Anthony J. Thody

MSH peptides are present in mammalian skin

Immunoreactive alpha-MSH was found in human skin and the skin of numerous other mammals. After hypophysectomy the concentration of alpha-MSH in rat skin showed little change suggesting that the pituitary is not the source of this MSH. In human skin the highest concentration was found in the epidermis and HPLC revealed four peaks of immunoreactive alpha-MSH. Two of these co-eluted with mono- and des-acetyl alpha-MSH standards. An earlier peak probably represented an oxidized MSH and a later running peak, diacetylated alpha-MSH. Although no differences were found in alpha-MSH content of skin from albino and pigmented rats or between involved and non-involved epidermis of patients with vitiligo, its predominance in human epidermis could suggest a relationship with the melanocyte or its melanin. Whether alpha-MSH in the skin has any pigmentary significance or any other role has yet to be established.

Melanocyte Function and Its Control by Melanocortin Peptides

Melanocytes are cells of neural crest origin. In the human epidermis, they form a close association with keratinocytes via their dendrites. Melanocytes are well known for their role in skin pigmentation, and their ability to produce and distribute melanin has been studied extensively. One of the factors that regulates melanocytes and skin pigmentation is the locally produced melanocortin peptide α-MSH. The effects of α-MSH on melanogenesis are mediated via the MC-1R and tyrosinase, the rate-limiting enzyme in the melanogenesis pathway. Binding of α-MSH to its receptor increases tyrosinase activity and eumelanin production, which accounts for the skin-darkening effect of α-MSH. Other α-MSH-related melanocortin peptides, such as ACTH1–17 and desacetylated α-MSH, are also agonists at the MC-1R and could regulate melanocyte function. Recent evidence shows that melanocytes have other functions in the skin in addition to their ability to produce melanin. They are able to secrete a wide range of signal molecules, including cytokines, POMC peptides, catecholamines, and NO in response to UV irradiation and other stimuli. Potential targets of these secretory products are keratinocytes, lymphocytes, fibroblasts, mast cells, and endothelial cells, all of which express receptors for these signal molecules. Melanocytes may therefore act as important local regulators of a range of skin cells. It has been shown that α-MSH regulates NO production from melanocytes, and it is possible that the melanocortins regulate the release of other signalling molecules from melanocytes. Therefore, the melanocortin signaling system is one of the important regulators of skin homeostasis.

Proopiomelanocortin (POMC), the ACTH/ melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin‐1 receptor (MC‐1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melano‐cytes in culture. Much lower levels of α‐MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and α‐MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone conver‐tases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and α‐MSH, and this was enhanced in response to corti‐cotrophin‐releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably trans‐fected with the MC‐1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, α‐MSH, and β‐MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC‐1R highlight the importance of POMC processing as a key regulatory event in the skin.—Rousseau, K., Kauser, S., Pritchard, L. E., Warhurst, A., Oliver, R. L., Slominski, A., Wei, E. T., Thody, A. J., Tobin, D. J., White, A. Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and mela‐nocytes and stimulates melanogenesis. FASEB J. 21, 1844–1856 (2007)

Production of catecholamines in the human epidermis.

Cell-free extracts from human full thickness skin (i.e., epidermis and dermis), suction blister roofs (i.e., epidermis) and from human keratinocytes express biopterin-dependent tyrosine hydroxylase a well as phenylethanolamine-N-methyl transferase, both representing key enzymes for the biosynthesis of epinephrine. These enzyme activities could not be detected in cell extracts from human melanocytes and human fibroblasts. Since keratinocytes in the human epidermis, and in cell cultures, express a high density of beta-2-adrenoceptors, and this signal transduction system regulates intracellular calcium homeostasis, it can be concluded that epinephrine production in the epidermis activates calcium transport via the beta-2-adrenoceptor system. Our results show for the first time that the human epidermis has the capacity to independently produce epinephrine.

The expression of functional MSH receptors on cultured human melanocytes

Although α-MSH increases skin darkening in humans, there are several reports that it fails to have melanogenic effects on human melanocytes in vitro. The purpose of this study was to see whether cultured human melanocytes express MSH receptors. Human melanocytes were grown in the absence of artificial mitogens such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin (CT) and incubated for 2 h at room temperature with increasing amounts of 125I-labelled Nle4DPhe7-α-MSH with and without excess cold peptide. Binding was saturable and specific: Scatchard analysis gave a Kd of 4.9×10−11M and ~ 700 binding sites/cell. Human keratinocytes and fibroblasts showed no specific binding. The addition of 1 mM dibutyryl cAMP to the culture medium caused a 62% increase in MSH binding to human melanocytes. A smaller increase (25%) was seen with 10−9M CT while 25 mM TPA caused a 24% decrease. These results show that human melanocytes in culture express MSH receptors and that this expression can be modulated by mitogens.

α-MSH and the Regulation of Melanocyte Function

ABSTRACT: α‐MSH, has numerous actions in the skin and by activating the MC1 receptor (MC1‐R) on melanocytes it stimulates melanogenesis. Rather than producing large increase in melanin production α‐MSH acts specifically to stimulate eumelanin synthesis. Although this could be important in determining skin color and tanning there is debate as to the pigmentary significance of α‐MSH in humans. Circulating levels of α‐MSH are negligible and although it is produced in the skin by different cell types, including melanocytes, the major skin form is desacetyl α‐MSH, and this is a weak agonist at MC1‐R. Certain ACTH peptides, notably ACTH1–17, are more potent agonists at the MC1‐R and, since their skin concentrations exceed those of α‐MSH, they could serve as natural ligands at this receptor an regulate pigmentary responses in humans. Activation of MC1‐R does, however, produce other responses in human melanocytes. Thus, α‐MSH stimulates melanocyte dendricity and attachment to extracellular matrix proteins. It also protects melanocytes from the damaging effects of oxidative stress, and regulates their production of NO by modulating the induction of iNOS‐as it does within macrophages. α‐MSH clearly affects various aspects of melanocyte behavior and its melanogenic effects could be the consequence of a more fundamental role in the melanocyte. The precise nature of this role is unclear, but it could be part of a generic role that α‐MSH and other POMC peptides have in skin homeostasis.

The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases

Sunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B‐induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids, and their subsequent metabolism by cyclooxygenases (COXs) and lipoxygenases (LOXs) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and, for 72 h, examined expression of proinflammatory and antiinflammatory eicosanoids using LC/ESI‐MS/MS, and examined immunohistochemical expression of COX‐2, 12‐LOX, 15‐LOX, and leukocyte markers, while quantifying clinical erythema. We show that vasodilatory prostaglandins (PGs) PGE2, PGF2a, and PGE3 accompany the erythema in the first 24–48 h, associated with increased COX‐2 expression at 24 h. Novel, potent leukocyte chemoattractants 11‐, 12‐, and 8‐monohydroxy‐eicosatetraenoic acid (HETE) are elevated from 4 to 72 h, in association with peak dermal neutrophil influx at 24 h, and increased dermal CD3+ lymphocytes and 12‐ and 15‐LOX expression from 24 to 72 h. Anti‐inflammatory metabolite 15‐HETE shows later expression, peaking at 72 h. Sunburn is characterized by overlapping sequential profiles of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution.—Rhodes, L. E., Gledhill, K., Masoodi, M., Haylett, A. K., Brownrigg, M., Thody, A. J., Tobin, D. J., Nicolaou, A. The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases. FASEB J. 23, 3947–3956 (2009). www.fasebj.org

An Improved Radioimmunoassay for α-Melanocyte-Stimulating Hormone (α-MSH) in the Rat: Serum and Pituitary α-MSH Levels after Drugs Which Modify Catecholaminergic Neurotransmission

A modification of our previous radioimmunoassay (RIA) for α-melanocyte-stimulating hormone (α-MSH) is described that permits the measurement of circulating levels in the rat without the need for an extraction procedure. Using this method, serum and neurointermediate lobe () immunoreactive α-MSH levels were measured in rats after administration of haloperidol, 2-bromo α-ergocryptine (CB 154), and α-methyl-p-tyrosine (α-MPT). Haloperidol caused a rapid increase, α-MPT a slow increase, and CB 154 a rapid decrease in serum α-MSH. At the time intervals studied none of the drugs had any significant effect on α-MSH content. It was concluded from the results of the above drug treatments that modulation of dopaminergic neurotransmission both pre- and postsynaptically produces changes in serum immunoreactive α-MSH levels. This supports the suggestion that circulating α-MSH in the rat is under an inhibitory control by a catecholaminergic system.

Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes

Melanocyte cultures were obtained from a modification of the keratinocyte culture system MCDB153. Either promelanocytes or mature melanocytes were selected from epidermal cell primary cultures. Pure subcultures of actively dividing melanocytes of both types were grown in a low-serum medium totally deprived of TPA and cholera toxin called melanocyte growth medium (MGM). Early passaged cells from MGM primary cocultures were similar to normal adult human melanocytes in vivo, exhibiting numerous melanosomes, strong dopa positivity and a high dendricity. The ability of MGM to support melanocyte growth was mainly a consequence of its basic composition, combined with a low serum concentration. Bovine pituitary extract significantly enhanced melanocyte growth. Using complete MGM, in the absence of mitogens and keratinocytes, cell growth was maintained, but the differentiation of melanocytes decreased. The presence of keratinocytes was found to promote melanocyte growth. The coculture system used strongly suggests the action of soluble keratinocyte-derived factors. Keratinocyte contact was necessary to sustain melanocyte dendricity and melanization. Melanization and dendricity behaved mostly as independent features when keratinocyte influence was withheld. Our results underline the essential role of keratinocytes in the regulation of melanocyte growth and differentiation in a physiological culture system.

Co-culture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation

Melanocytes grown in pure monolayer culture lack the three-dimensional organization and many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. In this study skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in various ratios onto de-epidermized dermis. They were cultured in DMEM/Hams F12 (3∶1) for 3 days and then lifted to the air-liquid interface and maintained for 11 days. Histological examination revealed a structure that closely resembled human interfollicular epidermis. Melanocytes, identified by their dendritic appearance, positive dopa reaction and positive staining with a melanocyte-specific antibody (MEL5), were located in the basal layer. Melanin was seen both in melanocytes and in neighbouring keratinocytes. Whilst the skin equivalent became more pigmented following UV irradiation (total UVB 4760 J/m2 over 3 days), the quantity and distribution of melanin at the light microscopic level appeared to be unchanged. However, the number and dendricity of melanocytes increased, as did their staining with dopa and MEL5. These results indicate that melanocytes are functional and capable of responding to UV irradiation.