Purification and growth of melanocortin 1 receptor (<Emphasis Type="Italic">Mc1r</Emphasis>)-defective primary murine melanocytes is dependent on stem cell factor (SFC) from keratinocyte-conditioned media |
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Authors: | Timothy L Scott Kazumasa Wakamatsu Shosuke Ito John A D’Orazio |
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Institution: | (1) Department of Pediatrics, The Markey Cancer Center, University of Kentucky College of Medicine, Combs Research Building 204, 800 Rose Street, Lexington, KY 40536-0096, USA;(2) Fujita Health University School of Health Sciences, Toyoake Aichi, 470-1192, Japan;(3) The Graduate Center for Toxicology, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA; |
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Abstract: | The melanocortin 1 receptor (MC1R) is a transmembrane Gs-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-of-function 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. |
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