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.
Chavez-Muñoz
Claudia
C
Department of Surgery, BC Professional Burn and Wound Healing Research Lab., University of British Columbia, Vancouver, British Columbia, Canada.
Kilani
Ruhangiz T
RT
Ghahary
Aziz
A
D048948:14-3-3 Proteins / Q000378:metabolism; D001681:Biological Assay; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D055354:Exosomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics; D011994:Recombinant Proteins / Q000494:pharmacology
D048948:14-3-3 Proteins; D007476:Ionophores; D020033:Protein Isoforms; D011506:Proteins; D011994:Recombinant Proteins; D020781:Matrix Metalloproteinase 1