0 1991 MUNKSGAARD
Pigment Cell Research 4:193-198 (1991)
Evidence for Alpha-MSH Binding Sites on Human Scalp Hair Follicles: Preliminary Results P.B. NANNINGA,’ G.E. GHANEM,2 F.J. LEJEUNE,2 J.D. BOS,’ AND w.WESTERHOF’ ’Department of Dermatology, Academisch Medisch Centrum, University of Amsterdam, Amsterdam, The Netherlands; ’Laboratoire d’ Oncologie et de Chirurgie Experimentale, Institut Jules Bordet, Faculte de Medecine, Universite Libre de Bruxelles, Bruxelles, Belgium
Alpha-MSH, considered an important pigmentation hormone, binds to melanocytes and is thought to stimulate melanogenesis through a cyclic-AMP-dependent mechanism. The binding of alpha-MSH to follicular melanocytes has been investigated in human hair of different colors, ranging from black to blond and senile white. Hairs were plucked, the follicles were cut off, and an alpha-MSH binding assay, using a radiolabeled alpha-MSH analogue, was performed on these bulbs. As controls of each assay, fragments of hairs of the same person were used. The results show a dose-response relationship and the assay seems to be specific for alpha-MSH, because other peptides such as ACTH, beta-LPH and beta-endorphins do not compete for binding sites as alpha-MSH does. These binding sites seem to be present only on melanin synthesizing melanocytes, since the controls and follicles of senile white hair, which do not contain active melanocytes, show negative results. All the assays were performed on raw material, i.e., whole plucked hair follicles. This is the first time that binding sites for alpha-MSH have been demonstrated on human scalp hair follicles. In addition, their presence was found to be associated with active melanin production; their absence was demonstrated on senile white hair follicles. Key words: Alpha-MSH binding sites, Hair follicles, Senile greying
INTRODUCTION Normally, senile greying of human scalp hair is related to aging and is a common occurrence in man. Visible senile “greying”of the skin normally does not takeplace. However, the number of active epidermal melanocytes decreases with age at a rate of approximately 10% per decade (Ortonne et al., 1983). Melanocytes that populate the epidermis and hair folliclesare morphologicallysimilar, but physiologically they may act independently. The physiological mechanisms of senile greying are not clearly understood. The most accepted explanation for this process is a combination of a decrease of tyrosinase activity and a reduction in the number of follicular melanocytes (Herzberg and Guser, 1970; Lloyd et al., 1987). In white hair follicles, active melanocytes are absent. However, under certain circumstance+i.e., inflammatory conditions, radiotherapy, sympathectomy, and under the influence of some drugs-a temporary darkening of previously white hair has been observed (Cline, 1988; Lerner 1966; Rook and Dawber, 1991). This implies that even in the completely white hair, the follicle contains inactive melanocytes that can be reactivated. At the transition from the anagen to the catagen phase in a normal hair growth cycle, the follicles of the scalp show
a lightening of pigment at the base of the hair shaft. The follicular melanocytes cease producing melanin, retract their dendrites, and become recognizable as clear cells between the surrounding keratinocytes. Such clear cells have been demonstrated in animal as well as human hair follicles (Sato et al., 1973; Sugiyama and Kukita, 1976). At this stage no tyrosinase activity is detectable. This cyclic character of melanocytes during the hair growth cycle points out that there must exist certain stimuli to reactivate this “dormant”melanocyte, because at the beginning of the new anagen the melanocytes reappear, duplicate, and renew their melanin synthesis. Why follicularmelanocytes do not reappear in the follicle at a certain moment, resulting in white hair, remains unclear. Alpha-MSH is considered as the main pigmentation hormone in many vertebrates, including humans. Recently, the binding of alpha-MSH to Address reprint requests toW. Westerhof, Academisch Medisch Centrum, Department of Dermatology, Meibergdreef 9,1105 AZ Amsterdam, The Netherlands. Received July 23, 1991; accepted October 2,1991. Preliminary results of this work were presented at the 14‘h International Pigment Cell Conference (1990) in Kobe, Japan.
Nanninga et al.
U C -d
m 0 -rl
cc .d u al a
a Specific Binding (cprn ~ 1 0 0 0 )
Blond hair follicles
1Black hair follicles
Log a-MSH Molarity
Fig. 1. a: Time course of binding of the iodine-labeled alpha-MSH analogue after incubation at 37°C. F’rom these results it was decided to set the incubation time at 1 hr, since longer incubation does not increase the binding significantly. b: Displacement curves obtained from an alpha-MSH analogue binding assay with black ( + ) and blond (0)hair bulbs. The a-MSH molarity on the horizontal axis is of the cold hormone. Black hair bulbs show more binding than blond ones with ratio percentages from 18%to 56%, respectively.
the cell surface of melanocytes was studied in mouse melanoma and in human melanoma cell lines (Legros et al., 1981; Ghanem et al., 1988, 1989; Siegrist et al., 1988). In these studies evidence was found for the existence of specific alpha-MSH binding sites or receptors on melanoma cells in culture. The process of senile greying could be the result of a reduced response of melanocytes to alpha-MSH (Levine et al., 1987). Therefore, we investigated whether human scalp hair follicles in pigmented hairs have alpha-MSH binding sites. Furthermore, senile white hairs were investigated to ascertain the absence of binding sites. A radiolabeled alpha-MSH analogue (Libert et al., 1989)was used
for the first time in receptor binding assays on human hair bulbs. This is a very attractive approach, because a method is used that does not depend on the chemical and enzymatical reactions in pigment synthesis.
MATERIALS AND METHODS Scalp hairs of different colors, ranging from black t o senile white, were randomly plucked with epilating forceps (Peereboom-Wynia,1982).The hair bulbs were cut off with fine scissors in such a way that approximately 0.5 cm of the proximal hair, the bulb included, was collected. Fifty bulbs were collectedin each polypropylenetube (Bio-Freeze Vials, Cambridge, MA, USA), previously filled with 1.8
Alpha-MSH and Hair Follicles Binding (cpm ~ 1 0 0 0 )
\ alpha-MSH -14
Log Peptide Molarity Fig. 2. Speciiicity study in binding assays showing alpha-MSH @); desacetyl-alpha-MSH ( + );
ACTA,-% (*); beta-LPH ( x ) and beta-Endorphin (0).The peptide molarities on the horizontal axis are of the different cold hormones. This figure is a compilation of different experiments, thus explaining the different amounts of cpm's.
ml minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), (pH 7.5) and tightly closed.
As control in each experiment, a tube filled with 50 fragments of terminal hair (0.5 cm long) of the same person was used. These tubes were filled with medium of the same composition as described above. Alpha-MSH Binding Assay
All tubes were centrifuged for 30 min at 3500g at room temperature. The supernatants were discarded and 100 I-L~ of MEM, containing 10% FCS, were added to each tube followed by 350,000to 1,000,000cpm of an alpha-MSH analabeled with 125-iodine logue (Nle4-DPhe7)-alpha-MSHl-13 as described before (Ghanem, 1988).In each assay the Same amount of radioactive a-MSH analogue in all the tubes was used. The amount of tracer differed however, in the various experiments, because they were performed as quickly as possible after collecting the hair bulbs. The incubation was performed at 3'7°C in a water bath for 60 min, at the end of which different dilutions of the "cold" analogue were added (finalconcentrations:10- 14, lo-", lo-", lo-', and M, respectively). The tubes were incubated under the same conditionsfor an additional 60 min with the "cold" peptide and then centrifuged as stated above. The control tube, containing 50 fragments of hair without hair bulbs, was treated with the 10 - l4 M dilution. For the specificity studies the same method was used. Hair bulbs were collected from subjects with dark blond hair. After incubation with the labeled alpha-MSH analogue, the second incubation was carried out with the following
peptides: desacetyl-alpha-MSH,beta-endorphin, ACTH1-= and beta-LPH. The same final concentrations,as described above, of these peptides were added in the relevant assays. After one washing with 1.5 ml of phosphate-buffered saline, containing 1%bovine serum albumin, the tubes were centrifuged and the pellets' associatedradioactivity counted in a gamma counter.
RESUEIS "b investigate whether any binding would occur, 50 hairs
were plucked at random from five healthy volunteers with hair colors ranging from black to blond. By plucking the hairs at random, about 85%of the follicles will be in anagen. The results we obtained showed that significant binding took place. Time course of binding and dose-response curves were established in 10 tubes with black hair bulbs and 10 tubes with blond hair bulbs, each with their controls. The result of the time course of binding is shown in Figure la. Black hair bulbs show more binding than blond ones, with ratio percentages from 18%to 56%, respectively (Fig. lb). Peptide specificity was studied with the following pep tides: alpha-MSH, desacetyl-alpha-MSH, ACTH1-=, betaendorphin and beta-LPH. In Figure 2 the results of the various assays are shown. Alpha-MSH could reproducibly displace the labeled hormone from its binding sites. An assay with desacetyl-alpha-MSH gives a similar curve, ACTHI-, s h m a slight displacement. Beta-endorphin and beta-LPH do not seem to compete with alpha-MSH binding sites (Fig. 2). In all the experiments the controls did not show any displacement. Results of the assay with senile
Nanninga et al.
196 cpm (Thousands) 3 1
Log a-MSH Molarity Fig. 3. Alpha-MSH binding site assay on senile white hair follicles obtained from three different persons. The a-MSH molarity on the horizontal axis is of the cold hormone. The mean (solid line) and the higMow (dashed lines) range are shown.
white hairs of three different persons (Fig. 3) show no displacement in any of the series tested. In Figure 3 the mean and higMow values are shown. On two occasions Scatchard plots were calculated from the binding assays. The displacement curves reveal two binding sites, the first one is within the usual range of affinities and the other is very low (Fig. 4; only one Scatchard plot is shown). From the Kd values (Kdl: 3.9 x lo-’ and and 4.2 x an average 1.4 x lop8, Kd2: 2.2 x number of binding sites was calculated; 2,500 binding sites per 50 hair follicles. Because the assays were performed on plucked hairs, i.e., on raw material, leaving the bulbs as they appeared, this number of binding sites refers to the number of cells exposed in 50 hair follicles.
DISCUSSION Alpha-MSH is an important pigmentation hormone in many vertebrates, including humans. Darkening of human skin after injection of alpha-MSH was reported by Lerner (Lerner et al., 1961). Experiments were carried out in numerous animal models in which a positive effect of alphaMSH on the hair color in mice and guinea pigs was described (Clive and Snell, 1967; Geschwind, 1966, Geschwind et al., 1972; Levine et al., 1987). In short-term cultures of hair follicles of hamsters, alpha-MSH stimulates melanogenesisthrough a cyclic-AMP dependent mechanism by which an increased tyrosinase activity is obtained (Weatherhead and Logan, 1981). In this study hair bulbs from human plucked hairs were used. Experiments carried out by Burchill et al. (Burchill et al., 1986, Burchill and Thody, 1986)on mouse skin and hair follicles, in which the effect of alpha-MSH on tyrosinase
synthesis was measured, showed an increase of the enzyme, especially in young mice, which produce eumelanin. In older mice, with a phaeomelanin production, no increase in tyrosinase was observed and there was no response to alpha-MSH. Although evidence was found for the existence of alphaMSH specific binding sites on human malignant melanocytes and melanoma extracts in vitro, these binding sites or possible receptors have as yet not been identified on normal human melanocytes (Ghanem et al., 1988and 1989). The results of the experiments of the present study show a reproducible displacement of labeled alpha-MSH analogue by the “cold”hormone in different assays. Desacetylalpha-MSH shows similar results. The other peptides (beta-LPH and beta-endorphin) seem not to compete in the same manner with the alpha-MSH binding sites. Whereas ACTHI-=, shows a minimal displacement, which is seen only at certain (around lo-’’ M) concentrations of the hormone. The main problem encountered in this assay is the fact that the small pieces of tissue (whole hair bulbs) that are used, contain epidermal and dermal cells, fragments of hairs, debris, etc., and not a homogenous pure cell population of follicular melanocytes. In the pigmented hair follicles, the presence of melanin might interfere by binding the peptide or radioactive iodine. However, in the controls, in fragments of pigmented hairs that contain melanin as well, no binding was observed. The amount of tracer bound to the hair bulbs was found to be sensitive in some extent to variations in the acidity of the medium, mainly due to the addition of hormones stored in acidic solutions. This variation was found only at
Alpha-MSH and Hair Follicles
Fig. 4. Scatchard plot calculated from one assay. See the discussion for explanation.
high concentrations M), i.e., at the level of the nonspecific binding, and was corrected for afterwards. At low concentrations(10-'4-10-8) no problems wereencountered. pH values below 7.4 resulted in an increase of the binding in relation to the increase of acidity. On the other hand, nonspecificbinding obtained from hair fragments and from the maximum displacement of the tracer a t lop6M, differed in general. This may be explained by the fact that the assay was performed on raw material, increasing in this way the nonspecific binding values. In the Scatchard plot analysis two binding sites were revealed (Fig. 4). The exact meaning of this phenomenon is not clear. Possible explanations could be that there exist high and low affinity binding sites or binding occurs on keratinocytes as well. By labeling, the alpha-MSH analogue molecule could have changed and is less or differently recognized by the binding site, or that binding sites are blocked by some autocrine hormone. Last, the fact that raw material was used might play a role. Alpha-MSH analogue binding sites seem to be present exclusively on hair bulbs with active melanocytes, i.e., in pigmented hairs, since the assay was negative when performed on the controls', hair fragments without any bulbs and on senile white hair follicles. In the latter no active melanocytes are present. Furthermore, black hairs with a high metabolic activity in their melanocytes, show more binding than those that are less active (blond hairs). These results indicate that repigmentation as found sometimes in completely white hair (Cline, 1988; Lerner, 1966; Rook and Dawber, 1991)is probably not achieved by the stirnulatory effect of alpha-MSH, since no binding sites for alphaMSH could be demonstrated on white hair follicles. The melanocytes in white hair follicles may have lost their bind-
ing sites due to a transition to a dormant state (Levine et al., 1987). In these melanocytes the genes coding for the alpha-MSH binding sites could be blocked or shut off. Aging of the follicular melanocyte population may play an important role in these processes. The overall result is a nonpigmented hair. In some cases the inactive melanocytes are reactivated after appropriate stimuli and the binding sites reappear. However, the exact mechanisms in the process of transition and reactivation have to be elucidated.
ACKNOWLEDGMENTS This research was supported by a grant from the Lawrence M. Gelb Research Foundation, Stamford Connecticut, USA. REFERENCES Burchill S.A. and A.J. Thody (1986) Melanocyte-stimulating hormone and the regulation of tyrosinase activity in hair follicular melanocytes of the mouse. J. Endocrinol., 111225-232. Burchill S.A., A.J. Thody, and S. Ito (1988)Melanocyte-stimdationhormone, tyrosinaseactivity and the regulation of eumelanogenesis and phaeomelanogenesisin the hair follicular melanocytes of the mouse. J. Endocrinol. 10915-21. Cline D.J. (1988)Changes in hair color. Dermatol. Clin. 6295-303. Clive D. and R. Snell(1967) Effect of the alpha-melanocytestimulating hormone on mammalian hair color. J. Invest. Dermatol. 49:314-321. Geschwind 1.1. (1966) Change in hair color in mice induced by injection of alpha-MSH. Endocrinology 791165-1167. Geschwind I.I., R.A. Huseby, and R. Nishioka (1972) The effect of alpha-MSH on coat color in the mouse. Rec. Prog. Horm. Res. 28:01-120. Ghanem G.E., G. Comunale, A. Libert. A. Vercammen-Grandjean, and F.J. Lejeune (1988) Evidence for alpha-melanocytestimulating hormone (alpha-MSH)receptorson human malignant melanoma cells. Int. J. Cancer 41248-255.
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