- Email: [email protected]
ARA55 in balding dermal papilla cells
Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151
Naoki Oiso Department of Dermatology, Kinki University School of Medicine, Osaka, Japan Atsushi Tanemura Department of Dermatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Ichiro Katayama Department of Dermatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan *Corresponding author. Tel.: +81 6 6879 3031 E-mail address: [email protected] (M. Wataya-Kaneda)
Akira Kawada Department of Dermatology, Kinki University School of Medicine, Osaka, Japan Tamio Suzuki Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan
149
Received 4 April 2011 Revised 17 August 2011 Accepted 21 August 2011 doi:10.1016/j.jdermsci.2011.08.006
Letter to the Editor Androgen receptor transactivity is potentiated by TGF-b1 through Smad3 but checked by its coactivator Hic-5/ARA55 in balding dermal papilla cells We previously reported that TGF-b1 is a paracrine mediator from dermal papilla to hair follicle epithelium in the pathogenesis of androgenetic alopecia (AGA) [1,2]. Because TGF-b1 can induce catagen in hair cycling [3], it has been suggested that it functions as a paracrine pathogenic mediator from dermal papilla in AGA. On the other hand, TGF-b1 reportedly modulates androgen receptor (AR) transactivation in the monkey kidney cell line CV-1 as well as the human prostate cell lines PC-3 and DU145 cells [4,5]. However, it depends on the cell type or conditions whether TGFb1 potentiates [5] or represses AR [4]. It is therefore of considerable interest to examine potential modulation by TGFb1 and its downstream signaling for AR transcriptional activity in balding dermal papilla cells (bald DPCs). To address this issue, we used mouse mammary tumor virus long-terminal repeat (MMTV)-luciferase assays to examine whether TGF-b1 can alter AR transactivation in bald DPCs. The DPCs obtained at passages 4– 6 from an AGA bald frontal scalp were cultured on a 12-well plate in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% charcoal-treated fetal calf serum (FCS) (JRH Biosciences, Lenexa, KS, USA), penicillin (50 units/ml) and streptomycin (50 mg/ml) at 37 8C in a humidified atmosphere of 95% O2 and 5% CO2. At subconfluency, cells were transiently transfected by means of Fugene 6 (Roche Diagnostic Corp., Indianapolis, IN) with 0.1 mg pSG5-AR, 0.3 mg MMTV-luciferase reporter plasmid and 0.1 mg pRL-CMV vector as an internal control and 24 h later the medium was refreshed and 1 nM R1881 and 0.2 or 2.0 ng/ml of human recombinant TGF-b1 (R&D Systems Inc., Minneapolis, MN) or the corresponding mocks were added to the culture. After incubation for 24 h, the cells were harvested and subjected to luciferase assays using the DualLuciferase reporter assay system (Promega, Madison, WI). The results showed that 0.2 or 2.0 ng/ml of TGF-b1 can significantly enhance AR activity by a factor of 1.9 or 2.3 (Fig. 1), respectively, indicating that TGF-b1 signaling positively stimulates AR transactivation in bald DPCs. Next, to investigate the need for Smad3 to obtain this effect by TGF-b1, we examined the effect of Smad3 knockdown by siRNA on TGF-b1-induced AR transactivation. The bald DPCs were transiently transfected with 0.1 mg pSG5-AR, 0.3 mg MMTV-luciferase reporter plasmid, 0.1 mg pRLCMV, and 100 pg/ml siRNA against Smad3 (siTrio, NM_005902; BBridge International, Inc., Cupertino, CA) or control RNA (siTrio
negative control). Twenty hours later, the medium was refreshed and 1 nM R1881, 0.2 ng/ml human recombinant TGF-b1 or one of the corresponding mocks was added to the culture. After incubation for 24 h, the cells were harvested and subjected to luciferase assays. The results demonstrated that knockdown of Smad3 eliminated the effect of TGF-b1 on MMTV-luciferase activity (Fig. 2A, upper panel), indicating that Smad3 is necessary for TGF-b1 to exert its effect. The successful knockdown of Smad3 by siRNA was confirmed in this experiment (Fig. 2A, lower panel). In addition, because interaction of Smad3 and Hic-5/ARA55, which we previously reported is an androgen sensitivity regulator in DPCs [6], has been proven [7,8], we studied the effect of TGF-b1 on AR activity by using the MMTV-luciferase assays for bald DPCs
Fig. 1. Effect of TGF-b1 on transfected androgen receptor transactivity in balding dermal papilla cells (DPCs). The DPCs from AGA bald frontal scalp at subconfluency in a 12-well plate were transiently transfected at passages 4–6 with 0.1 mg pSG5AR, 0.3 mg MMTV-luciferase reporter plasmid and 0.1 mg pRL-CMV vector using Fugene 6 as an internal control. At 24 h after transfection, 1 nM R1881 (lanes 2–4), synthetic androgen, or an ethanol mock solution (lane 1), and TGF-b1 at the indicated concentration (lanes 3 and 4) or a corresponding mock solution (4 mM HCl/0.1% BSA) (lanes 1 and 2) was added to the culture. After incubation for 24 h, the cells were harvested and subjected to luciferase assays. Each luciferase activity (relative LUC) is shown relative to the mean transactivation observed in the absence of TGF-b1 and the presence of R1881 (lane 2). Bars represent the mean standard deviations of three independent experiments. *p < 0.05; n.s., not significant (p > 0.05); Mann–Whitney’s U test.
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Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151
Fig. 2. Effect of Smad3 knockdown and Hic-5/ARA55 overexpression on transfected androgen receptor transactivity in balding dermal papilla cells. (A, upper panel) The bald DPCs were transiently transfected with pSG5-AR, MMTV-luciferase reporter plasmid, pRL-CMV (lanes 1–4), and 100 pg/ml siRNA against Smad3 (lane 4) or control RNA (lanes 1–3), as mentioned in Fig. 1. At 24 h after transfection, 1 nM R1881 (lanes 2–4), synthetic androgen, or an ethanol mock solution (lane 1), and 0.2 ng/ml TGF-b1 (lanes 3 and 4) or a corresponding mock solution (4 mM HCl/0.1% BSA) (lanes 1 and 2) was added to the culture. After 24 h incubation, the cells were harvested and subjected to the luciferase assays. (A, lower panel) The knockdown of Smad3 was confirmed by semiquantitative RT-PCR. The following oligonucleotide primers were used: for Smad3 as sense primer: 50 -GAGTAGAGACGCCAGTTCTACC-30 and as antisense primer: 50 -GGTTTGGAGAACCTGCGTCCAT-30 ; for glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3phosphate dehydrogenase (G3PDH) as internal control: 50 -CCCATCACCATCTTCCAG-30 and 50 -CCTGCTTCACCACCTTCT-30 . PCR amplification was performed as 30 cycles of denaturation at 94 8C for 15 s, annealing at 62 8C for 30 s and extension at 72 8C for 2 min for Smad3 or 23 cycles of denaturation at 94 8C for 30 s, annealing at 55 8C for 30 s, and extension at 72 8C for 30 s for G3PDH. (B, upper panel) The bald DPCs were transiently transfected with pSG5-AR, MMTV-luciferase reporter plasmid, pRL-CMV and 0.5 mg pSG5-ARA55 (lanes 1–3). At 24 h after transfection, 1 nM R1881 (lanes 2 and 3), or mock ethanol (lane 1), and 0.2 ng/ml TGF-b1 (lane 3) or mock solution (4 mM HCl/0.1% BSA) (lanes 1 and 2) were added to the culture. (B, lower panel) The bald DPCs transiently transfected with 0.5 mg pSG5 mock vector (left lane) or pSG5-ARA55 (right lane) were lysed in 1% Nonidet P-40, 0.4 M NaCl and aprotinin and 5 mg of cell lysate protein per lane were loaded onto Novex1 4–12% Tris-glycine gel (EC60352box) (Invitrogen, CA, USA) and transferred to nitrocellulose membranes. The membrane was soaked in 5% skimmed milk in phosphate-buffered saline/0.05% Tween 20 for 2 h at room temperature and then incubated with anti-Hic-5 monoclonal IgG antibody (BD transduction laboratories, San Jose, CA) at a 1:500 dilution or monoclonal anti-b-actin IgG antibody (Sigma–Aldrich, St. Louis, MO) at a 1:15,000 dilution in Can get signal1 immunoreaction enhancer solution (Toyobo life science, Tokyo, Japan) for 2 h at room temperature. After being washed three times at intervals of 10 min with phosphate-buffered saline/0.05% Tween 20, the membranes were incubated with horseradish peroxidase linked sheep anti-mouse IgG (NA934V and NA931V, GE Healthcare, Piscataway, NJ) at a 1:10,000 dilution for 1 h at room temperature. Each luciferase activity (relative LUC) is shown relative to the mean transactivation observed in the absence of TGF-b1 and the presence of R1881 (lane 2 in upper panels of A and B). Bars represent the mean standard deviation of three independent experiments. *p < 0.05; n.s., not significant (p > 0.05); Mann–Whitney’s U test (upper panels of A and B).
overexpressing Hic-5/ARA55 as a result of transient transfection with its expression vector. The assay results showed that TGF-b1 did not have any significant effect on AR transcription (Fig. 2B, upper panel), suggesting that Hic-5/ARA55 impeded the enhancement of AR activity by TGF-b1. The successful overexpression of Hic-5/ARA55 generated by its expression vector was confirmed in this experiment (Fig. 2B, lower panel). Our data presented here suggest that TGF-b1 can enhance androgen sensitivity through Smad3 in the dermal papilla of AGA in an autocrine manner. Because TGF-b1 from bald DPCs inhibits hair follicle epithelial cell growth in a paracrine manner [1], TGFb1 exerts its pathogenic roles with dual secretion, autocrine and paracrine, between epithelium and dermal papilla in AGA. On the other hand, although Hic-5/ARA55 upregulates androgen sensitivity via coactivation for AR in DPCs [6], the data obtained in our current study indicated that this molecule impedes the AR stimulation by TGF-b1. This may be due to crosstalk between Hic5/ARA55 and Smad3 [8] or possibly the attenuated effect of TGF-b1 on the high expression of Hic-5/ARA55, which is reportedly increased by TGF-b1 [9]. Given that Hic-5/ARA55 is highly expressed in the androgen-sensitive DPCs from AGA [6], a complex compensatory mechanism through reciprocal interaction must be in place between TGF-b-Smad and androgen-AR signaling pathways in the hair follicles of AGA. Because the function of crosstalk
between the AR-Hic-5/ARA55 and TGF-b-Smad pathways depends on the cell type or organ [5,8], the interaction or crosstalk of these and various other molecules supposedly determines whether Hic-5/ARA55 and Smad3 function as positive or negative modulators. According to a very recent report, normal testis growth and maturation require coordinated and interdependent activin/TGF-b and androgen signaling with tightly regulated production of Smad3 in order to control the balance between cell growth, differentiation, and maturation [10]. Putting these findings together suggests that a properly balanced crosstalk may be required for normal physiology of hair cycling as well as pathogenesis of AGA. In conclusion, the findings of our in vivo investigation of AGA indicate that TGF-b1 produced by androgen from bald DPCs suppresses epithelial cell growth and simultaneously enhances androgen sensitivity in bald DPCs in an autocrine manner. Acknowledgement We thank Dr. Chawnshang Chang at University of Rochester for his kind gift of the plasmids and Mrs. Naoko Yamada for her excellent technical assistance. This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.
Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151
References [1] Inui S, Fukuzato Y, Nakajima T, Yoshikawa K, Itami S. Androgen-inducible TGFbeta1 from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of androgen on human hair growth. FASEB J 2002;16:1967–9. [2] Inui S, Itami S. Molecular basis of androgenetic alopecia: from androgen to paracrine mediators through dermal papilla. J Dermatol Sci 2011;61:1–6. [3] Foitzik K, Lindner G, Mueller-Roever S, Maurer M, Botchkareva N, Botchkarev V, et al. Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo. FASEB J 2000;14:752–60. [4] Hayes SA, Zarnegar M, Sharma M, Yang F, Peehl DM, ten Dijke P, et al. SMAD3 represses androgen receptor-mediated transcription. Cancer Res 2001;61: 2112–8. [5] Kang HY, Lin HK, Hu YC, Yeh S, Huang KE, Chang C. From transforming growth factor-beta signaling to androgen action: identification of Smad3 as an androgen receptor coregulator in prostate cancer cells. Proc Natl Acad Sci USA 2001;98:3018–23. [6] Inui S, Fukuzato Y, Nakajima T, Kurata S, Itami S. Androgen receptor coactivator Hic-5/ARA55 as a molecular regulator of androgen sensitivity in dermal papilla cells of human hair follicles. J Invest Dermatol 2007;127: 2302–6. [7] Shibanuma M, Kim-Kaneyama JR, Sato S, Nose K. A LIM protein. Hic-5, functions as a potential coactivator for Sp1. J Cell Biochem 2004;91: 633–45.
151
[8] Wang H, Song K, Sponseller TL, Danielpour D. Novel function of androgen receptor-associated protein 55/Hic-5 as a negative regulator of Smad3 signaling. J Biol Chem 2005;280:5154–62. [9] Fujimoto N, Yeh S, Kang HY, Inui S, Chang HC, Mizokami A, et al. Cloning and characterization of androgen receptor coactivator. ARA55, in human prostate. J Biol Chem 1999;274:8316–21. [10] Itman C, Wong C, Hunyadi B, Ernst M, Jans DA, Loveland KL. Smad3 dosage determines androgen responsiveness and sets the pace of postnatal testis development. Endocrinology 2011;152:2076–89.
Shigeki Inui*, Satoshi Itami Department of Regenerative Dermatology Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita-shi, Osaka 565-0871, Japan *Corresponding author. Tel.: +81 6 6879 3031; fax: +81 6 6879 3039 E-mail address: [email protected] (S. Inui). 19 July 2011 doi:10.1016/j.jdermsci.2011.08.010
Naoki Oiso Department of Dermatology, Kinki University School of Medicine, Osaka, Japan Atsushi Tanemura Department of Dermatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Ichiro Katayama Department of Dermatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan *Corresponding author. Tel.: +81 6 6879 3031 E-mail address: [email protected] (M. Wataya-Kaneda)
Akira Kawada Department of Dermatology, Kinki University School of Medicine, Osaka, Japan Tamio Suzuki Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan
149
Received 4 April 2011 Revised 17 August 2011 Accepted 21 August 2011 doi:10.1016/j.jdermsci.2011.08.006
Letter to the Editor Androgen receptor transactivity is potentiated by TGF-b1 through Smad3 but checked by its coactivator Hic-5/ARA55 in balding dermal papilla cells We previously reported that TGF-b1 is a paracrine mediator from dermal papilla to hair follicle epithelium in the pathogenesis of androgenetic alopecia (AGA) [1,2]. Because TGF-b1 can induce catagen in hair cycling [3], it has been suggested that it functions as a paracrine pathogenic mediator from dermal papilla in AGA. On the other hand, TGF-b1 reportedly modulates androgen receptor (AR) transactivation in the monkey kidney cell line CV-1 as well as the human prostate cell lines PC-3 and DU145 cells [4,5]. However, it depends on the cell type or conditions whether TGFb1 potentiates [5] or represses AR [4]. It is therefore of considerable interest to examine potential modulation by TGFb1 and its downstream signaling for AR transcriptional activity in balding dermal papilla cells (bald DPCs). To address this issue, we used mouse mammary tumor virus long-terminal repeat (MMTV)-luciferase assays to examine whether TGF-b1 can alter AR transactivation in bald DPCs. The DPCs obtained at passages 4– 6 from an AGA bald frontal scalp were cultured on a 12-well plate in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% charcoal-treated fetal calf serum (FCS) (JRH Biosciences, Lenexa, KS, USA), penicillin (50 units/ml) and streptomycin (50 mg/ml) at 37 8C in a humidified atmosphere of 95% O2 and 5% CO2. At subconfluency, cells were transiently transfected by means of Fugene 6 (Roche Diagnostic Corp., Indianapolis, IN) with 0.1 mg pSG5-AR, 0.3 mg MMTV-luciferase reporter plasmid and 0.1 mg pRL-CMV vector as an internal control and 24 h later the medium was refreshed and 1 nM R1881 and 0.2 or 2.0 ng/ml of human recombinant TGF-b1 (R&D Systems Inc., Minneapolis, MN) or the corresponding mocks were added to the culture. After incubation for 24 h, the cells were harvested and subjected to luciferase assays using the DualLuciferase reporter assay system (Promega, Madison, WI). The results showed that 0.2 or 2.0 ng/ml of TGF-b1 can significantly enhance AR activity by a factor of 1.9 or 2.3 (Fig. 1), respectively, indicating that TGF-b1 signaling positively stimulates AR transactivation in bald DPCs. Next, to investigate the need for Smad3 to obtain this effect by TGF-b1, we examined the effect of Smad3 knockdown by siRNA on TGF-b1-induced AR transactivation. The bald DPCs were transiently transfected with 0.1 mg pSG5-AR, 0.3 mg MMTV-luciferase reporter plasmid, 0.1 mg pRLCMV, and 100 pg/ml siRNA against Smad3 (siTrio, NM_005902; BBridge International, Inc., Cupertino, CA) or control RNA (siTrio
negative control). Twenty hours later, the medium was refreshed and 1 nM R1881, 0.2 ng/ml human recombinant TGF-b1 or one of the corresponding mocks was added to the culture. After incubation for 24 h, the cells were harvested and subjected to luciferase assays. The results demonstrated that knockdown of Smad3 eliminated the effect of TGF-b1 on MMTV-luciferase activity (Fig. 2A, upper panel), indicating that Smad3 is necessary for TGF-b1 to exert its effect. The successful knockdown of Smad3 by siRNA was confirmed in this experiment (Fig. 2A, lower panel). In addition, because interaction of Smad3 and Hic-5/ARA55, which we previously reported is an androgen sensitivity regulator in DPCs [6], has been proven [7,8], we studied the effect of TGF-b1 on AR activity by using the MMTV-luciferase assays for bald DPCs
Fig. 1. Effect of TGF-b1 on transfected androgen receptor transactivity in balding dermal papilla cells (DPCs). The DPCs from AGA bald frontal scalp at subconfluency in a 12-well plate were transiently transfected at passages 4–6 with 0.1 mg pSG5AR, 0.3 mg MMTV-luciferase reporter plasmid and 0.1 mg pRL-CMV vector using Fugene 6 as an internal control. At 24 h after transfection, 1 nM R1881 (lanes 2–4), synthetic androgen, or an ethanol mock solution (lane 1), and TGF-b1 at the indicated concentration (lanes 3 and 4) or a corresponding mock solution (4 mM HCl/0.1% BSA) (lanes 1 and 2) was added to the culture. After incubation for 24 h, the cells were harvested and subjected to luciferase assays. Each luciferase activity (relative LUC) is shown relative to the mean transactivation observed in the absence of TGF-b1 and the presence of R1881 (lane 2). Bars represent the mean standard deviations of three independent experiments. *p < 0.05; n.s., not significant (p > 0.05); Mann–Whitney’s U test.
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Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151
Fig. 2. Effect of Smad3 knockdown and Hic-5/ARA55 overexpression on transfected androgen receptor transactivity in balding dermal papilla cells. (A, upper panel) The bald DPCs were transiently transfected with pSG5-AR, MMTV-luciferase reporter plasmid, pRL-CMV (lanes 1–4), and 100 pg/ml siRNA against Smad3 (lane 4) or control RNA (lanes 1–3), as mentioned in Fig. 1. At 24 h after transfection, 1 nM R1881 (lanes 2–4), synthetic androgen, or an ethanol mock solution (lane 1), and 0.2 ng/ml TGF-b1 (lanes 3 and 4) or a corresponding mock solution (4 mM HCl/0.1% BSA) (lanes 1 and 2) was added to the culture. After 24 h incubation, the cells were harvested and subjected to the luciferase assays. (A, lower panel) The knockdown of Smad3 was confirmed by semiquantitative RT-PCR. The following oligonucleotide primers were used: for Smad3 as sense primer: 50 -GAGTAGAGACGCCAGTTCTACC-30 and as antisense primer: 50 -GGTTTGGAGAACCTGCGTCCAT-30 ; for glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3phosphate dehydrogenase (G3PDH) as internal control: 50 -CCCATCACCATCTTCCAG-30 and 50 -CCTGCTTCACCACCTTCT-30 . PCR amplification was performed as 30 cycles of denaturation at 94 8C for 15 s, annealing at 62 8C for 30 s and extension at 72 8C for 2 min for Smad3 or 23 cycles of denaturation at 94 8C for 30 s, annealing at 55 8C for 30 s, and extension at 72 8C for 30 s for G3PDH. (B, upper panel) The bald DPCs were transiently transfected with pSG5-AR, MMTV-luciferase reporter plasmid, pRL-CMV and 0.5 mg pSG5-ARA55 (lanes 1–3). At 24 h after transfection, 1 nM R1881 (lanes 2 and 3), or mock ethanol (lane 1), and 0.2 ng/ml TGF-b1 (lane 3) or mock solution (4 mM HCl/0.1% BSA) (lanes 1 and 2) were added to the culture. (B, lower panel) The bald DPCs transiently transfected with 0.5 mg pSG5 mock vector (left lane) or pSG5-ARA55 (right lane) were lysed in 1% Nonidet P-40, 0.4 M NaCl and aprotinin and 5 mg of cell lysate protein per lane were loaded onto Novex1 4–12% Tris-glycine gel (EC60352box) (Invitrogen, CA, USA) and transferred to nitrocellulose membranes. The membrane was soaked in 5% skimmed milk in phosphate-buffered saline/0.05% Tween 20 for 2 h at room temperature and then incubated with anti-Hic-5 monoclonal IgG antibody (BD transduction laboratories, San Jose, CA) at a 1:500 dilution or monoclonal anti-b-actin IgG antibody (Sigma–Aldrich, St. Louis, MO) at a 1:15,000 dilution in Can get signal1 immunoreaction enhancer solution (Toyobo life science, Tokyo, Japan) for 2 h at room temperature. After being washed three times at intervals of 10 min with phosphate-buffered saline/0.05% Tween 20, the membranes were incubated with horseradish peroxidase linked sheep anti-mouse IgG (NA934V and NA931V, GE Healthcare, Piscataway, NJ) at a 1:10,000 dilution for 1 h at room temperature. Each luciferase activity (relative LUC) is shown relative to the mean transactivation observed in the absence of TGF-b1 and the presence of R1881 (lane 2 in upper panels of A and B). Bars represent the mean standard deviation of three independent experiments. *p < 0.05; n.s., not significant (p > 0.05); Mann–Whitney’s U test (upper panels of A and B).
overexpressing Hic-5/ARA55 as a result of transient transfection with its expression vector. The assay results showed that TGF-b1 did not have any significant effect on AR transcription (Fig. 2B, upper panel), suggesting that Hic-5/ARA55 impeded the enhancement of AR activity by TGF-b1. The successful overexpression of Hic-5/ARA55 generated by its expression vector was confirmed in this experiment (Fig. 2B, lower panel). Our data presented here suggest that TGF-b1 can enhance androgen sensitivity through Smad3 in the dermal papilla of AGA in an autocrine manner. Because TGF-b1 from bald DPCs inhibits hair follicle epithelial cell growth in a paracrine manner [1], TGFb1 exerts its pathogenic roles with dual secretion, autocrine and paracrine, between epithelium and dermal papilla in AGA. On the other hand, although Hic-5/ARA55 upregulates androgen sensitivity via coactivation for AR in DPCs [6], the data obtained in our current study indicated that this molecule impedes the AR stimulation by TGF-b1. This may be due to crosstalk between Hic5/ARA55 and Smad3 [8] or possibly the attenuated effect of TGF-b1 on the high expression of Hic-5/ARA55, which is reportedly increased by TGF-b1 [9]. Given that Hic-5/ARA55 is highly expressed in the androgen-sensitive DPCs from AGA [6], a complex compensatory mechanism through reciprocal interaction must be in place between TGF-b-Smad and androgen-AR signaling pathways in the hair follicles of AGA. Because the function of crosstalk
between the AR-Hic-5/ARA55 and TGF-b-Smad pathways depends on the cell type or organ [5,8], the interaction or crosstalk of these and various other molecules supposedly determines whether Hic-5/ARA55 and Smad3 function as positive or negative modulators. According to a very recent report, normal testis growth and maturation require coordinated and interdependent activin/TGF-b and androgen signaling with tightly regulated production of Smad3 in order to control the balance between cell growth, differentiation, and maturation [10]. Putting these findings together suggests that a properly balanced crosstalk may be required for normal physiology of hair cycling as well as pathogenesis of AGA. In conclusion, the findings of our in vivo investigation of AGA indicate that TGF-b1 produced by androgen from bald DPCs suppresses epithelial cell growth and simultaneously enhances androgen sensitivity in bald DPCs in an autocrine manner. Acknowledgement We thank Dr. Chawnshang Chang at University of Rochester for his kind gift of the plasmids and Mrs. Naoko Yamada for her excellent technical assistance. This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.
Letters to the Editor / Journal of Dermatological Science 64 (2011) 142–151
References [1] Inui S, Fukuzato Y, Nakajima T, Yoshikawa K, Itami S. Androgen-inducible TGFbeta1 from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of androgen on human hair growth. FASEB J 2002;16:1967–9. [2] Inui S, Itami S. Molecular basis of androgenetic alopecia: from androgen to paracrine mediators through dermal papilla. J Dermatol Sci 2011;61:1–6. [3] Foitzik K, Lindner G, Mueller-Roever S, Maurer M, Botchkareva N, Botchkarev V, et al. Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo. FASEB J 2000;14:752–60. [4] Hayes SA, Zarnegar M, Sharma M, Yang F, Peehl DM, ten Dijke P, et al. SMAD3 represses androgen receptor-mediated transcription. Cancer Res 2001;61: 2112–8. [5] Kang HY, Lin HK, Hu YC, Yeh S, Huang KE, Chang C. From transforming growth factor-beta signaling to androgen action: identification of Smad3 as an androgen receptor coregulator in prostate cancer cells. Proc Natl Acad Sci USA 2001;98:3018–23. [6] Inui S, Fukuzato Y, Nakajima T, Kurata S, Itami S. Androgen receptor coactivator Hic-5/ARA55 as a molecular regulator of androgen sensitivity in dermal papilla cells of human hair follicles. J Invest Dermatol 2007;127: 2302–6. [7] Shibanuma M, Kim-Kaneyama JR, Sato S, Nose K. A LIM protein. Hic-5, functions as a potential coactivator for Sp1. J Cell Biochem 2004;91: 633–45.
151
[8] Wang H, Song K, Sponseller TL, Danielpour D. Novel function of androgen receptor-associated protein 55/Hic-5 as a negative regulator of Smad3 signaling. J Biol Chem 2005;280:5154–62. [9] Fujimoto N, Yeh S, Kang HY, Inui S, Chang HC, Mizokami A, et al. Cloning and characterization of androgen receptor coactivator. ARA55, in human prostate. J Biol Chem 1999;274:8316–21. [10] Itman C, Wong C, Hunyadi B, Ernst M, Jans DA, Loveland KL. Smad3 dosage determines androgen responsiveness and sets the pace of postnatal testis development. Endocrinology 2011;152:2076–89.
Shigeki Inui*, Satoshi Itami Department of Regenerative Dermatology Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita-shi, Osaka 565-0871, Japan *Corresponding author. Tel.: +81 6 6879 3031; fax: +81 6 6879 3039 E-mail address: [email protected] (S. Inui). 19 July 2011 doi:10.1016/j.jdermsci.2011.08.010