Red hair, fair skin and melanoma – melanocortin 1 receptor

POMC processing in human melanocytes has been widely documented, and the α-MSH/MC1R/cAMP cascade has been implicated in the control of pigmentation. Only very recently, a role of β-endorphin, one cleavage product of β-LPH, has been demonstrated to influence melanocyte growth, dendricity and melanin biosynthesis via the µ-opiate receptor. However, much earlier, it was shown that β-MSH, the other cleavage product of β-LPH, controls melanogenesis and melanin transfer in amphibians. To date, a specific receptor for β-MSH has not been identified. Earlier POMC processing has been found in melanosomes. Therefore, an MC1R-independent role of α-MSH was postulated and demonstrated in control of 6-tetrahydrobiopterin (6BH₄)-inhibited tyrosinase. Utilizing the depigmentation disorder vitiligo, we were now able to follow the fate of epidermal POMC processing in the presence of mM levels of hydrogen peroxide (H₂O₂). In vitiligo epidermal PC2 and 7B2 protein expression is increased, whereas α-MSH, β-MSH and β-endorphin are significantly decreased. Analysis of the peptide sequences revealed in all three cases H₂O₂ oxidation targets such as methionine and tryptophan yielding significant structural alterations. Moreover, we have identified a new function of β-MSH due to its capacity to bind the important cofactor 6BH₄ as well as its isomer 7BH₄. Hence, we propose for the first time that β-MSH can control both the supply of l -tyrosine from l -phenylalanine via phenylalanine hydroxylase and l -Dopa synthesis via tyrosinase hydroxylase in melanocytes and keratinocytes. Therefore, both melanogenesis and catecholamine synthesis could be regulated by this peptide.     

Processing of POMC peptides by dermal microvascular endo-thelial cells: implication for EC biologic functions and skin inflammation

Dermal microvascular endothelial cells (ECs) are both source and target of the pro-opiomelanocortin (POMC) peptides ACTH and α-melanocyte-stimulating hormone (α-MSH). The availability of neuropeptides as important modulators of innate and adaptive immune responses is controlled by neuropeptide-specific peptidases such as neutral endopeptidase (NEP) or angiotensin-converting enzyme (ACE). In this study, we have tested the possibility that NEP or ACE expressed by ECs may influence the local bioavailability of POMC peptides. Incubation of ACTH₁₋₃₉ with cell membranes prepared from the high NEP-/low ACE-expressing microvascular EC line 1 (HMEC-1) or from low NEP-/high ACE-expressing primary human dermal ECs (HDMECs) for 30–480 min resulted in a decrease in ACTH immunoreactivity (IR) over time in membrane supernatants that could be partially blocked with NEP inhibitors as detected by radioimmunoassay. In parallel, α-MSH IR increased peaking after 60 min. Fragments generated by incubation of HMEC-1 or HDMEC membranes with ACTH₁₋₃₉, ACHT₁₋₂₄ or α-MSH for 1–120 min were further analysed by mass spectroscopy. HMEC-1 membranes generated peptide products which could be altered by inhibition of NEP, but not ACE. Likewise, HDMEC membranes fragmented ACTH similar to HMEC-1 membranes in the presence of NEP inhibitors. Some of the proteins can be assigned to regular proteolytic cleavage, while others seem to be modified. Importantly, HMEC-1 and HDMEC membranes also slowly degraded α-MSH, suggesting that EC proteolytic peptidases locally control ACTH/α-MSH bioavailability, which may be important in controlling cutaneous inflammation.     

The Ca2+ binding capacity of epidermal furin is disrupted by H2O2-mediated oxidation in vitiligo

It was previously established that epidermal keratinocytes and melanosomes express the Ca²⁺-dependent precursor convertase furin. All prohormone convertases including furin are regulated by Ca²⁺. Meanwhile, it is noteworthy that proopiomelanocortin (POMC) cleavage is processed by furin, in addition to the classical PC1 and PC2 convertases, leading to the production of ACTH, β-LPH and β-endorphin. Since numerous epidermal peptides and enzymes are affected by H₂O₂-mediatedoxidation, including the POMC derived peptides α-MSH and β-endorphin, as shown in the epidermis of patients with vitiligo, it was of interest whether furin could also be a possible target for this oxidation mechanism. Confirming the presence of furin in epidermal keratinocytes and melanocytes using immunoflurescence, RT-PCR, and Western blotting, we further demonstrate significantly decreased in situ immunoreactivity of furin in the epidermis of patients with progressive vitiligo suggesting H2O₂-mediated oxidation. This was substantiated by [⁴⁵Ca²⁺] binding studies with human recombinant furin identifying the loss of one Ca²⁺-binding site from the enzyme after oxidation with H₂O₂. Computer simulation supported alteration of one of the two Ca²⁺-binding sites on furin. Overall, our results demonstrate that the Ca²⁺ dependent proteolytic activity of this convertase is a target for H₂O₂-mediated oxidation which in turn could contribute to the reduced epidermal expression of the POMC derived peptides α-MSH and β-endorphin as documented earlier in patients with vitiligo.     

Human hair follicle pigmentary unit as a direct target for modulators of melanogenesis, as studied by [14C]-2-Thiouracil incorporation

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  μ m 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  μ m ). Incubation with the MC1-R agonist α-MSH (0.2  μ m ) 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.     

Antioxidant and anti-inflammatory activities of melanocortin peptides

The molecular pathways regulating ultraviolet (UV) radiation-induced apoptosis of melanocytes, a cell population crucially involved in the protection of epidermal keratinocytes against the harmful effects of UV light, are poorly characterized. We show that the α-melanocyte-stimulating hormone (α-MSH) blocks UVB-induced apoptosis of normal human melanocytes in vitro . The effect of α-MSH is not restricted to melanocytes but is also operative in cells that do not produce melanin, for example in human epidermal keratinocytes and in dermal fibroblasts. α-MSH not only delays but also protects melanocytes from UVB-induced cell death. The anti-apoptotic activity of α-MSH is not mediated by a filtering effect or induction of melanin synthesis. α-MSH also does not induce changes in the cell cycle distribution or expression of Bcl₂, Bcl_x, CD95 (Fas/APO-1) and FasL. In contrast, α-MSH markedly reduces the formation of cyclobutane pyrimidine dimers induced by UVB radiation. Human dermal fibroblasts carrying a defective XPA gene are not protected from UVB-induced apoptosis by α-MSH. These results highlight a novel biological activity of α-MSH as well as novel regulatory pathways within the UV response of skin cells targeted by this neuropeptide.     

Solar-simulated, ultraviolet radiation-induced, upregulation of the melanocortin-1 receptor, pro-opiomelanocortin, and α-melanocyte- stimulating hormone in human epidermis in vivo

Ultraviolet (UV) light is one of the most crucial environmental factors with regard to its capacity to induce skin cancer, premature aging of the skin, and immunosuppression. Although UV directly affects the function of epidermal cells, many of these effects are mediated by the induction of cytokines, growth factors, and neuropeptides such as α-melanocyte-stimulating hormone (α-MSH). Recently, in addition to its well-known pigmentation inducing activity, a strong anti-inflammatory as well as an immunomodulatory potential of α-MSH has been recognized. The aim of this study was to determine whether UV irradiation affects the expression of both α-MSH and the melanocortin-1 receptor (MC-1R) in human epidermis in vivo . The volar aspects of the forearms were exposed to twice the minimal erythema dose of solar simulating radiation (SSR). Three, 6, and 24 h after irradiation, the pro-opiomelanocortin (POMC) and interleukin-10 (IL-10) mRNA levels in suction blister-induced epidermal sheets were considerably upregulated as detected by semiquantitative RT-PCR. Furthermore, α-MSH and IL-10 protein levels in blister fluids were significantly increased 24 h after UV irradiation, an effect which could be abolished by the application of the broadspectrum sunscreen AnthéliosXL^® prior to UV (SSR) exposure. In addition, enhanced MC-1R mRNA and receptor protein expression upon SSR was ascertained by RT-PCR and immunohistochemistry of the epidermal sheets, respectively. POMC-derived neuropeptides such as α-MSH may therefore play an important role in modulating UV-induced inflammation.     

Expression of MC-R, POMC and POMC peptides and evidence for a immunoregulatory role of α-MSH in human dermal papilla cells

Pro-opiomelanocortin (POMC)-derived peptides are well-known regulators of pigmentation and proliferation of epidermal and hair follicle-derived melanocytes. We demonstrated that human dermal papilla cells (DPCs), a distinct myofibroblastic cell population of the hair follicle, participate in the cutaneous POMC system. DPCs in vitro and in situ expressed the melanocortin receptor-1 (MC-1R) as well as MC-4R as shown by RT-PCR, immunofluorescence and immunohistochemistry. Expression of POMC but not agouti signalling protein, a natural MC-1R/MC-4R antagonist, was also detectable in DPC. Generation of POMC peptides by DPCs in vitro was demonstrated by immunofluorescence and ELISA studies revealing the expression of both adrenocorticotropin and β-endorphin. To investigate the functional relevance of MC-R expression in DPCs, we examined the effect of α-MSH on interferon-γ (IFN-γ)-induced expression of intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule upregulated in inflammatory disorders of the hair follicle such as alopecia areata. α-MSH markedly suppressed the IFN-γ-mediated upregulation of ICAM-1 in DPCs as shown by real-time PCR studies, while α-MSH alone did not have any effect. Our data suggest that melanocortins such as α-MSH mediate paracrine and autocrine effect in the dermal papilla whose disruption may contribute to inflammatory diseases of the hair follicle.     

The α-melanocyte stimulating hormone-related tripeptide K(D)PT stimulates human hair follicle pigmentation in situ under proinflammatory conditions

Background  α-Melanocyte stimulating hormone (α-MSH) is a well-tolerated immunomodulator with cytoprotective and anti-inflammatory effects that is known to stimulate melanogenesis and proliferation of follicular melanocytes. As human hair follicles (HFs) locally synthesize α-MSH, pharmacologically more easily handled α-MSH-related tripeptides, such as K(D)PT, may imitate this endogenous regulation, and may show a favourable side-effect profile on clinical use.
Objectives  To investigate the effect of the synthetic, α-MSH-related peptide K(D)PT [which is identical to interleukin (IL)-1β_193–195] on melanogenesis in human anagen HFs, under normal and proinflammatory growth conditions.
Methods  Normal human anagen VI scalp HFs were microdissected and organ cultured with different concentrations of K(D)PT with or without coadministration of a proinflammatory, catagen-inducing stimulus, interferon (INF)-γ. Masson–Fontana histochemistry and NKI/beteb immunohistochemistry were employed to assess changes in the degree of human HF pigmentation and melanocyte dendricity.
Results  As confirmed by quantitative (immuno-)histomorphometry, compared with controls, K(D)PT alone did not affect human HF pigmentation in organ culture. However, in the presence of a strong, prototypic proinflammatory stimulus (IFN-γ), K(D)PT significantly stimulated HF melanin content and melanocyte dendrite formation in situ .
Conclusions  The IL-1β- and α-MSH-related tripeptide, K(D)PT, displays interesting hair pigmentation-stimulatory activities under proinflammatory conditions. These might become exploitable for innovative antigreying strategies, notably in postinflammatory poliosis (regrowth of white hair, e.g. during recovery from alopecia areata), where no effective clinical therapy is yet available.     

α-melanocyte stimulating hormone inhibits tumour necrosis factor-α stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines

α-Melanocyte stimulating hormone (α-MSH) was found significantly to reduce tumour necrosis factor-α (TNF-α) stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in normal adult cutaneous melanocytes. The maximum inhibitory response to α-MSH was obtained at around 10⁻¹⁰ mol/L α-MSH when cells were coincubated with α-MSH and TNF-α for 24 h. α-MSH had little or no effect on basal ICAM-1 expression in melanocytes and the effects of α-MSH could be mimicked with 3-isobutyl-1-methylxanthine (IBMX). Preliminary data in three human melanoma cell lines also showed α-MSH and forskolin to be effective in significantly reducing TNF-α stimulated ICAM-1 expression over 24 h. The extent of the inhibition varied from cell line to cell line and was greatest in those cells with the highest number of α-MSH receptors. These data suggest that α-MSH has the ability to oppose the action of the pro-inflammatory cytokine TNF-α on melanocytes and melanoma cells.     

p-Coumaric acid, a constituent of Sasa quelpaertensis Nakai, inhibits cellular melanogenesis stimulated by alpha-melanocyte stimulating hormone

Background   Recent study has demonstrated that Sasa quelpaertensis (Korean name, Jeju-Joritdae) extracts inhibit cellular melanogenesis implicating potential use in the control of skin pigmentation.
Objectives   The purpose of the present study was to elucidate the active constituents of this plant inhibiting melanogenesis and the associated mechanism.
Methods   The effect of the plant-derived materials on melanin production and/or tyrosinase expression was examined in murine melanoma B16/F10 cells and neonatal human melanocytes.
Results   When tested in melanoma B16/F10 cells treated with the α-melanocyte stimulating hormone (α-MSH), the aqueous ethanol extract of S . quelpaertensis culm inhibited the cellular melanogenesis more effectively than its leaf extract. A major active compound was isolated from the culm extract by solvent fractionation and column chromatography, and identified to be p -coumaric acid by spectroscopic and chromatographic analyses. The compound ( p -coumaric acid) inhibited α-MSH-stimulated cellular melanogenesis more effectively than arbutin or other structurally similar compounds including 3-(4-hydroxyphenyl) propionic acid, cinnamic acid and caffeic acid. It also attenuated α-MSH-dependent increase of tyrosinase protein. The antimelanogenic effect of p -coumaric acid was also verified in neonatal human melanocytes.
Conclusions   The present study identified p -coumaric acid as a main constituent of S . quelpaertensis inhibiting cellular melanogenesis. Because of its structural similarity, p -coumaric acid may interfere with l -tyrosine action in the control of tyrosinase expression in response to α-MSH.     

Effect of UVB radiation emitted from the narrowband TL‐01 lamp (311 nm) on the calcitriol synthesis in organotypic cultures of keratinocytes

UV‐exposition is considered as the main reason for the development of cancers of the skin. However, 90 to 100% of the Vitamin‐D reqirement is formed within the skin through the action of sunlight. Considering the results of epidemiological studies, that have detected an association of Vitamin‐D deficiency with various types of cancer (e.g. colon‐, prostate‐ and breast cancer), this is a real dilema. The cancer protective effect of vitamin‐D is contributed to the extra renal, local production of 1α,25(OH)₂D₃ by the 25‐hydroxyvitamin D‐1α‐hydroxylase, which has been detected in various tissues. In respect of the novel functions of vitamin‐D and the risk of adverse consequences in case of deficiency we have screened sun deprived risk groups ( A : patients with genodermatoses connected with defects in sun‐induced DNA repair: n = 4: 3 patients with xeroderma pigmentosum and 1 patient with basal cell nevus syndrome; and B : non vitamin‐D substituted renal transplant recipients under immunosuppressants: n = 33) for their vitamin‐D status. As measure of the vitamin‐D store and as substrate for the 25‐hydroxyvitamin D‐1α‐hydroxylase basal 25(OH)D₃ serum levels (Nichols Institute Diagnostika GmbH, Bad Nauheim, Germany) have been analysed. In both groups decreased basal 25(OH)D₃ serum levels were detected. Therefore we demand a monitoring of vitamin‐D status in patients practising sun protection, in case of vitamin‐D deficiency an oral substitution should be recommended.     

Tenascin is overexpressed in vitiligo lesional skin and inhibits melanocyte adhesion

The aetiology of vitiligo remains obscure. In this study, the role of integrins in the observed inability of melanocytes to repopulate lesional skin was investigated. Antibodies directed to α₂, α₃, α₅, α_v, α₆, β₁ and β₃ integrin subunits were used. Immunohistology revealed no marked differences in the overall levels of expression of integrins between control, non-lesional, perilesional or lesional skin. Moreover, no differences were noted in the level of expression of integrins or the adhesive capacity between cultured control cells derived from three separate donors and vitiligo-derived melanocytes from two donors. Rather, it was clearly observed that towards the lesion, vitiligo skin contains increasing amounts of tenascin in the basal membrane and papillary dermis in five patients employing T2H5 antihuman tenascin antibody. The anti-adhesive effect observed in vitro for this extracellular matrix molecule using normal melanocytes may contribute to loss of pigment cells in vitiligo or to ineffective repopulation of the lesions.     

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Abstract:  Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT-PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG-CoA reductase and transport cholesterol via LDL/Apo-B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time- and dose-dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor β, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo-B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.     

1α,24-Dihydroxyvitamin D3 (tacalcitol) is effective against Hailey–Hailey disease both in vivo and in vitro

We report a case of Hailey–Hailey disease (HHD) in which 1α,24-dihydroxyvitamin D₃ (tacalcitol) was effective both clinically ( in vivo ) and in explant cultures ( in vitro ) of a skin lesion. The patient was a 65-year-old man with HHD lesions in the axillary and inguinal areas bilaterally. We applied ointment containing 1α,24-dihydroxyvitamin D₃ (tacalcitol), an analogue of active vitamin D₃, to the lesions and assessed its clinical effectiveness. The HHD lesions in both groins disappeared after treatment with the 1α,24-dihydroxyvitamin D₃ ointment, and the remission has continued to the present. A punch biopsy specimen of the lesion that had remitted showed no acantholysis. In addition, dissociation of migrating keratinocytes was observed when biopsy specimens of the HHD skin lesion were cultured in medium without 1α,24-dihydroxyvitamin D₃, but inhibition of keratinocyte dissociation was observed in medium containing it. These results suggest the effectiveness of 1α,24-dihydroxyvitamin D₃ against HHD both in vivo and in vitro .