Cyclic mechanical stretch decreases cell migration by inhibiting phosphatidylinositol 3-kinase- and focal adhesion kinase-mediated JNK1 activation

Abstract

Epithelial cell migration during wound healing requires coordinated signaling pathways that direct polarization of the leading and trailing ends of the cells, cytoskeletal organization, and remodeling of focal adhesions. These inherently mechanical processes are disrupted by cyclic stretch (CS), but the specific signaling molecules involved in this disruption are not well understood. In this study, we demonstrate that inhibition of phosphatidylinositol 3-kinase (PI3K) or expression of a dominant-negative form of PI3K caused inhibition of airway epithelial cell wound closure. CS caused a sustained decrease in activation of PI3K and inhibited wound healing. Expression of constitutively active PI3K stimulated translocation of Tiam1 to the membrane, increased Rac1 activity, and increased wound healing of airway epithelial cells. Increased Rac1 activity resulted in increased phosphorylation of JNK1. PI3K activation was not regulated by association with focal adhesion kinase. Restoration of efficient cell migration during CS required coexpression of constitutively active PI3K, focal adhesion kinase, and JIP3.

FIGURE 1.

PI3K regulates migration of 16HBE14o⁻ cells. Scrape-wounded 16HBE14o⁻ cells grown on collagen IV (A) or laminin-5 (B) matrix were exposed to static conditions or cyclic mechanical stretch (CS) for 9 h. Multiple wounds were created in each well, and measurements were made on three wounds/well and averaged. A, treatment of static cells with the PI3K inhibitor LY294002 (20 μm) inhibited cell migration to the same extent as cells exposed to CS. B, cells were infected with adenovirus expressing EGFP, CA-PI3K, or DN-PI3K, and wound widths were measured. CA-PI3K stimulated cell migration, and DN-PI3K inhibited cell migration to the same extent as CS. Data are expressed as means ± S.E. (n = three independent experiments). *, p < 0.05, significant differences from static untreated cells.

FIGURE 2.

Wounding stimulates PI3K activation, and CS inhibits activation. A, wounding of 16HBE14o⁻ cells caused a sustained increase in PI3K activation, and activity was increased in cells grown on laminin-5 relative to collagen IV. Multiple wounds were applied to confluent cells, and cell lysates were collected after 2 and 6 h. *, p < 0.05, versus unwounded cells on the same matrix. B, CS inhibited the activation of PI3K in wounded cells on both collagen IV and laminin-5. Multiple wounds were applied to confluent cells, and cell lysates were collected after 2 and 6 h of CS or static conditions. *, p < 0.05, versus static cells on the same matrix. PI3K activity was estimated based on the formation of phosphatidylinositol 3,4,5-trisphosphate (PIP₃) as described under “Experimental Procedures.”

FIGURE 3.

Expression of CA-PI3K prevents CS-induced loss of PI3K activity and partially restores cell migration. Cells were grown on laminin-5 matrix and treated with adenovirus expressing CA-PI3K, DN-PI3K, or EGFP (not shown) for 48 h prior to wounding. A, multiple wounds were applied, and cell lysates were collected at 6 h for PI3K activity. B, wound closure was measured over 9 h. (Similar results were obtained using cells grown on collagen IV.) *, p < 0.05, significant difference from static untreated cells. PIP₃, phosphatidylinositol 3,4,5-trisphosphate.

FIGURE 4.

PI3K regulates Rac1 activation. Cells were grown on laminin-5 matrix and treated with adenovirus expressing CA-PI3K, DN-PI3K, or EGFP for 48 h prior to wounding. Multiple wounds were applied, cells were exposed to CS or static conditions for 6 h, and Rac1 activity (GTP-Rac1) was assessed via a pulldown assay. The blot is representative of four independent experiments, and the bar graph summarizes the densitometry data expressed as means ± S.E. Values are expressed as the ratio of active GTP-bound Rac1 to total Rac1. *, p < 0.05, significant difference from the static control. (Similar results (not shown) were obtained after 2 h.)

FIGURE 5.

PI3K regulates localization of the Rac1 guanosine exchange factor Tiam1. Cells were grown on laminin-5 matrix and treated with adenovirus expressing CA-PI3K, DN-PI3K, or EGFP for 48 h prior to wounding. Multiple wounds were applied, and cells were exposed to CS or static conditions for 2 or 6 h. Cells were lysed, aliquots of the total fraction were withdrawn, and cell lysates were further subjected to high speed centrifugation to prepare cytosolic and membrane fractions. The total, cytosolic (A), and membrane (B) levels of Tiam1 were assessed by Western blotting. The ratio of cytosolic or membrane-associated Tiam1 to total Tiam1 was then normalized to the ratio for static control cells at that time. The blots are representative of four different experiments, and the bar graphs summarize the densitometry data expressed as means ± S.E. (n = four independent experiments). *, p < 0.05, significant difference from the static control.

FIGURE 6.

Activated Rac induces phosphorylation of JNK1. Representative immunoblots of equal amounts of cell lysates from multiple wounded monolayers grown on laminin-5 matrix expressing WT-Rac1 or DN-Rac1 were probed with anti-phosphorylated and anti-total JNK1 antibodies. The bar graph summarizes the densitometry data expressed as means ± S.E. (n = four independent experiments). *, p < 0.05, significant difference from the untreated control. HA, hemagglutinin.

FIGURE 7.

PI3K activation is independent of FAK phosphorylation. Confluent cells grown on collagen IV (C) or laminin-5 (L) matrix were treated with adenovirus expressing WT-FAK or FRNK for 48 h prior to application of multiple wounds. Cells were exposed to static or CS conditions for 6 h. A, PI3K activity was not affected by expression of WT-FAK or FRNK. *, p < 0.05, significant difference from the static control. PIP₃, phosphatidylinositol 3,4,5-trisphosphate. B and C, cells were lysed and immunoprecipitated (IP) with anti-FAK (B) or anti-JIP3 (C) antibody and then immunoblotted (IB) with anti-PI3K antibody. Representative Western blots from four different experiments are shown. Jurkat (J) cell lysate was used as a positive control.

FIGURE 8.

Coexpression of WT-FAK, JIP3, and CA-PI3K restores migration of cells exposed to CS to the level in static cells. Cells were grown on laminin-5 matrix and treated with adenovirus expressing WT-FAK, JIP3, and CA-PI3K for 48 h prior to wounding. Multiple wounds were applied, and cells were exposed to CS or static conditions for 9 h. *, p < 0.05, significant difference from the static control.

FIGURE 9.

Schematic representation of pathways affected by cyclic strain causing inhibition of migration of AECs. After injury, signals from integrin receptors in the extracellular matrix activate two signaling pathways, one that leads to phosphorylation of FAK and one that leads to activation of Rac via activation of PI3K. Both lead to phosphorylation of JNK and thus to cell migration. Both pathways may be down-regulated by cyclic mechanical stretch, and loss of either pathway slows cell migration.