Abstract
Keratinocytes and neutrophils are the main cellular components in wound healing during re-epithelization and inflammation. Free fatty acids such as linoleic acid (LA) present beneficial properties for wound healing by modulating the inflammatory response. LA is a natural ligand of free fatty acids receptor 1 (FFA1), a G protein-coupled receptor (GPCR), able to modulate inflammatory process; however, the role of FFA1 in keratinocytes and wound healing remains poorly understood. In this study, we investigated the role of FFA1 signaling in migration, matrix metalloproteinase-9 (MMP-9) activity, and IL-8 expression induced by LA in keratinocytes. We confirmed that HaCaT cells, a human keratinocyte cell line, expresses the FFA1 receptor and GW1100, a selective antagonist of FFA1, decreased LA-induced migration of HaCaT cells. Also, GW9508, a synthetic agonist of FFA1, increased migration of these cells. Furthermore, ERK1/2 and p38 MAPK inhibitors abolished the LA-induced increase in cell migration. Besides, HaCaT cells stimulated with LA or GW9508 increased the activity of MMP-9 and the expression of IL-8. GW1100 partially inhibited both responses. We further evaluated the effects of HaCaT cells conditioned media stimulated with LA or GW9508 on neutrophil chemotaxis. Conditioned media induced neutrophil chemotaxis. Furthermore, IL-8 secreted by HaCaT cells stimulated with LA or GW9508, contributed to neutrophil chemotaxis. In conclusion, LA increased migration, MMP-9 activity, and expression of IL-8 from HaCaT cells via FFA1. Hence, these results showed that the effects induced by LA in keratinocytes can be mediated through FFA1, thus explaining a possible mechanism by which this fatty acid could accelerate wound healing.
Highlights
Wound healing consists of four phases coordinated temporally and spatially: hemostasis, inflammation, proliferation, and remodeling (Gurtner et al, 2008; Makrantonaki et al, 2017)
Our results showed that HaCaT cells stimulated with 50 μM linoleic acid (LA) or 100 μM LA for 24 h migrated toward cell-free areas, resulting in significantly better coverage of the wound compared with control (0.1% DMSO) (Figure 1A)
LA-induced HaCaT cell migration was further confirmed by transwell migration assay in which the number of cells that migrated through the pores of the membranes were counted after being stimulated with 50 μM LA or 100 mM LA for 24 h (Figure 1C)
Summary
Wound healing consists of four phases coordinated temporally and spatially: hemostasis, inflammation, proliferation, and remodeling (Gurtner et al, 2008; Makrantonaki et al, 2017). Migration of keratinocytes from the wound edge over the provisional matrix is critical to ensure correct closure of the wound (Pastar et al, 2008). Growth factors, such as epidermal growth factor (EGF) and fibroblast growth factor (FGF), accelerate keratinocyte migration and re-epithelialization during wound healing (Yurko et al, 2001; Cheong et al, 2005). Another essential component in keratinocyte migration is secretion of MMP-9, which contributes to basal keratinocyte detachment from the basement membrane (Greenlee et al, 2006; Dufour et al, 2010; Jiang et al, 2014)
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