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 Int. J. Mol. Sci. 2018, 19, 556; doi:10.3390/ijms19020556 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 12 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. Int. J. Mol. Sci. 2018, 19, 556 3 of 12 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 12 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.