β1 integrin signaling in asymmetric migration of keratinocytes under mechanical stretch in a co-cultured wound repair model - PubMed (original) (raw)

β1 integrin signaling in asymmetric migration of keratinocytes under mechanical stretch in a co-cultured wound repair model

Dongyuan Lü et al. Biomed Eng Online. 2016.

Abstract

Background: Keratinocyte (KC) migration in re-epithelization is crucial in repairing injured skin. But the mechanisms of how mechanical stimuli regulate the migration of keratinocytes have been poorly understood.

Methods: Human immortalized keratinocyte HaCaT cells were co-cultured with skin fibroblasts on PDMS membranes and transferred to the static stretch device developed in-house for additional 6 day culture under mechanical stretch to mimic surface tension in skin. To detect the expression of proteins on different position at different time points and the effect of β1 integrin mechanotransduction on HaCaT migration, Immunofluorescence, Reverse transcription-polymerase chain reaction, Flow cytometry, Western blotting assays were applied.

Results: Mechanical receptor of β1 integrin that recognizes its ligand of collagen I was found to be strongly associated with migration of HaCaT cells since the knockdown of β1 integrin via RNA silence eliminated the key protein expression dynamically. Here the expression of vinculin was lower but that of Cdc42 was higher for the cells at outward edge than those at inward edge, respectively, supporting that the migration capability of keratinocytes is inversely correlated with the formation of focal adhesion complexes but positively related to the lamellipodia formation. This asymmetric expression feature was further confirmed by high or low expression of PI3K for outward- or inward-migrating cells. And ERK1/2 phosphorylation was up-regulated by mechanical stretch.

Conclusion: We reported here, a novel mechanotransduction signaling pathways were β1 integrin-dependent pattern of keratinocytes migration under static stretch in an in vitro co-culture model. These results provided an insight into underlying molecular mechanisms of keratinocyte migration under mechanical stimuli.

Keywords: Fibroblast; Keratinocyte; Mechanical stretch; Mechanotransduction; β1 integrin.

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Figures

Fig. 1

Fig. 1

Mechanical stimuli used to examine signaling proteins in HaCaT cells migration. a Image of an in-house developed static stretch device by applying mechanical stimuli via a stretchable silicone membrane to the cells. b Schematic of cell migration under tensile stress on silicone membrane at a typical 20% strain. HaCaT and HF cells were seeded in two separated regions, and the migration distance L (away from HF cells) or L’ (towards HF cells) and the migration leading edge of HaCaT cells were illustrated

Fig. 2

Fig. 2

Alteration of β1 integrin expression on WT- and β1 integrin knockdown- HaCaT cell migration under coculture and mechanical stretch. a RT-PCR analysis of β1 integrin in WT- and silenced- (Sil-) HaCaT cells. Red lines indicated the molecular weight of the target fragments. b WB analysis of β1 integrin in WT- and Sil-HaCaT cells. c Comparison of β1 integrin expression between WT- (open bar) and Sil- (solid bar) HaCaT cells. WT-cells transfected via plain plasmid (grey bar) was used as control. d, e Time courses of β1 integrin expression in WT- (d) or Sil- (e) HaCaT cells under cocultured with fibroblasts and mechanical stretch. Data were presented as the mean ± standard error (SE) of normalized fluorescence intensity (FI) fold of totally >9 cells at the leading edge

Fig. 3

Fig. 3

Expression of vinculin in WT- and Sil-HaCaT cells in four migration patterns at different time points. a, b IF images and time courses of vinculin expression in WT-HaCaT cells (a). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge. ††The level of statistical significance of difference in normalized mean FI at t = 1 h between C/S IN and C/S OUT patterns in WT-HaCaT cells (b). c, d IF images and time courses of vinculin expression in Sil-HaCaT cells (c). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge (d). Scale bar 50 μm

Fig. 4

Fig. 4

Expression of Cdc42 in WT- and Sil-HaCaT cells in four migration patterns at different time points. a, b IF images and time courses of Cdc42 expression in WT-HaCaT cells (a). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge. ††The level of statistical significance of difference in normalized mean FI at t = 1 h between C/S IN and C/S OUT patterns in WT-HaCaT cells (b). c, d IF images and time courses of Cdc42 expression in Sil-HaCaT cells (c). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge (d). e Optical images of pseudopodium formation in WT-HaCaT cells. Arrows indicate the pseudopodium at day 2. Scale bar 50 μm

Fig. 5

Fig. 5

Expression of PI3K in WT- and Sil-HaCaT cells migration in four migration patterns at different time points. (a, b) IF images and time courses of PI3K expression in WT-HaCaT cells (a). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge. †The level of statistical significance of difference in normalized mean FI at t = 1 h between C/S IN and C/S OUT patterns in WT-HaCaT cells (b). (c, d) IF images and time courses of PI3K expression in Sil-HaCaT cells (c). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge (d). Scale bar 50 μm

Fig. 6

Fig. 6

Expression of phosphorylated ERK1/2 in WT- and Sil-HaCaT cells in four migration patterns at different time points. a, b IF images and time courses of phosphorylated ERK1/2 expression in WT-HaCaT cells (a). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge. ††The level of statistical significance of difference in normalized mean FI at t = 24 between C/S IN and C/S OUT patterns in WT-HaCaT cells (b). (c, d) IF images and time courses of phosphorylated ERK1/2 expression in Sil-HaCaT cells (c). Data were presented as the mean ± SE of normalized FI of totally >9 cells at the leading edge (d). Scale bar 50 μm

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