Human hair follicle pigmentary unit as a direct target for modulators of melanogenesis, as studied by [14C]-2-Thiouracil incorporation
Abstract
Abstract: The purpose of this study was to evaluate human hair follicle melanogenic activity using the [14C]-2-thiouracil, which was known to incorporate into nascent melanins. Results obtained on pigmented, grey and non-pigmented hair follicles demonstrated that [14C]-2-TU incorporation was restricted to the melanogenic compartment with a strong accumulation located around dermal papilla and within the fibre of pigmented hair follicles. Quantitative analysis of [14C]-2-TU incorporation showed a significant increase in pigmented hair follicles upon stimulation with 1 μm forskolin concomitant to an increase in tyrosinase levels. A strong significant decrease in [14C]-2-TU incorporation was noted, when hair follicles were incubated with the tyrosinase competitive inhibitor kojic acid (200 μm). Incubation with the MC1-R agonist α-MSH (0.2 μm) did not induce a significant stimulation of hair melanogenesis. The present model could thus represent a useful new tool to identify modulators of human hair pigmentation.
Introduction
Hair pigmentation involves a population of differentiated melanocytes located in the basal layer around the upper part of dermal papilla. These active melanocytes synthesize melanins within melanosomes and transfer them to the hair fibre precortical keratinocytes (1). Previous studies performed in skin organ cultures, in cultured melanocytes and in melanocytes–keratinocytes co-cultures have used [14C]-2-TU as a specific pigmentation marker of synthesis of nascent melanins (2–5). After injection in mice, high selective retention of 2-[14C]-TU also has been shown in melanoma cells, eyes and hair follicles (6). Recently, assays performed on anagen human hairs using Fontana–Masson staining in vitro have demonstrated a possible stimulation of hair melanogenesis by T-oligos (7), but such studies are scarce and well known potent modulators of pigmentation remain to be assessed using a quantitative method on whole hair follicles in vitro.
Question addressed
Using 2-[14C]-thiouracil the aim of our study was (i) to specify and quantify the synthesis of nascent melanin in the anagen hair follicle pigmentation unit in vitro and (ii) to test the effect on hair pigmentation of PKA pathway activators such as forskolin and α-MSH as well as the tyrosinase inhibitor, kojic acid.
Experimental design
Human hair follicles were microdissected from temporal region biopsies obtained from dark hair caucasian female volunteers (>48 years old) after informed consent (8), donor 1 (75), donor 2 (57), for assays on non-pigmented HF versus fully pigmented HF (60, 60), for assays with kojic acid (donor 1 and a 48-year-old woman). Isolated fully pigmented, grey and non-pigmented human hair follicles were incubated with compounds of interest for various durations in William’s E medium supplemented with 3 μCi/ml [14C]-2-Thiouracil (60 mCi/mmol; Amersham Biosciences, Piscataway, NJ, USA).
Qualitative analysis
Hair follicles were embedded in Tissue-Tek OCT compound (Miles, Naperville, IL, USA). Longitudinal cryosections were prepared on a CM3050 cryostat (Leica Microsystèmes, Rueil-Malmaison, France), covered with a scintillator sheet (Biospace Mesures, Paris, France) and analysed using a Micro-Imager M25 equipment (Biospace Mesures) (9).
Quantitative analysis
Experiments were performed on non-pigmented or fully pigmented anagen hair follicles. 24 hair follicles were assigned to each group, incubated as above, treated for 3–5 days and then lysed in soluen-350 (Packard instrument Company, Meriden, CT, USA). Optiphase hisafe supermix scintillation cocktail (Wallac, Turku, Finland) was then added and counts (cpm) were measured on a 1450 Trilux Scintillation Counter (Perkin-Elmer, Courtaboeuf, France).
Western blot analysis of tyrosinase
Fully pigmented human hair follicles were treated with 1 μm forskolin (3 and 5 days). Proteins were then extracted and analysed (30 μg) by SDS–PAGE and Western blot using a monoclonal antibody against human tyrosinase (NCL-tyros, Novocastra, Newcastle, UK) and a polyclonal antibody against human P42/P44 MAP kinase (#9102-Cell Signaling Technology, Ozyme, St Quentin Fallavier, France).
Results
In fully pigmented hair follicles (FHF), merged pictures (M, Fig. 1, right) showed a high [14C]-2-TU incorporation in the melanogenic compartment and its transfer to the fibre. In grey hair, the [14C]-2-TU incorporation seemed to be correlated with the number of melanocytes (Fig. 1, central). Abnormal signal was sometimes also observed in outer root sheath, reflecting ectopic location of some still active melanocytes. As expected, the grey hair pigmentation unit remained active when melanocytes were properly located. However, a strong variability was noted in the signal intensity, disqualifying grey hairs for quantitative assays. In non-pigmented hair follicle, only a weak incorporation of [14C]-2-TU with no transfer to the hair fibre was observed (Fig. 1, left). In FHF treated with 200 μm tyrosinase inhibitor kojic acid, a significant reduction in [14C]-2-TU incorporation (−56%, p < 0.001, Mann–Whitney test) was observed, whereas a significant increase in melanogenesis (+29% to +49% in two representative experiments, p < 0.05, Kruskall–Wallis and Mann–Whitney test) was obtained under 1 μm forskolin stimulation. Of note, when used at this concentration, forskolin did not inhibit hair growth. This increase seemed to be linked to an increase in tyrosinase levels as shown by Western blot analysis (Fig. 2). A weak but non significant stimulation of [14C]-2-TU incorporation was measured with 0.2 μmα-MSH (+17%, p > 0.05, Mann–Whitney test).
[14C]-2-Thiouracil incorporation in vitro is linked to the potential of human hair follicle pigmentation. Upper panel: Bright field microscope picture of non-pigmented human hair bulb (top left), grey hair follicles with reduced number of melanocytes (<5 and >5 top centre) and fully pigmented hair follicles (top right). (A) autoradiographic pictures obtained from longitudinal sections of non-pigmented, grey and fully pigmented human hair follicles, after a 4-day incubation with [14C]-2-TU in vitro. (M): merged pictures of bright field and autoradiographic pictures. Lower panel: Quantitative measurement of [14C]-2-TU incorporation in non-pigmented and fully pigmented hair follicles of 24 individual hair counts (cpm ± standard deviation for each histogram).
Effect of pigmentation modulators on [14C]-2-Thiouracil incorporation in fully pigmented human hair follicle in vitro. (a) Representative experiment showing the effects of forskolin (1 μm) and α-MSH (0.2 μm) on hair melanogenesis in vitro, as quantitatively assessed by [14C]-2-TU incorporation. Measurement was performed in 24 individual hair follicles for each treatment (cpm ± standard deviation for each histogram, *p < 0.05, ***p < 0.001). Hair follicles were treated for either 3 days (donor 1) or 5 days (donor 2) and Western blot analysis for tyrosinase enzyme was performed on 30 μg crude protein extracts of forskolin treated (F) and not treated (C) hair follicles. ERK protein was chosen as control. (b) Effect of 200 μm kojic acid on hair follicle melanogenesis after a 5-day treatment (similar conditions).
Conclusion
Our results confirmed that the anagen human hair follicle pigmentation unit remained active for several days in vitro (7). We showed a gradual [14C]-2-TU incorporation from a low signal in non-pigmented hair follicle to a high signal coupled with an intense melanogenic activity in FHFs (Fig. 1). Our results (Fig. 2) showed that human hair melanogenesis could be modulated in vitro as illustrated by the inhibitory effect of kojic acid and the stimulatory effect of 1 μm forskolin, which permanently activated adenylate cyclase and hence cAMP production. This stimulation was correlated to an increase in tyrosinase synthesis. This result was not unexpected, as the PKA pathway is known to upregulate Microphthalmia associated transcription factor (MITF) expression (10,11) and consequently tyrosinase synthesis. In contrast, under our experimental conditions, the main representative Pro-opiomelanocortin-derived peptide (POMC) α-MSH (0.2 μm) failed to give an effect similar to that of forskolin. Although additional assays need to be performed with other POMC derivatives (12,13) as well as other potent cAMP activators, this may indicate that because of only transient signalling activation, intracellular cAMP increase in human hair follicle did not reach the threshold of efficacy in vitro. This low effect of α-MSH thus confirmed the very low processing machinery for α-MSH/MC1R signalling in the human hair follicle pigmentation unit (14). In addition, experiments performed on red hair carrying the R151C MC1R loss-of-function have shown that this mutation did not decrease the level of tyrosinase and Trp-1 enzymes (15). Interestingly, the variability of the results obtained with forskolin probably illustrated that inter-individual difference in hair melanogenesis in vitro was linked to various intrinsic/individual hair pigmentation potential in vivo. During ageing, hair pigmentation declines (16) because of a progressive exhaustion of both melanotic melanocytes and progenitor cells located in the outer root sheath (17). Our results suggest that as long as a few melanotic melanocytes remain active in a proper location, the use of melanin synthesis stimulators (18) could be a means to delay the perception of hair greying.
Acknowledgements
The authors thank Dr C. Bouillon and Dr M. Donovan for critical reading of the manuscript, Dr C. Duval, P. Sextius and P. Bastien for their help and advice.
