pharmacologic-induction-epidermal-melanin-protection-against-sunburn

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

University of Kentucky College of Medicine

Ultraviolet-light (UV)-induced tanning is defective in numerous 'fair-skinned' individuals, many of whom contain functional disruption of the melanocortin 1 receptor (MC1R). Although this suggested a critical role for the MC1R ligand melanocyte stimulating hormone (MSH) in this response, a genetically controlled system has been lacking in which to determine the precise role of MSH-MC1R. Here we show that ultraviolet light potently induces expression of MSH in keratinocytes, but fails to stimulate pigmentation in the absence of functional MC1R in red/blonde-haired Mc1r(e/e) mice. However, pigmentation could be rescued by topical application of the cyclic AMP agonist forskolin, without the need for ultraviolet light, demonstrating that the pigmentation machinery is available despite the absence of functional MC1R. This chemically induced pigmentation was protective against ultraviolet-light-induced cutaneous DNA damage and tumorigenesis when tested in the cancer-prone, xeroderma-pigmentosum-complementation-group-C-deficient genetic background. These data emphasize the essential role of intercellular MSH signalling in the tanning response, and suggest a clinical strategy for topical small-molecule manipulation of pigmentation.

Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning.

Every living organism is exposed to numerous genomic insults on a daily basis as a consequence of cellular metabolism and exposure to environmental agents capable of interacting with the genome (e.g. chemicals, toxins, pollutants, UV and ionizing radiation) (1). Maintenance of the integrity of the genome is paramount to the survival and propagation of a species and involves the continuous activity of a variety of DNA repair pathways. Inherited mutations in genes involved in DNA damage recognition and repair lead to disease by destabilization of the genome and increased mutagenesis. In fact, it is common for cancer cells to exhibit loss of genomic stability presumably as a result of clonally acquired mutations in DNA repair genes (2). Currently, roughly 150 DNA repair genes have been identified in humans (3) and a variety of familial cancer predisposition and/or premature aging syndromes are now linked to various loss-of-function mutations in these genes (4). Genetic interaction between DNA repair pathways and global cell differentiation pathways is supported by phenotypic similarities between inactivating mutations in a DNA repair, cell cycle arrest and apoptosis proteins. Though there is clearly some degree of functional redundancy between DNA repair pathways for correction of specific DNA lesions, the particular clinical characteristics of a repair defect can be predicted by the specific repair pathway affected (5). Patients with cancer predisposition syndromes often have multiple family members affected by cancer, develop cancer at an early age, and are at risk for developing multiple primary tumors over time (6, 7). Though patients with identifiable cancer predisposition syndromes are rare, defining their molecular defects has led to widespread applicability by uncovering relevant molecular pathways that are perturbed via somatic (non-inherited) mutations in the majority of sporadic cancers. In this review, we describe general molecular mechanisms of major forms of DNA repair and illustrate clinical consequences of deficiencies in these pathways. For more in depth detail, the reader is referred to several recent reviews and texts (2, 8-13).

DNA repair pathways and hereditary cancer susceptibility syndromes.

Though infantile leukemia has a historically poor prognosis, there may be a subset of patients with cutaneous disease whose disease will resolve without therapy. The authors report a case of infantile leukemia cutis who presented with a single subcutaneous chloroma that spontaneously resolved over the course of several weeks and who remains without evidence of disease nearly two years later. After reviewing the literature of congenital leukemia cutis, the authors conclude that withholding chemotherapy in infants with cutaneous myeloid leukemia in the absence of known negative prognostic factors (MLL or BCR-ABL translocations) or progressive disease is clinically indicated.

Spontaneous resolution of a single lesion of myeloid leukemia cutis in an infant: case report and discussion.

We previously reported that topical application of forskolin to the skin of fair-skinned MC1R-defective mice with epidermal melanocytes resulted in accumulation of eumelanin in the epidermis and was highly protective against UV-mediated cutaneous injury. In this report, we describe the long-term effects of chronic topical forskolin treatment in this animal model. Forskolin-induced eumelanin production persisted through 3 months of daily applications, and forskolin-induced eumelanin remained protective against UV damage as assessed by minimal erythematous dose (MED). No obvious toxic changes were noted in the skin or overall health of animals exposed to prolonged forskolin therapy. Body weights were maintained throughout the course of topical forskolin application. Topical application of forskolin was associated with an increase in the number of melanocytes in the epidermis and thickening of the epidermis due, at least in part, to an accumulation of nucleated keratinocytes. Together, these data suggest in this animal model, short-term topical regular application of forskolin promotes eumelanin induction and that over time, topical forskolin treatment is associated with persistent melanization, epidermal cell accumulation, and skin thickening.

Prolonged treatment of fair-skinned mice with topical forskolin causes persistent tanning and UV protection.

Acute myelogenous leukemia presenting as acute infectious meningitis in a 7-year-old boy.

An in vitro skin diffusion study of pure forskolin (1) versus a 1-containing Plectranthus barbatus root extract (P. barbatus extract) in hairless guinea pig skin and human skin in a flow-through diffusion cell system was conducted and is being reported for the first time. Both topical agents were formulated in a solution of 70% ethanol and 30% propylene glycol (v/v). The results showed that forskolin can be delivered through the stratum corneum and that the flux of this compound was enhanced when 1 was delivered as a constituent of the P. barbatus extract as compared to an equivalent amount in pure form. These results suggest that the P. barbatus extract used contains permeation enhancement activity from other compound(s) contained in the crude root extract. It is possible that P. barbatus root extract may be used as an economical source of 1 to perform topical chemical manipulation of pigmentation in high-risk populations.

In vitro skin diffusion study of pure forskolin versus a forskolin-containing Plectranthus barbatus root extract.

The melanocortin 1 receptor (MC1R) is a transmembrane G(s)-coupled surface protein found on melanocytes that binds melanocyte-stimulating hormone and mediates activation of adenylyl cyclase and generation of the second messenger cyclic AMP (cAMP). MC1R regulates growth and differentiation of melanocytes and protects against carcinogenesis. Persons with loss-offunction polymorphisms of MC1R tend to be UV-sensitive (fair-skinned and with a poor tanning response) and are at high risk for melanoma. Mechanistic studies of the role of MC1R in melanocytic UV responses, however, have been hindered in part because Mc1r-defective primary murine melanocytes have been difficult to culture in vitro. Until now, effective growth of murine melanocytes has depended on cAMP stimulation with adenylyl cyclase-activating or phosphodiesterase-inhibiting agents. However, rescuing cAMP in the setting of defective MC1R signaling would be expected to confound experiments directly testing MC1R function on melanocytic UV responses. In this paper, we report a novel method of culturing primary murine melanocytes in the absence of pharmacologic cAMP stimulation by incorporating conditioned supernatants containing stem cell factor derived from primary keratinocytes. Importantly, this method seems to permit similar pigment expression by cultured melanocytes as that found in the skin of their parental murine strains. This novel approach will allow mechanistic investigation into MC1R's role in the protection against UV-mediated carcinogenesis and determination of the role of melanin pigment subtypes on UV-mediated melanocyte responses.

Purification and growth of melanocortin 1 receptor (Mc1r)- defective primary murine melanocytes is dependent on stem cell factor (SFC) from keratinocyte-conditioned media.

The K14-SCF transgenic murine model of variant pigmentation is based on epidermal expression of stem cell factor (SCF) on the C57BL/6J background. In this system, constitutive expression of SCF by epidermal keratinocytes results in retention of melanocytes in the interfollicular basal layer and pigmentation of the epidermis itself. Here, we extend this animal model by developing a compound mutant transgenic amelanotic animal defective at both the melanocortin 1 receptor (Mc1r) and tyrosinase (Tyr) loci. In the presence of K14-Scf, tyrosinase-mutant animals (previously thought incapable of synthesizing melanin) exhibited progressive robust epidermal pigmentation with age in the ears and tails. Furthermore, K14-SCF Tyr(c2j/c2j) animals demonstrated tyrosinase expression and enzymatic activity, suggesting that the c2j Tyr defect can be rescued in part by SCF in the ears and tail. Lastly, UV sensitivity of K14-Scf congenic animals depended mainly on the amount of eumelanin present in the skin. These findings suggest that c-kit signaling can overcome the c2j Tyr mutation in the ears and tails of aging animals and that UV resistance depends on accumulation of epidermal eumelanin.

Stem cell factor rescues tyrosinase expression and pigmentation in discreet anatomic locations in albino mice.

Geneticists estimate that 5% to 10% of all cancers diagnosed in the pediatric age range occur in children born with a genetic mutation that directly increases their lifetime risk for neoplasia. However, despite the fact that only a fraction of cancers in children occur as a result of an identified inherited predisposition, characterizing genetic mutations responsible for increased cancer risk in such syndromes has resulted in a profound understanding of relevant molecular pathways involved in carcinogenesis and/or resistance to neoplasia. Importantly, because most cancer predisposition syndromes result in an increased risk of a small number of defined malignancies, personalized prophylactic surveillance and preventive measures can be implemented in affected patients. Lastly, many of the same genetic targets identified from cancer-prone families are mechanistically involved in the majority of sporadic cancers in adults and children, thereby underscoring the clinical relevance of knowledge gained from these defined syndromes and introducing novel therapeutic opportunities to the broader oncologic community. This review highlights the clinical and genetic features of many of the known constitutional genetic syndromes that predispose to malignancy in children and young adults.

Inherited cancer syndromes in children and young adults.

B12 deficient megaloblastic anemia in a toddler with a history of gastroschisis.

Chest wall Ewing sarcoma presenting as abdominal pain in a 12-year-old boy.

Intracranial hemorrhage and a white blood cell count of almost 1 million cells/Î¼L.

Primary immune thrombocytopenia (ITP) is one of the most common bleeding disorders of childhood. In most cases, it presents with sudden widespread bruising and petechiae in an otherwise well child. Thought to be mainly a disorder of antibody-mediated platelet destruction, ITP can be self-limited or develop into a chronic condition. In this review, we discuss current concepts of the pathophysiology and treatment approaches to pediatric ITP.

ITP in children: pathophysiology and current treatment approaches.

People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r(e/e) background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1r(e/e) mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1r(e/e) mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.

Article	Year
Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning. Nature\| PubMed ID: 16988713	2006
DNA repair pathways and hereditary cancer susceptibility syndromes. Frontiers in bioscience : a journal and virtual library\| PubMed ID: 17485367	2007
Spontaneous resolution of a single lesion of myeloid leukemia cutis in an infant: case report and discussion. Pediatric hematology and oncology\| PubMed ID: 18569848	2008
Prolonged treatment of fair-skinned mice with topical forskolin causes persistent tanning and UV protection. Pigment cell & melanoma research\| PubMed ID: 19087231	2009
Acute myelogenous leukemia presenting as acute infectious meningitis in a 7-year-old boy. Clinical pediatrics\| PubMed ID: 19224868	2009
In vitro skin diffusion study of pure forskolin versus a forskolin-containing Plectranthus barbatus root extract. Journal of natural products\| PubMed ID: 19281221	2009
Purification and growth of melanocortin 1 receptor (Mc1r)- defective primary murine melanocytes is dependent on stem cell factor (SFC) from keratinocyte-conditioned media. In vitro cellular & developmental biology. Animal\| PubMed ID: 19633898	2009
Stem cell factor rescues tyrosinase expression and pigmentation in discreet anatomic locations in albino mice. Pigment cell & melanoma research\| PubMed ID: 19682281	2009
Inherited cancer syndromes in children and young adults. Journal of pediatric hematology/oncology\| PubMed ID: 20186103	2010
B12 deficient megaloblastic anemia in a toddler with a history of gastroschisis. The Journal of pediatrics\| PubMed ID: 20961567	2011
Chest wall Ewing sarcoma presenting as abdominal pain in a 12-year-old boy. The Journal of pediatrics\| PubMed ID: 21962600	2011
Intracranial hemorrhage and a white blood cell count of almost 1 million cells/Î¼L. The Journal of pediatrics\| PubMed ID: 22947653	2013
ITP in children: pathophysiology and current treatment approaches. Journal of pediatric hematology/oncology\| PubMed ID: 23073045	2013
An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background. Nature\| PubMed ID: 23123854	2012

John A. D'Orazio

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