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Androgen-inducible TGF-ß1 from balding dermal papilla cells inhibits epithelial cell growth: a clue to understand paradoxical effects of androgen on human hair growth ¹

Department of Dermatology, Course of Molecular Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan

²Correspondence: Department of Dermatology, Course of Molecular Medicine, Graduate School of Medicine, Osaka University, 2–2, Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: itami{at}derma.med.osaka-u.ac.jp

SPECIFIC AIMS

The objective of this study is to establish an in vitro coculture system using human dermal papilla cells (DPCs) from androgenetic alopecia (AGA) and keratinocytes (KCs) to explore the role of androgens in the hair growth regulation.

PRINCIPAL FINDINGS

1. No significant effect of androgen on the growth of KCs cocultured with DPCs from AGA
We cocultured KCs and DPCs from AGA using transwell culture dishes and examined the effect of R1881 (methyltrienolone, synthetic androgen) on the growth of KCs. The addition of 10^-9 M R1881 showed no significant influence on the proliferation of KCs in the coculture. We then assumed that in vitro cultivation of DPCs might alter the expression level of androgen receptor (AR). Semiquantitative RT-PCR showed that AR mRNA in DPCs from AGA was decreased during subcultivation from the third to ninth passage. To solve this problem, AR was overexpressed in the DPCs by transfecting the AR expression vector pSG5-AR.

2. Androgen shows distinct effects on the growth of KCs cocultured with DPCs transfected with AR
After DPCs from beard, bald frontal, non-bald frontal, or occipital scalp were transiently transfected with the AR expression vector, DPCs and KCs were cocultured for 4 days in the presence of 10^-9 M R1881 or ethanol. R1881 stimulated the growth of KCs by 41% when cocultured with beard DPCs (Fig. 1 A, lane 1 vs. 2). In contrast, R1881 suppressed the growth of KCs by 50% in the coculture with DPCs from AGA (lane 3 vs. 4). R1881 showed no significant effect on the growth of KCs cocultured with non-bald frontal or occipital DPCs (lane 5 vs. 6 and lane 7 vs. 8). In the cocultures, R1881 did not show any significant effect on the proliferation of DPCs. These results suggested that this in vitro coculture of DPCs and KCs recapitulates well the in vivo interaction between DPCs and follicular epithelial cells especially in terms of the site-specific effect of androgens on hair growth. To rule out the possibility that androgen/AR complex in DPCs from non-bald frontal scalp has no effect on the growth of KCs due to the low expression or activity of transfected AR, we compared the expression level and transcriptional activity of AR in AR-transfected DPCs from bald and non-bald frontal scalp. Western blot analysis and reporter assays using MMTV-Luc showed that the expression level and transcriptional activity of AR were similar in AR-transfected DPCs from bald and non-bald frontal scalp (Fig. 1B ).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of androgen on the proliferation of KCs cocultured with DPCs from various body sites transiently transfected with pSG5-AR. A) KCs were cocultured with beard (lanes 1 and 2), bald frontal (lanes 3 and 4), non-bald frontal (lanes 5 and 6), or occipital DPCs (lanes 7 and 8) transfected with pSG5-AR. Cells were cultured in the absence (ethanol mock treatment: lanes 1, 3, 5, and 7) or presence of 10-9 M R1881 (lanes 2, 4, 6, and 8) for 4 days. B) In the upper panel, 40 µg of the cell lysates from DPCs from bald (left lane) or non-bald frontal scalp (right lane) transiently transfected with pSG5-AR were subjected to Western blot analysis with polyclonal antibodies against AR. The arrow indicates AR protein. In the lower panel, DPCs from bald frontal scalp (lanes 1 and 2) and non-bald frontal scalp (lanes 3 and 4) were transiently transfected with 0.1 µg of pSG5-AR, 0.3 µg of the reporter plasmid MMTV-Luc, and 0.1 µg of pRL-CMV vector. The cells were administered 10-9 M R1881 (lanes 2 and 4) or ethanol as a mock treatment (lanes 1 and 3). Luciferase activities were determined and expressed as relative activity of each reporter in the presence of ethanol only (lanes 1 or 3). Results are summarized from 3 independent sets using DPCs from the fifth to seventh passage and presented as mean ± SD *P < 0.05, **P < 0.01 vs. the respective controls.

3. TGF-ß1 is up-regulated by androgen in DPCs of human AGA
In the coculture of KCs and DPCs from AGA, androgen may alter expressions of some soluble factors that can be involved in the growth regulation of KCs. This idea prompted us to search responsible factors for the androgen-induced inhibition of KC growth. TGF-ß1 is known to inhibit human KC growth in vitro and induce catagen progression in hair follicles. Therefore, we focused on TGF-ß1 as a candidate mediator for the androgen-induced growth suppression of KCs. Semiquantitative RT-PCR showed that expression of TGF-ß1 in the DPCs from AGA in the coculture was significantly up-regulated by androgen.

4. Secretion of TGF-ß1 in the medium of the coculture of KCs and DPCs from AGA is up-regulated by androgen
To examine whether the up-regulation of TGF-ß1 by androgen can occur at the protein level, we measured the concentration of TGF-ß1 protein in the conditioned medium after the coculture of KCs and DPCs from AGA for 4 days by ELISA assays specific for active TGF-ß1. For the assays, we prepared the samples with or without activating latent TGF-ß1 by HCl to measure both total (active and latent) and active TGF-ß1 in the conditioned medium. The concentration of total TGF-ß1 in the conditioned medium treated with 10^-9 M R1881 was increased by 2.68-fold compared with the control (Fig. 2 , lanes 1 and 2). Anti-androgen CPA at the concentration of 10^-6 M completely diminished this increase, demonstrating that AR is involved in the induction of TGF-ß1 (lane 3). The concentration of active TGF-ß1 was 174.1 pg/mL in the medium treated with 10^-9 M R1881 but 12.9 pg/mL in the medium treated with ethanol (lanes 4 and 5). Likewise, this induction of active TGF-ß1 was reversed by 10^-6 M CPA (lane 6). From these results, TGF-ß1 activity is regulated by androgens through its production and activation. When DPCs from the non-bald frontal scalp were cocultured with KCs, 10^-9 M R1881 did not show any significant effect on the concentration of total and active TGF-ß1 in the conditioned medium (lanes 7–10). In the coculture of DPCs from AGA without transfection of AR and KCs, R1881 did not show any significant effect on the amount of active and total TGF-ß1 (lanes 11–14), suggesting that transfection of exogenous AR is necessary for the induction of TGF-ß1.

View larger version (30K): [in this window] [in a new window]   Figure 2. Androgen increases TGF-ß1 in the medium of the coculture of KCs and DPCs from AGA. After coculturing KCs and DPCs transfected with pSG5-AR from AGA (lanes 1–6), the non-bald frontal scalp (lanes 7–10), or pSG-5 as a mock vector (lanes 11–14) in the presence of 10-9 M R1881 (lanes 2, 5, 8, 10, 12, and 14), 10-9 M R1881 and 10-6 M CPA (lanes 3 and 6) or ethanol as a mock (lanes 1, 4, 7, 9, and 11) for 4 days, concentrations of total (active and latent) (lanes 1, 2, 3, 7, 8, 11, and 12) and active (lanes 4, 5, 6, 9, 10, 13, and 14) TGF-ß1 in the conditioned medium were measured by ELISA assays. In each case, the mean ± SD of 3 independent experiments. *P < 0.03 vs. the respective controls.

5. Androgen-induced growth inhibition of KCs is reversed by TGF-ß1 neutralization
To confirm the involvement of TGF-ß1 in androgen-induced growth inhibition of KCs, we examined the effect of the neutralizing antibody against human TGF-ß1 on this growth suppression of KCs in the coculture. The neutralizing antibody against TGF-ß1 at the concentration of 5.0 µg/mL almost completely reversed the inhibition of proliferation of KCs by 10^-9 M R1881 in this coculture system, demonstrating that TGF-ß1 mediates the signal from DPCs to KCs for their growth suppression.

CONCLUSION AND SIGNIFICANCE

Although androgen stimulates beard growth, it paradoxically suppresses the growth of frontal scalp hair of AGA in vivo. We first demonstrated the differences in biological characteristics among human DPCs from bald and other sites using coculture systems and subsequently established the in vitro culture system using human DPCs, which provides a clue to solve androgen paradox in hair growth regulation. Moreover, our results demonstrate that androgen-inducible TGF-ß1 derived from DPCs from AGA is responsible for the androgen-induced epithelial cell growth inhibition in the coculture. For the beard growth stimulation by androgen, insulin-like growth factor-I is known as the paracrine mediator from DPCs to epithelial cells (Fig. 3 ). However, such a paracrine mediator in the pathogenesis of AGA has never been identified until now. Here our data suggest that TGF-ß1 is a paracrine mediator for AGA (Fig. 3) . Our data also suggested that activation process of TGF-ß1 is involved in the androgen-induced suppression of epithelial cell growth. Further studies are therefore required to explore the activation mechanism.

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0043fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0043fje

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