ABSTRACT The TGFβ pathway plays a critical role in maintaining the homeostasis and overall health of articular cartilage. However, the diminished expression of the TGFβ receptor (Tgfbr2) in osteoarthritis (OA) cartilage hampers the utilization of TGF-β1 as a therapeutic treatment for OA. Therefore, the goal of this proposal is to identify the key downstream mediators as well as potential therapeutic targets that are regulated by TGFβ1. We performed an unbiased multi-omics screening combining RNA-seq, ATAC-seq as well as metabolomics, and establish that TGFβ1 maintains articular chondrocyte homeostasis at least partially through suppression of lipid metabolism. We have shown that Nuclear Factor I A (NFIA) is a crucial transcription factor in the regulation of lipid metabolism in articular chondrocytes. Specifically, TGFβ1 reduces Nfia expression in murine articular chondrocyte, while increased Nfia expression is observed in Tgfbr2 loss-of-function (LOF) chondrocytes and human OA articular chondrocytes. Mechanistically, Nfia physically binds to the promoter regions of Acetyl-CoA carboxylase a (Acaca) and Carnitine palmitoyltransferase 2 (Cpt2) genes, which are rate-limiting enzymes for fatty acid synthesis and oxidation, respectively. Consequently, Nfia overexpression in articular chondrocytes induces the expression of both Acaca and Cpt2. Conversely, Nfia inhibition results in the suppression of the elevated expression of Acaca and Cpt2 in Tgfbr2 LOF chondrocytes. More importantly, NFIA LOF restores lipid metabolism and cellular homeostasis in human OA articular chondrocytes, and Nfia gene ablation in articular cartilage attenuates injury-induced OA progression in mice. Thus, Nfia represents a potential therapeutic target for OA. Based on these novel findings, our central hypothesis proposes that TGFβ1 maintains articular cartilage homeostasis by suppressing lipid metabolism through Nfia inhibition, and lipid metabolism is a viable and highly innovative target for OA treatment. Two Specific Aims are proposed. Specific Aim 1 will establish Nfia as a downstream mediator of the TGFβ pathway in regulating articular chondrocyte lipid metabolism and homeostasis in mice. Complementary in vitro and in vivo genetic approaches along with metabolomics analyses will be employed to establish TGFβ/Nfia-regulated lipid metabolism as a critical pathway necessary for articular chondrocyte homeostasis. Specific Aim 2 will define Nfia-mediated lipid metabolism as a potential therapeutic target for OA treatment. We will investigate if cartilage specific Nfia LOF abolishes the increase in lipid metabolism and attenuates OA progression associated with obesity and aging in mice. Single cell RNA-seq and metabolomics will be used to establish the mechanism behind the whole joint protection. In summary, this proposal will advance our understanding of mechanisms regulating lipid metabolism in OA disease and define Nfia mediated lipid metabolism as a novel target to trea...