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Am J Physiol Lung Cell Mol Physiol 291: L38-L45, 2006. First published March 1, 2006; doi:10.1152/ajplung.00287.2004
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Stretch-induced phosphorylation of focal adhesion kinase in endothelial cells: role of mitochondrial oxidants

Mir H. Ali, Paul T. Mungai, and Paul T. Schumacker

Department of Medicine, Section of Pulmonary and Critical Care, The University of Chicago, Chicago, Illinois

Submitted 27 July 2004 ; accepted in final form 13 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2'7'-dichlorofluorescin (DCFH), increased 152 ± 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 µM) or rotenone (2 µM) attenuated this increase, whereas L-nitroarginine (100 µM), allopurinol (100 µM), or apocynin (30 µM) had no effect. The antioxidants ebselen (5 µM) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 µM) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 µM) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretch-induced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or Gö-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.

reactive oxygen species; superoxide; cytoskeleton; cyclic stretch; protein kinase C

ENDOTHELIAL DYSFUNCTION CONTRIBUTES to the pathogenesis in a wide range of diseases (9, 20). In the lung, mechanical strain and shear stress represent normal stimuli to endothelial cells. However, excessive strain encountered during mechanical ventilation with large tidal volumes exacerbates lung injury and inflammation in patients with acute respiratory failure (7). An improved understanding of the mechanisms by which mechanical strain affects endothelial cell function could help to clarify how excessive lung strain exacerbates preexisting lung injury.

Mechanical stretch has been shown to increase reactive oxygen species (ROS) production in endothelial cells, leading to the upregulation of cell adhesion molecules and chemokines (13, 51). Recent studies have implicated mitochondria as a source of ROS responsible for mediating flow-induced dilation in coronary arteries (32) and intercellular communication in vascular smooth muscle cells subjected to stretch (16). Endothelial cells lacking a functional electron transport chain lose the ability to increase oxidant signaling in response to cyclic stretch and fail to activate NF-{kappa}B, yet they retain the ability to respond to other stimuli such as lipopolysaccharide (1). In yeast, mitochondria anchor to actin filaments (5). Although details regarding mitochondria-actin interactions have not been reported for mammalian cells, it is conceivable that strain applied to integrins at the plasma membrane could be transmitted to the mitochondria through cytoskeletal proteins. A strain-induced perturbation of mitochondria could then trigger release of ROS to the cytosol, thereby activating downstream effector molecules involved in the mechanotransduction signaling pathway.

Endothelial cells respond to mechanical stimulation by altering gene expression, modifying cell shape, and by activating signal transduction systems (15, 17). A major target in the mechanotransduction cascade has been focal adhesion kinase (FAK), which becomes phosphorylated in response to mechanical strain (43). Studies reveal that oxidative stress triggers FAK phosphorylation. For example, in bovine pulmonary artery endothelial cells (BPAEC), Vepa et al. (48) found that hydrogen peroxide (H2O2) caused a rapid increase in FAK phosphorylation in the absence of mechanical stimulation. Similarly, human umbilical vein endothelial cells (HUVEC) exposed to exogenous oxidant stress exhibited rapid increases in FAK phosphorylation (4). Given that oxidant signals are generated during mechanical strain and that exogenous oxidants can trigger phosphorylation of FAK, we hypothesized that ROS arising from the mitochondrial electron transport chain could mediate the increase in FAK phosphorylation in strained endothelial cells.

Increases in FAK phosphorylation during strain may also involve protein kinase C (PKC). Several studies implicate PKC in the signaling pathway leading to FAK phosphorylation in various cell types (30, 38, 39, 43, 44), and ROS are capable of activating PKC (29). We hypothesized that mitochondrial ROS production induced by cyclic strain could lead to the activation of PKC, which might subsequently trigger FAK phosphorylation in endothelial cells.

The present study therefore sought to determine the source of ROS production in BPAEC subjected to mechanical stretch, the relationship between this oxidant signal and the phosphorylation of FAK, and the requirement for PKC in this process.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Reagents were obtained from Sigma Chemical except where otherwise noted. L-nitroarginine (L-NA) was purchased from Calbiochem-Novabiochem and 2'7'-dichlorofluorescin (DCFH) diacetate was obtained from Molecular Probes. The Flexercell 3000 strain unit and BioFlex culture plates were from Flexcell International.

Cell culture. Bovine pulmonary artery endothelial cells (BPAEC CPAE; ATCC CCL-209) were cultured in minimal essential media-{alpha} supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% heat-inactivated serum (GIBCO). The phenotype of the BPAEC was confirmed by morphological cobblestone appearance in culture in addition to positive staining for angiotensin converting enzyme (sc-12187; Santa Cruz) and negative staining for {alpha}-smooth muscle actin (Sigma). Cells were grown to 80–100% confluence on collagen I-coated plates for stretch experiments (Bioflex 3001C, Flexcell International). To standardize comparisons between treated cells, results were normalized for total protein content as determined by Bradford analysis. Cells were used for experiments at passages ranging from 2 to 6.

Stretch apparatus. Intermittent cyclic stretch was applied to BPAEC as previously described (35). In brief, confluent Bioflex plates were placed on a Flexercell 3000 base plate in the incubator, and membranes at the bottom of the plates were subjected to a vacuum of –20 kPa at a frequency of 15 cycles/min for up to 6 h. During each cycle, cells were stretched for 3 s and relaxed for 1 s. A vacuum of –20 kPa was found to produce membrane strain of ~25% when the loading posts were installed beneath the wells. Similarly confluent cells in identical plates were placed in the incubator without stretch to serve as time-matched controls. BPAEC were preincubated with experimental treatment media (containing inhibitors, antioxidants, cytochalasin D, nocodazole, etc.) 2 h before strain treatments. The doses of pharmacological inhibitors were chosen after dose-response experiments identified the lowest concentration necessary to inhibit fluorescence in unstrained cells; these doses coincided with a 10-fold increase from the dose reported to inhibit 50% of enzyme activity for the targeted enzyme systems [apocynin for NADPH oxidase, allopurinol for xanthine oxidase, N-nitro-L-arginine for endothelial nitric oxide (NO) synthase, rotenone for mitochondrial complex I, and antimycin for mitochondrial complex III].

Measurement of ROS. ROS signaling was assessed using DCFH diacetate. Oxidants produced in the cells cause oxidation of DCFH, yielding the fluorescent compound 2'7'-dichlorofluorescein (DCF). DCFH was added to the media at a concentration of 10 µM immediately before experimentation at time = 0. To assess the intracellular levels of DCF after 6 h of stretch, the media were removed, and the cells were immediately lysed and centrifuged to remove debris. DCF fluorescence was measured in the cell lysate (excitation = 488 nm, emission = 530 nm; Perkin-Elmer LS-5). Some increases in DCF fluorescence were detected in unstrained cells, presumably due to nonspecific oxidation of the DCFH dye. To correct for this effect, data were normalized to unstrained controls and expressed as a percent change from unstretched controls.

Western blotting for FAK. Treated BPAEC were rinsed twice with ice-cold PBS. One milliliter of ice-cold PBS was added to each well, and the adherent cells were scraped off. The cell suspension was centrifuged for 5 min at 1,200 rpm at 4°C. The BPAEC pellet was then resuspended in 300 µl of RIPA buffer (50 mM Tris·HCl, pH 7.4, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 mM NaVO4, and 1 mM NaF). This suspension was passed through a 27-gauge needle to promote cell lysis. After 15 min on ice, the suspension was spun at 14,000 rpm for 10 min at 4°C; any resulting pellet was discarded, and the supernatant was diluted with 4x reducing dye and heated at 95°C for 5 min. When cooled, samples were stored at –20°C.

Western blots for FAK and phospho-FAK utilized 50 µg of protein per well. These were loaded onto a 7.5% Tris·HCl polyacrylamide gel (Bio-Rad) and electrophoresed at 100 V for 1.5 h. After being transferred to ECL membrane (Amersham Pharmacia) via semidry membrane transfer for 40 min at 15 V, the membrane was incubated in 5% milk in Tris-buffered saline with Tween 20 (TBS-T) for 2 h for blocking. After blocking, the 1° antibody was added to the membrane in 5% milk in TBS-T and incubated overnight at 4°C. For total FAK, the antibody was diluted 1:1,000, and for phospho-FAK the antibody was diluted 1:500. After overnight incubation, the membrane was rinsed with 5% milk in TBS-T 3x for 15 min each. The 2° FITC-conjugated antibody was then added to the membrane in 5% milk in TBS-T and incubated at room temperature for 4–6 h. After 2° incubation, the membrane was washed 3x in TBS-T and autoradiograph developed using ECL Western Blotting Kit (RPN2209, Amersham Pharmacia). All extractions and Western blots were performed a minimum of three times to ensure consistency of results.

Cytoskeleton experiments. Two hours before mechanical stretch, BPAEC were preincubated in media containing cytochalasin D (0.5–10 µM). The lowest concentration that returned strain-induced DCF fluorescence to baseline levels was used for subsequent experiments (5.0 µM for BPAEC). Similar experiments were conducted using nocodazole (4.0–40 µM).

Statistical analysis. The quantitative data acquired from the DCF fluorescence experiments were subjected to a two-way analysis of variance. After confirming the absence of nonrandom tendencies in the data, we confirmed the normal distribution via graphical techniques (histogram of residuals, residuals vs. normality, and residuals vs. fit). A t-test then was conducted to detect statistical differences between control unstrained cells and control strained cells. Further pairwise t-tests were then conducted to identify sources of variance in strained and unstrained groups. The groups of interest were those in which the difference in strained and unstrained cells was abolished. Significance was defined as P < 0.05, and all computations were conducted via Minitab statistical software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ROS signaling during stretch. To assess the source(s) of oxidant production during cyclic stretch, confluent BPAEC were incubated with DCFH, which becomes fluorescent (DCF) when oxidized. After 1.5 h (Fig. 1, A and B), nonspecific increases in DCF fluorescence were noted even in unstretched controls. However, the extent of DCFH oxidation was significantly increased in cells subjected to 1.5 h of stretch (152 ± 16%) compared with unstretched controls (P < 0.05). This response was attenuated by the mitochondrial complex I inhibitor rotenone (2 µM) (64 ± 17%), but not by the NADPH oxidase inhibitor apocynin (30 µM) (166 ± 13%, Fig. 1B) or the xanthine oxidase inhibitor allopurinol (100 µM) (161 ± 11%, data not shown). Antimycin A inhibits mitochondrial electron transport and increases mitochondrial ROS generation by prolonging the lifetime of ubisemiquinone in complex III (21). As expected, antimycin A increased DCFH oxidation in unstretched cells, but it did not further amplify the increase in DCF oxidation elicited by stretch. Diphenylene iodonium (DPI) is a flavoprotein inhibitor that blocks electron transport in a broad range of systems including mitochondrial complex I, NAD(P)H oxidases, NO synthase, and other systems (31). DPI (5 µM) virtually abolished increases in DCF fluorescence in unstretched cells and significantly attenuated the stretch-associated increase in DCF fluorescence compared with controls (35 ± 16%, P < 0.05). These findings suggest that functional flavoproteins are required for the DCF fluorescence response to stretch.


Figure 1
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  		Fig. 1. Effects of mechanical strain (25%) on dichlorofluorescin (DCF) fluorescence in bovine pulmonary artery endothelial cells (BPAEC) over 6 h. A: effects of pharmacological inhibitors of mitochondrial electron transport on strain-induced DCF fluorescence (n = 8). DPI, diphenylene iodonium. B: effects of antioxidants on strain-induced DCF fluorescence (n = 7). NAC, N-acetylcysteine; DDC, dithiodidiethyldithiocarbamate; L-NA, L-nitroarginine. C: effects of protein kinase inhibitors on strain-induced DCF fluorescence in BPAEC (n = 5). *P < 0.05 comparison between strained and unstrained cells within the same intervention group. {dagger}P < 0.05 comparison between strained control cells and strained group with intervention.

 
To clarify the nature of the reactive molecule(s) produced during cyclic stretch, DCF fluorescence was measured in BPAEC in the presence of various intracellular antioxidant compounds (Fig. 1B). Stretch-induced DCF oxidation was significantly diminished in the presence of the glutathione peroxidase mimetic ebselen (50 µM) and by N-acetylcysteine (NAC, 1 mM), a substrate for glutathione synthesis (P < 0.05). Dithiodidiethyldithiocarbamate (DDC, 1 mM), an inhibitor of Cu/Zn superoxide dismutase, also attenuated the stretch-induced increases in DCF fluorescence.

To clarify the requirement for protein phosphorylation in the response to stretch, BPAEC subjected to stretch were administered the tyrosine kinase inhibitor genistein (100 nM). In those experiments, increases in DCF fluorescence were not attenuated (Fig. 1C). The role of PKC was studied using the PKC inhibitors Ro-31-8220 and Gö-6976. Ro-31-8220 (100 nM), a selective and competitive inhibitor of PKC (36), did not diminish the strain-induced increase in DCF fluorescence. Gö-6976 (100 nM), a selective inhibitor of Ca2+-dependent PKC-{alpha} (12), also did not attenuate the BPAEC response over 1.5 h. The activation of PKC by phorbol-12-myristate-13-acetate (PMA, 100 nM) did not change the stretch-induced DCF response in BPAEC over 1.5 h. Moreover, treatment of unstretched cells with PMA did not increase the DCF signal over 1.5 h. These findings indicate that neither increases nor decreases in PKC activity alter mitochondrial ROS production during stretch.

Cytoskeletal effects on stretch-induced ROS production. To explore the involvement of the cytoskeleton in the stretch-induced mitochondrial ROS signaling, BPAEC were treated with cytochalasin D to disrupt the actin cytoskeleton 2 h before strain. After 1.5 h of stretch, DCF fluorescence was measured (Fig. 2A). In the presence of cytochalasin D (5.0 µM), stretch-induced increases in DCF fluorescence were virtually abolished. However, these cells retained the ability to augment mitochondrial ROS generation in response to antimycin A, indicating that cytochalasin D did not inhibit mitochondria directly. To evaluate the role of the tubulin cytoskeleton in stretch-induced ROS signaling, confluent cells were treated with nocodazole before cyclic strain. In BPAEC monolayers the oxidation of DCFH in response to stretch or antimycin A was not significantly altered by nocodazole (40 µM), but the signal was attenuated by rotenone and DPI (Fig. 2B).


Figure 2
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  		Fig. 2. Effects of cytoskeletal disruption on strain-induced DCF fluorescence in BPAEC over 6 h. A: effects of cytochalasin D (Cyto D) on strain-induced DCF fluorescence (n = 6). B: effects of nocodazole (Noco) on strain-induced DCF fluorescence (n = 6). *P < 0.05 comparison between strained and unstrained cells within the same intervention group. {dagger}P < 0.05 comparison between strained control cells and strained group with intervention.

 
Phosphorylation of FAK by mitochondrial ROS. Oxidant stress has been shown to induce activation of FAK (for review, see Ref. 4). We sought to determine the signaling consequences of strain-induced mitochondrial ROS generation in terms of its regulation of FAK phosphorylation. Western blots were performed using protein extracts from BPAEC stretched for 15 min. Consistent with previous studies, cyclic stretch triggered an increase in FAK phosphorylation compared with unstretched control BPAEC (Fig. 3A). A similar increase was observed in unstretched cells treated with antimycin A to augment ROS generation. Strain-induced increases in FAK phosphorylation were attenuated by rotenone and by DPI. To further implicate ROS in this activation, BPAEC were treated with antioxidants and then subjected to 15 min of cyclic stretch. Antioxidants that had decreased DCFH oxidation in the cell (ebselen, NAC, and DDC) all inhibited strain-induced FAK phosphorylation (Fig. 3B and data not shown). By contrast, neither apocynin nor allopurinol attenuated the observed response to strain.


Figure 3
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  		Fig. 3. Phosphorylation of FAK during 15 min of mechanical strain in BPAEC. A: Western blotting demonstrates a strain-induced increase in focal adhesion kinase (FAK) phosphorylation at Tyr397 that is abolished in the presence of rotenone or DPI. A similar increase was observed when unstrained cells were treated with antimycin A for 15 min. B: effects of antioxidants on stain-induced phosphorylation of FAK (PFAK). The concentrations of inhibitors were as follows: antimycin (2 µM), rotenone (2 µM), DPI (5 µM), L-NA (100 µM), allopurinol (100 µM), apocynin (30 µM), ebselen (50 µM), and NAC (1 mM). NOS, nitric oxide synthase; XO, xanthine oxidase.

 
Stretch-induced phosphorylation of FAK and the role of PKC. BPAEC monolayers were treated with PMA (100 nM) for 2 h to determine the effects of PKC activation on FAK phosphorylation. That treatment caused an increase in FAK phosphorylation (Fig. 4A). The increase in PKC-mediated FAK phosphorylation was not inhibited by the antioxidant ebselen (50 µM) but was inhibited by a 2-h pretreatment with the PKC inhibitor Ro-31-8220 (100 nM). When cells were stretched for 15 min, BPAEC that had been pretreated with Ro-31-8220 or Gö-6976 (100 nM) did not exhibit the characteristic increase in FAK phosphorylation. This indicates that PKC-mediated changes are involved in the phosphorylation of FAK in response to cyclic stretch (Fig. 4B).


Figure 4
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  		Fig. 4. Effects of PKC on mechanical strain-induced FAK phosphorylation. A: effects of PMA (100 nM) on FAK phosphorylation in unstrained BPAEC. B: effects of PKC inhibition on FAK phosphorylation during 15 min of mechanical strain. The concentrations of inhibitors were as follows: antimycin (2 µM), ebselen (50 µM), Gö-6976 (100 nM), and Ro-31-8220 (100 nM).

 
Oxidant-mediated phosphorylation of FAK and the role of PKC. To test whether FAK phosphorylation is augmented by oxidants, BPAEC monolayers were treated with varying concentrations of H2O2 for 15 min. Western blotting demonstrated that concentrations of 25, 50, and 100 µM H2O2 were sufficient to increase FAK phosphorylation (Fig. 5A). This increase in phosphorylated FAK was not prevented by rotenone pretreatment but was abolished when BPAEC were pretreated with Gö-6976 (Fig. 5B). This suggests that the oxidant-mediated increase by H2O2 depends on the PKC signaling pathway, particularly a Ca2+-dependent PKC-{alpha}. To test whether the effects of mitochondrial oxidants were mediated by PKC, cells were treated with antimycin A (2 µM) for 2 h. The observed increase in FAK phosphorylation in response to antimycin A was prevented in the presence of Ro-31-8220 or Gö-6976. This indicates that PKC acts downstream of the mitochondrial ROS to increase FAK phosphorylation in response to cyclic stretch (Fig. 5C).


Figure 5
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  		Fig. 5. Effects of ROS on FAK phosphorylation over 15 min. A: effects of exogenous H2O2 on FAK phosphorylation in unstrained BPAEC. B: effects of ROS or PKC inhibition on peroxide-induced FAK phosphorylation. C: effects of PKC inhibition on antimycin A-induced FAK phosphorylation. The concentrations of inhibitors were as follows: antimycin (2 µM), rotenone (2 µM), Gö-6976 (100 nM), and Ro-31-8220 (100 nM).

 
Cytoskeletal effects on stretch-induced FAK phosphorylation. BPAEC were treated with cytochalasin D for 2 h before exposure to 15 min of stretch. Western blotting demonstrated that disruption of the actin cytoskeleton prevented a stretch-induced increase in FAK phosphorylation (Fig. 6). Cytochalasin D pretreatment also prevented antimycin A-induced FAK phosphorylation over the course of 2 h, suggesting that the actin cytoskeleton is required for FAK phosphorylation, regardless of the ROS stimulus.


Figure 6
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  		Fig. 6. Effects of cytoskeletal disruption via Cyto D (5 µM) on mechanical strain-induced and antimycin A-induced FAK phosphorylation.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
These experiments examined the role of mitochondrial ROS in triggering stretch-induced changes in FAK phosphorylation in pulmonary artery endothelial cells. After 1.5 h of cyclic stretch, DCF fluorescence increased in confluent BPAEC monolayers. These responses were blocked by inhibitors of mitochondrial complex I (rotenone) or flavoproteins (DPI), indicating that mitochondrial electron transport is required for the increases in oxidation of the dye induced by stretch. This response cannot be explained by a loss of mitochondrial ATP production, because antimycin A, which also blocks electron transport while simultaneously increasing ROS production by complex III (46), increased DCF fluorescence in unstretched cells. The opposite effects of the two mitochondrial inhibitors also implicate electron transport in the proximal but not the distal region of the electron transport chain in the response to stretch. The importance of electron transport appears to relate to the production of ROS at complex III. In that regard, antioxidants blocked FAK phosphorylation during stretch, while exogenous H2O2 induced it in unstretched cells, indicating that ROS are both necessary and sufficient to trigger that downstream response. Disrupting the tubulin cytoskeleton or inhibiting cellular kinases did not prevent the stretch-induced ROS generation, but disassembly of the actin cytoskeleton using cytochalasin D attenuated the response in a dose-dependent manner. This suggests that the stretch-induced increase in mitochondrial ROS production requires an intact actin cytoskeleton. Western blotting demonstrated that the changes in DCF fluorescence are paralleled by changes in FAK phosphorylation. Mitochondrial complex I inhibition prevented stretch-induced FAK phosphorylation at Tyr397. This phosphorylation was induced in unstretched cells by antimycin A, suggesting that mitochondrial ROS are sufficient to trigger changes in FAK phosphorylation. The ability to mimic this pattern of FAK phosphorylation by activating PKC and the ability to block stretch-induced FAK phosphorylation with pharmacological inhibitors of PKC indicate that PKC is involved in the oxidant signaling pathway upstream of FAK phosphorylation at Tyr397 (Fig. 7). This conclusion is supported by the finding that while exogenous H2O2 caused FAK phosphorylation, this effect was not observed in cells pretreated with Gö-6976 before H2O2 exposure.


Figure 7
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  		Fig. 7. Proposed pathway of mechanotransduction in BPAEC. Cyclic stretch increases mitochondrial ROS production via the actin cytoskeleton. The mitochondrial ROS act as signaling molecules, activating PKC. PKC then phosphorylates FAK at Tyr397. This phosphorylation acts as the beginning of a wide variety of endothelial responses to mechanical stimuli.

 
Endothelial cells release NO in response to shear stress (8), and NO can potentially contribute to the oxidation of DCFH. Therefore, the participation of reactive nitrogen species (RNS) in the response to stretch was evaluated by inhibiting NO synthesis with L-NA. In both stretched and unstretched cells, L-NA attenuated but did not abolish the DCF fluorescence signal compared with their respective controls (Fig. 1B). To clarify the relative roles of NO and H2O2 in this response, free hemoglobin (Hb) was added to the media in an attempt to scavenge NO. Incubation with Hb produced a modest attenuation of the DCF response to stretch (data not shown), suggesting that NO is a minor contributor to the strain-induced oxidant signal in these cells. These findings suggest that both ROS and RNS participate in the response to stretch but that ROS are the primary species responsible for the DCF fluorescence changes.

The primary tools employed in this study were pharmacological. Although the concentrations used were selected based on previous studies examining the dose-response relationships, there is always a possibility that pharmacological agents may exhibit nonspecific effects on cells. Future studies using genetic models will be necessary to provide a more selective confirmation of the results obtained using these tools.

FAK is a nonreceptor tyrosine kinase that is essential for cell motility and cell survival (40), and targeted disruption of the FAK gene results in embryonic lethality (26). FAK undergoes changes in phosphorylation at six residues in response to various stimuli. One of these, Tyr397, is critical to the biochemical functions of FAK due to its proximity to the catalytic domain (41). This domain phosphorylates downstream target proteins such as paxillin and p130cas and binds the Src homology 2 domains of phosphatidylinositol 3-kinase, phospholipase C{gamma}, and Grb7 (22, 37, 52). Through autophosphorylation of the other residues and the assembly of these signaling complexes, FAK mediates changes in Ras/MAP kinase signaling (27, 42), Rho signaling (23), cell motility (34), and apoptosis (19, 24, 25, 50). Exogenous ROS induce tyrosine phosphorylation of FAK in BPAEC, although no increase in in vitro autophosphorylation activity was detected in H2O2-treated endothelial cells (48). Studies by Chiarugi et al. (14) indicate that ROS are capable of increasing FAK phosphorylation by inhibiting protein tyrosine phosphatase activity. Cyclic stretch induces phosphorylation of Tyr397 and Tyr925 (6), and cyclic stretch induces ROS production in endothelial cells (1). These observations led us to hypothesize that oxidant signals induced by stretch may trigger tyrosine phosphorylation of FAK. Accordingly, we assessed Tyr397 phosphorylation as a single marker of activation in BPAEC subjected to cyclic stretch. However, it is likely that other ROS-dependent responses, such as phosphorylation of Tyr925, may also have occurred. Moreover, although phosphorylation of Tyr395 is associated with activation of FAK, we did not directly measure the activity of the kinase in this study.

PKC activity may bridge the gap between mitochondrial ROS and FAK phosphorylation. H2O2 is known to activate PKC (3, 28, 45, 49), and PKC has been shown in some studies to mediate tyrosine phosphorylation of FAK in many cell types (30, 39), although ROS-dependent tyrosine phosphorylation of FAK was found to be independent of PKC in other studies (48). Our study suggests that an oxidant-mediated activation of PKC is responsible for the phosphorylation of Tyr397 of FAK in response to mechanical stretch. However, given the markedly different conclusions regarding the role of PKC in ROS-dependent tyrosine phosphorylation of FAK among previous reports employing pharmacological inhibitors of PKC, it seems likely that future studies using genetic suppression of specific PKC isoforms are needed to fully clarify the role of PKC in the stretch- and ROS-dependent phosphorylation of FAK.

The role of mitochondrial ROS as signaling agents has become a focus of investigation recently (33). ROS generated from mitochondria have been shown to mediate hypoxia inducible factor-1-mediated transcriptional activation of vascular endothelial growth factor-1 and erythropoietin during hypoxia (10). ROS also serve as important signaling molecules that are required for cardiomyocyte preconditioning (47). In the endothelium, mitochondria-derived ROS produced during prolonged hypoxia mediate changes in IL-6 production as well as increases in the paracellular permeability of endothelial monolayers (2); these results indicate that ROS function as important secondary signaling molecules in a variety of cell types in response to environmental stimuli (11).

A role for mitochondrial ROS in mediating stretch-induced transcriptional responses in umbilical vein endothelial cells has been recently described (1). In combination with the present report, these studies support a model in which externally applied mechanical strain is transmitted to the mitochondria via the actin cytoskeleton. This transmission of force appears to stimulate either the increased generation or the increased release of superoxide or H2O2 to the cytosol, which then acts as a signaling molecule to activate diverse functional responses in the cell (3, 18). It is conceivable that this signaling pathway contributes to the increased inflammatory response and lung injury observed in patients ventilated with high tidal volumes. The potential clinical relevance of these findings therefore merits further investigation.


   GRANTS
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-35440, HL-32646, and HL-66315.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. T. Schumacker, Dept. of Pediatrics, Div. of Neonatology, 303 E. Chicago Ave., Ward Bldg. Rm. 12-191, Chicago, IL 60611 (e-mail: p-schumacker{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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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