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A role of melatonin in neuroectodermal-mesodermal interactions: the hair follicle synthesizes melatonin and expresses functional melatonin receptors

Hiromi Kobayashi^*,, Arno Kromminga^,1, Thomas W. Dunlop^,1, Birte Tychsen^*, Franziska Conrad^*, Naoto Suzuki^||, Ai Memezawa, Albrecht Bettermann^*, Setsuya Aiba, Carsten Carlberg and Ralf Paus^*,2

^* Department of Dermatology, and
Department of Dermatology, University Hospital Schleswig-Holstein, Campus Lübeck, University of Lübeck, Lübeck, Germany;
Institute for Immunology, Clinical Pathology & Molecular Medicine, Hamburg, Germany;
Deptartment of Biochemistry, University of Kuopio, Kuopio, Finland; and
^|| Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan

² Correspondence: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Lübeck, University of Lübeck, D-23538 Lübeck, Germany. E-mail: ralf.paus{at}derma.uni-luebeck.de

SPECIFIC AIMS

Given that the hair follicle (HF) is a prototypic neuroectodermal-mesodermal interaction system, in which the peripheral functions of melatonin in situ can be dissected under physiologically relevant conditions, we studied whether the HF is a site of melatonin synthesis and whether it expresses functional receptors that respond to melatonin stimulation.

PRINCIPAL FINDINGS

We used immunohistochemistry, radioimmunoassay, and liquid chromatography/mass spectrometry (LC/MS/MS) to detect melatonin in murine and/or human HFs and to compare results with the melatonin serum level. We also probed whether norepinephrine (NE), the key endogenous stimulus for pineal melatonin synthesis, further stimulates the melatonin content of isolated HFs. The skin and HF expression of cognate melatonin receptors in murine skin (i.e., the recognized melatonin membrane receptors [MT1, MT2], and the nuclear mediator of melatonin signaling, ROR) were assessed by immunohistology and/or real-time PCR. Finally, the functionality of these receptors was probed in organ-cultured murine skin and human HFs by assessing whether melatonin administration altered HF growth parameters, pigmentation, and/or estrogen receptor- expression.

1. Melatonin-like immunoreactivity (IR) is expressed both in human scalp HFs and in murine HFs, where melatonin-like IR is hair cycle-dependent
By immunohistochemistry, both human and murine HFs showed prominent melatonin-like IR (Fig. 1 ). Melatonin-like IR was detected in outer root sheath (ORS) and selected inner root sheath (IRS) keratinocytes of murine HFs in the growth and regression stages of the hair cycle (anagen, catagen), while it appeared absent in "resting" HFs (telogen) (Fig. 1A ). Therefore, melatonin-like IR in murine HFs appears to be hair cycle-dependent. Melatonin-like IR was also seen in the sebaceous gland (SG) (Fig. 1A ) at a level clearly above background (Fig. 1A+B ; positive control: murine pineal gland, Fig. 1E+F ).

View larger version (79K): [in this window] [in a new window]   Figure 1. Melatonin like-IR in mouse and human HFs. Melatonin-like IR was seen both in murine HFs during different stages of the depilation-induced murine hair cycle (A; anagen IV: mid-growth phase; catagen VI: late regression phase; Telogen: "quiescence" phase) and in human scalp HF (C) in anagen (stained by the avidin-biotin-peroxidase complex (ABC) method using Fast Red (red) as substrate and Mayer’s hemalaun for counterstaining). As a positive control, mouse pineal gland shows melatonin-like IR (E). Higher magnification is shown in the square. Background controls: B, D, F. The summary of hair cycle-dependent changes in melatonin-like IR in EP, lower part of IRS and ORS keratinocytes of anagen and catagen HFs and in the SG (red). EP, epidermis; Club, telogen shaft base; DP, dermal papilla; IRS, inner root sheath; ORS, outer root sheath; SG, sebaceous gland; CTS, connective tissue sheath; DP, dermal papilla. Scale bars: A, B) 10 µm; C–F) 50 µm; (square in panel E) 10 µm.

2. The melatonin content of murine skin and mouse vibrissae follicles is substantially higher than that of serum
Radioimmunoassay and LC/MS/MS were used as independent, mutually confirmatory techniques to detect whether the HF is an extrapineal site of melatonin synthesis after it had been separated from its normal vascular and neural connections.

In organ-cultured skin of C57BL/6 mice with all HFs in anagen, the melatonin level was 5- to 10-fold higher than the serum level measured (mean: 0.5 pg/mg total protein; peak level: 1.1 pg/mg). This is striking since this mouse strain has long been claimed to be a "natural melatonin knockout." In microdissected vibrissae follicles of C57 BL/6 mice, the melatonin level was even 50- to 100-fold higher than the peak serum level. Similar findings were made with microdissected human scalp HFs compared with the recognized peak melatonin serum level in humans (0.1–0.5 pg/mg total protein). This suggests that murine skin and HFs as well as human HFs are important extrapineal sites of melatonin synthesis.

3. NE stimulation increases the melatonin content of organ cultured murine skin, vibrissae follicles, and human HFs
The above data might still be explained by a passive accumulation of serum-derived melatonin in the skin and its appendages (e.g., via cutaneous melatonin receptors). We probed whether the melatonin content of skin and/or HFs that had been separated from any vascular or neural connections could be further stimulated in vitro by the key endogenous stimulus for pineal melatonin synthesis, NE. This was indeed the case. This was independently confirmed by LC/MS/MS: a much more prominent melatonin-associated peak was detected in NE-stimulated human HFs than in vehicle-treated humans HFs. This provides further evidence that murine and human skin, in particular its HFs, are a prominent site of extrapineal melatonin synthesis.

4. Murine skin transcribes the MT2 gene in a hair cycle-dependent manner
To study whether murine HFs express transcripts for high-affinity melatonin cell surface receptors, real-time PCR for the membrane melatonin receptors [MT1, MT2] was performed on murine whole skin cDNA prepared from the back skin of C57BL/6 mice with all HFs in defined stages of the depilation-induced murine hair cycle. Real-time PCR revealed that murine skin only transcribes the MT2 gene in detectable amounts and that MT2 steady-state mRNA levels change in a hair cycle-dependent manner: MT2 transcripts are significantly up-regulated in late anagen and are maximal in murine back skin with all HF in catagen (for details, see full online text). This suggests an involvement of MT-2-mediated melatonin signaling in hair cycle control.

5. ROR is hair cycle-dependently expressed in murine skin
Murine skin also expresses a recently recognized mediator of nuclear melatonin signaling, the retinoid orphan receptor ROR. By real-time PCR, hair cycle-dependent intracutaneous transcription of the ROR gene was demonstrated (for details, see full online text). ROR mRNA steady state-levels were significantly down-regulated in late anagen but up-regulated in late catagen skin, suggesting an involvement of these nuclear melatonin receptors in the control of HF cycling. By immunohistochemistry of C57BL/6 mouse skin in different hair cycle stages, ROR-like IR was detected, suggesting that ROR transcripts are translated.

6. Melatonin inhibits murine keratinocyte apoptosis in situ
To obtain indications as to whether the detected melatonin receptors (MT2 and/or ROR) are functionally active, melatonin (1 or 0.01 nM for 6 or 12 h) was added to organ-cultured skin of C57BL/6 mice with all HFs in defined hair cycle stages. In telogen skin this significantly increased the percentage of TUNEL+ apoptotic cells in the HF epithelium compared with vehicle controls (P<0.01) whereas HF proliferation was unaffected.

7. Melatonin does not significantly modulate human HF growth or pigmentation in vitro
It has been reported that melatonin inhibits melanogenesis in cultured HFs of Siberian hamsters and promotes human hair growth in vivo after topical application. However, when melatonin was added to organ-cultured frontotemporal human scalp HFs in anagen VI (derived from female facelift patients), hair shaft elongation, hair bulb keratinocyte apoptosis and proliferation, HF cycling, and HF pigmentation in situ were not altered significantly by a wide range of melatonin concentration (1 µM to 1 pM) after 48 h or 6 days of HF organ culture. Thus, significant effects of melatonin on human hair shaft elongation or on human HF growth and pigmentation in vitro could not be revealed.

8. Melatonin down-regulates ER expression in murine HFs in situ
Since melatonin down-regulates ER expression in vitro and ER-mediated signaling is a powerful modulator of HF growth and cycling in vivo, we examined ER expression in mouse skin by real-time PCR and immunohistochemistry. ER receptors were found to be transcribed in a hair cycle-dependent manner. Compared with vehicle controls, ER-like IR was down-regulated in HF matrix and IRS keratinocytes as well as in dermal papilla fibroblasts in situ in melatonin-treated, organ-cultured murine skin. By real-time PCR, ER mRNA expression was found to be significantly decreased after melatonin treatment in organ-cultured telogen and anagen mouse skin.

CONCLUSIONS AND SIGNIFICANCE

These observations suggest that the HF is not only a target of melatonin bioregulation, but also an important extrapineal site of melatonin synthesis.

That both murine and human HFs are prominent sites of extrapineal melatonin synthesis (alongside and beyond such recognized sites as the retina, gut, liver, kidney, spleen, and ovary) confirms and extends the recent landmark studies by Slominski et al., who have shown that the serotoninergic and melatoninergic systems are expressed and functionally active in human skin cells in vitro. Our study provides the missing evidence that normal murine and human skin, most notably the HF, actually synthesize melatonin in situ and can be stimulated to do so by the same endogenous stimulus that promotes pineal gland melatonin synthesis (NE).

We show that if stringently controlled, quantitative PCR techniques are used, only MT2 and ROR are transcribed at detectable levels among the membrane melatonin receptors tested (MT1, MT2, ROR). This makes it unlikely that MT1 plays a significant role in murine skin and hair biology. In contrast, ROR, a newly recognized nuclear mediator of melatonin signaling, is prominently expressed in murine skin, both on the gene and protein level.

While the hair cycle-dependent changes in MT2 and ROR expression (Fig. 2 A, B) suggest that signaling through these receptors is somehow involved in the control of HF cycling, it remains to be systematically dissected in subsequent studies whether both ROR and MT2 are functionally important melatonin receptors in murine skin, and which of these melatonin receptors is responsible for defined functional effects of a defined concentration of melatonin.

View larger version (28K): [in this window] [in a new window]   Figure 2. Hair cycle-dependent changes in melatonin and ROR-like IR. In murine back skin sections, melatonin-like IR was detected in EP, lower part of IRS and ORS keratinocytes in a hair cycle-dependent manner with an apparent maximum in anagen and catagen HFs. ROR, the nuclear mediator of melatonin signaling, was most prominent in the mesenchymal DP of mid-anagen HFs (anagen IV) and was also seen in the IRS and ORS in the upper part of the HF epithelium as well as in the epidermis from mid-anagen to telogen. This study demonstrate that melatonin may play a role in hair cycle regulation, since HF is a both a substantial source of melatonin and an important target via its receptors whish are expressed in murine skin in a hair cycle-dependent manner.

A key challenge is to define the role of locally generated melatonin in skin and hair biology. Low doses of melatonin (0.01 and 1 nM) inhibited HF keratinocyte apoptosis in situ in short-term mouse skin organ culture. Given that the dissection and preparation of mouse skin for organ culture is traumatic and likely associated with the generation of large amounts of reactive oxygen species, the antiapoptotic effect of melatonin seen here could well be due to the antioxidant effects of melatonin as a potent free radical scavenger. This finding is in line with previous reports that melatonin inhibits apoptosis of cultured isolated keratinocytes, immune cells and neuronal cells in vitro.

We provide the first evidence that ER gene and protein expression in murine HFs in situ is down-regulated by melatonin. ER signaling is a key modulator of HF cycling and, by modulating TGF or IGF-I expression, has potent yet complex hair growth-modulatory properties in all mammalian species examined so far. Therefore, melatonin may exert substantial indirect hair growth- modulatory effects in humans and/or mice via down-regulating ER expression. In subsequent human HF organ culture assays, it therefore deserves to be tested whether melatonin is capable of modulating human follicle keratinocyte functions (e.g., apoptosis) in the presence of estrogen. This suggests that melatonin can indirectly modulate hair growth by desensitizing the HFs to the strong hair growth-regulatory effects of natural ER ligands like 17-ß-estradiol, which are synthesized and metabolized in the pilosebaceous unit itself.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2293fje;

¹ Both authors contributed equally to this study.

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