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Granzyme B mediates cleavage of Thrombospondin-2 : impact on keratinocyte viability and adhesion Wilhelm, Anna-Catharina

Abstract

The epidermis, consisting of keratinocytes, is the first defense against the exterior environment of the skin. Integral to maintaining the homeostasis of skin function by aiding in thermoregulation, fluid retention, and pathogen defense, the extracellular matrix composition aids in regulating keratinocyte migration after injury. Impaired healing processes can progress into chronic non-healing wounds, leading to infection and mortality. Granzyme B (GzmB) is a serine protease, significantly elevated in chronic non-healing wounds. GzmB impairment of wound healing is attributed to its ability to sustain its proteolytic activity and downstream inflammation, inhibiting wound repair and tissue remodeling following injury. GzmB is classically known for its intracellular role in cytotoxic lymphocyte-mediated apoptosis. Recent evidence demonstrates that extracellular GzmB has the ability to cleave a variety of important extracellular proteins. Thrombospondin-2 (TSP-2) is an extracellular glycoprotein secreted by a variety of cells known to be involved in cell-cell and cell-matrix interactions. TSP-2 has been reported to impair viability, adhesion, and migration in endothelial cells and squamous carcinoma cells. Although extensive studies have been done with TSP-2, literature investigating the role of TSP-2 in the skin has largely been neglected. I hypothesized that GzmB cleaves TSP2, and GzmB cleavage of TSP-2 mediates extracellular matrix disorganization, altered keratinocyte viability, and reduced adhesion. To investigate this hypothesis purposed two aims. In the first aim, cell-free protease assays were utilized to determine whether TSP-2 is a proteolytic substrate of GzmB. In the second aim, the impact of TSP-2-mediated cleavage was assessed using cultured human keratinocytes. TSP-2 was cleaved by GzmB in a time- and dose-dependent manner. Further, while GzmB-mediated TSP-2 cleavage did not affect cell viability, it did reduce cellular adhesion in a dose-dependent manner. In summary, TSP-2 is a proteolytic substrate of GzmB and such cleavage interferes with keratinocyte adhesion.

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