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Keratinocyte-releasable factors modulate extracellular matrix components in dermal fibroblasts

University of British Columbia

A fine balance between the synthesis and degradation of extracellular matrix (ECM) is required in maintaining the structural integrity of healing tissue. An imbalance in ECM expression leads to fibrotic conditions such as hypertrophic scars (HS). It has been demonstrated that keratinocyte-releasable factors can function as ECM modulating factors (MMP-1 and type I collagen) in fibroblasts. We have shown that Stratifin (SFN) is an MMP-1 stimulatory factor in fibroblasts that failed to suppress the expression of type I collagen in fibroblasts. SFN is an intracellular protein that lacks a signal peptide. As such, it is critical to explore its mechanism of release and identify the keratinocyte-derived collageninhibiting factor(s) for dermal fibroblasts. In this doctoral research project I hypothesize that keratinocyte-releasable factor(s) function as a stop signal(s) for wound healing by modulating the expression of key ECM components such as MMP-1 and type I collagen in fibroblasts. Two specific objectives were accomplished to address these issues. Under objective 1, the mechanism by which SFN is released has been explored. The findings demonstrate that SFN is released via exosomes in a Ca²⁺ dependant fashion. Moreover, only differentiated keratinocytes release SFN. Exosome-associated SFN exhibits a potent MMP-1 stimulatory effect. Under objective 2, using a series of systematic protein purification methods followed by mass spectroscopy, two proteins (SPARC and SFN) that inhibited collagen production by dermal fibroblasts were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby regulating type I collagen expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, the identification of the mechanism of release of SFN and the identification of SPARC/SFN complex contribute to the understanding of how these factors are involved in the wound healing process. Also, SPARC/SFN complex provides us with another anti-fibrogenic factor that may be used to generate an effective therapeutic agent to treat HS frequently developed following burn injury, deep trauma and/or surgical incisions.

Text

2012-08-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/37005

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/