Mechanical stretch decreases migration of alveolar epithelial cells through mechanisms involving Rac1 and Tiam1

Abstract

Mechanical ventilation can overdistend the lungs or generate shear forces in them during repetitive opening/closing, contributing to lung injury and inflammation in patients with acute respiratory distress syndrome (ARDS). Repair of the injured lung epithelium is important for restoring normal barrier and lung function. In the current study, we investigated the effects of cyclic mechanical strain (CS), constant distention strain (CD), and simulated positive end-expiratory pressure (PEEP) on activation of Rac1 and wound closure of rat primary alveolar type 2 (AT2) cells. Cyclic stretch inhibited the migration of wounded AT2 cells in a dose-dependent manner with no inhibition occurring with 5% CS, but significant inhibition with 10% and 15% CS. PEEP conditions were investigated by stretching AT2 cells to 15% maximum strain (at a frequency of 10 cycles/min) with relaxation to 10% strain. AT2 cells were also exposed to 20% CD. All three types of mechanical strain inhibited wound closure of AT2 cells compared with static controls. Since lamellipodial extensions in migrating cells at the wound edge were significantly smaller in stretched cells, we measured Rac1 activity and found it to be decreased in stretched cells. We also demonstrate that Tiam1, a Rac1-specific guanine nucleotide exchange factor, was expressed mainly in the cytosol of AT2 cells exposed to mechanical strain compared with membrane localization in static cells. Downregulation of Tiam1 with 100 microM NSC-23766 inhibited activation of Rac1 and migration of AT2 cells, suggesting its involvement in repair mechanisms of AT2 cells subjected to mechanical strain.

Fig. 1.

Mechanical strain inhibited migration of alveolar type 2 (AT2) cells. AT2 cells were plated (3.5 × 10⁶ cells/well) on 6-well Flexercell plates coated with RLF-6 matrix. Confluent monolayers were wounded with a pipette tip, cells were exposed to either static or stretch conditions immediately, and wound closure was monitored for 24 h. A: cyclic strain (CS) at 0–5% (30 cpm), 0–10% (30 cpm), 0–15% (10 cpm) compared with static cells. B: static cells compared with cells stretched with 20% constant distention (CD) or conditions simulating PEEP (10–15% CS, 10 cpm). #P < 0.05 vs. static control; measurements were made in at least 3 wells from 3 different isolations (n = 9).

Fig. 2.

Mechanical strain inhibited lamellipodial extensions in migrating AT2 cells. Confluent monolayers were wounded, subjected to 0–15% CS (10 cpm) for 16 h, and fixed and stained with rhodamine-phalloidin for F-actin. Images were acquired using Z series, and lamellipodial extensions at the wound edge were traced and quantitated using Metamorph software. A: low-magnification images of cells at the wound edge as well as higher-magnification views of the indicated regions. Lamellipodial width (μm) was calculated as lamellipodial area/wound edge length as indicated (A, bottom). B: the measured lamellipodial width for static and 0–15% CS cells. Data are expressed as means ± SE of 3 independent experiments. #P < 0.05 vs. static control.

Fig. 3.

Mechanical strain inhibited Rac1 activation. Confluent monolayers of AT2 cells on RLF matrix were exposed to mechanical strain for 2 and 12 h. GTP-Rac1 levels in AT2 cells exposed to static conditions, cyclic strain (0–15% CS) (A) and 10–15% CS (PEEP) conditions (B), and constant distention strain (CD) (C) were determined via a pull-down assay; a representative blot is shown at the top of each panel. The bar graphs summarize the data expressed as means ± SE, from 4 independent experiments, the values of which are expressed as the ratio of active GTP-bound Rac1 to total Rac1. #P < 0.05 vs. static controls at 2 and 12 h.

Fig. 4.

Mechanical strain caused translocation of Tiam1 from membrane to the cytosol. Confluent monolayers of AT2 cells on RLF-6 matrix were exposed to mechanical strain for 2 and 12 h. Cells were lysed, aliquots of total fraction withdrawn, and cell lysates further subjected to high-speed centrifugation to prepare the cytosolic and membrane fractions. The total, cytosolic (A), and membrane (B) levels of Tiam1 were assessed by Western blotting. The blots are representative of 3 independent experiments from separate isolations, and the bar graphs summarize the densitometry data expressed as means ± SE; n = 3 independent experiments. #P < 0.05 vs. static controls at 2 and 12 h.

Fig. 5.

Inhibition of Tiam1 downregulated both Rac1 activity and migration of AT2 and A549 cells. Confluent monolayers of AT2 cells were treated with the Tiam1 inhibitor NSC-23766. A: GTP-Rac1 levels in AT2 cells treated with 100 μM NSC-23766 for 2 and 12 h. Representative Western blot was probed for activated Rac1 and total Rac1 levels. B: wound closure of AT2 and A549 cells treated with 100 μM NSC-23766. Data are expressed as means ± SE of 4 independent experiments. #P < 0.05 vs. untreated corresponding condition.