UBC Theses and Dissertations
Identification and mechanism of action of keratinocyte-releasable factors in regulation of extracellular matrix expression Ghaffari, Abdi
The complexity of multi-cellular organisms demands a dynamic reciprocity between neighboring cells in any given tissue. The disruption in keratinocytes and fibroblasts cross-talk in skin has been linked to an imbalance in extracellular matrix (ECM) expression leading to the onset of fibrosis such as hypertrophic scarring. Our group has recently identified 14-3-3σ or stratifin (SFN) as a potent MMP-1 stimulatory factor in fibroblasts. In this doctoral research project, we hypothesized that SFN can modulate other ECM components and execute its transmembrane signaling through interaction with a receptor on the surface of fibroblasts. Three specific objectives were accomplished in this project. Under objective 1, ECM gene expression profile of fibroblasts treated with SFN or co-cultured with keratinocytes was characterized by an ECM-pathway specific gene array and revealed that SFN upregulates a wider range of MMPs such as MMP-3, 8, 10, and 24 other than MMP-1. As SFN was not responsible for the keratinocyte-mediated decrease in collagen expression, under objective 2 attempts were made to characterize the nature of a collagen inhibitory factor in keratinocyte-conditioned medium (KCM). Analysis of keratinocyte/fibroblast co-culture and KCM revealed a 30-50 kDa keratinocyte-derived collagen inhibitory factor with stable activity at high temperature (56 ºC) and acidic environment (pH=2, 30 min). Under objective 3, SFN’s transmembrane signaling mechanism was investigated by utilizing a combination of receptor ectodomain biotin labeling, serial affinity purification, and MS/MS to identify aminopeptidase N or CD13 (APN) as a potential SFN receptor in fibroblasts. APN/SFN binding was further confirmed by immunoprecipitation, cross-linking, and co-distribution studies. Expression of APN and SFN increased after wound closure in a rabbit ear fibrotic model as well as a longitudinal study in rats. The transient knockdown of APN blocked SFN-mediated p38 MAPK activation and MMP-1 expression. Collectively, the findings presented in this thesis provide further support for the importance of keratinocyte-releasable factors in the regulation of ECM and MMP expression in fibroblasts. We also identify APN as a novel cell surface receptor for SFN. Therefore, our findings may provide additional therapeutic tools for the regulation of MMP expression in dermal fibrosis and chronic wound healing disorders.
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