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

TL;DR

This study explores how TIMP-3 affects gene activity in mouse cartilage under different oxygen levels, revealing inflammation-related changes that could impact arthritis treatments.

Contribution

The study reveals TIMP-3's differential gene expression effects under normoxia and hypoxia, highlighting oxygen tension's role in cartilage biology.

Findings

TIMP-3 upregulates inflammation genes like Saa3 and IL-17 pathway genes under normoxia.

Hypoxia downregulates proliferative genes Pbk/Topk and Racgap1 when combined with TIMP-3.

Oxygen tension significantly influences TIMP-3's transcriptional effects in cartilage.

Abstract

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.

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).

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.

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.

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