Mechanisms of cAMP-dependent regulation of melanosome pH

摘要 黑素小体pH控制色素沉积并影响皮肤癌风险；然而，影响皮肤癌的信号通路 人们对这种重要的色素机制知之甚少。黑素皮质素1受体(MC1R)，通过 跨膜腺苷酸环化酶(TMAC)定义的cAMP信号通路在 色素沉着，并通过激活关键色素合成酶的表达而影响皮肤癌风险。 但MC1R信号是否影响黑素小体的pH仍不清楚。我们最近发现了一种新的 黑素细胞中的cAMP信号通路，由可溶性腺苷环化酶(SAC)定义，调节 黑素小体pH值。而依赖麦迪的cAMP的升高通过上调Key来增加真黑素 色素酶(如酪氨酸酶)，SAC依赖的cAMP的减少也通过 诱导黑素小体pH碱化，提高酪氨酸酶活性。因此，我们最重要的是 假说是SAC和tmACs调节黑素细胞中不同的cAMP信号级联反应，并在 控制色素沉着的音乐会。目前尚不清楚的是 控制黑素小体pH和色素沉着的SAC依赖性调节。在我们的第一个目标中，我们将使用人类 原代表达野生型MC1R或MC1R多态的黑素细胞与Mc1re小鼠 黑素细胞和一种新的Mc1re(e/e)条件性SAC基因敲除小鼠之间的相互作用 MC1R信号和SAC依赖的黑素小体pH和色素沉着的控制。在我们的第一个目标中，我们还将 评估碳酸氢盐，一种已知的SAC刺激物，与黑色素合成有关，是否影响 人和小鼠黑素细胞中黑素小体的pH值和色素沉着依赖于SAC的方式。在目标2中， 我们将确定SAC如何调节黑素小体的pH值和色素沉着。我们的初步数据表明 SAC激活cAMP激活的cAMP效应蛋白交换蛋白(EPAC)，进而刺激 黑素小体离子通道两孔通道2(TPC2)。利用遗传学和药理学方法 小鼠和人类黑素细胞，我们将确定EPAC亚型和黑素小体通道是 依赖SAC控制黑素小体的pH所必需的。最后，我们的初步数据表明，SAC 抑制修复OCA2缺失小鼠黑素细胞黑素小体pH和酪氨酸酶活性的缺陷 无论是在体外还是在小鼠身上。因此，药理上的SAC抑制剂是潜在的眼皮肤疗法。 白化病2型。我们将通过一种新的条件SAC基因敲除OCA2-/-进一步探索这种治疗的可能性 (p/p)小鼠模型。总体而言，这项提案中的实验将系统地检查依赖营地的人 调节黑素小体pH值和色素沉着的信号级联反应。拟议的研究将建立新的 将克服我们对cAMP信号在色素沉着中的研究的局限性的模型，将提供 更深入地了解控制黑素小体pH的cAMP依赖机制，并可能导致新的 色素沉着性疾病的治疗。

SUMMARY Melanosome pH controls pigmentation and affects skin cancer risk; however, the signaling pathways that affect this important pigment mechanism are poorly understood. The Melanocortin 1 Receptor (MC1R), through transmembrane adenylyl cyclase (tmAC)-defined cAMP signaling pathways, has an important role in pigmentation, and affects skin cancer risk by activating the expression of key pigment synthesizing enzymes. But whether MC1R signaling affects melanosome pH has remained unclear. We recently identified a new cAMP signaling pathway in melanocytes, defined by the soluble adenylyl cyclase (sAC), that regulates melanosome pH. Whereas elevation of tmAC-dependent cAMP increases eumelanin by upregulating key pigment enzymes (e.g., tyrosinase), a reduction in sAC-dependent cAMP also increases eumelanin by inducing the alkalization of melanosome pH and enhancing tyrosinase activity. Thus, our overarching hypothesis is that sAC and tmACs regulate distinct cAMP signaling cascades in melanocytes and function in concert to control pigmentation. What remains unclear are the upstream and downstream mechanisms that control sAC-dependent regulation of melanosome pH and pigmentation. In our first Aim, we will use human primary melanocytes expressing either wild type MC1R or MC1R polymorphisms along with Mc1re mouse melanocytes and a novel Mc1re (e/e) conditional sAC knockout mouse to determine the interplay between MC1R signaling and sAC-dependent control of melanosome pH and pigmentation. In our first Aim, we will also assess whether bicarbonate, a known stimulator of sAC that has been linked to melanin synthesis, affects melanosome pH and pigmentation in human and mouse melanocytes in a sAC-dependent manner. In Aim 2, we will determine how sAC regulates melanosome pH and pigmentation. Our preliminary data suggests that sAC activates the cAMP effector protein exchange protein activated by cAMP (EPAC), which then stimulates the melanosome ion channel two-pore channel 2 (TPC2). Using genetic and pharmacological methods in mouse and human melanocytes, we will establish which EPAC isoforms and melanosome channels are required for sAC-dependent control of melanosome pH. Finally, our preliminary data suggest that sAC inhibition rescues the defective melanosome pH and tyrosinase activity in Oca2 deleted mouse melanocytes both in vitro and in mice. Thus, pharmacological sAC inhibitors are potential therapeutics for oculocutaneous albinism type 2. We will further explore this therapeutic possibility with a new conditional sAC knockout Oca2-/- (p/p) mouse model. Overall, the experiments in this proposal will systemically examine the cAMP dependent signaling cascades that regulate melanosome pH and pigmentation. The proposed studies will establish new models that will overcome limitations in our investigation of cAMP signaling in pigmentation, will provide greater insight into the cAMP-dependent mechanisms that control melanosome pH, and may lead to new therapeutics for diseases of pigmentation.