Recessive dystrophic epidermolysis bullosa (RDEB) is a severe hereditary skin blistering disease. Clinically, individuals with RDEB are characterized by fragile skin and blister formations followed by devastating fibrosis. The fibrosis in RDEB has been reported to be driven by transforming growth factor-beta (TGF-) signaling and we previously demonstrated that reduced TGF-activity lead to significantly slower progression of fibrosis in RDEB model mice. Nevertheless, unbiased targeting of TGF- activity in skin could have dire consequences as TGF-signaling supports dermal-epidermal communication to main skin homeostasis.  Therefore, there is a need to better characterize the TGF- signaling-enhancing mechanisms in RDEB. Toward this end, we performed careful assessment of latent- TGF- activating mechanisms in dermal fibroblasts from human donors with RDEB. Using western blotting for pSMAD2/3 as a surrogate marker of TGF- activity, we showed that RDEB fibroblasts had an enhanced capacity to activate TGF- compared to control fibroblasts. Next, to investigate keratinocyte-fibroblast interaction in RDEB skin, we cocultured these two cell types. The level of pSMAD2/3 and expression of fibrosis-related proteins were elevated in RDEB fibroblasts when they were cocultured with RDEB keratinocytes compared to control keratinocytes. Additionally, inhibition of thrombospondin-1 (TSP-1), matrix metalloproteinases (MMPs) and RGD-binding integrins decreased the level of pSMAD2/3 and fibrosis related proteins in fibroblasts derived from some but not all RDEB donors in both monoculture and co-culture system. Our study reveals a complexity of the regulation of TGF- activity in RDEB skin with donor-dependent bias and variation in latent TGF- activating mechanisms, and the involvement of keratinocytes in initiating and progressing dermal fibrosis via TSP-1, MMPs, and RGD-binding integrins. These aspects need to be further explored in order to improve disease-modulating therapy strategies for RDEB.

Donor-dependent variation and bias of transforming growth factor-β activating mechanisms in recessive dystrophic epidermolysis bullosa