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Crosstalk between keratinocytes and dermal fibroblasts modulates the release of critical wound healing factors Carr, Matthew John
Abstract
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.
Item Metadata
Title |
Crosstalk between keratinocytes and dermal fibroblasts modulates the release of critical wound healing factors
|
Creator | |
Publisher |
University of British Columbia
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Date Issued |
2012
|
Description |
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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Genre | |
Type | |
Language |
eng
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Date Available |
2013-12-31
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Provider |
Vancouver : University of British Columbia Library
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Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
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DOI |
10.14288/1.0071845
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2013-05
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Campus | |
Scholarly Level |
Graduate
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Rights URI | |
Aggregated Source Repository |
DSpace
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Rights
Attribution-NonCommercial-NoDerivatives 4.0 International