Potential Role of Integrin α₅β₁/Focal Adhesion Kinase (FAK) and Actin Cytoskeleton in the Mechanotransduction and Response of Human Gingival Fibroblasts Cultured on a 3-Dimension Lactide-Co-Glycolide (3D PLGA) Scaffold

Abstract

BACKGROUND The stability of orthodontic treatment is thought to be significantly affected by the compression and retraction of gingival tissues, but the underlying molecular mechanism is not fully elucidated. The objectives of our study were to explore the effects of mechanical force on the ECM-integrin-cytoskeleton linkage response in human gingival fibroblasts (HGFs) cultured on 3-dimension (3D) lactide-co-glycolide (PLGA) biological scaffold and to further study the mechanotransduction pathways that could be involved. MATERIAL AND METHODS A compressive force of 25 g/m² was applied to the HGFs-PLGA 3D co-cultured model. Rhodamine-phalloidin staining was used to evaluate the filamentous actin (F-actin) cytoskeleton. The expression level of type I collagen (COL-1) and the activation of the integrin alpha₅ß₁/focal adhesion kinase (FAK) signaling pathway were determined by using real-time PCR and Western blotting analysis. The impacts of the applied force on the expression levels of FAK, phosphorylated focal adhesion kinase (p-FAK), and COL-1 were also measured in cells treated with integrin alpha₅ß₁ inhibitor (Ac-PHSCN-NH 2, ATN-161). RESULTS Mechanical force increased the expression of integrin alpha₅ß₁, FAK (p-FAK), and COL-1 in HGFs, and induced the formation of stress fibers. Blocking integrin alpha₅ß₁ reduced the expression of FAK (p-FAK), while the expression of COL-1 was not fully inhibited. CONCLUSIONS The integrin alpha₅ß₁/FAK signaling pathway and actin cytoskeleton appear to be involved in the mechanotransduction of HGFs. There could be other mechanisms involved in the promotion effect of mechanical force on collagen synthesis in addition to the integrin alpha₅ß₁ pathway.

Figure 1

Mechanical force induced the formation of stress fibers in HGFs. Rhodamine-phalloidin staining was used to evaluate the F-actin cytoskeleton in HGFs cultured on 3D PLGA scaffolds after applying a compressive force of 25 g/m² at different time points (red, actin; blue, nuclei).

Figure 2

Mechanical force increased integrin α₅β₁, FAK, p-FAK, and COL-1 expression in HGFs. mRNA expression: (A) integrin α₅, (C) integrin β₁, (E) FAK, (H) COL-1 and protein expression: (B) integrin α₅, (D) integrin β₁, (F) FAK, (G) p-FAK, (I) COL-1, were evaluated after applying a compressive force of 25 g/m² on HGFs at different time points. Data are presented as means±standard deviations, and were obtained from 3 independent experiments. * p<0.05 vs. 0 h control. ^# p<0.05 vs. mechanical stress group.

Figure 3

1 μM ATN-161 strongly inhibited integrin α₅β₁. (A, B) The cytotoxicity of ATN-161 was evaluated using Cell Counting Kit-8. The mRNA expression of (C) integrin α₅ and (D) integrin β₁ were evaluated using quantitative real-time polymerase chain reaction (RT-qPCR). Data are presented in the form of means±standard deviations, and were obtained from 3 independent experiments. * p<0.05 vs. 0 h control. ^# p<0.05 vs. mechanical stress group.

Figure 4

The mRNA expression: (A) FAK, (B) COL-1 and protein expression: (C) FAK, (D) p-FAK, (E) COL-1, were evaluated after treated with compressive force of 25 g/m² and integrin α₅β₁ inhibitor on HGFs at different time points. Data are presented as means±standard deviations, and were obtained from 3 independent experiments. * p<0.05 vs. 0 hour control. ^# p<0.05 vs. mechanical stress group.