TSP2 Deficiency Promotes Fibroblast Proliferation and Migration With Enhanced WNT4/β‐Catenin/TGFb3

ABSTRACT Chronic wounds, a major healthcare burden, are characterized by impaired fibroblast function and ECM remodeling. Thrombospondin‐2 (TSP2), a matricellular glycoprotein, has been shown to negatively regulate wound healing. Here, we investigated the cellular and transcriptomic consequences of TSP2 deficiency in dermal fibroblasts, key cells in tissue repair and extracellular matrix (ECM) remodeling. Using bulk RNA sequencing of wild‐type (WT) and TSP2 knockout (TSP2 KO) murine primary fibroblasts, we identified upregulation of pro‐regenerative molecules and signaling pathways, specifically TGF‐β3 and Wnt4/β‐catenin, in the latter. To overcome the inherent variability of primary cells and establish a robust model, we generated a stable CRISPR/Cas9‐engineered TSP2 knockout in NIH3T3 fibroblasts. This system confirmed that TSP2 depletion enhances fibroblast proliferation and migration, associated with increased activity of TGF‐β3 and Wnt/β‐catenin signaling pathways. These findings not only provide novel mechanistic insights into the role of TSP2 in regulating fibroblast function and ECM interactions during tissue repair, but also highlight TSP2 as a potential therapeutic target for promoting regeneration in healing‐impaired or chronic wounds.