Epigenetic Regulation of Lysosomal Ceramide Signaling and Function in Arterial Myocytes: Role of Kmt6 Gene

动脉肌细胞溶酶体神经酰胺信号和功能的表观遗传调控：Kmt6 基因的作用

• 批准号: 10298620
• 负责人: PinLan Li
• 金额: $ 62.89万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10298620
• 关键词: Abbreviations; Aging; Agonist; Applications Grants; Arteries; Atherosclerosis; Binding; Blood Vessels; Calcium; Calmodulin; Cathepsins B; Cell membrane; Cells; Ceramides; Chronic Kidney Failure; Code; Collagen; DNA; DNA Methylation; DNA Modification Methylases; Defect; Deposition; Development; Diabetes Mellitus; Disease; EZH2 gene; Early Endosome; Enzymes; Epigenetic Process; Extracellular Matrix; Face; Functional disorder; Gene Deletion; Gene Expression; Genes; Genetic Transcription; Histone-Lysine N-Methyltransferase; Histones; Hypertension; Image; Knock-out; Knockout Mice; Lead; Link; Lipids; Lysine; Lysosomes; Medial; Mediating; Membrane Proteins; Metabolism; Methylation; Methyltransferase; MicroRNAs; Molecular; Multivesicular Body; Mus; Muscle Cells; Pathologic; Pathologic Processes; Patients; Phenotype; Play; Process; Production; Proteins; Regulation; Reporting; Research; Role; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Sphingolipids; Sphingomyelinase; Sphingomyelins; Sphingosine; Stimulus; Testing; Time; Vascular calcification; Vesicle; Vitamin D; acid sphingomyelinase; arterial stiffness; base; calcification; cardiovascular disorder prevention; cardiovascular disorder risk; cardiovascular risk factor; enzyme activity; epigenetic regulation; exosome; galactosylgalactosylglucosylceramidase; gene repression; histone methylation; interstitial; late endosome; mineralization; new therapeutic target; novel; osteogenic; particle; patch clamp; prevent; programs; receptor; sensor; sphingosine 1-phosphate; trafficking; treatment strategy

Project Summary Arterial medial calcification (AMC) and arterial stiffening are a prevalent pathological process in different pathological conditions or diseases such as hypertension, aging, atherosclerosis, diabetes and chronic kidney disease. Enhanced exosome secretion by smooth muscle cells (SMCs) has been reported to be an essential mechanism for calcifying nidus formation and extracellular matrix mineralization in the arterial wall to result in AMC. Recent studies have also shown that lysosome function plays a critical role in controlling multivesicular body (MVB) fate and enhancing exosome secretion and thereby in the development of arterial calcification. However, it remains poorly understood how lysosome function is controlled to determine exosome secretion and thereby lead to AMC. This proposal seeks to explore a novel epigenetic mechanism that regulates lysosome trafficking and exosome secretion, which may contribute to the development of AMC. This epigenetic regulation of lysosome function may be associated with the lysine methyltransferase Kmt6- mediated repression of gene transcription of Smpd1, a lysosome enzyme that hydrolyzes sphingomyelin into ceramide. Kmt6 is considered as a crucial epigenetic regulator that represses the target gene expression by methylation of lysine residue in histone proteins. In preliminary studies, we demonstrated that SMC-specific Kmt6 gene deletion exacerbated AMC and arterial stiffening, which were associated with increased Smpd1 expression and ceramide production, reduced lysosome TRPML1 channel activity, and lysosome trafficking dysfunction. These observations led us to hypothesize that Kmt6 is an essential epigenetic regulatory enzyme that controls lysosomal Smpd1-mediated sphingolipid metabolism and thereby regulates lysosome trafficking or its fusion to MVBs and subsequent exosome secretion in SMCs. Kmt6 gene defect or functional deficiency may disturb lysosome-mediated degradation of MVBs leading to increased exosome secretion, calcifying nidus formation, osteogenic transition, and ultimately AMC in face of different pathological challenges. To test this hypothesis, the following Specific Aims are proposed. Aim 1 will determine loss of Kmt6 contributes to osteogenic transition and AMC in SMC-specific Kmt6 knockout mice with analysis of SMC phenotypes and calcification. Aim 2 attempts to test whether Kmt6-mediated epigenetic regulation of Smpd1 critically controls lysosome trafficking and exosome secretion by increasing TRPML1 channel activity and associated Ca2+ release using patch clamping of isolated lysosomes and lysosome-specific Ca2+ imaging. Aim 3 will explore the molecular mechanisms how Smpd1 gene is epigenetically regulated by Kmt6 with a focus on its action on histone and DNA methylation in cultured arterial SMCs. Our findings will for the first time define an epigenetic mechanism controlling Smpd1 expression and activity via Kmt6 in SMCs and reveal a novel role of epigenetic dysregulation of sphingolipid metabolism in the development of AMC and arterial stiffening. These findings may also identify novel therapeutic targets for the treatment of AMC under different pathological conditions.

项目摘要 动脉内侧钙化（AMC）和动脉僵硬是不同的病理过程 病理状况或疾病，例如高血压，衰老，动脉粥样硬化，糖尿病和慢性肾脏 疾病。据报道，增强平滑肌细胞（SMC）的外泌体分泌是必不可少的 钙化Nidus形成和动脉壁细胞外基质矿化的机制，导致 AMC。最近的研究还表明，溶酶体功能在控制多尖端方面起着至关重要的作用 身体（MVB）命运并增强外泌体分泌，从而在动脉钙化的发展中。 但是，它仍然不太了解如何控制溶酶体功能以确定外泌体分泌 从而导致AMC。该提议旨在探索一种调节的新型表观遗传机制 溶酶体贩运和外泌体分泌，这可能有助于AMC的发展。这 溶酶体功能的表观遗传调节可能与赖氨酸甲基转移酶KMT6-相关 SMPD1基因转录的介导的抑制作用，Smpd1是一种将鞘磷脂水解为的溶酶体酶 神经酰胺。 KMT6被认为是一种至关重要的表观遗传调节剂，可通过 赖氨酸残基在组蛋白蛋白中的甲基化。在初步研究中，我们证明了SMC特异性 KMT6基因缺失加剧了AMC和动脉僵硬，与SMPD1增加有关 表达和神经酰胺的产生，溶酶体TRPML1通道活性减少和溶酶体运输 功能障碍。这些观察结果使我们假设KMT6是必不可少的表观遗传调节酶 控制溶酶体SMPD1介导的鞘脂代谢，从而调节溶酶体贩运 或它与MVB的融合及其随后在SMC中的外泌体分泌。 KMT6基因缺陷或功能缺陷 可能会干扰MVB的溶酶体介导的降解导致外泌体分泌增加，钙化 面对不同的病理挑战，Nidus的形成，成骨过渡和最终AMC。测试 该假设提出了以下特定目标。 AIM 1将确定KMT6的损失有助于 SMC表型和SMC特异性KMT6基因敲除小鼠中的成骨过渡和AMC，分析SMC表型和 钙化。 AIM 2尝试测试KMT6介导的SMPD1的表观遗传调节是否严格控制 通过增加TRPML1通道活性和相关的Ca2+，溶酶体运输和外泌体分泌 使用分离的溶酶体和溶酶体特异性CA2+成像的斑块夹紧。 AIM 3将探索 分子机制SMPD1基因如何表观遗传受到KMT6的调节，重点是其对其对作用的作用 培养的动脉SMC中的组蛋白和DNA甲基化。我们的发现将首次定义表观遗传学 通过SMC中KMT6控制SMPD1表达和活性的机制，并揭示了表观遗传学的新作用 鞘脂代谢在AMC和动脉僵硬的发展中的失调。这些发现 还可以鉴定出在不同病理条件下治疗AMC的新型治疗靶标。