title: Rho kinase signaling pathways during stretch in primary alveolar epithelia.
abstract: Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.
authors.affiliation: Department of Bioengineering, University of Pennsylvania, 210 South 33rd St., Philadelphia, PA 19104-6321, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: DiPaolo
authors.forename: Susan S
authors.initials: SS
authors.lastname: Margulies
mesh_terms: D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D024722:Cardiac Myosins / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D008297:Male; D055595:Mechanical Phenomena; D040542:Mechanotransduction, Cellular; D008954:Models, Biological; D018994:Myosin Light Chains / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D012121:Respiration, Artificial; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D000199:Actins; D000577:Amides; D001381:Azepines; D004791:Enzyme Inhibitors; D006576:Heterocyclic Compounds, 4 or More Rings; D018994:Myosin Light Chains; D009281:Naphthalenes; D011725:Pyridines; C107184:myosin light chain 2; C070571:ML 7; C108830:Y 27632; C472645:blebbistatin; D054460:rho-Associated Kinases; D024722:Cardiac Myosins; D020742:rhoA GTP-Binding Protein