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

Our group has previously demonstrated the capacity of human keratinocytes to release 14‐3‐3σ 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 Ca2+) and diff‐K (high Ca2+) 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 (β, σ, η, ε, τ, ζ, and γ) 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 (β, η, ζ, and γ) were detected. Ca2+ 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 14‐3‐3σ 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 14‐3‐3β and η, tested in this study, in dermal fibroblasts, suggests additional anti‐fibrogenic factors other than 14‐3‐3σ. 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. J. Cell. Physiol. 221: 221–231, 2009. © 2009 Wiley‐Liss, Inc