Reactive oxidant and p42/44 MAP kinase signaling is
necessary for mechanical strain-induced proliferation in
pulmonary epithelial cells
PATRICIA R CHESS 1,2, MICHAEL A O’REILLY 1,3, FREDRICK SACHS4, and JACOB N
Departments of Pediatrics1, Biomedical Engineering2, Environmental Medicine3, University of
Rochester, Rochester, New York, 14642 and Department of Physiology and Biophysics4, State
University of New York (SUNY) Buffalo, Buffalo, New York, 14214.
Running title: Signaling of strain-induced proliferation in lung
Address for correspondence:
Patricia R Chess
601 Elmwood Avenue
Rochester, New York 14642
Mechanical strain is necessary for normal lung growth and development. Individuals with
respiratory failure are supported with mechanical ventilation, leading to altered lung growth and
injury. Understanding signaling pathways initiated by mechanical strain in lung epithelial cells will
help guide development of strategies aimed at optimizing strain-induced lung growth while
mitigating ventilator-induced lung injury. To study strain-induced proliferative signaling, focusing
on the role of reactive oxidant species (ROS) and p42/44 MAP kinase, human pulmonary epithelial
H441 and MLE15 cells were exposed to equibiaxial cyclic mechanical strain. ROS were increased
within 15 minutes of strain. N -acetylcysteine blocked strain-induced ROS production, p42/44 MAP
kinase phosphorylation, and strain-induced proliferation. PD98059 and UO126, p42/44 MAP kinase
inhibitors, blocked strain-induced proliferation. To verify the specificity of p42/44 MAP kinase
inhibition, cells were transfected with dominant negative MEK1 plasmid DNA. Transfected cells
did not proliferate in response to mechanical strain. To determine if strain-induced tyrosine kinase
activity is necessary for strain-induced ROS- p42/44 MAP kinase signaling, genistein, a tyrosine
kinase inhibitor, was used. Genistein did not block strain-induced ROS production or p42/44 MAP
kinase phosphorylation. Gadolinium, a stretch-activated ion channel inhibitor, blocked strain-
induced ROS production and p42/44 MAP kinase phosphorylation, but not strain-induced tyrosine
phosphorylation. This data support ROS production and p42/44 MAP kinase phosphorylation being
involved in a common strain-induced signaling pathway, necessary for strain-induced proliferation
in pulmonary epithelial cells, with a parallel strain-induced tyrosine kinase pathway.
Key words: mechanical strain, pulmonary epithelium, ROS, p42/22 MAP kinase
Mechanical ventilation is frequently used as a life-saving intervention in respiratory failure.
Infants born prematurely often receive prolonged pulmonary support resulting in potentially fatal
pulmonary lung injury . Adults with trauma, aspiration or pneumonia resulting in respiratory
failure often progress to Acute Respiratory Distress Syndrome (ARDS), with a mortality of 40-70%
. The interventions initiated to support patients with respiratory failure in ARDS, including
mechanical ventilation, have been implicated in initiating a systemic response contributing to this
mortality [3-6]. Understanding the signaling cascades initiated by mechanical strain could help
optimize beneficial strain responses while mitigating detrimental effects of this therapy.
Mechanical strain is known to induce proliferation in lung cells [7,8]. The signaling
molecule p42/44 MAP kinase, activated by mechanical strain in lung epithelial cells, is a key
effector in signaling proliferative responses in other cell types [9,10]. Strain has also been found to
initiate an inflammatory response in isolated mouse lungs . Reactive oxygen species (ROS)
production is known to be a signaling molecule involved in proliferation in a variety of cell types
[12-16] . ROS also plays a role in signaling of inflammatory cytokines , and has been linked to
p42/44 MAP kinase signaling . We have demonstrated a strain-induced oxidant response in
pulmonary epithelial cells .
The current work investigates the hypothesis that enhanced ROS production is involved in
strain-induced signaling in lung epithelial cells, and that this ROS production mediates strain-
induced p42/44 MAP kinase activation and ultimately strain-induced proliferation. While cell-cell
interactions can be studied more effectively in whole animal models, in vitro studies allow
investigation of specific cell types in complex signal transduction cascades. Experiments in this
paper focus on in vitro studies that address pulmonary epithelial cell mechanical strain-induced
MATERIALS AND METHODS
Cell culture: Human pulmonary adenocarcinoma H441 cells and MLE15 cells were obtained from
ATCC (Rockville, Maryland). Cells were plated in RPMI 1640 or DMEM/F12 respectively (Sigma,
St Louis MO) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) /0.1% 10mg/ml
Gentamicin (Gibco BRL, Grand Island, NY), at 2x105 cells/well on flexible bottom, type I collagen
coated, silicone elastomer 6-well Bioflex culture plates (Flexcell Corp, McKeesport. PA) for 24h,
serum starved for 24 hours, then treated as described. For epidermal growth factor (EGF)
experiments, cells were incubated with 10 ng/ml EGF (Upstate Biotechnology, Waltham, Mass).
Cell number was determined after cells were removed from the wells by trypsin digestion followed
by hemocytometer cell count.
Inhibitor studies: Inhibitor concentrations were determined based on literature review, toxicity as
determined by trypan blue dye exclusion assessment, and activity determination. For NAC
experiments, cells were pretreated with 20µM NAC (Sigma-Aldrich, St. Louis, MO) while serum
starving for 24 hours prior to strain. For other inhibitor experiments, cells were pretreated for 2
hours prior to strain. The following concentrations were used: 10 µM PD98059 (Calbiotech-
Novabiochem, LaJolla, CA), 10 µM UO126 (Calbiotech-Novabiochem, LaJolla, CA), 10 µM
cyclohexamide (Sigma-Aldrich, St. Louis, MO), 100 µM gadolinium chloride (Sigma-Aldrich, St.
Louis, MO), 10 µM genistein (Calbiotech-Novabiochem, LaJolla, CA), and 500 µM allopurinol
(Sigma-Aldrich, St. Louis, MO). The stretch-activated ion channel blocker GsMtx-4 isolated from
tarantula toxin was added at a concentration of 5 µM  just prior to initiating strain.