1 Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611; and 2 Centro de Investigación del Cáncer, Universidad de Salamanca, 37007 Salamanca, Spain
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ABSTRACT |
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Mechanical stimuli are transduced into intracellular signals in lung alveolar epithelial cells (AEC). We studied whether mitogen-activated protein kinase (MAPK) pathways are activated during cyclic stretch of AEC. Cyclic stretch induced a rapid (within 5 min) increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in AEC. The inhibition of Na+, L-type Ca2+ and stretch-activated ion channels with amiloride, nifedipine, and gadolinium did not prevent the stretch-induced ERK1/2 activation. The inhibition of Grb2-SOS interaction with an SH3 binding sequence peptide, Ras with a farnesyl transferase inhibitor, and Raf-1 with forskolin did not affect the stretch-induced ERK1/2 phosphorylation. Moreover, cyclic stretch did not increase Ras activity, suggesting that stretch-induced ERK1/2 activation is independent of the classical receptor tyrosine kinase-MAPK pathway. Pertussis toxin and two specific epidermal growth factor receptor (EGFR) inhibitors (AG-1478 and PD-153035) prevented the stretch-induced ERK1/2 activation. Accordingly, in primary AEC, cyclic stretch activates ERK1/2 via G proteins and EGFR, in Na+ and Ca2+ influxes and Grb2-SOS-, Ras-, and Raf-1-independent pathways.
mechanotransduction; mechanical stress; mitogen-activated protein kinase; lung injury; epidermal growth factor receptor
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INTRODUCTION |
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MECHANICAL VENTILATION with high lung volumes and pressures causes ventilator-induced lung injury characterized by increased permeability pulmonary edema and inflammation (26, 35). During mechanical ventilation with high tidal volumes, alveolar epithelial cells (AEC) can be stretched. These cells are responsible for the normal function of the alveolar capillary barrier, alveolar fluid absorption, and the synthesis of pulmonary surfactant. It is known that AEC respond to mechanical stretch in different ways. A single stretch of AEC caused cytosolic Ca2+ to increase, followed by a stimulation of surfactant secretion at a single cell (37) and at the whole organ level (1).
Stretching AEC increased Na+-K+-ATPase activity (34) and, in fetal lung cells, increased [3H]thymidine incorporation, cAMP levels, and synthesis of surfactant-related phospholipids (25) and extracellular matrix modulation (38).
The kinases in the mitogen-activated protein kinase (MAPK) family are ubiquitous serine/threonine kinases, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2). These cascades play an important role in the transduction of mitogenic and differentiation signals, leading to activation of transcription factors (4). Several cells respond to mechanical forces by activating MAPK pathways, but little is known about how mechanical stimuli are converted into biochemical signals in AEC (18). Mechanical stress has been shown to stimulate ERK1/2 in endothelial cells (11, 29), cardiac myocytes (41), vascular smooth muscle cells (VSMC) (39), and in H441 cells (3). Another component of the MAPK family, the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), is activated by cyclic stretch in A549 cells (21), and stretching these cells induces an interleukin-8 release (33). Because mechanical stress is an important regulator of cell growth (16, 23) and because ERK1/2 are believed to be vital components of proliferative pathways (36), the goal of this study was to determine whether ERK1/2 are activated by cyclic stretch of AEC and study the pathways participating in this mechanotransduction.
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METHODS |
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Materials. U-0126 and polyclonal anti-ERK1/2 antibodies were purchased from Promega (Madison, WI); monoclonal anti-phosphorylated ERK1/2 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Farnesyl transferase inhibitor (FTI-277), PD-153035, pertussis toxin (PTx), hSOS n10 Sh3 binding peptide (SH3b-p), and nifedipine were purchased from Calbiochem-Novabiochem (La Jolla, CA); Rat IgG, gadolinium, and forskolin were purchased from Sigma (St. Louis, MO).
Cell isolation and culture. AEC were isolated from pathogen-free male Sprague-Dawley rats (200-225 g) as previously described (22). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase (3 U/ml; Worthington Biochemical, Freehold, NJ). The alveolar type II (ATII) cells were purified by differential adherence to IgG-pretreated dishes, and cell viability was assessed by trypan blue exclusion (>95%). Cells were resuspended in Dulbecco's modified Eagle's medium (DMEM, Cellgro) containing 10% fetal bovine serum (Hyclone) with 2 mM glutamine, 100 U/ml penicillin, 0.25 µg/ml amphotericin B, and 100 µg/ml streptomycin. Cells were seeded in six-well, 35-mm, elastomer-bottomed, and laminin-coated Bioflex plates (Flexcell International, McKeesport, PA) at a density of 0.5 × 106 cells/cm2. Cells were incubated in a humidified atmosphere of 5% CO2-95% air at 37°C. The day of isolation and plating is designated cultured day 0. All experimental conditions were tested in 24-h serum-starved cells when the cells achieved confluence, which occurred at day 4.
Cyclic stretch. Serum-starved AEC were subjected to mechanical stretch using the Cyclic Strain Unit (FX-3000 Flexercell Strain Unit, Flexcell), which consists of a controlled vacuum unit and a base plate to hold the culture plates. A vacuum (22.3 kPa) was cyclically applied to the elastomer-bottomed plates via the base plate. The vacuum produced a 30% elongation on the flexible bottom membranes at a frequency of 30 cycles/min, with a stretch/relaxation relation of 1:1. Cells were harvested at different durations of cyclic exposure. To assess the effect of smaller magnitudes of stretch on MAPK activation, 5% elongation was also applied, and cells were harvested at different time points.
Total protein isolation.
After cyclic stretch exposure, medium was aspirated and AEC were washed
twice in ice-cold PBS (Cellgro) and scraped in lysis buffer (LB)
containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM Na3VO4, 1 mM NaF, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells extracts were
sonicated and centrifuged for 15,000 rpm for 10 min, and supernatants
were collected. Protein content was determined by the Bradford
technique, using a Bio-Rad protein assay system (Bio-Rad, Hercules, CA).
Western blot analysis. Total protein (35 µg) from ATII cells was resolved in a 12% polyacrylamide gel and transferred onto nitrocellulose membranes (Optitran, Schleicher & Schuell, Keene, NH) using a semidry transfer apparatus (Bio-Rad). Incubation with a monoclonal antibody that specifically recognizes the dually phosphorylated active form of ERK1 and ERK2 (Cell Signaling Technology) was performed overnight at 4°C. Blots were developed with an enhanced chemiluminescence detection kit (ECL+, Amersham, Buckinghamshire, UK) used as recommended by the manufacturer. The bands were quantified by densitometric scan (Eagle Eye II, Stratagene, La Jolla, CA). To ensure equal loading and protein transfer, the blots were stripped and probed with a polyclonal antibody that recognizes both phosphorylated and nonphosphorylated p44/p42 MAPK (Promega). Blots were developed and quantified as described above. The densitometric values for the phosphorylated form of ERK1/2 were normalized according to the values for the total ERK1/2.
ERK assay.
The ERK activity was determined as described in the p44/p42 MAPK assay
kit manual by Cell Signaling Technology. Briefly, 200 µg of
total protein from the lysates were immunoprecipitated with an
immobilized phosphospecific p44/p42 MAP kinase (Thr202/Tyr204) monoclonal antibody by overnight incubation at 4°C with gentle rocking. After washing the beads twice with LB and twice with kinase
reaction buffer (KRB: 25 mM Tris, pH 7.5, 5 mM -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM
MgCl2), we resuspended the beads in 50 µl of KRB
supplemented with 200 µM ATP and 2 µg Elk-1 fusion protein.
Reaction was performed at 30°C for 30 min and terminated with 25 µl
of 3× SDS loading buffer (187 mM Tris · HCl, pH 6.8, 10%
glycerol, 2% SDS, 5%
-mercaptoethanol, and 1.25 × 10
3% bromphenol blue). Samples (30 µl) were
fractionated in a 12.5% SDS-PAGE gel and analyzed by immunoblotting
using a phosphospecific Elk-1 antibody (New England Biolabs, Beverly,
MA) as a probe. Blots were developed and quantified as described above.
Activated Ras affinity precipitation assay. A functional assay for the activated Ras was done with an affinity precipitation assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions and as described elsewhere (28). Briefly, serum-starved day 4 AEC were subjected to the stretch protocol or incubated with epidermal growth factor (EGF) for 5 min. After treatment, cells were washed twice with cold PBS and lysed in 200 µl of Mg2+ lysis/wash buffer (MLB) containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 1 mM NaF, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF. Fresh cell lysates were diluted to ~1 µg/ml total cell protein with MLB, and the lysates were precleared with glutathione agarose. After that, 500 µg-1 mg of cell lysate was incubated with 15 µl Raf-1 Ras binding domain agarose conjugate per assay, and the mixture was gently rocked at 4°C for 30 min. Agarose beads were collected by pulsing, and the supernatant was drained off. The beads were washed three times with MLB, resuspended in an appropriate amount of Laemmli sample buffer, and boiled for 5 min. Supernatants were collected and loaded on a 12% SDS-PAGE gel. The gel was transferred to a nitrocellulose membrane and probed with 1 µg/ml of anti-Ras, clone RAS10 (Upstate Biotechnology) overnight at 4°C. A dilution of horseradish peroxidase-conjugated anti-mouse antibody was used as the secondary antibody, and the ECL reagents were used for the final protein detection.
Lactate dehydrogenase assay. AEC were exposed to 30 min of cyclic stretch (0.5 Hz, 30% amplitude) and assayed for lactate dehydrogenase (LDH) release into the culture media, a marker of cell death. LDH assay was performed according to the manufacturer's instructions [Cytotoxicity Detection Kit (LDH), Roche Diagnostics].
Propidium iodide assessment of cell death. Serum-starved AEC were exposed for 60 min to cyclic stretch (0.5 Hz, 30% stretch). After cyclic stretch exposure, medium was aspirated and AEC were washed twice in warm PBS and detached by using 0.6 ml of trypsin per well. Cells were centrifuged, the cell pellet was resuspended in complete DMEM, and 2 µg/ml of propidium iodide (PI) was added. Cell count was done by flow cytometry in a laser Beckman Coulter Epics XL-MCL with a laser excitation wavelength of 488 nm and PI fluorescence emission at 620 nm. A quantitative measurement of cell death was obtained by counting the PI-positive-staining cells (dead cells).
Statistics. All data were expressed as means ± SE. A paired Student's t-test was used to assess differences between two groups. One-way analysis of variance was performed when more than two groups were compared, followed by a multiple comparison test (Tukey) when the F statistic indicated significance. A P value of <0.05 was considered significant.
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RESULTS |
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Cyclic stretch activates MAPK in AEC. As shown in Fig.
1, cyclic stretch stimulated MAPK in rat
AEC in a time-dependent manner. MAPK activation was assessed by an
ERK1/2 in vitro assay that measured the state of phosphorylation of the
transcription factor Elk-1, a specific ERK1/2 substrate. ERK1/2
activation was significantly increased after 5 min of stretch and
reached a peak at 10 min, returning to basal levels by 30 min. The
specific MAP/ERK kinase (MEK) inhibitor U-0126 abolished the
stretch-induced MAPK activation.
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To verify the amount of stretch time necessary to trigger the MAPK
activation, AEC were submitted to 1-min cyclic stretch (30 cycles/min)
or to a single stretch of 10 s and 30% amplitude (Fig.
2, A and B). Cell
lysates obtained 10 min after the initial stretch of 10 s revealed
a significant increase in ERK1/2 phosphorylation compared with static
controls.
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To verify the magnitude of stretch necessary to trigger MAPK activation, AEC were subjected to a 5% stretch (30 cycles/min), and cells were harvested at different durations of cyclic exposure. Five percent stretch elicited ERK1/2 phosphorylation of similar magnitude as 30% stretch, but at a delayed peak (15 min) (Fig. 2C).
Thirty percent cyclic stretch did not cause cell damage in AEC. Cyclic
stretch of AEC is associated with cell death (6, 30). To
assess whether 30% cyclic stretch is injurious to AEC in our system,
an LDH assay was performed after 30 min of cyclic stretch, and flow
cytometry count of the PI-positive-staining cells (dead cells) was done
after 60 min of cyclic stretch. No increase in percentage of cell death
compared with control was found with either method (Fig.
3).
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Stretch-induced MAPK activation is independent of
Ca2+ and
Na+ influx.
Activation of mechanosensitive ion channels has been proposed as the
transduction mechanism between mechanical stress and cell function
regulation (1, 16, 23, 29). To study the participation of
Ca2+ and Na+ influx in the stretch-induced MAPK
activation, AEC were treated with 100 µM gadolinium
(Gd3+) (Fig. 4, A
and B), a blocker of cation-selective stretch-activated (SA)
ion channels; amiloride (1 µM) (Fig. 4, A and
B), a sodium channel blocker; or nifedipine (5 µM) (Fig.
4C), an L-type Ca2+ channel blocker for 30 min
before the 10-min stretch protocol. Cell lysates were immunoblotted
with an antibody that specifically recognizes the dually phosphorylated
active form of ERK1/2 (top blot in Fig. 4A).
Blots were stripped and probed to an anti-ERK1/2 antibody
(bottom blot in Fig. 4A). Inhibition of
amiloride-sensitive, Gd3+-sensitive (see Fig.
4B), and L-type Ca2+ channels had no effect on
stretch-induced ERK1/2 phosphorylation.
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Stretch-induced ERK1/2 phosphorylation is Grb2-SOS, Ras, and
Raf-1 independent.
To determine whether the ERK1/2 activation induced by
cyclic stretch shares the classical pathway characterized for receptor tyrosine kinases (RTK), we conducted experiments to explore the participation of the adaptor protein Grb2 and the guanine nucleotide exchange protein SOS and its downstream signaling proteins, Ras and
Raf-1 kinase. AEC cells were incubated for 2 h with a peptide corresponding to the SH3 binding sequence of SOS
{[1,149-1,158],N10 SH3 binding domain
(SH3b-p)}, which has a strong affinity for the
NH2-terminal SH3 domain of Grb2, blocking the Grb2-SOS
interaction (17) and preventing Ras activation via this
interaction. Stimulation with EGF was used as a positive control,
because EGF activates ERK1/2 via the Grb2-SOS, Ras, Raf1 pathway. As
shown in Fig. 5A, the
stretch-induced ERK1/2 phosphorylation was not inhibited by SH3b-p, but
the EGF-induced ERK1/2 phosphorylation was abolished by the
preincubation with SH3b-p, suggesting that Grb2-SOS association is not
necessary for the mechanotransduction pathway that leads to ERK1/2
phosphorylation. To assess the participation of Ras and Raf-1 in the
stretch-induced ERK1/2 activation, we incubated AEC cells with the Ras
inhibitor FTI-277 (10 µM) (14) or the Raf-1 inhibitor
forskolin (50 µM) (5) before applying cyclic stretch. As
shown in Fig. 5C, neither FTI-277 nor forskolin blocked the
stretch-induced ERK phosphorylation. Moreover, a functional pull-down assay for the detection of activated Ras [guanosine 5'-triphosphate (GTP)-Ras] showed that GTP-Ras increased in the EGF-stimulated (200 ng/ml), but not in the mechanically stretched, AEC
(Fig. 6). Together, these data suggest
that Grb2-Sos, Ras, and Raf-1 do not participate in the
mechanotransduction pathways that lead to ERK1/2 phosphorylation.
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Stretch-induced ERK1/2 phosphorylation is PTx sensitive and is
dependent on the EGF receptor tyrosine kinase activity.
Several cell types respond to mechanical stress via activation of the
heterotrimeric G proteins (9, 13) or activation of growth
factors receptors (10, 12). To determine whether the
heterotrimeric G protein Gi participates in
mechanotransduction pathways in our system, we incubated AEC cells with
100 ng PTx 18 h before applying cyclic stretch. Pretreatment with
PTx blocked the stretch-induced ERK phosphorylation, suggesting that
Gi is involved in transducing mechanical stimulation to the
MAPK pathway in AEC (Fig. 7). To
determine whether EGF receptor (EGFR) mediated ERK1/2 activation by
cyclic stretch, AEC were incubated with 0.25 nM of PD-153035 or 250 nM
of tyrphostin AG-1478, both specific inhibitors of the tyrosine kinase
activity of the EGFR (7, 8, 15). Both PD-153035 and
AG-1478 inhibited the stretch-induced ERK1/2 activation, suggesting
that EGFR participates in the mechanical stress-induced activation of
MAPK. (Fig. 8).
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DISCUSSION |
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The data presented here demonstrate that mechanical stretch induces a rapid MAPK-ERK1/2 activation in rat AEC. Interestingly, a single large stretch (30%) of 10 s and even low-magnitude cyclic elongation (5%) are enough to trigger the ERK1/2 phosphorylation in AEC (see Fig. 2). Searching for fast-responder mechanotransducers in the plasma membrane, we inhibited mechanosensitive ion channels, which have been related to participate in transduction of mechanical stimuli (1, 16, 23, 29). However, the stretch-induced ERK1/2 activation appears to be independent of SA ion channel, nifedipine-sensitive Ca2+, or amiloride-sensitive Na+ influxes (see Fig. 4). These data are in agreement with the findings in rat neonatal cardiomyocytes and bovine aortic endothelial cells, where inhibition of SA ion channels with gadolinium did not block the stretch-induced MAPK activation (11, 40). However, in VSMC, ERK1/2 activation induced by mechanical stress was dependent on SA ion channel-mediated Ca2+ influx (12), suggesting that stretch-induced ERK1/2 activation may be dependent or independent of SA ion channels in a cell-specific manner. Although a single stretch of AEC increased intracellular Ca2+ (37), the stretch-induced ERK1/2 activation does not appear to depend on activation of mechanosensitive ion channels.
Variable percentages of cell death (30) and a low percentage of apoptosis (6) have been reported in AEC submitted to mechanical stress. However, in our system, up to 60 min of 30% cyclic stretch did not increase cell damage (see Fig. 3).
It has been demonstrated that mechanical stress-induced MAPK activation regulates several cell functions in endothelial cells, cardiac myocytes, and VSMC (16, 23, 29). Few studies have explored the mechanical stress-induced MAPK pathway in lung epithelial cells (18). Quinn et al. (21) reported SAPK/JNK and p38 activation in A549 cells subjected to stretch, and Chess et al. (3) have demonstrated that stretch rapidly activates ERK1/2 and elicits a proliferative response in H441 cells. The present study is the first to report stretch-induced ERK1/2 activation in primary AEC, an important finding, as ERK1/2 phosphorylation results in activation of the transcription factor Elk-1 and may lead to selective gene regulation of immediate-early response genes (c-fos, c-jun) and modulation of AEC responses to mechanical stress.
The data presented in Figs. 7 and 8 demonstrate that mechanical stretch activated ERK1/2 via G protein- and EGFR tyrosine kinase-dependent pathways. Interestingly, our data suggest that EGFR and G proteins are each necessary for transducing mechanical signals to MAPK. Interaction between G proteins and growth factor receptors have been described in ligand-mediated signals, but little is known about the coordinated action of G proteins and EGFR in mechanical stress-mediated signals. Both, independently, have been related to transduce mechanical signals (9, 12, 13), but there is no definitive report associating G proteins and EGFR in mechanotransduction. Jo et al. (13) have reported the participation of Gi protein and an unidentified tyrosine kinase in shear stress-mediated MAPK activation in endothelial cells, and Iwasaki et al. (12) have reported the participation of EGFR in mechanical stress-mediated signals, but these reports do not explore the concomitant participation of G proteins and EGFR.
Although stretch-induced ERK1/2 activation in our system is dependent
on EGFR tyrosine kinase activity, the mechanotransduction in AEC does
not share the classically described RTK-MAPK pathway (Grb2-Sos, Ras,
Raf-1), typically involved in growth factor signaling (17). The lack of inhibition of the RTK-MAPK pathway at
three different steps (see Fig. 5) is corroborated by the absence of stretch-induced Ras activation (see Fig. 6) and suggests that the
classical Grb2-SOS, Ras, Raf1 pathway does not participate in
mechanotransduction in AEC (see Fig. 9).
Additional pathways involving EGFR and G proteins leading to
stretch-induced ERK1/2 phosphorylation may include other adaptor
proteins, such as the small GTP-binding protein Rap1 and its downstream
serine/threonine kinase B-Raf (24). However, studies
pertaining to this pathway are out of the scope of this report.
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Recent data suggest that EGFR can activate signaling pathways in a
growth factor-independent way, as a point of convergence for mitogenic
signals arising from G protein-coupled receptor (GPCR), cytokine
receptors, and cell adhesion (2, 20). It has become
apparent that EGFR takes part in signaling networks activated by
stimuli not initiated by EGF (2). Recently, it has been
shown that 2-adrenergic receptor mediates ERK1/2 activation via
assembly with the EGFR (20) and that GPCR can account for Gi-mediated tyrosine phosphorylation of adapter proteins to
the membrane using EGFR as a scaffold (19). More studies
are warranted to understand whether mechanical stress-mediated signals
transmitted through growth factor receptors and G proteins (9,
10, 12) share the same pathways characterized for
ligand-mediated signals.
Our demonstration that a single stretch induces ERK1/2 phosphorylation (see Fig. 2) is compatible with the fast response of the heterotrimeric G protein to mechanical stress in other cells (9, 13). G proteins participate in signal transduction pathways in skeletal muscle cells (32), cardiac myocytes (23), cardiac fibroblasts (9), and endothelial cells (13), where mechanical signals can regulate growth and differentiation. Activation of ERK1/2 by shear stress in endothelial cells was found to be PTx sensitive (13) and insensitive (31), and there is evidence that mechanical forces may activate a distinct MAPK pathway through differential G protein activation (13).
The stretch-induced ERK1/2 activation may be of physiological relevance in AEC. First, these cells are essential in maintaining the integrity of the air-blood barrier and are submitted continuously to mechanical forces, especially during acute respiratory failure, where mechanical ventilation imposes a significant stress to the alveoli. A recent report has shown MAPK activation in AEC of rats submitted to a high stretch mechanical ventilation strategy (27), and these responses are attenuated by a low (protective) stretch strategy. Second, a growing body of evidence implicates mechanical forces regulating distinct cellular functions, and MAPK pathways appear to play a role in many of these responses. The identification of G proteins and EGFR as potential mechanosensors transducing ERK1/2 activation in AEC exposed to stretch expands the body of knowledge of how mechanical stimuli are transduced in these cells. Nonreceptor protein kinase, phospholipase C, protein kinase C, and phosphatidylinositol 3 kinase are among the potential components of a mechanotransduction pathway that may lead to MAPK activation; moreover, cell-matrix interaction via integrins, proteoglycans, and focal contacts may transduce mechanical signals (16, 18, 23, 29).
In summary, the data presented here demonstrate that cyclic stretch of AEC induces a rapid ERK1/2 activation, which is transduced via G proteins and EGFR tyrosine kinase. Stretch-induced MAPK activation is independent of Na+ and Ca2+ influxes and the Grb2-SOS, Ras, Raf-1 pathway.
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ACKNOWLEDGEMENTS |
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We thank Drs. Emilia Lecuona, Laura Dada, and Karen Ridge for helpful discussions, Kevin Fennel for excellent technical assistance, and Mary Paniagua and Mehrnoosh Abshari from the flow cytometry facility of the Robert H. Lurie Cancer Center for the flow cytometry studies.
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FOOTNOTES |
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This work was supported by National Heart, Lung, Blood Institute Grant HL-48129, Cornelius Crane Asthma Center, Northwestern University Grant IBNAM0104, and Fundação de Amparo a Pesquisa do Estado de São Paolo Grant 99/03107-1.
Address for reprint requests and other correspondence: J. I. Sznajder, Pulmonary & Critical Care Medicine, Northwestern Univ., 300 E. Superior St., Tarry Bldg., 14-707, Chicago, IL 60611 (E-mail: j-sznajder{at}northwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00203.2001
Received 4 June 2001; accepted in final form 24 August 2001.
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