Metabolic Regulation of Articular Cartilage and Joint Homeostasis

关节软骨的代谢调节和关节稳态

• 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
• 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

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.

摘要 转化生长因子是关节软骨细胞/软骨内稳态的重要调节因子。但是，减少/缺失 转化生长因子受体(TGFBR2)在增龄、关节损伤和骨关节炎(OA)中的表达阻止转化生长因子1的使用 作为治疗骨性关节炎的一种临床方法。因此，这项提议的目标是确定关键基因、途径和 受转化生长因子1调控的潜在治疗靶点。 我们的初步数据显示，转化生长因子1调节软骨细胞的动态平衡和合成代谢的生物合成。 通过刺激葡萄糖摄取、糖酵解和合成己糖胺生物合成途径(HBP)。 具体地说，我们发现转化生长因子通过tak1信号途径，通过上调3key来诱导高血压。 基因/靶点：i)Glut1，参与葡萄糖吸收的主要酶；ii)Gfpt2(谷氨酰胺-果糖-6-磷酸 氨基转移酶-2，HBP的限速酶)，以及III)，关键的线粒体柠檬酸运输的SLC25A1 提供产生UDP-GlcNAc所需的细胞质乙酰辅酶A来源的蛋白质。UDP-GlcNAc 是HBP途径中的终末代谢物，是合成透明质酸和 糖胺聚糖(GAG)。我们的质谱学(MS)数据证实转化生长因子-1促进了 并增加了来自放射性标记葡萄糖的UDP-GlcNAc中碳的比例。 此外，我们的rna-seq数据和额外的体外数据表明，igf1是转化生长因子1的关键下游靶点。 自从诱导糖代谢、糖酵解基因表达、葡萄糖摄取、HBP和蛋白多糖 在转化生长因子1基因缺失的关节软骨细胞中，产生的产物被取消。相比之下，Igf1 过度表达模拟转化生长因子1对糖代谢和软骨合成代谢的影响 动态平衡。总之，这些新的发现表明转化生长因子/igf1信号轴的存在。 这一轴的调节可能是治疗骨性关节炎的一种很有前途的治疗策略。 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 途径是转化生长因子介导的关节软骨内稳态的关键机制。 针对TGFBR2、Tak1、Glut1、Gfpt2和SLC25A1的体外和体内互补遗传途径 由于高效液相色谱-质谱法将被用来建立对HBP的调节，作为必要的合成代谢途径 关节软骨细胞动态平衡。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信号作为转化生长因子调节血糖的下游效应 代谢与关节软骨动态平衡。总之，拟议的研究将把转化生长因子/igf1定义为一种 新途径轴在调节糖代谢、HBP和关节软骨细胞内稳态中的作用 办公自动化的背景。这项工作将加深我们对办公自动化调控机制的理解，并提供新的目标 用于创新的治疗方法。