Metabolic Regulation of Articular Cartilage and Joint Homeostasis

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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.