Mechanisms of Cytokine-Mediated Integrin Activation and Force Transmission in Airway Smooth Muscle

PROJECT SUMMARY/ABSTRACT Exaggerated smooth muscle contraction is one of the hallmarks of asthma. To transmit enough force to deform the airway lumen, smooth muscle depends on firm tethering to the surrounding tissue. These tethering pathways play critical roles in connecting intracellular actin filaments to the extracellular matrix and adjacent cells to efficiently transmit force. We have shown that targeting specific tethering proteins in the integrin and cadherin families have functional effects on force transmission in smooth muscle without altering actin-myosin crossbridge cycling. We have further shown that pro-inflammatory cytokines enhance the activity of tethering proteins to promote adhesion. The long-term goal of this proposal is to understand the mechanisms regulating force transmission by tethering proteins in airway smooth muscle. The overall objectives of this application are to explore how the pro-inflammatory cytokines IL-13 and IL-17A regulate activation of β1 integrins and to apply advanced imaging methods to analyze cell-matrix and cell-cell tethering in tissue. The central hypothesis is that cytokine- mediated integrin activation enhances force transmission in the airways, and that cell-matrix and cell-cell tethering pathways have a significant impact on the exaggerated airway narrowing that characterizes asthma. These hypotheses are built on data demonstrating that (1) integrin α5β1, integrin α2β1, and cadherin-11 all regulate force transmission in disease-relevant models of asthma; (2) mitigation of exaggerated force transmission does not alter classical actin-myosin pathways; (3) integrins are differentially activated in airway samples from asthmatics; (4) IL-13 and IL-17A activate β1 integrins in a manner dependent on Rho kinase; and (5) integrin activation has functional effects on force transmission even in the absence of cytokine. These hypotheses will be tested through two specific aims: 1) Evaluate the mechanisms by which pro- inflammatory stimuli regulate β1 integrin activation; and 2) Apply advanced imaging tools to analyze cell-matrix and cell-cell tethering in tissue. Aim 1 will examine the time course and conformation states of integrins during cytokine-mediated activation, quantify differences in integrin activation from airway biopsies of asthmatics and controls, and determine signaling mechanisms by which cytokines regulate Rho kinase, and Rho kinase regulates physiologic integrin activation. Aim 2 will use two-photon microscopy and confetti mice to quantify changes intercellular tethering induced by ligation of cadherin-11, explore dynamic interplay between cell- matrix and cell-cell tethering pathways, as well as investigate the influence of pro-inflammatory cytokines on cadherin-mediated tethering. The proposed research is innovative because it explores two novel ideas (force transmission pathways and their activation by inflammatory stimuli) using cutting-edge tools. The prosed research is significant because it highlights an underappreciated contributor to asthma and lays the foundational mechanistic studies to one day identify novel therapeutics.