Stem cell factor/c-Kit signalling in normal and androgenetic alopecia hair follicles.

Androgens stimulate many hair follicles to alter hair colour and size via the hair growth cycle; in androgenetic alopecia tiny, pale hairs gradually replace large, pigmented ones. Since stem cell factor (SCF) is important in embryonic melanocyte migration and maintaining adult rodent pigmentation, we investigated SCF/c-Kit signalling in human hair follicles to determine whether this was altered in androgenetic alopecia. Quantitative immunohistochemistry detected three melanocyte-lineage markers and c-Kit in four focus areas: the epidermis, infundibulum, hair bulb (where pigment is formed) and mid-follicle outer root sheath (ORS). Colocalisation confirmed melanocyte c-Kit expression; cultured follicular melanocytes also exhibited c-Kit. Few ORS cells expressed differentiated melanocyte markers or c-Kit, but NKI/beteb antibody, which also recognises early melanocyte-lineage antigens, identified fourfold more cells, confirmed by colocalisation. Occasional similar bulbar cells were seen. Melanocyte distribution, concentration and c-Kit expression were unaltered in balding follicles. Androgenetic alopecia cultured dermal papilla cells secreted less SCF, measured by ELISA, than normal cells. This identifies three types of melanocyte-lineage cells in human follicles. The c-Kit expression by dendritic, pigmenting, bulbar melanocytes and rounded, differentiated, non-pigmenting ORS melanocytes implicate SCF in maintaining pigmentation and migration into regenerating hair bulbs. Less differentiated, c-Kit-independent cells in the mid-follicle ORS stem cell niche and occasionally in the bulb, presumably a local reserve for long scalp hair growth, implicate other factors in activating stem cells. Androgens appear to reduce alopecia hair colour by inhibiting dermal papilla SCF production, impeding bulbar melanocyte pigmentation. These results may facilitate new treatments for hair colour changes in hirsutism, alopecia or greying.

Decreased phenylalanine uptake and turnover in patients with vitiligo.

The human epidermis has the full machinery for autocrine L-phenylalanine turnover to L-tyrosine in keratinocytes and melanocytes. Phenylalanine hydroxylase (PAH) activities increase linearly with inherited skin colour (skin phototype I-VI, Fitzpatrick classification) yielding eightfold more activities in black skin compared to white skin. Moreover, UVB irradiation (1 MED) significantly increases epidermal PAH activities 24 h after exposure. Importantly, L-phenylalanine uptake and turnover in the pigment forming melanocytes is vital for initiation of melanogenesis. In this context it was shown that the uptake of this amino acid is regulated by calcium. The depigmentation disorder vitiligo provides a unique model to follow impaired L-phenylalanine turnover in the skin as well as in serum because affected individuals hold an impaired epidermal 6BH4 de novo synthesis/recycling and regulation including low epidermal PAH activities. After overnight fasting and oral loading with L-phenylalanine (100 mg/kg body weight), 29.6% of 970 patients tested (n=287/970) yielded serum phenylalanine/tyrosine ratios >or=4 and 35.3% (n=342/970) had mild to moderate hyperphenylalaninaemia (HPA), while 9.3% (n=90/970) had both serum L-phenylalanine levels >or=2.0 mg/dl and phe/tyr ratios >or=4.0. Isolated HPA was found in 26% (n=252/970), whereas 20.3% had only increased ratios (n=197/970). None of the patients had phenylketonuria and the family history for this metabolic disease was negative. The IQ followed normal Gaussian distribution. In vitro L-phenylalanine uptake/turnover studies on primary epidermal melanocytes originating from these patients demonstrated a significantly decreased calcium dependent L-phenylalanine uptake and turnover compared to healthy control cells. Based on our observation, we would like to propose that phenylalanine uptake/turnover is under tight control by calcium which in turn could offer an additional novel mechanism in the aetiology of HPA.

Autocrine catecholamine biosynthesis and the beta-adrenoceptor signal promote pigmentation in human epidermal melanocytes.

Earlier it has been shown that human proliferating/undifferentiated basal keratinocytes hold the full capacity for autocrine catecholamine synthesis/degradation and express beta2-adrenoceptors (beta2-AR). In this report, we show that human melanocytes also express all of the mRNA and enzymes for autocrine synthesis of norepinephrine but fail to produce epinephrine. So far, it was established that human melanocytes express alpha1-AR which are induced by norepinephrine yielding the inosine triphosphate diacylglycerol signal. The presence of catecholamine synthesis and the beta2-AR signal escaped definition at that time. Using RT-PCR, immunofluorescence and radioligand binding with the beta2-AR antagonist (-)-[3H]CGP 12177, we show here that human melanocytes express functional beta2-AR (4230 receptors per cell) with a Bmax at 129.3 and a KD of 3.19 nM but lack beta1-AR expression. beta2-AR stimulation with epinephrine 10(-6) M and salbutamol 10(-6)-10(-5) M yielded a strong cyclic adenosine monophospate (cAMP) response in association with upregulated melanin production. Taken together these results indicate that the biosynthesis and release of epinephrine (10(-6) M) by surrounding keratinocytes can provide the cAMP response leading to melanogenesis in melanocytes via the beta2-AR signal. Moreover, the discovery of this catecholaminergic cAMP response in melanocytes adds a new source for this important second messenger in melanogenesis.

Tyrosine hydroxylase isoenzyme I is present in human melanosomes: a possible novel function in pigmentation.

Both human epidermal melanocytes and keratinocytes have the full capacity for de novo synthesis of 6(R) L-erythro 5,6,7,8, tetrahydrobiopterin, the essential cofactor for the rate limiting step in catecholamine synthesis, via tyrosine hydroxylase. Catecholamine synthesis has been demonstrated in proliferating keratinocytes of the epidermis in human skin. This study presented herein identified for the first time the expression of tyrosine hydroxylase isozyme I mRNA within the melanocyte. The location of the enzyme was demonstrated in both the cytosol and melanosomes of human epidermal melanocytes, using immunohistochemistry and immunofluorescence double staining as well as immunogold electron microscopy. High-performance liquid chromatography (HPLC) analysis of pure melanosomal extracts from the human melanoma cell line, FM94, confirmed the production of L-dopa within these organelles. In addition, enzyme activities for both tyrosine hydroxylase and tyrosinase were measured in the same preparations, by following the catalytic release of tritiated water from L-[3,5-3H]tyrosine. The melanosomal membrane location of tyrosine hydroxylase together with tyrosinase implies a coupled interaction, where L-dopa production facilitates the activation of tyrosinase. Our results support a direct function for tyrosine hydroxylase in the melanosome via a concerted action with tyrosinase to promote pigmentation.

In situ and in vitro evidence for DCoH/HNF-1 alpha transcription of tyrosinase in human skin melanocytes.

Human epidermal melanocytes hold the full capacity for autocrine de novo synthesis/regulation/recycling of the essential cofactor 6-tetrahydrobiopterin (6BH(4)) for conversion of L-phenylalanine via phenylalanine hydroxylase to L-tyrosine and for production of L-Dopa via tyrosine hydroxylase to initiate both pigmentation and catecholamine synthesis in these neural crest-derived cells. Earlier we have demonstrated pterin-4a-carbinolamine dehydratase (PCD) mRNA and enzyme activities in epidermal melanocytes and keratinocytes. This protein dimerises also the transcription factor hepatocyte nuclear factor 1 (HNF-1), leading to activation of multiple genes. This study demonstrates for the first time DCoH/HNF-1 alpha expression and transcriptional activity in human epidermal melanocytes in vitro and in situ and identified tyrosinase, the key enzyme for pigmentation, as a new transcriptional target. Specific binding of DCoH/HNF-1 complex to the human tyrosinase promoter was confirmed by gel shift analysis. These results provide a novel mechanism in the regulation of skin pigmentation.