Amniotic membrane promotes focal adhesion remodeling to stimulate cell migration

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.

Figure 1

Amniotic membrane (AM) promotes cell protrusion generation and Paxillin expression in migrating Mv1Lu cells. (a) Detailed pictures of the migrating edge of artificial wound assays treated with AM in combination with inhibitors. Scale Bar 50 µm. (b) Western Blot of total protein extracts from sub-confluent Mv1Lu cells cultured in the presence of AM and/or inhibitors and collected after 24 hours. The dashed grey lines indicate that two distant parts of the very same blot were put together. ß-actin was used as loading control. (‡) Unspecific bands. (c) Relative protein level plots generated from Western Blot quantification. C: serum starvation; JNKi: SP600125; MEKi: PD98059. Asterisks denote statistically significant differences between conditions according to ANOVA statistical analysis: (***) p < 0.001; (ns) not significant. All experiments were repeated at least three times. Representative pictures and results are shown.

Figure 2

Amniotic membrane (AM) treatment alters Paxillin distribution and focal structures arrangement in migrating Mv1Lu cells. (a) Confocal microscopy images of Mv1Lu cells at the migrating edge of artificial wound assays. Scale Bar 50 µm. Juxtaposed field images were taken to show migration front from a wider perspective. (b) 2.5x magnified detail from merged pictures in (a). Scale Bar 10 µm. (c) Plots for average number and size of focal structures detected at the migrating leading edge. Asterisks denote statistically significant differences between conditions according to ANOVA statistical analysis: (*) p < 0.05, (**) p < 0.005 and (***) p < 0.001; (ns) not significant. Paxillin: green; Actin: red; Nuclei: blue; C: serum starvation; JNKi: SP600125; MEKi: PD98059; FS: focal structures. All the experiments were repeated at least three times. Representative pictures are shown.

Figure 3

Amniotic membrane (AM) promotes cell protrusion generation and Paxillin phosphorilation in migrating HaCaT cells. (a) Migrating HaCaT cells, protrusions are indicated by arrows. Scale Bar 50 µm. (b) Western Blot of total protein extracts from sub-confluent HaCaT cells cultured in the presence of AM and/or inhibitors and collected after 24 hours. ß-actin was used as loading control. (‡) Unspecific bands. (c) Relative protein level plots generated from Western Blot quantification data. C: serum starvation; JNKi: SP600125; MEKi: PD98059. Asterisks denote statistically significant differences between conditions according to ANOVA statistical analysis: (***) p < 0.001; (ns) not significant. All experiments were repeated at least three times. Representative pictures and results are shown.

Figure 4

Amniotic membrane (AM) treatment increases the detection focal structures in migrating HaCaT cells. (a) Confocal microscopy images of HaCaT cells at the migrating edge of artificial wound assays. Scale Bar 50 µm. (b) 2.5x magnified detail from merged pictures in (a). (c) Plots for average number and size of focal structures detected at the migrating leading edge. Asterisks denote statistically significant differences between conditions according to ANOVA statistical analysis: (*) p < 0.05, (**) p < 0.005 and (***) p < 0.001; (ns) not significant. Paxillin: green; Actin: red; Nuclei: blue; C: serum starvation; JNKi: SP600125; MEKi: PD98059; FS: focal structures. All the experiments were repeated at least three times. Representative pictures are shown.

Figure 5

Amniotic membrane (AM) promotes FAK activation and detection at focal structures in migrating HaCaT cells. (a) HaCaT cells at the migrating edge of artificial wound assays show increased p-tyr-100 immune-staining when AM was present. P-tyr-100 immune-staining signal was converted into pseudo-color rainbow scale to display fluorescence-intensity variations. Fluorescent signal was converted using Rainbow feature from ZEN software, on a linear mode and covering the full range of the data. Co-staining with phalloidin and Hoechst-33258 was used to show the cell structure and nuclei, respectively. P-tyr-100: pseudo-color; p-tyr-100: green; Actin: red; Nuclei: blue. Scale Bar 25 µm. (b) Relative fluorescence level plot generated from average p-tyr-100 signal intensity data obtained at the migrating leading edge. (c) Western Blot of total protein extracts from sub-confluent HaCaT cells cultured in the presence of AM and/or different inhibitors collected for a 24 hours time course. ß-actin was used a loading control. Relative protein level plots were generated from Western Blot quantification data. C: serum starvation; JNKi: SP600125; MEKi: PD98059. Asterisks denote statistically significant differences between conditions according to ANOVA statistical analysis: (***) p < 0.001; (ns) not significant. All the experiments were repeated at least three times. Representative pictures and results are shown.