Exploratory transcriptomic analysis of mouse articular cartilage in response to tissue inhibitor of metalloproteinase 3 identifies inflammation-associated gene expression changes.

INTRODUCTION

Tissue inhibitor of metalloproteinase 3 (TIMP-3) is a broad-spectrum inhibitor of matrix metalloproteinases (MMPs) and ADAM/ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family enzymes that regulate extracellular matrix (ECM) homeostasis. Because these enzymes play key roles in articular cartilage turnover, TIMP-3-mediated inhibition protects against cartilage degradation, a hallmark of osteoarthritis (OA), and has been explored as a therapeutic target. Nonetheless, unexpected detrimental effects of TIMP-3 on bone mass and structure have been reported in transgenic mice overexpressing TIMP-3 in cartilage. Mechanistically, TIMP-3 binds catabolic enzymes and blocks their active sites but also interacts with low-density lipoprotein receptor-related protein 1 (LRP-1) and sulfated proteoglycans in the ECM, processes that regulate its half-life through a balance between endocytosis and ECM retention and may influence cell signaling.

METHODS

We investigated whether TIMP-3 affects gene expression in ex vivo mouse articular cartilage explants under normoxia or physiological hypoxia (3% O2). Femoral head cartilage explants were treated with recombinant TIMP-3 and then processed for RNA sequencing (RNA-seq).

RESULTS

Hypoxia alone induced a strong transcriptional response, confirming the model's responsiveness, whereas TIMP-3 altered the expression of only a small subset of genes. RT-qPCR validation confirmed TIMP-3-mediated upregulation of inflammation-associated genes, including Saa3 under both oxygen conditions, and IL-17 signaling pathway genes (Il17b, Mmp3 and Lcn2) under normoxia, and downregulation of proliferative genes Pbk/Topk and Racgap1 under hypoxia. Hypoxia alone downregulated all these genes.

DISCUSSION: The distinct transcriptional effects observed under normoxia and hypoxia highlight the importance of accounting for oxygen tension in cartilage studies. Potential inflammation-associated gene expression responses to TIMP-3 should be considered in its therapeutic development for arthritic disease and may inform optimization of treatment strategies.