Primary human keratinocytes externalize stratifin protein via exosomes

Although, stratifin (SFN) is externalized by keratinocytes and stimulates the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts, its mechanism of externalization is not known. Here, we hypothesize that keratinocytes have a capacity to release stratifin through externalization of exosomes. To test this hypothesis, exosomes were purified from human keratinocyte conditioned medium (KCM) and analyzed for the presence of SFN by Western blot analysis using lysosomal-associated membrane protein 2 (LAMP-2) and heat shock cognate 70 (hsc70) as exosomal markers. The results showed the presence of SFN in keratinocyte lysate, concentrated KCM and exosomes, but not in concentrated unconditioned medium. Transmission electron microscopic examination revealed the presence of unique "saucer-like" structures characteristic of exosomes whose diameters were <100 nm. Similar to the recombinant SFN, the exosomes associated proteins stimulated MMP-1 expression in fibroblasts. Depletion of the exosomes markedly reduced this MMP-1 stimulatory effect. To further statistically confirm these findings, fibroblasts were treated with three different exosome preparations and the finding showed more than 7.4-fold increase in the level of MMP-1 in the treated cells. Furthermore, we found that approximately 1% of the total proteins contained in exosomes correspond to SFN. In conclusion, this study is the first report showing that keratinocytes have the capacity to produce exosomes through which some intracellular proteins such as SFN, with MMP-1 stimulating activity for fibroblasts, is externalized into keratinocyte microenvironment.