Human scalp hair follicles are both a target and a source of prolactin, which serves as an autocrine and/or paracrine promoter of apoptosis-driven hair follicle regression

Abstract

The prototypic pituitary hormone prolactin (PRL) exerts a wide variety of bioregulatory effects in mammals and is also found in extrapituitary sites, including murine skin. Here, we show by reverse transcriptase-polymerase chain reaction and immunohistology that, contrary to a previous report, human skin and normal human scalp hair follicles (HFs), in particular, express both PRL and PRL receptors (PRL-R) at the mRNA and protein level. PRL and PRL-R immunoreactivity can be detected in the epithelium of human anagen VI HFs, while the HF mesenchyme is negative. During the HF transformation from growth (anagen) to apoptosis-driven regression (catagen), PRL and PRL-R immunoreactivity appear up-regulated. Treatment of organ-cultured human scalp HFs with high-dose PRL (400 ng/ml) results in a significant inhibition of hair shaft elongation and premature catagen development, along with reduced proliferation and increased apoptosis of hair bulb keratinocytes (Ki-67/terminal dUTP nick-end labeling immunohistomorphometry). This shows that PRL receptors, expressed in HFs, are functional and that human skin and human scalp HFs are both direct targets and sources of PRL. Our data suggest that PRL acts as an autocrine hair growth modulator with catagen-promoting functions and that the hair growth-inhibitory effects of PRL demonstrated here may underlie the as yet ill-understood hair loss in patients with hyper-prolactinemia.

Figure 1

A–D: PRL-like immunoreactivity (A, C) and PRL-R-like immunoreactivity (B, D) in isolated human anagen (A, B) and catagen (C, D) HFs were stained using the ABC method with AEC+ as substrate and hematoxylin (blue) for counterstaining. IRS, inner root sheath; ORS, outer root sheath; M, melanocytes; DP, dermal papilla; HS, hair shaft; MK, matrix keratinocytes. Arrows designate PRL and PRL-R staining. E–H: Macroscopic and histological demonstration of human HFs after 8 days in culture. E and G: Light microscopy; E and F: vehicle control; F and H: H&E staining (anagen VI); G and H: 400 ng/ml PRL (catagen III). PRL induced cessation of pigmentation, the shape of the dermal papilla changed into a more condensed shape, the volume of the hair matrix diminishes, and the pigmented lower end of the hair shaft moved upward in isolated HFs. All are characteristic events in catagen development.

Figure 2

a: Hair shaft elongation of PRL-treated HFs compared to vehicle control (n = 63 HFs per group) after 0, 2, 4, 6, and 8 days in culture. ***P < 0001. b: Percentage of HFs at defined hair cycle stages after 8 days in culture were assessed and statistical significance was calculated using Mann-Whitney U-test. *P < 0.05. c: Calculation of the HCS. All HFs of each group were staged and each stage of the hair cycle has been scored as follows: anagen VI = 100, early catagen = 200, mid catagen = 300. The HCS indicates the mean of the stages of all HFs per group. *P < 0.05.

Figure 3

a: Total mean number of Ki-67⁺ and TUNEL⁺ cells after 8 days in culture. PRL-treated HFs show a down-regulation of proliferating cells and an increase of apoptotic cells. *P < 0.05, **P < 0.01. b: Outer root sheath of a control HF. c: Outer root sheath of a HF of the PRL-treated group. d: Hair bulb vehicle control. e: Hair bulb PRL-treated group. Ki-67/TUNEL double staining of anagen VI follicles after 8 days in culture. Red fluorescence, Ki-67⁺ cells; green fluorescence, TUNEL⁺ cells; blue fluorescence, DAPI counterstaining.

Figure 4

RT-PCR of PRL (a) and GAPDH (b). Total RNA was extracted from a pituitary gland, 30 freshly isolated human HFs, and full-thickness scalp skin. Lane 1, specific PCR signal using pituitary gland cDNA; lane 2, specific PCR signal with scalp skin cDNA; lane 3, isolated human HFs; lane 4, negative control.