Elevation of NTN4 Expression and Its Possible Regulation by Tumor Necrosis Factor-Alpha (TNF-α) in a Rat Model of Rotator Cuff Tear

TL;DR

This study finds that NTN4, a protein linked to inflammation and tissue breakdown, is elevated in rat models of rotator cuff tears and may be regulated by TNF-α, offering a new target for improving tendon healing.

Contribution

The study identifies NTN4 as a novel TNF-α-dependent mediator of extracellular matrix degradation in tendon healing.

Findings

Ntn4 gene expression significantly increases from day 14 post-injury and remains elevated.

TNF-α induces Ntn4 expression in a dose-dependent manner in cultured tenocytes.

Exogenous NTN4 increases MMP-3 expression, linking it to matrix degradation.

Abstract

Introduction: Rotator cuff tears are a major cause of shoulder dysfunction and pain, particularly in older adults, with re-tear rates remaining high despite surgical advances. Persistent inflammation and dysregulated extracellular matrix (ECM) remodeling contribute to impaired healing. Tumor necrosis factor-α (TNF-α) is known to drive tendon inflammation, but the downstream mediators linking TNF-α signaling to matrix degradation remain incompletely understood. Netrin-4 (NTN4), a laminin-related protein, has been implicated in inflammatory and ECM-modulating processes. We hypothesized that NTN4 is upregulated following tendon tear in a TNF-α-dependent manner and contributes to sustained ECM degradation.

Methods: A rat infraspinatus and supraspinatus full-thickness tendon tear model was established, and tendon tissues were harvested at 0 (intact), 7, 14, 28, and 56 days post-injury (n = 8/time point). Gene expressions of Ntn4, Il6, Tnfa, Mmp1, Mmp3, and Cxcl1 were quantified by qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction). Primary rat tenocytes were stimulated in vitro with recombinant TNF-α (0-10 ng/mL) or NTN4 (0-500 ng/mL), and target gene expression and protein secretion were assessed by qRT-PCR and ELISA (enzyme-linked immunosorbent assay). Statistical comparisons were performed using Kruskal-Wallis with Dunn's post-hoc tests.

Results: In vivo, Ntn4 expression significantly increased from day 14 onward (P < 0.001), peaking at day 28 and remaining elevated at day 56. Tnfa and Il6 were upregulated earlier (days 7-28), and Mmp3 showed sustained induction (days 7-28), while Mmp1 rose later (day 28). In vitro, TNF-α induced Ntn4 in a dose-dependent manner (P < 0.001). Exogenous NTN4 induced MMP-3 expression at both transcript and protein levels, while no significant changes were observed in MMP-1, TNF-α, or IL-6 protein levels under these experimental conditions.

Conclusions: Our findings identify NTN4 as a TNF-α-induced effector that contributes to sustained MMP-3-mediated matrix degradation following rotator cuff tear. By linking inflammatory cytokine signaling to prolonged ECM remodeling, the TNF-α/NTN4/MMP-3 axis may represent a potential therapeutic target for improving tendon healing and reducing re-tear risk.

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