Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce catagen

Abstract

Here, we provide the first study of prolactin (PRL) and prolactin receptor (PRLR) expression during the nonseasonal murine hair cycle, which is, in contrast to sheep, comparable with the human scalp and report that both PRL and PRLR are stringently restricted to the hair follicle epithelium and are strongly hair cycle-dependent. In addition we show that PRL exerts functional effects on anagen hair follicles in murine skin organ culture by down-regulation of proliferation in follicular keratinocytes. In telogen follicles, PRL-like immunoreactivity was detected in outer root sheath (ORS) keratinocytes. During early anagen (III to IV), the developing inner root sheath (IRS) and the surrounding ORS were positive for PRL. In later anagen stages, PRL could be detected in the proximal IRS and the inner layer of the ORS. The regressing (catagen) follicle showed a strong expression of PRL in the proximal ORS. In early anagen, PRLR immunoreactivity occurred in the distal part of the ORS around the developing IRS, and subsequently to a restricted area of the more distal ORS during later anagen stages and during early catagen. The dermal papilla (DP) stayed negative for both PRL and PRLR throughout the cycle. Telogen follicles showed only a very weak PRLR staining of ORS keratinocytes. The long-form PRLR transcript was shown by real-time polymerase chain reaction to be transiently down-regulated during early anagen, whereas PRL transcripts were up-regulated during mid anagen. Addition of PRL (400 ng/ml) to anagen hair follicles in murine skin organ culture for 72 hours induced premature catagen development in vitro along with a decline in the number of proliferating hair bulb keratinocytes. These data support the intriguing concept that PRL is generated locally in the hair follicle epithelium and acts directly in an autocrine or paracrine manner to modulate the hair cycle.

Figure 1.

PRL-like immunoreactivity during the depilation-induced murine hair cycle was stained by the ABC method using AEC+ (red) as substrate and hematoxylin for counterstaining. A, Telogen; B, anagen III hair follicles; C, anagen IV; D, anagen VI; E, catagen III; and F, catagen VII. MC, germinal matrix cells. Bottom: Schematic representation of immunoreactivity patterns of PRL during the murine hair cycle. Black: PRL expression.

Figure 2.

PRLR immunoreactivity during the depilation-induced murine hair cycle. A, Telogen; B, anagen III; C, anagen IV; D, anagen VI; E, catagen III; and F, catagen VII. MC, germinal matrix cells. Bottom: Schematic representation of immunoreactivity patterns of PRLR during the murine hair cycle. Black: PRLR expression.

Figure 3.

Real-time PCR analysis of expression of PRLRs and their ligands in murine skin. Amplification of product was measured in real time and the cycle number at which the reaction entered exponential phase growth was detected. These values are compared to show relative changes in specific mRNA concentration and normalized to the expression of GAPDH. Each data point is the mean of three mice, except for day 3 (n = 6) and day 5 (n = 8). Error bars are SEM. The major stages of the hair follicle growth cycle are indicated at the bottom.

Figure 4.

H&E staining for quantitative histomorphometry after 72 hours in culture. A: Control hair follicles in late anagen VI (arrow points to DP) and early catagen II. B: PRL-treated follicles in catagen III to IV (arrowhead points to DP). C: Schematic drawing of control hair follicles mostly in late anagen VI. D: Schematic drawing of PRL (400 ng/ml)-treated hair follicles in catagen III to IV.

Figure 5.

A: The percentage of hair follicles in defined hair-cycle stages were assessed by histomorphometry, and the statistical significance was calculated using the Mann-Whitney U-test: *, P <0.05; **, P <0.01. B: Calculation of the hair-cycle score: all hair follicles of each group were classified and each stage of the hair cycle has been scored as follows: anagen VI/catagen I = 100, catagen II to III = 200, catagen IV to VI = 300. The hair-cycle score indicates the mean of the stages of all hair follicles per group. C: Total mean number of Ki-67+ cells in the hair bulbs after 72 hours of skin organ culture. PRL-treated hair follicles show a down-regulation of proliferating cells.

Figure 6.

Quantitative histomorphometry of hair follicles after 72 hours in culture directly after depilation. The percentage of hair follicles in defined hair-cycle stages were assessed by histomorphometry, and the statistical significance was calculated using Mann-Whitney U-test: *, P <0.05; **, P <0.01.