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: 10450193
• 负责人: PinLan Li
• 金额: $ 61.31万
• 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/10450193
• 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 动脉中膜钙化（AMC）和动脉硬化是一种普遍的病理过程，在不同的 病理状况或疾病，如高血压、衰老、动脉粥样硬化、糖尿病和慢性肾病 疾病据报道，平滑肌细胞（SMC）分泌的增强的外泌体是平滑肌细胞（SMC）分泌外泌体的必要条件。 动脉壁中钙化病灶形成和细胞外基质矿化的机制， AMC。最近的研究还表明，溶酶体功能在控制多泡囊泡形成中起着关键作用。 在动脉钙化的发展中，MVB的命运和增强外泌体的分泌，从而促进动脉钙化。 然而，对于溶酶体的功能是如何被控制来决定外泌体分泌的， 从而导致AMC。这项提案旨在探索一种新的表观遗传机制， 溶酶体运输和外泌体分泌，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Kmt 6- 介导Smpd 1基因转录的抑制，Smpd 1是一种溶酶体酶，可将鞘磷脂水解为 神经酰胺Kmt 6被认为是一个重要的表观遗传调节因子，通过抑制靶基因的表达， 组蛋白中赖氨酸残基的甲基化。在初步研究中，我们证明了SMC特异性 Kmt 6基因缺失加重AMC和动脉硬化，与Smpd 1增加相关 表达和神经酰胺产生，降低溶酶体TRPML 1通道活性，以及溶酶体运输 功能障碍这些观察使我们假设Kmt 6是一种重要的表观遗传调节酶 控制溶酶体Smpd 1介导的鞘脂代谢，从而调节溶酶体运输 或其与MVB的融合以及随后在SMC中的外泌体分泌。Kmt 6基因缺陷或功能缺陷 可能干扰溶酶体介导的MVB降解，导致外泌体分泌增加，钙化 病灶形成、成骨转化和最终AMC面临不同的病理挑战。测试 根据这一假设，提出了以下具体目标。目标1将确定Kmt 6的损失有助于 SMC特异性Kmt 6基因敲除小鼠的成骨转变和AMC，分析SMC表型， 钙化目的2试图测试Kmt 6介导的Smpd 1表观遗传调控是否严格控制 通过增加TRPML 1通道活性和相关的Ca 2+， 使用分离的溶酶体的膜片钳和溶酶体特异性Ca 2+成像释放。Aim 3将探索 Smpd 1基因如何被Kmt 6表观遗传调控的分子机制，重点是其对 组蛋白和DNA甲基化。我们的发现将首次定义一个表观遗传 通过Kmt 6在SMC中控制Smpd 1表达和活性的机制，并揭示了表观遗传学的新作用。 在AMC和动脉硬化的发展中鞘脂代谢失调。这些发现 还可以鉴定用于在不同病理条件下治疗AMC的新的治疗靶点。