Mechanisms of cAMP-dependent regulation of melanosome pH

总结 黑素体pH值控制色素沉着并影响皮肤癌风险;然而，影响皮肤癌的信号通路 这一重要的颜料机理知之甚少。黑皮质素1受体（MC 1 R），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来影响皮肤癌的风险。 但MC 1 R信号是否影响黑素体pH值仍不清楚。我们最近发现了一种新的 黑素细胞中的cAMP信号通路，由可溶性腺苷酸环化酶（sAC）定义，其调节 而tmAC依赖性cAMP的升高通过上调黑素小体的关键酶而增加真黑素。 色素酶（例如，酪氨酸酶），sAC依赖性cAMP的减少也增加了真黑素， 诱导黑素体pH的碱化和增强酪氨酸酶活性。因此， 假设sAC和tmAC调节黑素细胞中不同的cAMP信号级联， 音乐会，以控制色素沉着。目前尚不清楚的是上游和下游机制， 控制黑素体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 表达野生型MC 1 R或MC 1 R多态性的原代黑素细胞与Mc 1 re小鼠沿着 黑素细胞和一种新的Mc 1 re（e/e）条件性sAC敲除小鼠，以确定 MC 1 R信号传导和黑素体pH和色素沉着的sAC依赖性控制。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 黑素体pH和人和小鼠黑素细胞中的色素沉着以sAC依赖的方式。在目标2中， 我们将确定sAC如何调节黑素体pH和色素沉着。我们的初步数据显示， sAC激活由cAMP激活的cAMP效应蛋白交换蛋白（EPAC），然后刺激 黑素体离子通道双孔通道2（TPC 2）。利用遗传学和药理学方法， 小鼠和人类黑素细胞，我们将建立哪些EPAC亚型和黑素体通道， 最后，我们的初步数据表明，sAC 在Oca 2缺失的小鼠黑素细胞中抑制拯救有缺陷的黑素体pH和酪氨酸酶活性 无论是在体外还是在小鼠中。因此，药理学sAC抑制剂是眼皮肤炎的潜在治疗剂。 白化病2型。我们将进一步探索这种治疗的可能性与新的条件sAC敲除Oca 2-/- （p/p）小鼠模型。总的来说，本提案中的实验将系统地检查cAMP依赖性的 调节黑素体pH和色素沉着的信号级联。这些研究将建立新的 模型将克服我们在色素沉着中cAMP信号转导研究的局限性，将提供 更深入地了解控制黑素体pH值的cAMP依赖性机制，并可能导致新的 色素沉着疾病的治疗剂。

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.