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UBC Theses and Dissertations

Crosstalk between keratinocytes and dermal fibroblasts modulates the release of critical wound healing factors Carr, Matthew John


Epidermal-mesenchymal communication plays a critical role in cutaneous integrity during both normal and pathological wound healing processes. We have previously demonstrated that crosstalk between keratinocytes and dermal fibroblasts has the capacity to influence the expression of key extracellular matrix (ECM) components including members of the matrix metalloproteinase (MMP) and 14-3-3 protein families. In this Masters project, we hypothesized that bidirectional communication between these cell types plays a central role in regulating local expression of critical wound healing factors. To address this question, two specific objectives were accomplished. Under Objective 1, we investigated whether fibroblasts are capable of modulating the expression of keratinocyte-releasable anti-fibrogenic 14-3-3 proteins by examining the levels of four 14-3-3 family members (β, η, γ, σ) by western blot and quantitative real-time polymerase chain-reaction (qPCR). Gene analysis revealed upregulation of all four 14-3-3 isoforms of interest in co-cultured compared to mono-cultured keratinocytes. Additionally, co-cultured keratinocytes expressed significantly higher levels of intracellular 14-3-3 γ and σ proteins. Under Objective 2, we examined the impact of keratinocyte-releasable factors upon the fibroblast expression profile of key cytokines and growth factors using DNA microarray analysis. Fibroblasts co-cultured with keratinocytes demonstrated significantly upregulated colony-stimulating factor 3 (CSF3) expression, which was further verified by RT-PCR and western blot. Keratinocyte-conditioned medium (KCM) from undifferentiated keratinocytes was observed to have higher CSF3-stimulatory potential than that of differentiated cells. Partial-characterization of the CSF3-stimulatory factor(s) was achieved by treating fibroblasts with KCM subjected to thermal stability and ammonium sulfate precipitation analyses. Additionally, to investigate the CSF3-stimulatory potential of interleukin-1 (IL-1) upon fibroblasts, we utilized an IL-1 receptor antagonist (IL-1ra) and revealed that keratinocyte-releasable IL-1 may be one of the factors responsible for stimulating CSF3 expression in fibroblasts. Our studies concluded that dermal fibroblasts regulate the expression of several 14-3-3 isoforms (notably γ and σ) in keratinocytes, and conversely, that keratinocyte-releasable factors such as IL-1 modulate the expression of CSF3 in fibroblasts. These findings underline the critical nature of crosstalk between keratinocytes and fibroblasts, suggesting that bidirectional communication between these cells in vivo plays a prominent regulatory role in the cytokine production required for wound healing and maintaining a normal skin environment.

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