862 Absence of collagen VII binding to thrombospondin 1 promotes activation of TGF-beta in recessive dystrophic epidermolysis bullosa

Abstract

Mutations in the gene encoding collagen VII (C7) cause the devastating blistering disease recessive dystrophic epidermolysis bullosa (RDEB). This disease is characterized by severe skin fragility and non-healing wounds aggravated by scarring and fibrosis. The altered skin architecture in RDEB also allows for development of fatal squamous cell carcinoma. We previously demonstrated that the matricellular protein thrombospondin-1 (TSP1) is increased in RDEB fibroblasts and that C7 expression modulates TSP1. Because TGF-beta is increased in RDEB and correlates with disease severity, and TSP1 is shown to activate TGF-beta, we investigated the role of TSP1 in TGF-beta activation in RDEB patient cells. Knock-down of TSP1 reduced phosphorylation of SMAD3 (a downstream target of TGF-beta signaling) in RDEB patient fibroblasts. In contrast, no reduced SMAD3 phosphorylation was observed in control cells. Over-expression of C7 in RDEB fibroblasts reduced TSP1 and phosphorylation of SMAD3. Furthermore, inhibition of TSP1 binding to the latency-associated protein (LAP)/ TGF-beta complex decreased fibrosis in engineered extracellular matrix derived from RDEB fibroblasts, as evaluated by picrosirius red staining and analyses of birefringent collagen fibrillar deposits. Using immuno-precipitation, proximity ligation assay and real-time, labelfree analysis of binding interactions, we demonstrate that C7 binds TSP1, thereby likely preventing TSP1-LAP association and subsequent TGF-beta activation. Our study suggests a novel mechanism for increased TGF-beta signaling in the absence of C7 in RDEB patient skin. Moreover, our data identify TSP1 as a possible target for reducing fibrosis in the tumor-promoting dermal microenvironment in RDEB patients.