Exogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro

Abstract

Background:Collagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders like Epidermolysis bullosa, EDS the ability to offer such stability is lost either due to mutations in collagens or defect in the chaperones involved in collagen assembly, which leads to chronic wounds, skin fragility, and blisters. Existing approaches to study and develop therapy against such conditions are the use of small molecules like 4-phenylbutyrate (4-PBA) or growth factors like TGF-β. However, these approaches are not collagen specific resulting in unsolicited responses. Therefore, a collagen specific booster is required to guide the correct folding and deposition of collagen in a highly regulated manner. Hsp47 is a chaperone with a major role in collagen biosynthesis.Expression levels of Hsp47 correlate with collagen production. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) in skin cells, including specific collagen subtypes quantification.Results: Here we quantify the collagen deposition level and the type of deposited collagens by different cell types from skin tissue (fibroblasts NHDF, L929 and MEF, keratinocytes HaCat and endothelial cells HDMEC) after Hsp47 stimulation. We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs and fibril-associated collagen XII were not affected by the increased Hsp47 intracellular levels. The deposition levels of fibrillar collagen were celldependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs. Conclusions:A 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders. Additional filesAdditional file 1: Figure S1 shows supplementary information on delivery of H47 to ER via KDEL receptor-mediated endocytosis to different cell types.Additional file 2: Figure S2 shows Z-stack orthogonal projection images of NHDF after incubation with EGFP for 3 h.Additional file 3: Figure S3 shows Immunostaining of COL I, III, IV, V and XII deposited in MEF, L929, HaCaT and HDMEC cultures 24 h with and without treatment of H47.Additional file 4: Figure S4 shows Stimulated deposition of COL I, III and V in MEF Hsp47 -/-cells after H47 uptake.Additional file 5: Figure S5 shows H47 binds to collagen on the matrix on L929 cells reaching confluency.