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Mechanisms of cAMP-dependent regulation of melanosome pH

cAMP 依赖性黑素体 pH 调节机制

基本信息

批准号：
10596538
负责人：
Jonathan Hale Zippin
金额：
$ 62.45万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
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/10596538
关键词：
5&apos -AMP-activated protein kinase Address Adenylate Cyclase Affect African African American population 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 Link Mass Spectrum Analysis Measures Melanins Melanocortin 1 Receptor Melanosomes Membrane Methods Microscopy Modeling Modification Monophenol Monooxygenase Mus Mutation Oculocutaneous albinism type 2 Organelles Pathway interactions 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 cancer risk carcinogenicity conditional knockout eumelanin experimental study in vivo inhibitor innovation insight loss of function melanocyte melanoma mouse model new therapeutic target novel novel therapeutics pharmacologic photoprotection skin cancer prevention solar ultraviolet radiation synergism tool transcription factor

项目摘要

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.

概括黑素体 pH 值控制色素沉着并影响皮肤癌风险；然而，影响的信号通路人们对这一重要的色素机制知之甚少。黑皮质素 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风险。但 MC1R 信号传导是否影响黑素体 pH 值仍不清楚。我们最近发现了一个新的黑素细胞中的 cAMP 信号通路，由可溶性腺苷酸环化酶 (sAC) 定义，可调节黑素体 pH 值。而 tmAC 依赖性 cAMP 的升高则通过上调关键的关键因子来增加真黑色素色素酶（例如酪氨酸酶），sAC 依赖性 cAMP 的减少也会增加真黑色素诱导黑素体pH值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，并在音乐会控制色素沉着。目前尚不清楚的是其上游和下游机制控制黑素体 pH 值和色素沉着的 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 小鼠的原代黑素细胞黑素细胞和新型 Mc1re (e/e) 条件 sAC 敲除小鼠以确定黑素细胞之间的相互作用 MC1R 信号传导和 sAC 依赖性黑素体 pH 值和色素沉着控制。在我们的第一个目标中，我们还将评估碳酸氢盐（一种已知的与黑色素合成有关的 sAC 刺激剂）是否会影响以 sAC 依赖性方式影响人类和小鼠黑素细胞中黑素体 pH 值和色素沉着。在目标 2 中，我们将确定 sAC 如何调节黑素体 pH 值和色素沉着。我们的初步数据表明 sAC 激活由 cAMP (EPAC) 激活的 cAMP 效应蛋白交换蛋白，然后刺激黑素体离子通道双孔通道 2 (TPC2)。利用遗传和药理学方法小鼠和人类黑素细胞，我们将确定哪些 EPAC 亚型和黑素体通道是 sAC 依赖性黑素体 pH 控制所需。最后，我们的初步数据表明 sAC 抑制可挽救 Oca2 缺失的小鼠黑素细胞中存在缺陷的黑素体 pH 值和酪氨酸酶活性体外和小鼠体内。因此，药理学 sAC 抑制剂是眼皮肤疾病的潜在治疗方法。 2 型白化病。我们将通过新的条件性 sAC 敲除 Oca2-/- 进一步探索这种治疗可能性 (p/p) 小鼠模型。总的来说，本提案中的实验将系统地检查 cAMP 依赖性调节黑素体 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在色素沉着中 cAMP 信号传导研究中的局限性的模型，将提供更深入地了解控制黑素体 pH 值的 cAMP 依赖性机制，并可能导致新的发现色素沉着疾病的治疗。