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.     

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.     

Imaging of melanin distribution using multiphoton autofluorescence decay curves

Background/purpose: Multiphoton fluorescence lifetime imaging (FLIM) is a technique that produces an image based on differences in the decay rate of fluorescence from a sample. Based on this method, the DermaInspect was developed to observe human skin components non-invasively. In this study, we used the DermaInspect to study melanin in skin.
Methods: A human three-dimensional skin model containing melanocytes was embedded in an OCT compound, frozen and sectioned at 10 μm. The melanin distribution in each section was visualized by the DermaInspect using time-resolved single-photon counting and near-infrared femtosecond laser pulse excitation. The melanin distribution of the same sections was then visualized using the Fontana-Masson staining method.
Results: High-resolution images were generated from the ratio of a ₁/ a ₂ ( a ₁e^{− t /120}+ a ₂e^{− t /1100} was chosen to express the exponential fluorescent decay curve) obtained using the DermaInspect. Granules with a high a ₁/ a ₂ ratio, approximately 1 μm in diameter, were observed. Fontana-Masson staining identified these granules as melanin. This new technique was then applied for in vivo observation of melanin in human skin. 'Melanin caps' were visualized in the basal cell layer around the nuclei in images derived from the a ₁/ a ₂ ratio.
Conclusion: Our study confirms that FLIM can non-invasively provide data of the melanin distribution with almost the same quality as the conventional Fontana-Masson staining method, and demonstrates that FLIM is useful for in vivo observation of melanin granules in human skin.     

Pathophysiology of scleroderma: an update

Objectives To review the pathophysiological background of systemic sclerosis in relation to the main, components involved: microvascular system, immunological system and fibroblasts of the connective tissue.
Background Although many particular aspects of the pathophysiology of systemic sclerosis have been investigated in recent years, the complexity of the pathogenesis and the important links between the components involved remain unclear.
Methods Literature review.
Results and conclusion Scleroderma is a connective tissue disorder resulting in a progressive fibrosis of skin and internal organs. The genetic background is not clear. The microvascular system is one of the first targets involved (damage of capillaries, enhanced expression of adhesion molecules interacting with lymphocytes, perivascular infiltrates as starting points for tissue fibrosis). The immune system is unbalanced (selection of T-cell subpopulations, elevated serum levels of several cytokines, occurrence of autoantigens to topoisomerase I, centromeric proteins and RNA polymerases). As far as autoimmunity is concerned the triggering autoantigen is still unknown. Development of connective tissue fibrosis is prominent (sub-populations of fibroblasts with disturbed regulation of collagen turnover by TGF- β , CTGF and collagen receptors (α₁β₁, α₂β₂)). Investigation of pathophysiology of scleroderma is effected by monitoring the serum levels for soluble mediators, by cell culture studies of affected and non-affected fibroblasts and EC, by studying environmentally induced forms of scleroderma and by studies using animal models.     

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.     

白癜风表皮黑素细胞超微结构及小眼畸形相关转录因子转录调控研究

目的 研究白癜风黑素细胞超微结构和小眼畸形相关转录因子 （MITF）及其转录调控的酪氨酸酶相关蛋白（TRP）与白癜风临床类型与病程的相关性。方法 选择不同病程的寻常型白癜风（VV）12例和节段型白癜风（SV）8例,分别取白斑区、白斑边缘正常肤色区和远离白斑正常肤色区的表皮片,经组织学确定其表皮的完整性。透射电镜观察10例患者（VV 6例,SV 4例）不同区表皮黑素细胞的超微结构特点。对所有20例远离白斑正常肤色区的表皮片黑素细胞进行培养,应用免疫印迹方法检测 MITF及其转录调控的酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TYRP1）和酪氨酸酶相关蛋白2（TYRP2）的表达水平。结果 白癜风表皮黑素细胞超微结构病理改变：10例中7例白斑区表皮内未见黑素细胞,1例短病程和2例长病程VV分别可见少量黑素体显著减少或缺失的黑素细胞;白斑边缘正常肤色区,6例VV中,3例病程小于15个月者可见黑素细胞超微结构异常,而4例SV中仅1例异常;远离白斑正常肤色区,10例黑素细胞超微结构均正常。白癜风表皮黑素细胞MITF及其转录调控TRP的表达：VV的MITF表达下调与TYR、TYRP1、TYRP2的表达下调一致;SV存在MITF显著表达下调,而TYR、TYRP1、TYRP2几均正常表达。结论 VV和SV可能存在不同的表皮黑素细胞超微结构病理改变和MITF转录调控机制。     

Characterization of angiotensin-converting enzyme expression during epidermis morphogenesis in humans: a potential marker for epidermal stem cells

Background  Recent evidence has revealed that angiotensin-converting enzyme (ACE) participates in cutaneous wound healing and contributes to the pathophysiological process of some skin diseases. However, little is known about the role of ACE in epidermis morphogenesis during development.
Objective  To clarify the expression pattern of ACE during embryonic development of human skin.
Methods  Skin samples were obtained from aborted fetuses at different gestational ages and from healthy individuals. Localization of ACE, together with β₁-integrin, keratin 19 (K19) and p63 was examined by immunofluorescence and immunohistochemical staining.
Results  In human fetal skin, at 11–13 weeks of gestation, ACE-positive cells were observed in the primitive epidermis. As the fetuses developed, ACE-positive cells appeared in all the epidermal layers. From 21 weeks of gestation, ACE expression was largely restricted to the basal layer of the fetal epidermis. In contrast, ACE-positive cells were found only in the adult skin basal layer which harbours epidermal stem cells. To explore the possible link between ACE and epidermal stem cells, we further examined the expression of β₁-integrin, K19 and p63, the putative markers for epidermal stem cells. Consistent with the results of ACE expression, from 21 weeks of gestation, the expression of β₁-integrin, K19 and p63 was mainly confined to the basal layer. Immunofluorescent double labelling revealed that ACE-positive cells substantially overlapped with β₁-integrin-, K19- and p63-positive cells.
Conclusions  Our results suggest that ACE may play a role in human epidermis morphogenesis during fetal life and serve as an unrecognized marker for keratinocyte progenitor cells.     

Computer simulation of heterogeneous single nucleotide polymorphisms in the catalase gene indicates structural changes in the enzyme active site,NADPH‐binding and tetramerization domains: a genetic predisposition for an altered catalase in patients with vitiligo?

Abstract:  Patients with vitiligo have low levels/activities of catalase in their lesional and non-lesional epidermis as well as in their epidermal melanocytes under in vitro conditions while the levels of catalase mRNA are unaltered. This defect leads to a build-up of hydrogen peroxide (H₂O₂) in the 10⁻³  m range in the epidermis of these patients. In this context, it was realized that 10⁻³  m H₂O₂ deactivates catalase. Along this line, it was also suspected that catalase in patients with vitiligo possesses a special sensitivity to this reactive oxygen species (ROS), and indeed several heterozygous single nucleotide polymorphisms (SNPs) have been documented in the cat gene of these patients. Based on the 3D structure of human catalase monomer, we have modelled the influence of three selected SNPs on the enzyme active site, on the NADPH- as well as the tetramerization-binding domains. Our results show that these SNPs severely alter catalase structurally, which in turn should make the enzyme more susceptible to ROS compared with wild-type enzyme. Taken together, the work presented herein together with the earlier results on SNPs in the cat gene suggests a genetic predisposition for an altered catalase in patients with vitiligo.     

An in vitro three-dimensional model of primary human cutaneous squamous cell carcinoma

Abstract:  Squamous cell carcinomas (SCC) represent a substantial clinical problem because of increases, frequent recurrences and successive de novo tumors, especially in organ transplant recipients. To improve upon the current surgical and other non-selective therapies, a validated organotypic in vitro model of primary human SCC needs to be developed. Such a model will have obvious advantages over current cell line and animal based approaches, and may render the latter partly obsolete. In a first approach, an explant technique of primary SCC biopsies onto dermal constructs was used to emulate tumor expansion in an in vitro model. Histological analysis revealed the formation of nests of squamous cells, mimicking an invasive morphological feature of primary SCC. Immunohistochemical analysis comprised an array of markers characteristic of keratinocyte (hyper) proliferation (K6, K16, K17 and Ki67), differentiation (K1, K10 and involucrin), basement membrane (collagen types IV and VII, integrins α₆ and β₄ and laminin 332) and SCC (K4, K13 and Axl). The generated human SCC models displayed disturbed differentiation and keratins associated with hyperproliferation, but a low frequency of Ki67 positive cells. Basement membrane composition of the in vitro SCC model resembled that of normal skin. These results show for the first time that in vitro modelling of three-dimensional growth of primary cutaneous human SCC is feasible. This model may provide a platform to develop refined preventive and curative treatments and thereby gain understanding of SCC pathogenesis.     

HLA and β2-microglobulin Expression in Basal and Squamous Cell Carcinomas of the Skin

Abstract: Histologic sections of seven squamous cell carcinomas (SCC), 13 basal cell carcinomas (BCC), and a Bowenoid actinic keratoses were examined for expression of HLA class 1 antigens (HLA-ABC) using a monoclonal antibody and an immunoperoxidase technique. Expression of β₂-microglobulin was examined with a polyclonal antibody method. Neither cell marker was detected within the Bowenoid actinic keratoses. Squamous cell carcinomas and basal cell carcinomas exhibited decreased expression of both HLA-ABC and β₂-microglobulin and often did not express these antigens at all. HLA-ABC was present in only two of 13 basal cell carcinomas and four of seven squamous cell carcinomas. β₂-microglobulin was present in one of 13 basal cell carcinomas and two of seven squamous cell carcinomas. When present, these antigens often were present in a few areas of the tumor, but absent in others. In both SCC and BCC. both antigens were usually lost simultaneously. In all tumors with β₂-microglobulin, HLA-ABC a/so was retained. There was no apparent relationship of anatomic site or type of tumor with retention of surface antigens. Since some tumors or portions of tumors retained HLA-ABC and β₂-microglobulin on their surfaces, the absence of these antigens is not an absolute marker for malignancy.     

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 .     

Suprabasal expression of epidermal α2β1 and α3β1 integrins in skin treated with topical retinoic acid

In normal adult human skin, expression of epidermal integrins is confined to keratinocytes in the basal layer. However, suprabasal expression of α2, α3 and β1 integrin subunits is noted in hyperproliferative epidermis in wound repair and psoriasis. In this study, we examined the effect of topical all- trans -retinoic acid (RA), known to induce epidermal hyperplasia, on expression of integrins in human epidermis. Immunostaining of vehicle-treated skin revealed expression of α2, α3 and β1, as well as α6 and β4 integrin subunits entirely on basal keratinocytes. Topical application of RA (0.1%) for 2 weeks resulted in marked suprabasal expression of α2, α3 and β1 integrin subunits, whereas α6 and β4 staining remained on basal keratinocytes. Staining for putative ligands of α2β1 and α3β1 integrins, i.e. type IV collagen, laminin-5 and fibronectin, was not detected in the epidermal layer in RA- or vehicle-treated skin. Treatment of HaCaT keratinocytes in culture with RA (1 μmol/L) enhanced α2 and β1 mRNA abundance. Furthermore, RA slightly up-regulated the expression of α2, α3 and β1 integrin subunits on primary epidermal keratinocytes and HaCaT cells in culture with no effect on cell proliferation. These results provide evidence that RA-elicited epidermal hyperplasia is associated with aberrant suprabasal expression of α2β1 and α3β1 integrins, and that this also involves direct stimulation of keratinocyte integrin expression by RA.     

The expression of the c-kit receptor by epidermal melanocytes may be reduced in vitiligo

Summary The proto-oncogene c-kit encodes the transmembrane tyrosine kinase receptor that has a role in the growth regulation of various cell types including melanocytes. In the present study we have examined the expression of the c-kit protein in the skin of seven patients with vitiligo. Melanocytes positive for c-kit protein were observed in the basal layer in non-lesional skin and the mean number of 25.8 ± 5·2 (per 200 basai ceils) compared with that of 21·8 ± 3·5 from six control subjects. In perilesional skin there was a reduction in the numbers of c-kit positive melanocytes (6·7±2·6) and this was especially noticeable in six of the seven patients. Such a reduction was less obvious following staining with MEL-5 and in only two subjects were the numbers of melanocytes below the normal range. This suggests that the reduction in c-kit staining was the result of decreased expression of the protein rather than a loss of melanocytes. No melanocytes. positive for c-kit protein, or after staining with MEL-5. were identified in lesional skin although isolated tyrosinase-positive melanocytes were seen in one subject. There was no apparent change in the numbers of mast ceils expressing c-kit protein and the intensity of staining in the dermis even in lesional skin was similar to that in the controls. These results demonstrate that c-kit protein is present on melanocytes in adult human skin and that in perilesional skin of some vitiligo patients there is a reduction in the numbers of melanocytes expressing this receptor. Whether this may contribute to the defective melanocyte growth and/or survival that occurs in vitiligo or whether it is a consequence of melanocyte damage remains to be seen.     

Prostanoid receptors in anagen human hair follicles

Abstract:  Prostanoid pathway in hair follicle gained closer attention since trichogenic side-effects on hair growth has been observed concomitantly with prostaglandin F_2α receptor (FP) agonist treatment of intraocular pressure. We thus investigated prostanoid receptor distribution in anagen hair follicle and different cell types from hair and skin. Using RT-PCR, Western blot and immunohistochemistry (IHC), we found that all receptors were present in hair follicle. This data shed new light on an underestimated complex network involved in hair growth control. Indeed most of these receptors showed a wide spectrum of expression in cultured cells and the whole hair follicle. Using IHC, we observed that expression of prostaglandin E₂ receptors (EP₂, EP₃, EP₄), prostaglandin D₂ receptor (DP₂), prostanoid thromboxane A₂ receptor (TP) and to a lesser extent EP₁ involved several hair follicle compartments. On the opposite, Prostaglandin I₂ receptor (IP) and DP₁ were more specifically expressed in hair cuticle layer and outer root sheath (ORS) basal layer, respectively. FP expression was essentially restricted to ORS companion layer and dermal papilla ( DP ). Although extracting a clear functional significance from this intricate network remains open challenge, FP labelling, i.e. could explain the biological effect of PGF_{2 α} on hair regrowth, by directly modulating DP function.     

Expression of α subunit of guanine nucleotide-binding protein Go in Merkel cell carcinoma

The α subunit of guanine nucleotide-binding protein G_o(G_oα), which was initially isolated from bovine brain, interacts with muscarinic cholinergic receptors and regulates neuronal calcium channels. G_oα is known to be localized in neural tissues, some endocrine cells, and neuroendocrine tumors. We have immunohistochemically investigated the expression of G_oα in 4 cases of Merkel cell carcinoma using the method of microwave treatment. In all cases of Merkel cell carcinoma, G_oα was consistently detected on the plasma membrane and cytoplasm of the tumor cells. Nerve fibers in the skin were also positive for G_oα, but other epidermal or dermal components such as keratinocytes, melanocytes, fibroblasts, or lymphoid cells were negative. Tumor cells of squamous cell carcinoma, cutaneous lymphoma, sweat gland carcinoma, and malignant melanoma were negative for G_o4aL. The present study indicates that G_oα may be a useful immunohistochemical marker of Merkel cell carcinoma.