evaluate the impact of cyclooxygenase-2 inhibition by celecoxib on the apoptosis of keratinocytes modulated by UVB, anandamide (AEA) and cannabidiol (CBD). For this purpose, keratinocytes were non-treated/treated with celecoxib and/or with UVB and CBD and AEA. Apoptosis was evaluated using microscopy, gene expressions using quantitate reverse-transcriptase polymerase chain reaction; prostaglandins using liquid chromatography tandem mass spectrometry and cyclooxygenase activity using spectrophotometry. UVB enhances the percentage of apoptotic keratinocytes, which can be caused by the increased prostaglandin generation by cyclooxygenase-2, or/and induced cannabinoid receptor 1/2 (CB1/2) expression. AEA used alone intensifies apoptosis by affecting caspase expression, and in UVB-irradiated keratinocytes, cyclooxygenase-2 activity is increased, while CBD acts as a cytoprotective when used with or without UVB. After COX-2 inhibition, UVB-induced changes are partially ameliorated, when anandamide becomes an anti-apoptotic agent. It can be caused by observed reduced generation of anandamide pro-apoptotic derivative prostaglandin-ethanolamide by COX. Therefore, products of cyclooxygenase-dependent lipid metabolism seem to play an important role in the modulation of UVB-induced apoptosis by cannabinoids, which is particularly significant in case of AEA as inhibition of cyclooxygenase reduces the generation of pro-apoptotic lipid mediators and thus prevents apoptosis.
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Androgenetic Alopecia (AGA) is by far the most common cause of hair loss in men, and its high prevalence has been reported in detail for many decades [1]. Several different terms in international medical bibliography have been suggested by several authors, such as androgenic alopecia, male pattern baldness, androgen-dependent alopecia, common baldness, and genetic hair loss. However, the term “Androgenetic Alopecia” is considered the most appropriate since it summarizes the etiology of the condition, with the term “andro-” referring to the hormonal and “-genetic”, implying the inherited parameter of its pathogenesis.
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Prostaglandin D2 (PGD2), one of the key lipid mediators of allergic airway inflammation, is increased in the airways of asthmatics. However, the role of PGD2 in the pathogenesis of asthma is not fully understood. In the present study, effects of PGD2 on smooth muscle contractility of the airways were determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In a murine model of allergic asthma, antigen challenge to the sensitized animals caused a sustained increase in PGD2 levels in bronchoalveolar lavage (BAL) fluids, indicating that smooth muscle cells of the airways are continually exposed to PGD2 after the antigen exposure. In bronchial smooth muscles (BSMs) isolated from naive mice, a prolonged incubation with PGD2 (10-5 M, for 24 h) induced an augmentation of contraction induced by acetylcholine (ACh): the ACh concentration-response curve was significantly shifted upward by the 24-h incubation with PGD2. Application of PGD2 caused phosphorylation of ERK1/2 and p38 in cultured BSM cells: both of the PGD2-induced events were abolished by laropiprant (a DP1 receptor antagonist) but not by fevipiprant (a DP2 receptor antagonist). In addition, the BSM hyperresponsiveness to ACh induced by the 24-h incubation with PGD2 was significantly inhibited by co-incubation with SB203580 (a p38 inhibitor), whereas U0126 (a ERK1/2 inhibitor) had no effect on it. These findings suggest that prolonged exposure to PGD2 causes the BSM hyperresponsiveness via the DP1 receptor-mediated activation of p38. A sustained increase in PGD2 in the airways might be a cause of the AHR in allergic asthmatics.
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Introduction: In the past thirty years, only two drugs have received FDA approval for the treatment of androgenetic alopecia reflecting a lack of success in unraveling novel targets for pharmacological intervention. However, as our knowledge of hair biology improves, new signaling pathways and organogenesis processes are being uncovered which have the potential to yield more effective therapeutic modalities. Areas covered: This review focuses on potential targets for drug development to treat hair loss. The physiological processes underlying the promise of regenerative medicine to recreate new functional hair follicles in bald scalp are also examined. Expert opinion: The discovery of promising new targets may soon enable treatment options that modulate the hair cycle to preserve or extend the growth phase of the hair follicle. This could also enable stimulation of progenitor cells and morphogenic pathways to reactivate miniaturized follicles in bald scalp or leverage the potential of wound healing and embryogenic development as an emerging paradigm to generate new hair follicles in barren skin.
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Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce β-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3β, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-β2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased β-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3β and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-β2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/β-catenin signaling, and that MPA stabilizes β-catenin by inhibiting GSK3β leading to increased β-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.
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Androgen receptor (AR)-mediated signaling is necessary for prostate cancer cell proliferation and an important target for therapeutic drug development. Canonically, AR signals through a genomic or transcriptional pathway, involving the translocation of androgen-bound AR to the nucleus, its binding to cognate androgen response elements on promoter, with ensuing modulation of target gene expression, leading to cell proliferation. However, prostate cancer cells can show dose-dependent proliferation responses to androgen within minutes, without the need for genomic AR signaling. This proliferation response known as the non-genomic AR signaling is mediated by cytoplasmic AR, which facilitates the activation of kinasesignaling cascades, including the Ras-Raf-1, phosphatidyl-inositol 3-kinase (PI3K)/Akt and protein kinase C (PKC), which in turn converge on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation, leading to cell proliferation. Further, since activated ERK may also phosphorylate AR and its coactivators, the non-genomic AR signaling may enhance AR genomic activity. Non-genomic AR signaling may occur in an ERK-independent manner, via activation of mammalian target of rapamycin (mTOR) pathway, or modulation of intracellular Ca2+ concentration through plasma membrane G proteincoupled receptors (GPCRs). These data suggest that therapeutic strategies aimed at preventing AR nuclear translocation and genomic AR signaling alone may not completely abrogate AR signaling. Thus, elucidation of mechanisms that underlie non-genomic AR signaling may identify potential mechanisms of resistance to current anti-androgens and help developing novel therapies that abolish all AR signaling in prostate cancer.
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Prostaglandins (PGs) are key inflammatory mediators involved in wound healing and regulating hair growth; however, their role in skin regeneration after injury is unknown. Using wound-induced hair follicle neogenesis (WIHN) as a marker of skin regeneration, we hypothesized that PGD(2) decreases follicle neogenesis. PGE(2) and PGD(2) were elevated early and late, respectively, during wound healing. The levels of WIHN, lipocalin-type prostaglandin D(2) synthase (Ptgds), and its product PGD(2) each varied significantly among background strains of mice after wounding, and all correlated such that the highest Ptgds and PGD(2) levels were associated with the lowest amount of regeneration. In addition, an alternatively spliced transcript variant of Ptgds missing exon 3 correlated with high regeneration in mice. Exogenous application of PGD(2) decreased WIHN in wild-type mice, and PGD(2) receptor Gpr44-null mice showed increased WIHN compared with strain-matched control mice. Furthermore, Gpr44-null mice were resistant to PGD(2)-induced inhibition of follicle neogenesis. In all, these findings demonstrate that PGD(2) inhibits hair follicle regeneration through the Gpr44 receptor and imply that inhibition of PGD(2) production or Gpr44 signaling will promote skin regeneration.Journal of Investigative Dermatology advance online publication, 29 November 2012; doi:10.1038/jid.2012.398.
Recent studies have showed that psychosocial stress causes elevated secretion of cortisol, the principal glucocorticoid (GC), and thus increases the extent of periodontal breakdown. In this study, we investigated whether stress-associated periodontal disturbance may be due to GC-induced changes in the periodontal ligament stem cells (PDLSCs), one of the most promising candidates for periodontal tissue regeneration. Our results in this study showed that dexamethasone (Dex) treatment causes the translocation of the glucocorticoid receptor (GR) into the nucleus and increases the expression of many genes, including dickkopf-1 (DKK-1) in PDLSCs. ELISA showed that DKK-1 is secreted from PDLSCs in response to Dex treatment. The GR antagonist RU486 attenuated the Dex-inducible DKK-1 messenger RNA (mRNA) expression. DKK-1 inhibited the growth of PDLSCs and suppressed Wnt-mediated activation of β-catenin signaling in PDLSCs. Our results strongly suggest that stress-associated periodontal disturbance may be due to GC-induced changes in the activity of PDLSCs via DKK-1 expression and might provide a possible explanation for the deteriorating effect of stress on periodontal breakdown.
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Clinical evidence has demonstrated that the accumulation of 5α‑dihydrotestosterone (DHT) in dermal papilla cells (DPCs) is implicated in androgenetic alopecia. Whether this accumulation in DHT may have direct cellular effects leading to androgenetic alopecia remains to be elucidated. The present study aimed to determine whether DHT affects cell growth, cell cycle arrest, cell death, senescence and the induction of reactive oxygen species (ROS), and whether these effects are mediated by microRNA (miRNA)‑dependent mechanisms. The cell viability and cell cycle were determined, levels of ROS were examined and senescence‑associated β‑galactosidase assays were performed in normal human DPCs (nHDPCs). Furthermore, miRNA expression profiling was performed using an miRNA microarray to determine whether changes in the expression levels of miRNA were associated with the cellular effects of DHT. The results revealed that DHT decreased cell growth by inducing cell death and G2 cell cycle arrest, and by increasing the production of ROS and senescence in the nHDPCs. In addition, 55 miRNAs were upregulated and 6 miRNAs were downregulated inthe DHT‑treated nHDPCs. Bioinformatic analysis demonstrated that the putative target genes of these upregulated and downregulated miRNAs were involved in cell growth, cell cycle arrest, cell death, senescence and the production of ROS. Specifically, the target genes of five highly upregulated and downregulated miRNAs were identified and were associated with the aforementioned effects of DHT. These results demonstrated that the expression of miRNA was altered in the DHT‑treated nHDPCs and suggest the potential mechanisms of DHT‑induced cell growth repression, cell cycle arrest, cell death, senescence and induction of ROS.
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Androgenetic alopecia is characterised by progressive, patterned hair loss from the scalp. Recently the pathogenesis and genetic basis of the hair loss have been better understood, as has the distress experienced by men who have lost their hair. There have also been breakthroughs in the treatment of androgenetic alopecia.The transition of some terminal hairs into vellus hairs is a universal physiological secondary sexual characteristic.1 Androgenetic alopecia becomes a medical problem only when the hair loss is subjectively seen as excessive, premature, and distressing.The prerequisites for premature androgenetic alopecia are a genetic predisposition and sufficient circulating androgens.2 Eunuchs do not go bald.3 Every white man possesses the autosomal inherited predisposition,4 and 96% lose hair to some degree,5 but because of the variabity of gene expression far fewer have appreciable premature hair loss. Summary points Androgenetic alopecia is a specific type of hair loss mediated by systemic androgens and genetic factorsRecent advances in understanding of the biology of hair follicles have shed light on the pathogenesis of androgenetic alopeciaThough most men learn to deal with their androgenetic alopecia without it impairing their psychosocial functioning, some men tolerate hair loss poorly and have a negative overall body image and diminished quality of lifeSafe and effective treatments are currently available for androgenetic alopecia, but advice and counselling remain the most important aspects of management
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Androgenic alopecia, a condition characterized by increased levels of DHT could have been selected for due to the benefits that prostaglandin D2 (PGD2) has on the prostate. A DHT metabolite can increase the transcription of prostaglandin D2 synthase through estrogen receptor beta. The increase of PGD2 can decrease the risk of prostate cancer and proliferation of prostate cancer cells. Therefore, the mechanisms behind male pattern baldness may also curtail the advancement of prostate cancer.
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In a recent study we have shown that prostaglandin D2 (PGD2) induces human osteoclast (OC) apoptosis through the activation of the chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2) receptor and the intrinsic apoptotic pathway. However, the molecular mechanisms underlying this response remain elusive. The objective of this study is to investigate the intracellular signaling pathways mediating PGD2-induced OC apoptosis. OCs were generated by in vitro differentiation of human peripheral blood mononuclear cells (PBMCs), and then treated with or without the selective inhibitors of mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase, (MEK)-1/2, phosphatidylinositol3-kinase (PI3K) and NF-κB/IκB kinase-2 (IKK2) prior to the treatments of PGD2 as well as its agonists and antagonists. Fluorogenic substrate assay and immunoblotting were performed to determine the caspase-3 activity and key proteins involved in Akt, ERK1/2 and NF-κB signaling pathways. Treatments with both PGD2 and a CRTH2 agonist decreased ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylation, whereas both treatments increased β-arrestin-1 phosphorylation (Ser412) in the presence of naproxen, which was used to eliminate endogenous prostaglandins production. In the absence of naproxen, treatment with a CRTH2 antagonist increased both ERK1/2 and Akt phosphorylation, and reduced the phosphorylation of β-arrestin-1. Treatment of OCs with a selective MEK-1/2 inhibitor increased caspase-3 activity and OC apoptosis induced by both PGD2 and a CRTH2 agonist. Moreover, a CRTH2 antagonist diminished the selective MEK-1/2 inhibitor-induced increase in caspase-3 activity in the presence of endogenous prostaglandins. In addition, treatment of OCs with a selective PI3K inhibitor decreased ERK1/2 (Thr202/Tyr204) phosphorylation caused by PGD2, whereas increased ERK1/2 (Thr202/Tyr204) phosphorylation by a CRTH2 antagonist was attenuated with a PI3K inhibitor treatment. The DP receptor was not implicated in any of the parameters evaluated. Treatment of OCs with PGD2 as well as its receptors agonists and antagonists did not alter the phosphorylation of RelA/p65 (Ser536). Moreover, the caspase-3 activity was not altered in OCs treated with a selective IKK2/NF-κB inhibitor. In conclusion, endogenous or exogenous PGD2 induces CRTH2-dependent apoptosis in human differentiated OCs; β-arrestin-1, ERK1/2, and Akt, but not IKK2/NF-κB are probably implicated in the signaling pathways of this receptor in the model studied.
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Retinoid-inducible gene 1 (RIG1), also called tazarotene-induced gene 3, belongs to the HREV107 gene family, which contains five members in humans. RIG1 is expressed in high levels in well-differentiated tissues, but its expression is decreased in cancer tissues and cancer cell lines. We found RIG1 to be highly expressed in testicular cells. When RIG1 was expressed in NT2/D1 testicular cancer cells, neither cell death nor cell viability was affected. However, RIG1 significantly inhibited cell migration and invasion in NT2/D1 cells. We found that prostaglandin D2 synthase (PTGDS) interacted with RIG1 using yeast two-hybrid screens. Further, we found PTGDS to be co-localized with RIG1 in NT2/D1 testis cells. In RIG1-expressing cells, elevated levels of prostaglandin D2 (PGD2), cAMP, and SRY-related high-mobility group box 9 (SOX9) were observed. This indicated that RIG1 can enhance PTGDS activity. Silencing of PTGDS expression significantly decreased RIG1-mediated cAMP and PGD2 production. Furthermore, silencing of PTGDS or SOX9 alleviated RIG1-mediated suppression of migration and invasion. These results suggest that RIG1 will suppress cell migration/invasion through the PGD2 signaling pathway. In conclusion, RIG1 can interact with PTGDS to enhance its function and to further suppress NT2/D1 cell migration and invasion. Our study suggests that RIG1-PGD2 signaling might play an important role in cancer cell suppression in the testis.