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Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol

Natsuho Ito^*, Taisuke Ito^*, Arno Kromminga, Albrecht Bettermann, Masahiro Takigawa^*, Frieder Kees, Rainer H. Straub and Ralf Paus^,1

^* Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan;
Department of Dermatology, University Hospital Hamburg-Eppendorf, University of Hamburg, Hamburg, Germany;
Institute for Immunology, Clinical Pathology and Molecular Medicine (IPM), Hamburg, Germany;
Department of Internal Medicine I, University Medical Center Regensburg, Regensburg, Germany; and
Department of Pharmacology, University of Regensburg, Regensburg, Germany

¹Correspondence: Department of Dermatology, University Hospital Hamburg-Eppendorf, University of Hamburg, Martinistraße 52, Hamburg D-20246, Germany. E-mail: paus{at}biochem.mpg.de

SPECIFIC AIMS

Previous research has shown that the skin and its major appendage, the hair follicle, are prominent target organs and potent sources of key players along the classical hypothalamic pituitary adrenal (HPA) axis [e.g., of corticotropin-releasing hormone (CRH) and of melanocortins like adrenocorticotropic hormone (ACTH) and -melanocyte-stimulating hormone (-MSH)]. Mammalian skin has been shown to possess the full enzymatic equipment for glucocorticoid synthesis. The aim of the present study, therefore, was to explore whether human skin, namely, normal human scalp hair follicles, contain a functional HPA axis, including the synthesis of cortisol and regulatory feedback loops as they characterize the central HPA axis, using microdissected, organ-cultured human scalp hair follicles stimulated with CRH, -MSH, ACTH, or hydrocortisone.

PRINCIPAL FINDINGS

1. POMC transcription and immunoreactivity (IR) for POMC products are up-regulated by CRH treatment
First, we studied whether CRH (comparable to the signaling that occurs between hypothalamus and pituitary gland) modulates the expression of proopiomelanocortin (POMC), from which ACTH and -MSH are generated by prohormone convertases. POMC mRNA expression in the outer root sheath (ORS) of organ-cultured human scalp hair follicles in situ and mRNA steady-state levels in proximal anagen VI hair follicles were significantly up-regulated by CRH treatment, compared with vehicle controls, as has been reported for sheet preparations of murine epidermis in vitro. -MSH- and ACTH-IR in the ORS was also significantly enhanced by CRH treatment (Fig. 1 A, B; Fig. 2 A, B). This implies that exogenous CRH up-regulates not only hair follicle POMC gene expression, but also POMC processing in human hair follicles via prohormone convertases (PC1, PC2), into functionally active POMC products (i.e., the melanocortins ACTH and -MSH, just as it happens in the pituitary gland after stimulation with hypothalamic CRH).

View larger version (82K): [in this window] [in a new window]   Figure 1. Immunoreactivity (IR) of key components of the HPA axis in organ-cultured human scalp hair follicles. A) ACTH-IR; B) -MSH-IR; C) CRH-R1/2-IR; D) cortisol-IR; E) GR-IR; F) CRH-IR. A–C) After CRH treatment, CRH-R1/2-IR, -MSH-IR, and ACTH-IR in the outer root sheath (ORS) were enhanced. D) After ACTH treatment, cortisol-IR in ORS was enhanced. E, F) After cortisol treatment, glucocorticoid receptor-IR in the ORS was enhanced while CRH-IR in basement membrane was down-regulated.

View larger version (32K): [in this window] [in a new window]   Figure 2. Semiquantitative analysis of key HPA axis elements in organ-cultured human hair follicles. The selected reference areas indicated in Fig. 3 were analyzed in the ORS by NIH image software. (Student’s t test; *significant differences to the vehicle control, *P<0.05) A) ACTH-IR; B) -MSH-IR; C) CRH-R1/2-IR; D) MC1R-IR; E) MC2R-IR; F) Cortisol-IR; G) GR-IR; H) CRH-IR. A–C) IR of CRH-R1/2 and POMC-derived peptides in ORS are up-regulated by CRH treatment. D, E) CRH treatment modulate melanocortin receptors (MC1-R, MC2-R) in ORS. F) ACTH up-regulates cortisol IR in ORS. G, H) Hydrocortisone up-regulated GR-IR in ORS, while hydrocortisone down-regulated CRH-IR in basement membrane.

2. CRH, -MSH, and ACTH modulate CRH- and melanocortin-receptor expression (CRH-R1/2, MC1-R, MC2-R)
We then asked whether stimulation of the cognate receptors for CRH, -MSH, and ACTH (CRH-R1/2, MC1-R, and MC2-R) by their specific ligands in human anagen hair follicles modulate the IR for these receptors in a manner comparable to what occurs in the pituitary gland or adrenal grand. CRH-R1/2-IR in ORS was significantly up-regulated by CRH treatment (Fig. 1C , Fig. 2C ). MC1-R- and MC2-R-IR in the ORS was enhanced by CRH treatment (Fig. 2D, E ). MC1-R-IR in the ORS was enhanced by -MSH, and MC2-R-IR in the ORS was enhanced by ACTH (data not shown), suggesting that CRH (directly or indirectly through ACTH and -MSH) modulated the expression of melanocortin receptors, just as it is seen in the central HPA axis.

3. ACTH up-regulates cortisol IR in human hair follicles
While the above data supported the existence of a peripheral equivalent of the hypothalamic-pituitary-axis in human hair follicles, evidence that CRH and/or ACTH actually affect the intrafollicular synthesis of cortisol, the prototype adrenal hormone along the classical HPA axis, was still missing. Therefore, we studied whether exogenous ACTH up-regulates cortisol-IR, using an anti-human cortisol antibody for radioimmunoassay (RIA) and appropriate positive and negative controls. Surprisingly, vehicle-treated control hair follicles from normal human scalp skin displayed prominent cortisol-IR in the ORS, hair matrix, dermal papilla, and connective tissue sheath (Fig. 1D ). After ACTH treatment, cortisol-IR was significantly up-regulated (Fig. 1D , Fig. 2F ), just as ACTH up-regulates cortisol production in the adrenal cortex.

4. CRH stimulates cortisol secretion by organ cultured human hair follicles
To further explore this immunohistological evidence for intrafollicular cortisol synthesis and its stimulation by ACTH, the cortisol level in medium conditioned by organ-cultured human anagen VI hair follicles was quantified by RIA. We tested the effects of CRH, rather than ACTH, since it is fair to assume that any observed increase in cortisol would be indirectly mediated via the previously documented up-regulation of intrafollicular ACTH processing from locally generated POMC prohormone. Testing CRH in this RIA assay allowed us to double-check and confirm the above semiquantitative cortisol immunohistochemistry data and to further corroborate the existence of a functional intrafollicular HPA axis equivalent.

Substantial levels of cortisol (above the level of hydrocortisone that is contained in the standard hair follicle organ culture medium) were already detected in the medium conditioned by control hair follicles. These cortisol levels were significantly up-regulated by CRH treatment compared with vehicle controls.

Using RIA analysis, we detected cortisol in homogenized hair follicle extract cultured without hydrocortisone for 6 days, suggesting that human hair follicles actually produce cortisol.

Taken together with the immunohistochemical evidence for up-regulation of cortisol-IR within the hair follicle itself (Fig. 1D , Fig. 2F ), these data collectively support the concept that human hair follicles actually synthesize and secrete cortisol and that this steroidogenesis is stimulated by CRH.

5. Human hair follicles have established HPA axis-like feedback systems
After hydrocortisone treatment, glucocorticoid receptor (GR)-IR was enhanced while CRH-IR was down-regulated (Fig. 1E, F , Fig. 2G, H ). This suggests that human hair follicles have established fully operational feedback systems.

6. CRH inhibits human hair follicle growth and stimulates hair follicle pigmentation
CRH (1x10^–7 M) significantly inhibited hair shaft production in vitro, induced premature hair follicle regression (catagen), inhibited hair matrix keratinocyte proliferation, and promoted apoptosis of hair follicle keratinocytes in organ-cultured human scalp hair follicles compared with vehicle control and ACTH- or hydrocortisone-treated groups.

CRH increased melanin production in the anagen hair bulb (as determined by Masson-Fontana histochemistry). This suggests that in addition to its role in the proposed HPA axis equivalent, intrafollicularly generated CRH can profoundly affect hair follicle growth, cycling, and pigmentation directly or indirectly via stimulation of POMC product synthesis in the hair follicle (e.g., of ACTH and/or -MSH).

CONCLUSIONS AND SIGNIFICANCE

Recently, the hair follicle and its associated sebaceous gland have become appreciated as major independent peripheral endocrine organs that not only synthesize a host of steroid and peptide hormones, including hypothamalic and pituitary hormones, but also metabolize them. Our study now shows that normal, organ-cultured human scalp hair follicles respond to CRH stimulation in a manner strikingly similar to what is seen in the classical HPA axis, including cortisol production and activation of regulatory feedback loops (Fig. 3 ).

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic diagram of the classical HPA axis and its proposed equivalent in the human hair follicle. The classical systemic pathway for response to physical and psychological stressors is the hypothalamic-pituitary-adrenal (HPA) axis. Hypothalamic production of the peptide hormone, CRH is the most proximal element of the HPA axis, and the major regulator for the pituitary expression of the proopiomelanocortin (POMC) gene. CRH controls the processing of the prohormone POMC into various POMC-derived neuropeptide hormones, such as adrenocorticotropic hormone (ACTH), and -melanocyte-stimulating hormone (-MSH) by prohormone convertase (PC1 or PC2). ACTH then stimulates the adrenal cortex to secrete the powerful antiinflammatory factor, cortisol. Glucocorticoid exerts its effects on target cells by interacting with intracellular glucocorticoid receptor (GR). Then cortisol terminates the stress response and attenuates CRH and POMC peptide production to close the feedback system. Based on the findings of the current study, we propose that the epithelium of human hair follicles has established a (more primitive?), noncompartmentalized, yet fully functional peripheral equivalent of the HPA axis. This serves as an extra-adrenal site of cortisol synthesis and employs complex regulatory feedback loops with striking similarities to the classical HPA axis, and might operate locally in the skin as coordinator and executor of peripheral stress responses.

This shows that human skin (namely, the hair follicle) has indeed established a fully functional peripheral equivalent of the classical HPA axis, and supports the concept of locally operating stress response systems in the skin, which execute and coordinate peripheral stress responses. In contrast to the central HPA axis, however, in the follicular HPA axis equivalent described here, individual levels of regulation (i.e., CRH release, CRH-R1/2 stimulation+ACTH secretion, melanocortin receptor stimulation+cortisol production and secretion), do not seem to be compartmentalized in distinct anatomical structures (comparable to hypothalamus, pituitary, and adrenal glands in the central HPA axis). Instead, all levels of regulation seem to colocalize and cooperate in the hair follicle epithelium. This raises the question whether similar, relatively primitive, and noncompartmentalized HPA axis equivalents operate locally in many other peripheral tissues besides the hair follicle.

The observed up-regulation of cortisol secretion of human scalp hair follicles by CRH is particularly noteworthy since it occurred long after these cutaneous miniorgans had been disconnected from the systemic HPA axis. To the best of our knowledge, this provides the first evidence that mammalian skin (specifically: human scalp hair follicles) in situ actually is capable of synthesizing and secreting cortisol. This is in line with previous reports that human skin expresses steroidogenic enzymes and relevant cofactors, and raises the possibility that follicle-derived cortisol may even exert para- and/or endocrine activities beyond the hair follicle.

Finally, the skin and its appendages deserve systematic exploration as potent sources for key players along the HPA axis (e.g., CRH, ACTH, and cortisol) whose intracutaneous expression might well be manipulated by topical application of drugs or cosmeceuticals in order to stimulate or suppress the endogenous synthesis and release pharmacologically relevant doses of these hormones in the skin, thereby hoping to significantly improve the effect-benefit-risk ratio compared with direct application of exogenous hormone.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1968fje

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