Keratinocyte-fibroblast Crosstalk Via Extracellular Vesicles Reveals Interplay Of Mirnas That Inhibits Kgf Signaling In Diabetic Foot Ulcers
Irena Pastar, Horacio A. Ramirez, Andrea F. Ferreira, Ivan Jozic, Marta Garcia-Contreras, Jeffrey McBride, Robert S. Kirsner, Marjana Tomic-Canic.
University of Miami Miller School of Medicine, Miami, FL, USA.
Growth factor-based therapies did not achieve expected therapeutic potential as they transitioned from pre-clinical to clinical testing. To better understand why keratinocyte growth factor (KGF/FGF7) failed to reach efficacy in clinical trial we used laser captured epidermis of wound edge tissue from diabetic foot ulcers (DFUs) and genomic profiling. We identified set of deregulated microRNAs (miRs) including miR-31-5p and miR-15-5b among the top induced. One of predicted targets of miR-31-5p is KGF/FGF7, a stimulator of keratinocyte migration produced by fibroblasts. Consistent with epidermal miR-31-5p induction, we found suppression of FGF7 in DFU fibroblasts and validated FGF7 as miR31-5p target by 3’ UTR luciferase reporter assays. Furthermore, overexpression of miR-31-5p into the target cells, primary fibroblasts, caused suppression of FGF7, even in the presence of major stimulator, IL1β. Thus, we postulated that sustained miR-31-5p overexpression by keratinocytes in DFUs targets FGF7 in fibroblasts in a paracrine manner. Indeed, we found miR-31-5p to be secreted in extracellular vesicles (EVs) from keratinocytes, verified by nanoparticle tracking analysis. EVs containing miR-31-5p were functionally active in suppressing FGF7 in fibroblasts and in organotypic human skin. KGF/FGF7 signaling targets keratinocytes via FGFR2. We also found down-regulation of FGFR2 in DFU epidermis. miR-15b-5p and miR-424-5p, over-expressed in epidermis of DFUs, both target FGFR2. Their induction and direct targeting of FGFR2 was verified in DFU tissue and by luciferase reporter assays, respectively. We conclude that KGF/FGF7 signaling is impaired in DFUs via complex mechanism that involves over-expression of miR-31 in keratinocytes that targets and suppresses FGF7 in fibroblasts via EVs. Furthermore, suppression of FGFR2 in keratinocytes via miR-15b-5p and miR-424-5p leads to complete impairment of KGF-mediated effects, which provides insights into molecular mechanisms that precluded successful outcomes of recombinant KGF therapies.
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