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

Dongyuan Lü,Yuxin Gao,Chunhua Luo,Zhan Li,Mian Long,Jiawen Wang,Lu Zheng,Fan Zhang,Shujin Sun

doi:10.1186/s12938-016-0263-1

Abstract

BackgroundKeratinocyte (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.MethodsHuman 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.ResultsMechanical 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.ConclusionWe 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.

Highlights

Keratinocyte (KC) migration in re-epithelization is crucial in repairing injured skin
The challenging issues for mechanotransduction mechanisms of keratinocytes migration in cutaneous wounds mainly rely on: Whether does mechanical stretch modulate dynamically the expression of key signaling proteins and how do the cells sense the mechanical signals? Whether do the other signaling factors affect the migration of keratinocytes and what are the potential mechanotransduction mechanisms? Here we developed an in vitro static stretch approach to quantify the mechanicallyinduced proteins expression of human keratinocytes on substrate coated by collagen I and in the presence of human fibroblasts
Our results provided the insight into the mechanotransduction pathways in manipulating keratinocyte migration under static stretch, which implies potential application in clinical treatment of wound repairing

Summary

Introduction

Keratinocyte (KC) migration in re-epithelization is crucial in repairing injured skin. Wound healing is an intricate process in which the skin repairs itself with a series of sequential cellular and biochemical events after injury [1]. It is usually divided into three or four sequential yet overlapping phases, including hemostasis, inflammation, granulation tissue formation and re-epithelialization, matrix formation and remodeling [2, 3]. Lü et al BioMed Eng OnLine 2016, 15(Suppl 2):130 All of these phases are highly coordinated physiological processes and require dynamic, coordinated intercommunication among different type cells in specific tissue regions. Keratinocyte migration dynamics acts as an excellent model for elucidating the wound healing both in vivo and in vitro

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