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

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; [email protected] or [email protected] (K.H.J.); [email protected] (J.H.J.); [email protected] (J.E.K.) * Correspondence: [email protected]; 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 (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.

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. 5α-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

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Int. J. Mol.Cotsarelis Sci. 2018, 19and, 556 colleagues reported that the levels of prostaglandin D2 synthase (PTGDS) and its2 of 12 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 throughthrough DP2 DP2 [6 ].[6]. The The biological biological effectseffects ofof PGD2PGD2 are usually usually mediated mediated by by its its two two G Gprotein-coupled protein-coupled receptorsreceptors (PGD2 (PGD2 receptor; receptor; PTGDR): PTGDR): prostaglandin prostaglandin receptor receptor 1 (DP1)1 (DP1) and and prostaglandin prostaglandin receptor receptor 2 (DP2,2 also(DP2, known also as known chemoattractant as chemoattractant homologous homologous receptor receptor expressed expressed on Th2 on cells; Th2 CRTH2). cells; CRTH2). The induction The of PGD2induction could of PGD2 result could from increasedresult from androgen increased levels, androgen since levels, androgens since androgens have been have shown been to shown stimulate PTGDSto stimulate [7]. Some PTGDS evidences [7]. Some suggest evidences that suggest PGD2 promotesthat PGD2 thepromotes onset ofthe catagen onset of phasecatagen and phase decreased and hairdecreased lengthening, hair lengthening, leading to anleading increase to an in increase telogen in follicles telogen andfollicle miniaturizations and miniaturization of the of hair the follicles, hair andfollicles, PGD2 alsoand inhibits PGD2 hairalso follicleinhibits regeneration hair follicle involved regeneration in wound involved healing in [6wound,8,9]. These healing ﬁndings [6,8,9]. have demonstratedThese findings the have effect demonstrated of PGD2 on hair the growtheffect of and PGD2 its roles on inhair AGA. growth However, and its our roles understanding in AGA. ofHowever, how the PGD2our understanding pathway functions of how the in PGD2 DPCs path of AGAway functions remains in limited. DPCs of Thus, AGA weremains focused limited. on the expressionThus, we offocused AR related on the genes expression by PGD2-DP2 of AR related at cellular genes by level. PGD2-DP2 at cellular level.

2. Results2. Results

2.1.2.1. Prostaglandin Prostaglandin D2 D2 Receptor Receptor 2 2 (DP2) (DP2) AntagonistAntagonist Regulates Dihydrotestosterone Dihydrotestosterone (DHT)-Induced (DHT)-Induced ProstaglandinProstaglandin D2 D2 (PGD2) (PGD2) Pathway Pathway ToTo determine determine whether whether DHT DHT affects affects the the PGD2 PGD2 pathway, pathway, human human dermal dermal papilla papilla cells cells (hDPCs) (hDPCs) were treatedwere withtreated various with doses various of DHTdoses for of 24 DHT h. Treatment for 24 h. with Treatment 100 nM DHTwith increased100 nM DHT cyclooxygenase-2 increased (COX2),cyclooxygenase-2 PTGDS and (COX2), DP2 mRNA PTGDS expression and DP2 (Figure mRNA1A–C). expression Moreover, (Figure the protein1A–C). levelMoreover, of DP2 the was increasedprotein level by 100 of nMDP2 ofwas DHT increased treatment by 100 at 3nM h (1.4-fold)of DHT treatment and 5 h at (1.9-fold), 3 h (1.4-fold) respectively and 5 h (1.9-fold), (Figure1D). Inrespectively addition, the (Figure levels 1D). of PGD2 In addition, receptor the were levels signiﬁcantly of PGD2 receptor upregulated were significantly upon treatment upregulated with high upon treatment with high concentrations of DHT, such as 100 nM and 1000 nM (Figure 1E). We concentrations of DHT, such as 100 nM and 1000 nM (Figure1E). We next examined whether a next examined whether a DP2 antagonist affects AR expression. hDPCs were pretreated with DP2 antagonist affects AR expression. hDPCs were pretreated with TM30089 (DP2 antagonist) at TM30089 (DP2 antagonist) at 20 μM for 1 h and then treated with 100 nM DHT for 5 h. Stimulation 20 µM for 1 h and then treated with 100 nM DHT for 5 h. Stimulation with TM30089 inhibited the with TM30089 inhibited the upregulation of AR and DP2 (Figure 1F,G). Additionally, upregulationimmunocytochemistry of AR and data DP2 showed (Figure 1thatF,G). DHT-stimulated Additionally, immunocytochemistryAR and DP2 expression datawas detected showed in that DHT-stimulatedthe nucleus, and AR the and expression DP2 expression of AR and was DP2 detected in the TM30089-treated in the nucleus, group and the was expression weaker than of AR that and DP2in inthe the DHT-treated TM30089-treated group (Figure group S1A,B). was weaker than that in the DHT-treated group (Figure S1A,B).

Figure 1. Cont.

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FigureFigure 1. 1.Prostaglandin Prostaglandin D2D2 receptorreceptor (DP2)(DP2) antagonist (TM30089) (TM30089) decreases decreases dihydrotestosterone dihydrotestosterone (DHT)-induced(DHT)-induced androgen androgen receptor receptor (AR) (AR) andand prostaglandinprostaglandin expression expression in in human human dermal dermal papilla papilla cells cells (hDPCs).(hDPCs). The The mRNA mRNA expression expression of of cyclooxygenase-2 cyclooxygenase-2 (COX2), (COX2), prostaglandin prostaglandin D2 D2 synthase synthase (PTGDS) (PTGDS) andand DP2 DP2 was was examined examined in in hDPCs hDPCs treatedtreated withwith DHT for for 24 24 h. h. The The mRNA mRNA expression expression of COX2 of COX2 (A), ( A), PTGDSPTGDS (B )( andB) and DP2 DP2 (C) was(C) was induced induced by 100 by nM100 DHT.nM DHT. DP2 proteinDP2 protein expression expression was strongly was strongly induced byinduced 100 nM DHTby 100 at nM 5h DHT (D). at hDPCs 5 h (D were). hDPCs cultured were cultured for 24 h withfor 24 DHT, h with as DHT, indicated. as indicated. The level The of level PGD2 receptorof PGD2 in thereceptor supernatant in the wassupernatant evaluated was in ev threealuated independent in three independent experiments experiments (E). The relative (E). mRNAThe relative mRNA levels were normalized to that of GAPDH. hDPCs were pretreated with 20 μM levels were normalized to that of GAPDH. hDPCs were pretreated with 20 µM TM30089 for 1 h and TM30089 for 1 h and then treated with 100 nM DHT for 5 h. The protein level of AR (F) and DP2 (G) then treated with 100 nM DHT for 5 h. The protein level of AR (F) and DP2 (G) was measured by was measured by western blot. TM30089 decreased the DHT-induced AR and DP2 expression. western blot. TM30089 decreased the DHT-induced AR and DP2 expression. β-actin served as a loading β-actin served as a loading control for protein normalization. The results are expressed as the control for protein normalization. The results are expressed as the mean ± SD of three independent mean ± SD of three independent experiments: CTL; control. * p < 0.05 compared with the control (0 experiments: CTL; control. * p < 0.05 compared with the control (0 nM DHT). # p < 0.05 compared with nM DHT). # p < 0.05 compared with the DHT 100 nM. the DHT 100 nM. 2.2. The Effects of PGD2 on AR Expression and hDPCs 2.2. The Effects of PGD2 on AR Expression and hDPCs To determine whether PGD2 directly regulates AR expression, hDPCs were stimulated with variousTo determine concentrations whether of PGD2 PGD2 in directly serum-free regulates medium AR for expression, 24 h. PGD2 hDPCsat 50 nM–1000 were stimulated nM induced with variousthe expression concentrations of AR. of At PGD2 200 innM serum-free in particular, medium PGD2 for treatment 24 h. PGD2 increased at 50 nM–1000 the expression nM induced of AR the expression(2.3-fold) ofmRNA AR. At at 20024 h nM compared in particular, with 0 PGD2nM group treatment (Figure increased 2A). The mRNA the expression expression of of AR AR (2.3-fold) was mRNAincreased at 24 at h compared24 h in compare with 0with nM PGD2 group treatment (Figure2 A).for The5 h group mRNA (Figure expression 2B). On of the AR other was increasedhand, the at 24 hprotein in compare level of with AR PGD2 was increased treatment at for 3 5h hand group 5 h (Figure(Figure2 B).2C). On We the examined other hand, whether the protein AR related level of ARfactors was increased are mediated at 3 h by and PGD2 5 h (Figure in hDPCs.2C). WeWe examinedobserved whetherthat the mRNA AR related expression factors areof AR mediated related by PGD2factors in hDPCs.(TGFβ1, WeCreb, observed LEF1, and that IGF-1) the mRNA was increased expression by PGD2 of AR treatment related factors (200 nM (TGF for β241, h) Creb, (Figure LEF1, 2D). We next examined whether PGD2 is involved in the growth inhibition of hDPCs. hDPCs were and IGF-1) was increased by PGD2 treatment (200 nM for 24 h) (Figure2D). We next examined whether treated with various concentrations of PGD2 (0 nM–1000 nM) for 72 h. PGD2 treatment PGD2 is involved in the growth inhibition of hDPCs. hDPCs were treated with various concentrations dose-dependently inhibited cell viability at 72 h (Figure 2E). Furthermore, the mRNA expression of of PGD2 (0 nM–1000 nM) for 72 h. PGD2 treatment dose-dependently inhibited cell viability at 72 h apoptosis-related genes, including caspase-1, -3, and -9, was dose-dependently increased by PGD2 (Figuretreatment2E). Furthermore, for 24 h (Figure the 2F). mRNA In addition, expression apoptosi of apoptosis-relateds in various concentratio genes,n includingof PGD2 treated caspase-1, with -3, andhDPCs -9, was detected dose-dependently by TUNEL increasedassay. We byfound PGD2 that treatment the number for 24of hapoptotic (Figure2 F).cells In dose-dependently addition, apoptosis in variousincreased concentration in the PGD2-treated of PGD2 groups treated (Figure with hDPCs S2A). detectedAlso, we byexamined TUNEL the assay. changes We found in protein that the numberlevels ofof apoptoticthe Bcl2 and cells Bax dose-dependently genes, which are increased known to in regulate the PGD2-treated apoptotic cell groups death. (Figure The Bax/Bcl2 S2A). Also, weratio examined was 3.5-fold the changes higher in in the protein PGD2 levels (1000 ofnM) the at Bcl224 h andcompared Bax genes, with 5 which h (Figure are S2B). 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).

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FigureFigure 2. PGD2 2. PGD2 regulates regulates the ARthe expression AR expression and viability and viab ofility hDPCs. of hDPCs. hDPCs hDPCs were cultured were cultured in serum-free in DMEMserum-free for 24 DMEM h, and thenfor 24 treated h, and withthen thetreated indicated with the concentrations indicated concentrations of PGD2 for of 24PGD2 h (A for). For 24 h optimal (A). conditionFor optimal of AR condition mRNA of expression, AR mRNA hDPCs expression, were hDPCs cultured were in serum-freecultured in serum-free DMEM for DMEM 24 h, andfor 24 then 200h, nM and PGD2 then 200 treated nM PGD2 in hDPCs treated for in 5 andhDPCs 24 hfor (B 5). and hDPCs 24 h were(B). hDPCs treated were with treated PGD2 with (200 PGD2 nM) for(200 the indicatednM) for timesthe indicated and harvested. times and The harvested. AR protein The AR level protein was level determined was determined using western using western blot analysis blot (C).analysis The mRNA (C). The expression mRNA expression of transforming of transforming growth factor growth beta factor 1 (TGF betaβ 1),1 (TGF Creb,β1), lymphoid Creb, lymphoid enhancer bindingenhancer factor binding 1 (LEF1) factor and 1 (LEF1) insulin-like and insulin-like growth growth factor factor 1 (IGF-1) 1 (IGF-1) was was measured measured by by qRT-PCR qRT-PCR (D ). Cell(D viability). Cell viability was determined was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)bromide assay (MTT) after incubationassay after withincubation different with concentrations different concentrations of PGD2 (0, of 50, PGD2 100, (0, 200, 50, 500, 100, 1000 200, nM) 500, for 72 h1000 (E). nM) The for mRNA 72 h ( expressionE). The mRNA of caspase-1, expression -3, of and caspase-1, -9 was -3, measured and -9 was by measured qRT-PCR (byF). qRT-PCR The results (F). are expressedThe results as the are mean expressed± SD as of the three mean independent ± SD of three experiments. independent * pexperiments.< 0.05, compared * p < 0.05, with compared the control (0 nMwith PGD2). the control (0 nM PGD2).

2.3. PGD2-Induced AR Expression is Regulated by AKT Signalling 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,To investigate hDPCs thewere association treated with of the PGD2 AKT andfor ARdifferent signalling amounts pathways of time, in PGD2-inducedup to 24 h. AKT hDPCs, hDPCsphsphorylation were treated was with observed PGD2 at for 3 and different 5 h (Figure amounts 3A). of Second, time, uphDPCs to 24 were h. AKT treated phsphorylation with LY294002 was observed(AKT inhibitor) at 3 and 5 at h (Figure20 μM 3forA). 1 Second,h before hDPCs AR, AKT were and treated GSK3 withβ (AKT/GSK3 LY294002β (AKT) phosphorylation inhibitor) at 20wasµ M foranalysed 1 h before using AR, AKT western and GSK3 blot.β (AKT/GSK3Stimulation β)with phosphorylation PGD2 increased was analysed AR and using AKT/GSK3 western blot.β Stimulationphosphorylation with PGD2 in hDPCs increased compared AR and with AKT/GSK3 control. Treatmentβ phosphorylation with LY294002 in hDPCs along compared with PGD2 with control.further Treatment decreased with AKT/GSK3 LY294002β phosphorylation along with PGD2 compared further decreased with PGD2 AKT/GSK3 treated groupβ phosphorylation (Figure 3B). comparedWe also withexamined PGD2 the treated mRNA group level (Figureof AR and3B). AKT We alsosignal examined related factors the mRNA LEF1, Creb, level and of AR IGF-1. and All AKT signalmRNA related of factorsexamined LEF1, molecules Creb, and related IGF-1. to All the mRNA AR were of examined blocked moleculesby treatment related with to theLY294002 AR were blocked(Figure by 3C). treatment with LY294002 (Figure3C).

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FigureFigure 3. PGD23. PGD2 regulates regulates the the AKT AKT signal. signal. TheThe AKT phosphorylation was was determined determined using using western western blotblot analysis. analysis. hDPCs hDPCs were were treatedtreated withwith PGD2 ( (200200 nM) nM) for for the the indicated indicated times times and and harvested harvested (A). ( A). hDPCs were pretreated with LY294002 for 1 h, and then PGD2 (200 nM) treatment for 5 h. The hDPCs were pretreated with LY294002 for 1 h, and then PGD2 (200 nM) treatment for 5 h. The protein protein levels of AR, and AKT/GSK3β/Creb phosphorylation was measured using western blot levels of AR, and AKT/GSK3β/Creb phosphorylation was measured using western blot analysis (B). analysis (B). The mRNA expression of AR, LEF1, Creb, and IGF-1 was measured using qRT-PCR The mRNA expression of AR, LEF1, Creb, and IGF-1 was measured using qRT-PCR (C). β-actin served (C). β-actin served as a loading control for protein normalization. GAPDH was used as an internal as a loading control for protein normalization. GAPDH was used as an internal control for mRNA control for mRNA normalization. The results are expressed as the mean ± SD of three independent normalization. The results are expressed as the mean ± SD of three independent experiments. * p < 0.05, experiments. * p < 0.05, compared with the control (0 nM PGD2), # p < 0.05 compared with PGD2. 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 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 (includingWe conﬁrmed LEF1, thatCreb, PGD2-DP2 and IGF-1). affects We hypothesized AR expression that via suppression AKT and of its DP2 involved would factors inactivate (including AR LEF1,expression Creb, and by IGF-1).inhibiting We AR-related hypothesized factors that and suppression AKT signalling. of DP2 wouldThus, inactivatewe examined AR expressionwhether byinhibition inhibiting of AR-related DP2 could factorsregulate and the AKTactivity signalling. of AR and Thus, its related we examined factors. TM30089 whether has inhibition been known of DP2 couldas a regulate highly thepotent activity antagonist of AR andon mouse its related CRTH2/DP2 factors. TM30089[10]. PGD2-induced has been known AR, DP2, as a highlyand COX2 potent antagonistmRNA expression on mouse CRTH2/DP2was reduced [by10]. TM30089 PGD2-induced (Figure AR, 4A–C). DP2, We and also COX2 found mRNA that expression the mRNA was reducedexpression by TM30089 of TGFβ1, (Figure Creb, 4LEF1,A–C). and We IGF-1, also found which that are therelated mRNA to the expression activity of of AR TGF andβ 1,AKT Creb, LEF1,signalling, and IGF-1, was which blocked are relatedby TM30089 to the activity(Figure of4D–G). AR and In AKT addition, signalling, protein was levels blocked of byAR TM30089 and phosphorylation of AKT/GSK3β was also reduced by the TM30089. (Figure 4H). PGD2-inhibited (Figure4D–G). In addition, protein levels of AR and phosphorylation of AKT/GSK3 β was also reduced cell viability was significantly recovered by 30% upon treatment with TM30089 compared with the by the TM30089. (Figure4H). PGD2-inhibited cell viability was signiﬁcantly recovered by 30% upon PGD2-treated group (Figure 4I). These results indicated that AR expression and hDPC viability treatment with TM30089 compared with the PGD2-treated group (Figure4I). These results indicated were regulated by PGD2 through DP2. that AR expression and hDPC viability were regulated by PGD2 through DP2.

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FigureFigure 4. 4.The The effect effect of of aa DP2DP2 antagonist on on PGD2-ind PGD2-induceduced AR AR related related genes genes and andAKT AKT signalling signalling in in hDPCs.hDPCs. hDPCs hDPCs were were pretreated pretreated with with TM30089 TM30089 (20 (20 μM)µM) or orLY294002 LY294002 (20 (20 μM)µM) for for1 h 1 and h and then then stimulatedstimulated with with 200 200 nM nM PGD2 PGD2 for for 24 24 h.h. TheThe mRNA expression expression of of AR, AR, DP2, DP2, COX2, COX2, IGF-1, IGF-1, LEF1, LEF1, Creb,Creb, and and TGF TGFβ1β was1 was measured measured by by qRT-PCRqRT-PCR ((AA–G). hDPCs in in serum-free serum-free DMEM DMEM were were pretreated pretreated with TM30089 (20 μM) for 1 h and then stimulated with 200 nM PGD2 for 5 h. The protein level of 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 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 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 densitometric analysis of three independent experiments (H). An MTT-based assay was performed to to determine the effects of PGD2 after 72 h of treatment. TM30089 (20 μM) restored the viability of determine the effects of PGD2 after 72 h of treatment. TM30089 (20 µM) restored the viability of hDPCs hDPCs that was inhibited by 200 nM PGD2 (I). β-actin served as a loading control for protein that was inhibited by 200 nM PGD2 (I). β-actin served as a loading control for protein normalization. normalization. GAPDH was used as an internal control for mRNA normalization. The results are GAPDHexpressed was as used the asmean an internal± SD of three control independent for mRNA experiments. normalization. TM; TheTM30089, results * p are < 0.05 expressed compared as the meanwith± theSD control of three (0 independentnM PGD2), # p experiments. < 0.05 compared TM; with TM30089, PGD2. * 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 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 transfectedNext, to study with DP2-targeting the involvement siRNA of DP2 (20 nM). in PGD2-induced Transfection with AR expression,DP2 siRNA hDPCssignificantly were transfectedknocked withdown DP2-targeting the protein siRNAlevel of (20 AR, nM). DP2, Transfection COX2 and withAKT/GSK3 DP2 siRNAβ/Creb signiﬁcantlyphosphorylation, knocked whereas down the the proteinnegative level control of AR, DP2,siRNA COX2 (siNC) and (20 AKT/GSK3 nM) had βno/Creb effect phosphorylation, (Figure 5A). We whereasalso confirmed the negative DP2 gene control siRNAsilencing (siNC) at (20the nM)mRNA had level. no effect PGD2-induced (Figure5A). the We target also of conﬁrmed AR or AKT DP2 genes gene (including silencing atAR, the COX2, mRNA level.DP2, PGD2-induced LEF1, and Creb) the targetand cell of ARapoptosis or AKT genes genes such (including as caspase-3 AR, COX2,and caspase-9 DP2, LEF1, were and markedly Creb) and attenuated by DP2-targeting siRNA transfection (Figure 5B). These data suggest that DP2 is cell apoptosis genes such as caspase-3 and caspase-9 were markedly attenuated by DP2-targeting important for PGD2-mediated AKT signal on AR expression in hDPCs. siRNA transfection (Figure5B). These data suggest that DP2 is important for PGD2-mediated AKT signal on AR expression in hDPCs.

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FigureFigure 5. 5.Knockdown Knockdown of of DP2 DP2suppress suppress ARAR relatedrelated genes and and AKT AKT signal. signal. After After transfection transfection with with negativenegative control control (siNC) (siNC) or or DP2 DP2 siRNA, siRNA, and and then then treated treated with with PGD2 PGD2 (200 (200 nM) nM) for for 5 h. 5 Theh. The protein protein levels of AR,levels DP2, of AR, COX2, DP2, and COX2, AKT/GSK3 and AKT/GSK3β/Crebβ phosphorylation/Creb phosphorylation was measured was measured using using western western blot analysis blot (A).analysis After transfection(A). After transfection with siNA with or DP2siNA siRNA or DP2 for siRNA 24 h, for and 24 thenh, and treated then treated with PGD2 with PGD2 (200 nM)(200 for nM) for 24 h. The mRNA expression of AR, DP2, COX2, LEF1, Creb, and caspases (-3, and -9) was 24 h. The mRNA expression of AR, DP2, COX2, LEF1, Creb, and caspases (-3, and -9) was measured measured by qRT-PCR (B). β-actin served as a loading control for protein normalization. GAPDH by qRT-PCR (B). β-actin served as a loading control for protein normalization. GAPDH was used as was used as an internal control for mRNA normalization. The results are expressed as the mean ± an internal control for mRNA normalization. The results are expressed as the mean ± SD of three SD of three independent experiments. * p < 0.05 compared with the siNC (siRNA negative control), independent experiments. * p < 0.05 compared with the siNC (siRNA negative control), # p < 0.05 # p < 0.05 compared with PGD2. compared with PGD2. 3. Discussion 3. Discussion Human dermal papilla cells (hDPCs) play an important role in hair follicle formation and hair regenerationHuman dermal and growth papilla [11]. cells In (hDPCs) particular, play the an regulation important of rolegrowth inhair and follicleapoptosis formation in hDPCs and has hair regenerationbeen reported and to growth be necessary [11]. In fo particular,r maintaining the regulation hair growth of growth[12]. Some and studies apoptosis have in suggested hDPCs has that been reportedthe factors to be secreted necessary from for maintaininghDPCs in response hair growth to DHT [12]. can Some induce studies male have hair suggested loss by affecting that the factorsthe secretedactivity from of various hDPCs genes in response in hair tofollicles DHT can[13,14] induce. DHT-induced male hair lossandrogens by affecting stimulate the activitythe secretion of various of geneshair in growth hair follicles inhibitory [13, 14factors]. DHT-induced such as transforming androgens growth stimulate factor the secretionbeta 1 and of 2 hair (TGF growthβ1/2) [15,16]. inhibitory factorsDHT such is involved as transforming in several growth cellular factor signalling beta 1me andchanisms. 2 (TGF βFor1/2) example, [15,16]. DHT DHT increases is involved cell in death several cellularand inhibits signalling the mechanisms. cell cycle [12]. For example,DHT modulates DHT increases hair growth, cell death hair andcycling, inhibits and the hair cell loss cycle in [12 ]. DHTAGA-susceptible modulates hair hair growth, follicles hair only cycling, [17]. andAlthou hairgh loss definitive in AGA-susceptible evidence has hairbeen follicles reported only for [17 ]. pathological mechanisms of AGA, the function of DPCs in AGA remain unclear. Although definitive evidence has been reported for pathological mechanisms of AGA, the function of DKK-1 and TGFβ1, which are cell death factors, are produced by DHT to destroy hair follicle DPCs in AGA remain unclear. cells and induce them to enter catagen stage, thereby causing hair loss [14,18]. Importantly, in DKK-1 and TGFβ1, which are cell death factors, are produced by DHT to destroy hair follicle cells susceptible individuals, DHT is also thought to precipitate an abbreviated anagen phase, as well as andstructural inducethem miniaturization to enter catagen in the hair stage, follic therebyle and causingassociated hair anatomical loss [14,18 structures.]. Importantly, in susceptible individuals,Interestingly, DHT is DHT also thoughtsimulated to pr precipitateostaglandin an D2 abbreviated signalling anagenthrough phase,the expression as well asof structuralCOX2, miniaturizationPTGDS, and DP2. in the Although hair follicle various and associatedstimuli may anatomical induce the structures. expression of COX2 in many cells [19,20],Interestingly, we used DHTDHT, simulated which promoted prostaglandin AR expression D2 signalling by affecting through DP2 the and expression COX2. We of also COX2, PTGDS,investigated and DP2. theAlthough changes the various activity stimuli of AR may by induce DP2 theantagonist. expression Our of results COX2 showed in many that cells DP2 [19,20 ], weantagonist used DHT, has which the potential promoted to suppress AR expression AR signal by by affecting reducing DP2 the protein and COX2. expression We also of DP2. investigated These thefindings changes indicated the activity that ofactivation AR by DP2of AR antagonist. is associated Our with results DHT as showed well as thatprostaglandin DP2 antagonist pathway. has the potentialCyclooxygenase-2 to suppress AR signal(COX2), by reducinga pro-inflammatory the protein inducible expression enzyme, of DP2. Theseis a key findings enzyme indicated in thatprostaglandin activation of (PG) AR isbiosynthesis associated that with converts DHT as arac wellhidonic as prostaglandin acid (AA) to pathway. PGG2 and subsequently to PGH2,Cyclooxygenase-2 which is metabolized (COX2), by various a pro-inflammatory PG synthases to other inducible PGs [21]. enzyme, PGs are is potent a key biologically enzyme in prostaglandinactive lipid mediators (PG) biosynthesis that are produced that converts from arachidonicAA by almost acid every (AA) cell to type PGG2 and and are subsequentlyknown to to PGH2,regulate which immune is metabolized responses. One by variousof them, PG PGD2 synthases is involved to other in wound PGs [21 healing]. PGs [8], are and potent hair biologically loss [6]

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 TGFβ1 and TGFβ2, 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 5αR and AR [23]. We found that PGD2 treatment enhances the expression of AR, Creb, TGFβ1, 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 GSK3β and GSK3α. Function of AKT and GSK3β were known as a key regulator of AR activation [29]. Activated GSK3β 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/GSK3β/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/GSK3β suppressed AR related gene expression [32]. These results suggest that AKT/GSK3β phosphorylation is involved in AR expression. Although the role of PGD2-mediated AKT/GSK3β/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/GSK3β/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% CO2. 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 × 104 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 µM) 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 µL/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 µL 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, GSK3β) (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 × 103 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 × 105 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% CO2 incubator until 70–80% conﬂuent. Thereafter, 2 µL DP2 siRNA duplex was diluted into 100 µL of siRNA transfection medium (Santa Cruz Biotechnology). In a separate tube, 2 µL of transfection reagent (Santa Cruz biotechnology) was diluted into 100 µL 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 × 104 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). p value < 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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