086 Biological Role and Pattern of 14-3-3 Proteins in Keratinocytes and Fibroblasts

Abstract

We have recently established a keratinocyte/fibroblast co-culture system and demonstrated a potent keratinocyte-derived antifibrogenic factor (KDAF) for dermal fibroblasts relative to that of control cells. Later experiments identified KDAF as being the keratinocytes releasable 14-3-3 sigma. It is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3 proteins in conditioned medium. We also suggested that fibroblasts are not the primary source of these proteins. To examine this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in our test medium consist of 49% keratinocyte serum-free medium (KSFM), 49% DMEM and 2% fetal bovine serum up to 24 days. When KCM was collected every other day and added to fibroblasts, the expression of collagenase mRNA was undetectable in cells receiving either fresh or 48 hr conditioned KSFM. However, the level of collagenase mRNA expression was markedly increased in fibroblasts receiving KCM collected at either early (day 2 and 4) or later (day 12–22) time points of replacing the KSFM with test medium. To examine whether fibroblasts also release different 14-3-3 isoforms, cultured conditioned media from both fibroblasts and keratinocytes were collected and evaluated for the presence of different isoforms of 14-3-3 proteins by western blot analysis. The results revealed that fibroblasts which are highly responsive to some of the 14-3-3 isoforms such as α/β and σ forms, are barely able to express and release these factors into conditioned medium. This was in sharp contrast to a very high level of 14-3-3 proteins found in KCM. In conclusion, keratinocytes, but not fibroblasts, release different forms of 14-3-3 proteins which may function as MMP-1 stimulating factors for fibroblasts. Acknowledgment: This work was supported by the Canadian Institute of Health Research (CIHR).