Cyclic Mechanical Stretch Decreases Cell Migration by Inhibiting Phosphatidylinositol 3-Kinase- and Focal Adhesion Kinase-mediated JNK1 Activation

Leena P Desai,Steven R White,Christopher M Waters

doi:10.1074/jbc.m109.084335

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.

Highlights

Airway epithelial cells (AECs) migration after injury occurs during a state of cyclic mechanical distention of the underlying substrate in the airways during respiration
Because focal adhesion kinase (FAK) has been shown to serve as a scaffold for interactions with other signaling molecules involved in cell migration [21, 22] and because Tyr397 has been identified as a binding site for phosphatidylinositol 3-kinase (PI3K) [23], we investigated whether PI3K activity is affected by changes in FAK signaling and further investigated the potential interaction in signaling via PI3K and Jun N-terminal kinase (JNK)
We showed previously that cell migration of 16HBE14oϪ cells is significantly faster on laminin-5 compared with collagen IV [12], and the results in Fig. 2 suggest that this difference may be due in part to enhanced activation of PI3K

Summary

EXPERIMENTAL PROCEDURES

Equal amounts of cell lysates were subjected to immunoprecipitation with either anti-FAK (BD Transduction Laboratories) or anti-JIP3 (Santa Cruz Biotechnology) antibody. For immunoblotting experiments to detect phosphorylated JNK1, 50 ␮g of the radioimmune precipitation assay cell lysates were loaded on gels for SDS-PAGE, transferred onto nitrocellulose membranes, and probed with anti-phospho-JNK1 (Abcam) and antiJNK1 antibodies. Rac Activation Assay—Activated Rac (GTP-Rac1) levels in multiple wounded 16HBE14oϪ cells, transfected with either CA-PI3K or DN-PI3K and exposed to static or CS conditions, were determined using the Rac activation assay kit from Upstate as described previously [32] using anti-Rac antibody (BD Transduction Laboratories). Cytosolic and Membrane Fraction Preparations—Multiple wounded 16HBE14oϪ cells expressing EGFP, CA-PI3K, or DN-PI3K were lysed, and the total, cytosolic, and membrane fractions for Tiam localization were prepared as described previously [32]. Significant differences were determined based on a threshold of p Ͻ 0.05

RESULTS

DISCUSSION

Matrix C C L L