Induction of the plasminogen activator system by mechanical stimulation of human bronchial epithelial cells.

Mechanical stimulation of the airway epithelium, as would occur during bronchoconstriction, is a potent stimulus and can activate profibrotic pathways. We used DNA microarray technology to examine gene expression in compressed normal human bronchial epithelial cells (NHBE). Compressive stress applied continuously over an 8-h period to NHBE cells led to the upregulation of several families of genes, including a family of plasminogen-related genes that were previously not known to be regulated in this system. Real-time PCR demonstrated a peak increase in gene expression of 8.0-fold for urokinase plasminogen activator (uPA), 16.2-fold for urokinase plasminogen activator receptor (uPAR), 4.2-fold for plasminogen activator inhibitor-1 (PAI-1), and 3.9-fold for tissue plasminogen activator (tPA). Compressive stress also increased uPA protein levels in the cell lysates (112.0 versus 82.0 ng/ml, P = 0.0004), and increased uPA (4.7 versus 3.3 ng/ml, P = 0.02), uPAR (1.3 versus 0.86 ng/ml, P = 0.007), and PAI-1 (50 versus 36 ng/ml, P = 0.006) protein levels in cell culture media. Functional studies demonstrated increased urokinase-dependent plasmin generation in compression-stimulated cells (0.0090 versus 0.0033 OD/min, P = 0.03). In addition, compression led to increased activation of matrix metalloproteinase (MMP)-9 and MMP-2 in a urokinase-dependent manner. In postmortem human lung tissue, we observed an increase in epithelial uPA and uPAR immunostaining in the airways of two patients who died in status asthmaticus compared with minimal immunoreactivity noted in airways from seven lung donors without asthma. Together these observations suggest an integrated response of airway epithelial cells to mechanical stimulation, acting through the plasminogen-activating system to modify the airway microenvironment.

Bronchial epithelial compression regulates epidermal growth factor receptor family ligand expression in an autocrine manner.

The epidermal growth factor receptor (EGFR), an important signaling pathway in airway biology, is stimulated by compressive stress applied to human airway epithelial cells. Although the EGFR ligand, heparin-binding epidermal growth factor-like growth factor (HB-EGF), is known to be released as a result of this stimulation, whether compressive stress enhances expression of other EGFR ligands, and the duration of mechanical compression required to initiate this response, is not known. Human airway epithelial cells were exposed to compressive stress, and expression of four EGFR ligands was examined by quantitative PCR. Cells were exposed to: (1) continuous compressive stress over 8 h, (2) compression with and without EGFR inhibitor (AG1478), or (3) time-limited compression (3.75, 7.5, 15, 30, and 60 min). Compressive stress produced a sustained upregulation of the EGFR ligands HB-EGF, epiregulin, and amphiregulin, but not transforming growth factor-alpha. Inhibition with AG1478 demonstrated that expression of HB-EGF, epiregulin, and amphiregulin is dependent on the signaling via the EGFR. Immunostaining for epiregulin protein demonstrated increased expression with compression and attenuation with EGFR inhibition. The response of all three EGFR ligands persisted long after the mechanical stimulus was removed. Taken together, these data suggest the possibility of a mechanically activated EGFR autocrine feedback loop involving selected EGFR ligands.

Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation.

The Th2 cytokines interleukin (IL)-4 and IL-13 are thought to play critical roles in the airway inflammation and hyperresponsiveness that characterize asthma. Recent evidence indicates that IL-13 can mediate these effects by acting directly on airway epithelial cells. Here we evaluated early [signal transducer and activator of transcription (STAT)6 phosphorylation] and delayed [granulocyte/macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta(2) (TGF-beta(2)) secretion] responses of airway epithelial cells to IL-4 and IL-13 stimulation and the dependence of these responses on the culture technique employed. As expected, normal human bronchial epithelial cells grown on microporous inserts at an air-liquid interface (ALI) expressed a well-differentiated mucociliary phenotype; in contrast, cells grown on plastic in submerged cultures were poorly differentiated. When stimulated with IL-4 or IL-13, the magnitude and duration of STAT6 phosphorylation under the differing culture conditions were statistically indistinguishable. In contrast, cytokine secretion responses to IL-4 and IL-13 were highly dependent on the culture technique; cells cultured on plastic exhibited significant concentration-dependent increases in GM-CSF and TGF-beta(2) secretion, whereas cells grown at ALI showed no statistically significant response. These results demonstrate that the coupling between early signal transduction responses to IL-4 and IL-13 and downstream functions such as cytokine secretion may be critically dependent on the cell culture technique employed and the resulting differentiation status of bronchial epithelial cells.