Hormone influence on melanogenesis and spots formation
Authors: Tadokoro T. , Ozawa K. , Bonnet‐Duquennoy M. , Bonté F. 
Published: September 2010

The biological effects of hormones on human melanocytes are still being investigated. Several kinds of hormones have mitogenic and/or melanogenic effects on human melanocytes. For example, melanotropic hormones, such as α‐melanocyte‐stimulating hormone (α‐MSH) and adrenocorticotrophic hormone (ACTH), were shown to induce skin darkening some time ago, and more recently, the mechanism of up‐regulation of tyrosinase activity in human normal melanocytes by melanotropins through the cyclic AMP (cAMP) signaling pathway has been reported. cAMP is now recognized to play a key role in the regulation of skin pigmentation. α‐MSH is known to elevate intracellular cAMP levels through the melanocortin 1 receptor (MC1R) and this plays a critical role in the regulation of melanogenesis. Recently it has been recognized that cAMP elevating agents up‐regulate transcription of the MITF gene that in turn stimulates expression of the tyrosinase gene via MITF binding to its M‐box.

Solar lentigo is a benign pigmented spot on sun‐exposed skin especially on the back of the hands, arms and on the forehead. We have compared melanogenesis histological parameters in solar lentigo and in normal skin of Japanese women. We chose to analyze melanin distribution and expression of α‐MSH as mediators of human pigmentation, in solar lentigo and normal skin biopsies of photo‐exposed dorsal forearm. Punch biopsies were fixed with 4% formaldehyde and sections were stained with Fontana‐Masson technique to evaluate melanin distribution. Immunohistochemistry was carried out to demonstrate α‐MSH protein expression using rabbit polyclonal antibody anti‐human α‐MSH. Melanin distribution and relative expression of α‐MSH were analyzed and quantified using semiquantitative computer‐assisted image analysis. With this technique, we found that solar lentigo skin possessed 60% more α‐MSH expression than normal skin (Fig. 1).

Quantification of alpha MSH expression in solar lentigo and normal skin for 10 japanese subjects. Immunolocalization of alpha MSH in solar lentigo and normal skin. (a) Representative image of alpha MSH expression in solar lentigo. (b) Representative image of alpha MSH expression in normal skin. Alpha MSH expression was determined from the integrated intensity given areas of epidermis reported to the total surface of epidermis in each section. Four selected areas of each section were photographed and quantified. Statistical differences were evaluated using the paired Student's t‐test. Data are presented as the mean of 10 biopsies.

image

While it has been reported that human skin color is changed by castration or by injection of androgen and a recent in vitro study in contrast suggested that testosterone might reduce tyrosinase activities in human melanocytes through the decrease of the mRNA expression of MC1R, the precise mechanism involved in the regulation of skin pigmentation by androgens has remained unclear. To date, there have been few reports about the effects of androgens on human melanocytes. In a previous study, we demonstrated that human genital melanocytes are androgen target cells. Those melanocytes had androgen receptors (ARs) in their nuclei as detected by immunohistochemistry and they also had high levels of 5α ‐reductase activities, the enzyme that converts testosterone to its more active form, 5α‐dihydrotestosterone (DHT). R1881 (methyltrienolone, a potent synthetic androgen) stimulated tyrosinase activity when melanocytes were cultured continuously with TPA, hydrocortisone and BPE and then were treated with FBS [1]. When melanocytes were deprived of FBS and BPE for a prolonged time, the effects of androgen on tyrosinase activity were reduced. Furthermore, when cultured without FBS, BPE or TPA, prolonged exposure to R1881 down‐regulated tyrosinase activity when added together with FBS and BPE. These data suggest that FBS or BPE contain elements that inhibit tyrosinase activity when contacting melanocytes even if only briefly [2].

Recent advances in the understanding of androgen signaling indicate the specific membrane‐signaling pathway involved in Sex hormone‐binding globulin (SHBG) as well as in the androgen‐AR mechanism, a classical pathway. SHBG is a glycoprotein found in human plasma that possesses a high affinity for binding of sex hormones. It has been recognized that unbound, free steroids function on target cells through their intracellular receptors. Recently, it has been speculated that SHBG participates in steroid signaling pathways by binding to SHBG‐receptors (RSHBG) located on the target cell membranes. SHBG that is bound to a steroid cannot bind to its receptor, but free SHBG that first binds to the RSHBG can subsequently bind steroids. Several studies on human tissues and cultured cells, such as prostate and breast cancer cells, have revealed that the SHGB–RSHBG complex is involved in the steroid‐activated signal transduction pathway that results in the accumulation of intracellular cAMP. SHBG–RSHBG complexes on the cell membrane combine with some types of sex hormones and then induce the accumulation of intracellular cAMP in those tissues and cells. Further, some steroids antagonize the activation of SHBG–RSHBG induced by other steroids. FBS contains high endogenous levels of SHBG, which can confuse the study of steroid effects. We formed the hypothesis that androgen function in melanocytes is effected through the cell membrane signaling pathway via the SHBG–RSHBG complex. Immunohistochemistry was used to demonstrate the co‐staining of SHBG and testosterone with specific monoclonal antibodies. There was a dramatic increase in positive staining following SHBG pre‐treatment. Generally, staining for testosterone is much stronger when staining for SHBG is stronger. These immunohistochemical results strongly suggest the existence of the RSHBG on the surface of normal human melanocytes.

The responses of intracellular levels of cAMP to various androgens and hydrocortisone were studied. After the saturation of RSHBG on the cell membranes by SHBG and then washing to remove excess SHBG, a decrease in intracellular cAMP levels within 30 min of incubation with testosterone was observed in a dose‐dependent manner, but not in the absence of SHBG pre‐treatment. Those findings suggest the possibility that normal human melanocytes might utilize the cell membrane transduction pathway of androgen via the SHBG–RSHBG complex to regulate intracellular cAMP. They further suggest that strong androgens, such as testosterone and DHT, might act as antagonists in this pathway. We have already reported that human melanocytes possess significant levels of 5α ‐reductase activities and that testosterone is predominantly metabolized to DHT, a stronger androgen. DHT might work as a negative feedback factor against cAMP accumulation via the SHBG–RSHBG complex, since the affinity of DHT to SHBG is higher than that of testosterone.

Tyrosinase activity was decreased by the physiological concentration of testosterone after 4 days of treatment. As the decreases in tyrosinase activity coincided with decreases of intracellular cAMP, those intracellular cAMP levels (regulated through cell membrane signaling) might regulate tyrosinase activity influenced by androgens after treatment with SHBG. Tyrosinase activity was stimulated by R1881 when the melanocytes were cultured continuously in medium containing FBS and hydrocortisone. FBS and hydrocortisone that are endogenous in the medium might work against the effect of strong androgens that may be antagonists in the cell membrane signaling pathway, because high concentrations of unbound SHBG found in FBS can easily bind to the cell surface and high concentrations of hydrocortisone added to the culture medium might have already occupied the SHBG–RSHBG complex on the cell membrane.

Further, we studied the expression of tyrosinase mRNAs by semi‐quantitative RT‐PCR. The expression of tyrosinase gene transcripts was not altered by the physiological concentration of testosterone in human plasma. No significant differences were also seen in tyrosinase mRNA expression between androgen treated cells and controls as measured by Northern blot analysis. While the classical androgen signal pathway (the androgen‐AR complex) may possibly play a role in human melanocytes, this pathway might not directly regulate the transcriptional level of the tyrosinase gene. Western blot analysis also suggests that strong androgens have no significant effect on the levels of tyrosinase protein, even though its catalytic function is affected. Taken together, we speculate that strong androgens and SHBG might function as antagonists of intracellular cAMP accumulation via the SHBG–RSHBG complex signaling pathway on the cell membrane, and might also regulate tyrosinase activity at the post‐transcriptional and/or post‐translational levels.

In conclusion, androgens may be another type of humoral hormone as well as α‐MSH that controls melanin production and these hormones, along with other factors, may collaborate to regulate skin pigmentation. In solar lentigo, α‐MSH may be a good mediator of human skin pigmentation. Sex hormones and α‐MSH can be interesting target for whitening and anti‐spot compounds.

Acknowledgment

The authors thank N. Lachmann and J. B. Grieu for their support, M. Juan, E. Noblesse and P. Gasser (Bio EC, Clamart, France) for assistance in the histological studies.

References

1. Tadokoro, T. et al. Human genital melanocytes as androgen target cells. J Invest Dermatol. 109, 513–517 (1997).

2. Tadokoro, T. et al. The inhibitory effect of androgen and sex‐hormone‐binding globulin on the intracellular cAMP level and tyrosinase activity of normal human melanocytes. Pigment Cell Res. 16, 190–197 (2003).