Stretch-Induced Activation of AMP Kinase in the Lung Requires Dystroglycan

Abstract

Lung cells are exposed to cyclic stretch during normal respiration and during positive pressure mechanical ventilation administered to support gas exchange. Dystroglycan is a ubiquitously expressed matrix receptor that is required for normal basement membrane formation during embryogenesis and for maintaining the function of skeletal muscle myocytes and neurons where it links cells to matrix. We previously reported that equibiaxial stretch of primary alveolar epithelial cells activated the MAP kinase pathway ERK1/2 through a mechanism that required an interaction between dystroglycan and matrix. We determined whether this mechanism of mechanotransduction activates other signaling cascades in lung epithelium. Exposure of rat epithelial alveolar type II cells (AEC) to cyclic mechanical stretch resulted in activation of 59 AMP-activated protein kinase (AMPK). This response was not affected by pretreatment of AEC with the ERK inhibitor PD98059 but was inhibited by knockdown in dystroglycan expression. Moreover, production of reactive oxygen species was enhanced in mechanically stimulated AEC in which dystroglycan was knocked down. This enhancement was reversed by treatment of AEC with an AMPK activator. Activation of AMPK was also observed in lung homogenates from mice after 15 minutes of noninjurious mechanical ventilation. Furthermore, knockdown of dystroglycan in the lungs of mice using an adenovirus encoding a dystroglycan shRNA prevented the stretch-induced activation of AMPK. These results suggest that exposure to cyclic stretch activates the metabolic sensing pathway AMPK in the lung epithelium and supports a novel role for dystroglycan in this mechanotransduction.

Plectin-containing, centrally-localized focal adhesions exert traction forces in primary lung epithelial cells

“…Primary AEC were isolated from pathogen-free, male Sprague-Dawley rats (200-225 g; for rAEC) or C57/BL6 mice (for mAEC) as previously described (Budinger et al, 2008;DeBiase et al, 2006;Eisenberg et al, 2011). The day of cell isolation is designated as day 0 in culture.…”

“…Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes and processed for immunoblotting as previously described (Budinger et al, 2008;Eisenberg et al, 2011). Immunoblots were scanned and quantified using ImageJ (National Institutes of Health, NIH; Bethesda, MD).…”

Lung-specific loss of the laminin α3 subunit confers resistance to mechanical injury

“…Stretch of the alveolar basement membrane can exceed this level in patients with respiratory failure who are subjected to positive pressure mechanical ventilation (Vlahakis and Hubmayr, 2005). In cultured primary rat alveolar epithelial cells, we have reported that the interaction between a3-subunit-containing laminins and the cell surface receptor dystroglycan induces signaling that might protect cells from the harmful effects of mechanical stimulation (Budinger et al, 2008). Thus, we hypothesized that in mice the loss of a3 laminin might not only alter the viscoelastic properties of the lung but also disrupt signaling pathways activated by ventilation.…”

Reproducible uniform equibiaxial stretch of precision-cut lung slices

“…Studies that simulate the mechanical stretch of the alveolar wall during VILI utilize different models ranging from pulmonary alveolar epithelial type II cell lines (3,29), primary alveolar epithelial type I and II cells (1,2,15), excised intact lungs (5,6), and living animals (28,32). Although VILI studies with stretched monolayers of cells or ventilated animals can be complicated, stretching precision-cut lung slices (PCLSs) presents additional technical challenges because of the need to fix the slices to a stretchable substrate or hold them in a way that allows deformation without rupture.…”