Metabolic Regulation of Articular Cartilage and Joint Homeostasis

摘要 TGF β是关节软骨细胞/软骨稳态的重要调节剂。减少/不存在 TGF β 1受体（Tgfbr 2）在衰老、关节损伤和骨关节炎（OA）中的表达阻止了TGF β 1的使用 作为OA的临床治疗。因此，本提案的目标是确定关键基因，途径， TGF β 1调节的潜在治疗靶点。 我们的初步数据表明，TGF β 1调节软骨细胞的稳态和合成代谢的生物合成 通过刺激葡萄糖摄取、糖酵解和合成代谢氨基己糖生物合成途径（HBP）。 具体地说，我们发现TGF β 1通过TAK 1信号传导，通过上调3个关键的HBP， 基因/靶标：i）Glut 1，参与葡萄糖摄取的主要酶; ii）Gfpt 2（谷氨酰胺-果糖-6-磷酸 酰胺转移酶-2，HBP的限速酶），和iii）Slc 25 a1，线粒体柠檬酸盐转运的关键 提供产生UDP-GlcNAc所必需的细胞质乙酰辅酶A来源的蛋白质。UDP-GlcNAc 是HBP途径中的末端代谢产物，是透明质酸基质合成所必需的， 糖胺聚糖（GAG）。我们的质谱（MS）数据证实TGF β 1促进了 的UDP-GlcNAc和增加的比例的碳在UDP-GlcNAc衍生自放射性标记的葡萄糖。 此外，我们的RNA-seq数据和额外的体外数据将Igf 1鉴定为TGF β 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和蛋白多糖 在TGF β 1处理的Igf 1 r基因缺失的关节软骨细胞中，相反，Igf 1 过度表达模拟TGF β 1对葡萄糖代谢以及软骨附着的作用， 体内平衡总的来说，这些新的发现表明，TGF β 1/IGF 1信号轴的存在， 软骨细胞，并且调节该轴可能是治疗OA的有前景的治疗策略。 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 HBP通路是参与TGF β 1介导的关节软骨稳态的关键机制。 靶向Tgfbr 2、Tak 1、Glut 1、Gfpt 2和Slc 25 a1的体外和体内互补遗传方法 因为HPLC-MS将用于建立HBP的调节，作为必需的合成代谢途径， 关节软骨细胞稳态具体目标2将利用Igf 1 r功能丧失和Igf 1功能获得 体外和体内模型，以建立Igf 1信号传导作为TGF β 1调节葡萄糖的下游效应物 代谢和关节软骨稳态。总之，拟议的研究将TGF β 1/IGF 1定义为 新的途径轴调节葡萄糖代谢，HBP，和关节软骨细胞的稳态， OA的背景。这项工作将提高我们对OA调控机制的理解，并提供新的靶点 创新的治疗方法。

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.