The Graduate Center for Toxicology, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA.
In Vitro Cell Dev Biol Anim. 2009 Dec;45(10):577-83. doi: 10.1007/s11626-009-9232-3.
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.
黑素皮质素 1 受体（MC1R）是一种位于黑素细胞上的跨膜 G(s)偶联表面蛋白，可与黑素细胞刺激素结合，并介导腺苷酸环化酶的激活和第二信使环磷酸腺苷（cAMP）的产生。MC1R 调节黑素细胞的生长和分化，并防止致癌作用。MC1R 功能丧失性多态性的个体往往对紫外线敏感（皮肤白皙，晒黑反应差），并且患黑色素瘤的风险很高。然而，MC1R 在黑素细胞对紫外线反应中的作用的机制研究在一定程度上受到阻碍，因为 Mc1r 缺陷型原代鼠黑素细胞难以在体外培养。到目前为止，鼠黑素细胞的有效生长依赖于 cAMP 刺激，使用腺苷酸环化酶激活剂或磷酸二酯酶抑制剂。然而，在 MC1R 信号传导缺陷的情况下，挽救 cAMP 可能会混淆直接测试 MC1R 功能对黑素细胞紫外线反应的实验。在本文中，我们报告了一种在没有药理学 cAMP 刺激的情况下培养原代鼠黑素细胞的新方法，方法是将来源于原代角质形成细胞的干细胞因子的条件培养基纳入其中。重要的是，这种方法似乎允许培养的黑素细胞与它们的亲本鼠系皮肤中的色素表达相似。这种新方法将允许对 MC1R 在保护免受 UV 介导的致癌作用中的作用进行机制研究，并确定黑色素色素亚型在 UV 介导的黑素细胞反应中的作用。
