Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts.
Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.
Ghahary
Aziz
A
Department of Surgery, Wound Healing Research Group, University of Alberta, Alberta, Canada.
Marcoux
Yvonne
Y
Karimi-Busheri
Feridoun
F
Li
Yunyaun
Y
Tredget
Edward E
EE
Kilani
Ruhangiz T
RT
Lam
Eugene
E
Weinfeld
Michael
M
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D020405:Dermis / Q000166:cytology*; D005092:Exonucleases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D005095:Exoribonucleases; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D020781:Matrix Metalloproteinase 1 / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human; D020781:Matrix Metalloproteinase 1