Profile of exosomes related proteins released by differentiated and undifferentiated human keratinocytes

Our group has previously demonstrated the capacity of human keratinocytes to release 14-3-3sigma into conditioned medium through the mechanism of exosome externalization. In this study the release of other proteins through the same mechanism and the differences in the profiles of 14-3-3 proteins between differentiated (diff-K) and undifferentiated keratinocytes (undiff-K) were investigated. The stimulatory effect of other 14-3-3 isoforms on the expression of MMP-1 in dermal fibroblasts was also evaluated. Exosomes isolated from undiff-K (low Ca(2+)) and diff-K (high Ca(2+)) were subjected to proteomic and Western blot analysis. The results showed that more than 50 different cytoplasmic proteins including all seven 14-3-3 protein isoforms (beta, sigma, eta, epsilon, tau, zeta, and gamma) were released from diff-K through the mechanism of exosome externalization. However, in exosomes of undiff-K only four of the 14-3-3 protein isoforms (beta, eta, zeta, and gamma) were detected. Ca(2+) treatment increased the release of exosomes from undiff-K by at least two times relative to the control. Consistent with this finding, the stimulatory effect of exosomes containing 14-3-3sigma from diff-K had higher MMP-1 stimulatory effect in fibroblasts relative to those exosomes isolated from undiff-K. MMP-1 stimulatory effect of recombinant 14-3-3beta and eta, tested in this study, in dermal fibroblasts, suggests additional anti-fibrogenic factors other than 14-3-3sigma. In conclusion, keratinocytes release many proteins through the mechanism of exosome externalization from which some such as 14-3-3 isoforms may function as extracellular matrix (ECM) modulating factors for dermal fibroblasts. These findings revealed the presence of a novel mechanism by which keratinocytes can potentially interact with fibroblasts.