Amniotic membrane promotes focal adhesion remodeling to stimulate cell migration

Ángel Bernabé-García,Gregorio Castellanos,Sergio Liarte,Jose M Moraleda,Francisco J Nicolás

doi:10.1038/s41598-017-15509-z

Abstract

During wound healing, the migration of keratinocytes onto newly restored extracellular matrix aims to reestablish continuity of the epidermis. The application of amniotic membrane (AM) to chronic, deep traumatic, non-healing wounds has proven successful at stimulating re-epithelialization. When applied on epithelial cell cultures, AM activates extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinases 1/2 (JNK1/2), with the overexpression and phosphorylation of c-Jun along the wound edge. The effect of AM on the migration of cells was investigated by studying critical proteins involved in the focal adhesions turn-over: Focal Adhesion Kinase (FAK), Paxillin and Vinculin. In Mv1Lu and HaCaT cells, validated models for cell migration and wound healing, AM affected the expression and activation of Paxillin, but did not affect Vinculin expression, both factors which integrate into focal adhesions. Moreover, AM regulation also affected FAK activity through phosphorylation. Finally, we have determined that AM regulation of focal adhesions involves both JNK and MEK MAP kinase signaling pathways. This data provides a molecular background to understand how AM regulates critical cell and molecular aspects of cell migration, organizing and directing the movement of cells by the continuous formation, maturation, and turnover of focal adhesion structures at the migration leading edge.

Highlights

During wound healing, skin integrity is restored by the actions of numerous cell types
When we studied Paxillin expression by performing WB on total protein extracts obtained from sub-confluent Mv1Lu cells, so that they would behave like a migratory edge[8], we detected that amniotic membrane (AM) treatment resulted in increased Paxillin levels (Fig. 1b and c)
In this paper, using two validated cell models for studying migration and wound healing, we have shown how the ability of AM to promote migration is sustained in the reorganization of cell focal structures, with the particular implication of Paxillin and Focal Adhesion Kinase (FAK)

Summary

Introduction

Skin integrity is restored by the actions of numerous cell types. For the case of deep-traumatic and chronic non-healing wounds, the application of amniotic membrane (AM) has proven to successfully encourage re-epithelialization, resulting in a suitable therapeutic alternative to avoid autologous skin transplantation[3,4] These properties derive from the ability of the AM to provide immuno-modulatory effects[5] and to supply helpful growth factors including EGF or TGF-ß, among others[6,7,8]. We applied the wound scratch assay method along with specific inhibitors for MEK-1 and JNK signaling pathways to study in situ the effect of AM on focal structures, either FCs or FAs, dynamism during the migration of two well established migration and wound healing cellular models, the Mink Lung Epithelial (Mv1Lu) cells and the spontaneously immortalized human keratinocyte (HaCaT) cells. By paying attention to focal structures and the proteins involved in this biological phenomenon, we describe the ability of AM treatment to stimulate the dynamics and turnover of focal structures contributing to enhanced migration of cells at the front edge of wounds

Methods

Results

Conclusion