title: Stretch-induced mitogen-activated protein kinase activation in lung fibroblasts is independent of receptor tyrosine kinases.
abstract: Lung growth and remodeling are modulated by mechanical stress, with fibroblasts thought to play a leading role. Little mechanistic information is available about how lung fibroblasts respond to mechanical stress. We exposed cultured lung fibroblasts to tonic stretch and measured changes in phosphorylation status of mitogen-activated protein kinases (MAPKs), selected receptor tyrosine kinases (RTKs), and phospholipase Cgamma1 (PLCgamma1) and activation of the small G-protein Ras. Human lung fibroblasts (LFs) were seeded on matrix-coated silicone membranes and exposed to equibiaxial 10 to 40% static stretch or 20% contraction. LFs were stimulated with EGF, FGF2, or PDGF-BB or exposed to stretch in the presence of inhibitors of EGFR (AG1478), FGFR (PD173074), and PDGFR (AG1296). Phospho-MAPK, phospho-RTK, and phospho-PLCgamma1 levels were measured by Western blotting. Active GTP-Ras was quantified by immunoblotting after pull-down with a glutathione S-transferase-Raf-RBD construct. Normalized p-ERK1/2, p-JNK, and p-p38 levels increased after stretch but not contraction. Ligands to RTKs broadly stimulated MAPKs, with the responses to EGF and PDGF most similar to stretch in terms of magnitude and rank order of MAPK responses. Stretching cells failed to elicit measurable activation of EGFR, FGFR (FRS2alpha phosphorylation), or PDGFR. Potent inhibitors of the kinase activity of each receptor failed to attenuate stretch-induced MAPK activation. PLCgamma1 and Ras, prominent effectors downstream of RTKs, were not activated by stretch. Our findings demonstrate that MAPKs are potently activated by stretch in lung fibroblasts, but, in contrast to stress responses observed in other cell types, RTKs are not necessary for stretch-induced MAPK activation in LFs.
authors.affiliation: Department of Environmental Health, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, USA.
authors.forename: Francis
authors.initials: F
authors.lastname: Boudreault
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Tschumperlin
mesh_terms: D004789:Enzyme Activation; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008168:Lung / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D008954:Models, Biological; D009119:Muscle Contraction; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D013314:Stress, Mechanical; D013997:Time Factors
chemical_list: D017468:Receptors, Fibroblast Growth Factor; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor
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