Metabolic Regulation of Articular Cartilage and Joint Homeostasis

关节软骨的代谢调节和关节稳态

基本信息

批准号：
10202074
负责人：
Regis J O'Keefe
金额：
$ 60.68万
依托单位：
WASHINGTON UNIVERSITY
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：
2021-07-08 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10202074
关键词：
Ablation Acetyl Coenzyme A Address Affect Aging Anabolism Attenuated Carbon Carrier Proteins Cartilage Catabolism Chondrocytes Citrates Clinical Treatment Data Degenerative polyarthritis Development Disease Enzymes Equilibrium Gene Deletion Gene Expression Gene Targeting Genes Genetic Glucose Glutamine Glycolysis Glycosaminoglycans Goals Hexosamines High Pressure Liquid Chromatography Homeostasis Hospital Costs Hyaluronic Acid IGF1 gene IGF1R gene In Vitro Injury Joints Knowledge Label Lead MAP3K7 gene Mass Spectrum Analysis Mediating Medical Medicare Medicine Metabolic Mitochondria Mus Pathway interactions Patients Pharmaceutical Preparations Production Proteoglycan Radiolabeled Regulation Replacement Arthroplasty Replacement Therapy Role Signal Transduction Source Symptoms Therapeutic Transforming Growth Factor alpha United States Up-Regulation Work articular cartilage blood glucose regulation fructose-6-phosphate gain of function genetic approach glucose metabolism glucose uptake in vitro Model in vivo innovation insight joint injury loss of function metabolomics novel overexpression prevent receptor response therapeutic target transcriptome sequencing uptake

项目摘要

ABSTRACT TGF is an essential regulator of articular chondrocyte/cartilage homeostasis. However, reduced/absent TGF receptor (Tgfbr2) expression with aging, joint injury, and in osteoarthritis (OA) prevents the use of TGF1 as a clinical treatment for OA. Therefore, the goal of this proposal is to identify the key genes, pathways, and potential therapeutic targets that are regulated by TGF1. Our preliminary data shows that TGF1 regulates chondrocyte homeostasis and anabolic biosynthesis through stimulation of glucose uptake, glycolysis and anabolic Hexosamine Biosynthetic Pathway (HBP). Specifically, we show that TGF, via TAK1 signaling, induces the HBP through upregulation of 3 key genes/targets: i) Glut1, the major enzyme involved in glucose uptake; ii) Gfpt2 (glutamine-fructose-6-phosphate amidotransferase-2, the rate limiting enzyme of HBP), and iii) Slc25a1, the key mitochondrial citrate transport protein that provides a source of cytoplasmic Acetyl CoA necessary for production of UDP-GlcNAc. UDP-GlcNAc is the terminal metabolite in the HBP pathway and is required for matrix synthesis of hyaluronic acid and glycosaminoglycans (GAGs). Our mass spectrometry (MS) data establish that TGF1 enhances the production of UDP-GlcNAc and increases the proportion of carbons in UDP-GlcNAc derived from radiolabeled glucose. Moreover, our RNA-seq data and additional in vitro data identify Igf1 as a critical downstream target of TGF1 since the induction of glucose metabolism, glycolytic gene expressions, glucose uptake, HBP, and proteoglycan production is abolished in in TGF1 treated articular chondrocytes with Igf1r gene deletion. In contrast, Igf1 over-expression mimics the effect of TGF1 on glucose metabolism as well as cartilage anabolism and homeostasis. Collectively, these novel findings indicate the existence of a TGF/IGF1 signaling axis in chondrocytes, and that modulation of this axis may be a promising therapeutic strategy to treat OA. Two Specific Aims are proposed. Specific Aim 1 will define the upregulation of Hexosamine Biosynthesis Pathway (HBP) as a key mechanism involved in TGF-mediated homeostasis of articular cartilage. Complementary in vitro and in vivo genetic approaches targeting Tgfbr2, Tak1, Glut1, Gfpt2 and Slc25a1 as well as HPLC-MS will be used to establish regulation of the HBP as an essential anabolic pathway necessary for articular chondrocyte homeostasis. Specific Aim 2 will utilize Igf1r loss-of-function and Igf1 gain-of-function models in vitro and in vivo to establish Igf1 signaling as a downstream effector of TGF regulation of glucose metabolism and articular cartilage homeostasis. In summary, the proposed studies will define TGF/IGF1 as a novel pathway axis in regulation of glucose metabolism, HBP, and articular chondrocytes homeostasis in the context of OA. This work will enhance our understanding of mechanisms regulating OA and provide novel targets for innovative therapeutic approaches.

抽象的 TGF 是关节软骨细胞/软骨稳态的重要调节剂，但其含量减少/缺失。 TGF 受体 (Tgfbr2) 在衰老、关节损伤和骨关节炎 (OA) 中的表达会阻止 TGF1 的使用因此，本提案的目标是确定关键基因、途径和途径。 TGFβ1 调节的潜在治疗靶点。我们的初步数据表明 TGF1 调节软骨细胞稳态和合成代谢生物合成通过刺激葡萄糖摄取、糖酵解和合成代谢己糖胺生物合成途径（HBP）。具体来说，我们表明 TGF 通过 TAK1 信号传导，通过上调 3 个关键因子来诱导 HBP 基因/靶标：i) Glut1，参与葡萄糖摄取的主要酶；ii) Gfpt2（谷氨酰胺-果糖-6-磷酸）酰胺转移酶-2（HBP 的限速酶）和 iii) Slc25a1（关键的线粒体柠檬酸转运）提供生产 UDP-GlcNAc 所需的细胞质乙酰辅酶 A 来源的蛋白质。是 HBP 途径中的终端代谢物，是透明质酸和基质合成所必需的我们的质谱 (MS) 数据表明 TGF1 可以增强糖胺聚糖 (GAG) 的产生。 UDP-GlcNAc 并增加源自放射性标记葡萄糖的 UDP-GlcNAc 中碳的比例。此外，我们的 RNA-seq 数据和其他体外数据将 Igf1 确定为 TGF1 的关键下游靶标由于诱导葡萄糖代谢、糖酵解基因表达、葡萄糖摄取、HBP 和蛋白聚糖在 TGFβ1 处理的 Igf1r 基因缺失的关节软骨细胞中，Igf1 的产生被消除。过度表达模拟 TGFβ1 对葡萄糖代谢以及软骨合成代谢的影响总的来说，这些新发现表明 TGFβ/IGF1 信号轴的存在。软骨细胞，调节该轴可能是治疗 OA 的一种有前途的治疗策略。提出了两个具体目标，具体目标 1 将定义己糖胺生物合成的上调。通路 (HBP) 作为参与 TGFβ 介导的关节软骨稳态的关键机制。针对 Tgfbr2、Tak1、Glut1、Gfpt2 和 Slc25a1 的补充体外和体内遗传方法因为 HPLC-MS 将用于建立 HBP 的调节，将其作为合成代谢途径所必需的具体目标 2 将利用 Igf1r 功能丧失和 Igf1 功能获得。体外和体内模型建立 Igf1 信号传导作为 TGFβ 葡萄糖调节的下游效应器总之，拟议的研究将 TGF/IGF1 定义为调节葡萄糖代谢、HBP 和关节软骨细胞稳态的新途径轴这项工作将增强我们对 OA 调节机制的理解并提供新的目标。创新的治疗方法。