Mechanisms of cAMP-dependent regulation of melanosome pH

cAMP 依赖性黑素体 pH 调节机制

• 批准号: 10211582
• 负责人: Jonathan Hale Zippin
• 金额: $ 63.37万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211582
• 关键词: Address; Adenosine Monophosphate; Adenylate Cyclase; Affect; African American; Albinism; Alleles; Bicarbonates; Cell Line; Cell model; Cells; Cutaneous; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Enzymes; Gene Expression; Genes; Genetic; Genetic Models; Genetic Polymorphism; Human; In Vitro; Investigation; Ion Channel; Knock-out; Knockout Mice; Lead; Link; Mass Spectrum Analysis; Measures; Melanins; Melanocortin 1 Receptor; Melanosomes; Methods; Microscopy; Modeling; Modification; Monophenol Monooxygenase; Mus; Mutation; Oculocutaneous albinism type 2; Organelles; Pathway interactions; Periodicity; Pharmacology; Pigmentation physiologic function; Pigments; Protein Isoforms; Proteins; Publishing; Receptor Activation; Receptor Signaling; Regulation; Risk; Role; Signal Pathway; Signal Transduction; Skin Cancer; Skin Pigmentation; Techniques; Testing; Therapeutic; Ultraviolet Rays; base; cancer risk; carcinogenicity; conditional knockout; eumelanin; experimental study; in vivo; inhibitor/antagonist; innovation; insight; loss of function; melanocyte; melanoma; mouse model; new therapeutic target; novel; novel therapeutics; photoprotection; skin cancer prevention; tool; transcription factor

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.

概括 黑色素体pH控制着色素沉着并影响皮肤癌风险；但是，影响的信号通路 这种重要的色素机制知之甚少。黑色皮质素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来影响皮肤癌的风险。 但是，MC1R信号是否影响黑色素体pH值仍不清楚。我们最近确定了一个新的 由可溶性腺苷环酶（SAC）定义的黑素细胞中的cAMP信号通路 黑色素体PH。而依赖TMAC的营地的升高会通过上调钥匙来增加Eumelanin 颜料酶（例如酪氨酸酶），SAC依赖性cAMP的降低也会增加依umelanin 诱导黑色素体pH的碱化并增强酪氨酸酶活性。因此，我们的总体 假设是SAC和TMAC调节黑素细胞中不同的cAMP信号级联和功能 控制色素沉着的音乐会。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MC1R或MC1R多态性以及MC1RE小鼠的主要黑素细胞 黑色素细胞和新型MC1RE（E/E）有条件的SAC基因敲除小鼠，以确定之间的相互作用 MC1R信号传导和SAC依赖性对黑色素体pH和色素沉着的控制。在我们的第一个目标中，我们也将 评估碳酸氢盐是已知的SAC的刺激剂，与黑色素合成有关，会影响 人和小鼠黑素细胞中的黑色素体pH和色素沉着以SAC依赖性方式。在AIM 2中， 我们将确定SAC如何调节黑色素体pH和色素沉着。我们的初步数据表明 SAC激活cAMP（EPAC）激活的营地效应蛋白交换蛋白，然后刺激 黑色素体离子通道两孔通道2（TPC2）。使用遗传和药理方法 小鼠和人类黑素细胞，我们将确定哪些EPAC同工型和黑色素体通道是 依赖于黑色素体pH的SAC依赖性控制所需。最后，我们的初步数据表明SAC 抑制作用挽救了OCA2中缺陷的黑色素体pH和酪氨酸酶活性删除的小鼠黑素细胞 体外和小鼠。因此，药理学SAC抑制剂是眼皮抑制剂的潜在治疗剂 白化病2型。我们将通过新的有条件的SAC敲除OCA2 - / - 进一步探索这种治疗可能性 （P/P）鼠标模型。总体而言，该提案中的实验将系统地检查营地依赖性 信号调节黑素体pH和色素沉着的信号级联反应。拟议的研究将建立新的 在我们调查色素沉着中的cAMP信号传导中将克服限制的模型将提供 对控制黑素体pH的cAMP依赖机制有更深入的了解，并可能导致新的 用于色素沉着疾病的治疗剂。