Mechanical strain-induced proliferation and signaling in pulmonary epithelial H441 cells

Patricia R. Chess1, Liana Toia1, and Jacob N. Finkelstein1,2

Departments of 1 Pediatrics and 2 Environmental Medicine, University of Rochester, Rochester, New York 14642


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pulmonary epithelial cells are exposed to mechanical strain during physiological breathing and mechanical ventilation. Strain regulates pulmonary growth and development and is implicated in volutrauma-induced fibrosis. The mechanisms of strain-induced effects are not well understood. It was hypothesized that mechanical strain induces proliferation of pulmonary epithelial cells and that this is mediated by signals initiated within seconds of strain. To test this hypothesis, human pulmonary adenocarcinoma H441 cells were strained in vitro. Cyclic as well as tonic strain resulted in increased cellular proliferation. Western blot analysis of strained cells demonstrated three newly phosphorylated tyrosine residues within 30 s of strain. Phosphorylation of mitogen-activated protein kinases p42/44 increased, electrophoretic mobility shift assay demonstrated activation of transcription factor activating protein-1, and immunohistochemistry demonstrated increased phosphorylation of c-jun in response to strain. The tyrosine kinase inhibitor genistein blocked the strain-induced proliferation. We conclude that strain induces proliferation in pulmonary epithelial cells and that tyrosine kinase activity is necessary to signal the proliferative response to mechanical strain.

lung; tyrosine kinase; mitogen-activating protein kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MECHANICAL STRAIN is an important regulator of normal and abnormal pulmonary growth and development. Pulmonary hypoplasia is a well-recognized phenomenon after insufficient breathing movements in utero (33). In addition, respiratory failure resulting in mechanical ventilation is a common clinical scenario that can lead to lung injury and fibrosis (40). When this intervention is required in the neonatal period, developing lungs that are still forming alveoli are at risk for altered growth and development as well as fibrosis (1, 10). Chest wall restriction in ventilated rabbits minimizes increased capillary permeability previously seen in response to ventilation at high pressures, providing evidence that lung injury after ventilation may be more related to volume-induced mechanical strain rather than barotrauma (15). Strain also has been shown to induce growth, calcium mobilization, surfactant secretion, and increased prostacyclin production in the lung (27, 37, 44). Knowledge of the cell signaling responses to strain that result in altered growth and development in the lung may aid in the development of therapeutic interventions to minimize the negative effects of volutrauma and allow for optimal utilization of the enhanced growth response to strain in pulmonary hypoplasia.

To better understand the mechanisms leading to mechanical strain responses in the lung, cell signaling pathways were studied. It is difficult to differentiate cell signaling responses of various cell types when simultaneously exposed to strain. Previous work studying strain effects in the lung used mixed cell types to allow for cell-cell communication (27, 45). The studies presented here focus on pulmonary epithelial cell strain effects. Although the Weibel folding schema of alveolar expansion suggests some protection from physiological strain, excessive strain forces may still result in epithelial cell injury (11). Pulmonary epithelia are important because pulmonary epithelial type I cells, responsible for gas exchange, cover more than 90% of alveolar surface area. Pulmonary epithelial type II cells produce surfactant and proliferate to repopulate damaged type I cells (8). Fresh isolates of type II cells generally result in a low level of contaminating nonrelevant cells and are difficult to maintain their phenotype in culture, making signaling experiments difficult to interpret. The human pulmonary epithelial cell line A549 was initially utilized for these experiments, but they had a high rate of basal proliferation, making it difficult to determine differences in proliferation rate between static and strained conditions. The human pulmonary adenocarcinoma cell line H441 has a longer doubling time. H441 cells also have type II and Clara cell characteristics and produce surfactant protein (SP) A and SP-B (32). Their signaling and proliferative responses to mitogenic growth factors are similar to isolated type II cells (7). H441 cells were therefore chosen to evaluate the effects of strain on proliferation and signaling on the pulmonary epithelium.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cultures. H441 cells were obtained from American Type Culture Collection (Manassas, VA). The doubling time of H441 cells was found to be ~48 h in 10% fetal bovine serum (FBS) and 72 h in 2% FBS, with a population doubling level of five to seven. Cells were trypsinized and plated at 1 × 106 cells/well to achieve 70% confluence 24 h after plating, at the beginning of strain exposure. Cells used in phosphotyrosine Western blotting experiments were plated at 2 × 106 cells/well (70% confluence) because these experiments were performed on cells plated for only 1 h to minimize constitutive phosphorylation. The variability in absolute cell numbers at the start of experiments ranged from 1.4 × 106 to 2.1 × 106 cells/well. This variability was believed to be secondary to variable plating efficiency between experiments. Inasmuch as the absolute cell number, confirmed by cell counting before strain application, was more consistent within an experiment, data are represented as percent of control for each experiment rather than total cell number. Unless otherwise stated in specific methods, cells were plated in RPMI 1640 medium (Sigma; St. Louis, MO) with 10% FBS on collagen-coated six-well plates. Cells plated in the presence of serum had high levels of constitutive tyrosine phosphorylation, making it extremely difficult to analyze differences in phosphorylation state between control and strain. If cells were plated in serum and then serum starved, again the basal phosphorylation state was high, presumably because of factors being released into the medium by the cells being plated. It was only when the cells were plated in serum-free medium that constitutive phosphorylation could be minimized, allowing analysis of differential strain response. These cells did not survive for the time period of the proliferation experiments in serum-free medium so that although the different serum conditions are not optimal, they were unavoidable to answer the questions being addressed by the different sets of experiments. The tyrosine kinase inhibitor genistein (10 µM; Calbiochem; San Diego, CA) was added to medium just before initiation of strain for inhibition experiments.

Strain application. Radial and circumferential strain was applied using a computer-driven Flexercell strain unit (Flexcell; McKeesport, PA). Cultured cells plated on flexible bottom, type I collagen-coated, silicone elastomer culture plates were strained at 14 kPa (~20% elongation at the periphery; Fig. 1) at 60 cycles/min to mimic ventilation conditions frequently seen in critically ill neonates or under constant tonic strain at 14 kPa to mimic positive end-expiratory pressure for the specified time. Control cells were also plated on elastomer plates to avoid variations based on tissue culture plates. The strain effect of this device is graduated across the membrane, with maximal strain at the periphery (12). In contrast to primary pulmonary epithelial type II cells, which lifted off the plate in response to the strain applied under these experimental conditions, the H441 cells did not detach.


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Fig. 1.   Bull's-eye grid used to randomly select 9 areas at predetermined distances from the center of the well. P, periphery; M, midway; C, center.

Cell counting. Cells were counted while plated using a gridded eyepiece. A bull's-eye grid was used to randomly select nine specific areas equidistant from the center of the well to allow analysis of graduated strain response (Fig. 1). Total number of cells and immunoreactive cells within each quadrant pictured were counted. For cell number, results are presented as percent control, with control assigned a relative value of 100. For immunohistochemistry (IHC), to take into account the greater number of cells in the wells that had been exposed to strain, results are presented as percent immunoreactive in strained wells divided by percent immunoreactive in unstrained cells, with control assigned a relative value of 100. Unless otherwise specified, results presented represent average cell counts of the four peripheral quadrants, averaged for three experiments. Of note, in addition to proliferation, increase in cell number could also represent variability in rate of apoptosis.

Thymidine incorporation studies. After 24 h in culture in RPMI 1640 medium (Sigma) with 10% FBS, incubated at standard conditions of 37°C, 5% CO2, H441 cells were changed to RPMI 1640 medium with 2% FBS for 24 h and then strained at 14 kPa, 60 cycles/min with 5 µCi/ml tritiated thymidine for 24-72 h. Tritiated thymidine incorporation into DNA was determined as described previously (6). Assays for lactate dehydrogenase (LDH) release (Sigma) were performed to determine cell viability.

5-Bromo-2'-deoxyuridine incorporation. Cells were plated in RPMI 1640 medium with 2% FBS. Twenty-four hours after plating, cells were placed in serum-free RPMI 1640 medium containing 1:100 diluted 5-bromo-2'-deoxyuridine (BrdU) labeling reagent (Zymed Laboratories; San Francisco, CA) and exposed to 20% elongation, cyclic or tonic strain, for 2 h. Cells were washed with PBS, fixed with 10% buffered neutral Formalin, and blocked in 3% H2O2 in methanol (36). Incorporation of BrdU into proliferating cells was detected using a biotinylated monoclonal anti-BrdU antibody and detected using diaminobenzidine tetrahydrochloride (DAB) as a chromogen (Zymed Laboratories).

Labeling index. Cells were plated as described in BrdU methods. Medium was then changed to RPMI 1640 medium with 2% FBS and 5 µCi/ml [3H]thymidine ([3H]TdR) and subjected to 60 cycles/min, 20% elongation for 24 h. The cells were then washed with PBS, fixed in 10% buffered neutral Formalin for 30 min, and then dehydrated in ethanol with serial washes from 30 to 100% as previously described (36). The wells were placed in photographic emulsion NTB-2 (Kodak; Rochester, NY) at 4°C in a dark box and air-dried for 2 h. After 5 days the wells were treated sequentially in D-19 developer for 2.5 min, distilled deionized water for 30 s, and fixative for 4 min and then stained with hematoxylin and eosin and dehydrated in increasing concentrations (30-100%) of ethanol. Experimental conditions were performed in triplicate.

Proliferating cell nuclear antigen. Cells were plated as described in BrdU methods. Cells were changed to RPMI 1640 medium with 2% FBS and strained at 20% elongation, 60 cycles/min, for 24 h. Cells were fixed in 10% buffered neutral Formalin, washed with Tris-buffered saline (TBS) for 20 min, and then washed with PBS for 10 min. After blocking for 20 min with 5% horse serum in TBS, cells were incubated with primary antibody PC-10 (Novocastra, UK) at a 1:100 dilution containing 2% horse serum in TBS overnight at 4°C. The cells were then washed with TBS for 10 min and incubated with secondary antibody, biotinylated horse anti-mouse IgG (Vector Laboratories; Burlingame, CA), at a 1:400 dilution in TBS containing 2% horse serum for 2 h. Cells were rinsed with TBS for 10 min and incubated in ABC Vectastain reagent (Vector Laboratories) and then DAB for 5 min. Cells were washed and dehydrated as previously described in labeling index methods.

Western blot analysis. Cells were plated for 1 h in serum-free RPMI 1640 medium for Western blotting utilizing anti-phosphotyrosine experiments to minimize tyrosine phosphorylation stimulated by growth factors present in serum or conditioned medium. Samples were exposed to mechanical strain for the specified times. Degree of elongation was adjusted utilizing the Flexcell system to manipulate vacuum pressure applied to the well. For mitogen-activated protein (MAP) kinase experiments, H441 cells were plated in RPMI 1640 medium, 2% FBS for 24 h. To minimize MAP kinase activation before strain, cells were then changed to serum-free RPMI 1640 medium for 2 h and exposed to 20% elongation, 60 cycles/min for the specified times. Samples were collected, and 200 µg of protein, measured by bicinchoninic acid quantification (Pierce; Rockford, IL), were loaded per lane, separated by 10% polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Equal transfer was confirmed with Ponceau S staining (Sigma), and Western blotting was performed as described previously (6) utilizing a mouse monoclonal anti-phosphotyrosine primary (Upstate Biotechnology; Lake Placid, NY) and goat anti-mouse IgG secondary antibodies (Cappel; Durham, NC), polyclonal rabbit p42/44 MAP kinase or polyclonal rabbit phospho-specific p42/44 MAP kinase primary (New England Biolabs; Beverly, MA), and secondary goat anti-rabbit antibodies (New England Biolabs) or monoclonal mouse anti-focal adhesion kinase (FAK) primary (Upstate Biotechnology) and goat anti-mouse IgG secondary antibodies (Cappel) (23). Immunoprecipitation of FAK with anti-FAK antibody was performed before Western blot analysis to detect FAK as described previously (7). Western blotting without primary antibody was performed to identify nonspecific binding of secondary antibody.

IHC. Cells were plated as described in BrdU methods. Cells were changed to serum-free RPMI 1640 medium for 2 h and exposed to the specified strain. Cells were fixed as previously described in BrdU methods. For MAP kinase, IHC cells were serially incubated in the primary antibody rabbit polyclonal p42/44 MAP kinase IgG, secondary antibody biotinylated goat anti-rabbit IgG, 0.6% H2O2, and ABC Vectastain (Vector Laboratories) and detected using DAB as a chromogen (Zymed Laboratories). For c-jun analysis, similar conditions were utilized with a rabbit polyclonal antibody against the phosphorylated serine-63 residue on c-jun as the primary antibody (New England Biolabs). Cells were also processed in the absence of primary antibody to verify the lack of nonspecific binding.

Electrophoresis mobility shift assay. Cells were cultured as described in BrdU methods. Nuclear extracts were isolated by suspending the cells in lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, and 0.1% Nonidet P-40, pH 7.9) for 10 min on ice. The lysate was microcentrifuged for 5 min at 12,000 g at 4°C, the nuclear pellet was washed with lysis buffer without Nonidet P-40 and resuspended in 20 µl of protein extraction buffer (420 mM NaCl, 20 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol, pH 7.9) for 10 min at 4°C. The sample was microcentrifuged for 5 min, and the supernatant was centrifuged for 5 min at 12,000 g at 4°C. Thirty microliters of dilution buffer (50 mM KCl, 20 mM HEPES, 0.2 mM EDTA, and 20% glycerol, pH 7.9) were added to the nuclear suspension. Dithiothreitol (DTT, 0.5 mM), phenylmethylsulfonyl fluoride (PMSF, 0.5 mM), and leupeptin (10 µg/ml) were added to the lysis and extraction buffers just before use. The diluting buffer contained the same concentration of DTT and leupeptin but only 0.2 mM PMSF. Five micrograms of nuclear extract were incubated with 4 µl of binding buffer [20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris · HCl, pH 7.5, 0.25 mg/ml poly(dI-dC) · poly(dI-dC)], and unlabeled competitor oligonucleotide as indicated. Binding reactions were initiated by the addition of 1 µl of 32P-labeled (1 × 105 counts/min) activating protein (AP)-1 oligonucleotide (21-base pair consensus sequence, which forms DNA-binding dimers with members of the AP-1 family through leucine zipper formation; Promega; Madison, WI) in a final volume of 20 µl. Complexes were resolved on a 4% polyacrylamide gel and subjected to autoradiography.

Data analysis. Student's t-test was used for discrete variables determining control vs. one degree of strain at one time point, and ANOVA with Scheffé's post hoc analysis was used for comparison of more than two groups. P < 0.05 was considered statistically significant. Values are shown as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A series of proliferation assays were performed to determine the proliferative effects of strain on pulmonary epithelial cells. Cells exposed to 20% elongation, 60 cycles/min, for 24 h resulted in a statistically significant increase in cell number, 178% of control, along the area of maximal strain (20%), the periphery, with less increase in the center and midway along the membrane where radial strain is decreased according to the Lagrangian equation: strain = change in length/original length (Figs. 1 and 2A). Those cells along the extreme periphery of the membrane where the flexible membrane attaches to the plate exhibited reorientation, with alignment of the cells along the strain force, whereas cells located over the rest of the membrane did not exhibit reorientation (see Figs. 6B and 7B). [3H]TdR incorporation was increased 120% (Fig. 2B). This more modest increase in proliferation compared with cell number was considered to be related to the differential strain distribution across the membrane, with [3H]TdR incorporation measuring change over the whole membrane, including areas exposed to lesser degrees of strain. To confirm the proliferative effect of strain, labeling index, proliferating cell nuclear antigen (PCNA) staining, and BrdU incorporation were utilized because these could be studied at the area of maximal strain, the periphery. These assays demonstrated an approximately twofold increase in proliferation in response to strain along the area of maximal strain (the periphery; Fig. 2, B and C). The center of the wells, which experience less strain as discussed previously, showed no statistically significant increase in labeling index, PCNA staining, or BrdU incorporation. In addition, 2 h of tonic strain resulted in increased BrdU incorporation at the periphery, suggesting that a single stretch can initiate the proliferative strain response (Fig. 2C). The tyrosine kinase inhibitor genistein blocked the proliferative response of strain as measured by cell number and BrdU incorporation (Fig. 2, A and C). LDH assays and trypan blue dye exclusion confirmed cell viability at each of the conditions tested, with a sensitivity of 1 × 105 cells for the LDH assay.


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Fig. 2.   H441 cells exposed to 20% elongation for 60 cycles/min for 24 h exhibit increased proliferation as measured by cell number (A), proliferating cell nuclear antigen (PCNA), [3H]thymidine ([3H]TdR) incorporation, and labeling index (B), and after 2 h of cyclic or tonic strain as measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation (C) over the area of maximal strain (periphery) compared with unstrained controls. Values are means ± SE; n = 3. * P < 0.05 compared with control. Dagger  P < 0.05 compared with cells strained in the absence of genistein. Periph/gen, cells analyzed at the periphery in the presence of genistein; cyclic+gen, cells analyzed with and without cyclic strain in the presence of genistein.

To develop an experimental system with which to study strain-induced signaling through tyrosine kinase pathways, a series of experiments was performed to determine maximal plating efficiency with minimal constitutive tyrosine phosphorylation. Whereas cells plated in 0-10% serum showed increasing tyrosine phosphorylation in response to strain, cells plated in serum-free medium had the lowest basal phosphorylation, resulting in the largest percent increase in phosphorylation in strained cells (data not shown). H441 cells plated for 1 h in serum-free RPMI 1640 medium exposed to 20% elongation at 60 cycles/min demonstrated an increase in tyrosine phosphorylation of a protein with molecular mass approximating 125 kDa within 30 s of strain application. A strain of 1 or 10% elongation did not result in tyrosine phosphorylation of this protein by 1 min (Fig. 3A). In addition, two other proteins, with molecular masses of ~97 and 14 kDa, appeared to be altered in response to strain; however, this was not as consistent as the 125-kDa protein. This variation was difficult to understand because bicinchoninic acid analysis was used to assure equal loading of samples, and Ponceau S staining was used to document equal transfer of protein to the membrane (data not shown). The tyrosine kinase inhibitor genistein at concentrations previously shown to inhibit growth factor-induced proliferation and tyrosine kinase receptor phosphorylation in isolated rabbit type II cells (6) inhibited, but did not completely block, the 125-kDa protein strain-induced tyrosine phosphorylation (Fig. 4). Because the high molecular mass protein was hypothesized to be p125FAK, an intracellular signaling molecule phosphorylated in response to activation by beta 1-integrin (14), immunoprecipitation of p125FAK after strain was performed, and tyrosine phosphorylation of the protein was assessed. p125FAK was minimally phosphorylated under control conditions, and the degree of phosphorylation did not increase significantly in response to strain (Fig. 5). This suggests that a pathway independent of p125FAK is involved in strain-induced proliferation.


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Fig. 3.   A: H441 cells exposed to 20% elongation for 60 cycles/min demonstrated an increase in tyrosine-phosphorylated proteins with molecular masses approximating 125, 97, and 14 kDa within 30 s of strain. H441 cells were plated for 1 h and then exposed to cyclic strain of 1, 10, and 20% elongation. Cells were collected in Laemmli sample buffer and subjected to SDS-PAGE using a 10% gel followed by Western blot analysis with an anti-phosphotyrosine antibody. Cells exposed to 1 and 10% elongation showed no increase in tyrosine phosphorylation of these proteins (n = 3). B: densitometric analysis of Western blots. * P < 0.05 compared with unstrained control cells.



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Fig. 4.   A: tyrosine kinase inhibitor genistein inhibits strain-induced phosphorylation of the 125-kDa protein (Sp125). H441 cells were cultured in serum-free RPMI 1640 medium for 1 h. Cells were then exposed to 20% elongation, 60 cycles/min for 5 min in the presence and absence of 10 µM genistein. C, unstrained control; S, strained for 5 min; C/G, unstrained control with genistein; S/G, strained for 5 min in the presence of genistein. Cells were collected in Laemmli sample buffer and subjected to SDS-PAGE using a 12% gel followed by Western blot analysis with an anti-phosphotyrosine antibody (n = 3). B: densitometric analysis of Western blots. * P < 0.05 compared with unstrained control cells.



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Fig. 5.   The tyrosine phosphorylation state of focal adhesion kinase (FAK) is not altered in response to strain in H441 cells. H441 cells were cultured in serum-free RPMI 1640 medium for 1 h. Cells were then exposed to 20% elongation, 60 cycles/min for 0-60 min (m). FAK was immunoprecipitated utilizing an anti-FAK antibody. Samples were separated on a 12% polyacrylamide gel, transferred to nitrocellulose, probed for FAK (T-FAK, total FAK), and then stripped and reprobed for tyrosine phosphorylation utilizing an anti-phosphotyrosine antibody (P-FAK, tyrosine-phosphorylated FAK). FAK-C, phosphorylated FAK-positive control (n = 3).

MAP kinases p42 and p44 are cytosolic secondary signaling molecules activated by phosphorylation in response to a variety of proliferation-inducing stimuli (46). To determine whether p42/44 MAP kinase is involved in mechanical strain-induced signaling in pulmonary epithelium, p42/44 MAP kinase phosphorylation was assessed by Western blot analysis. Figure 6A demonstrates a low basal phosphorylation of p42/44 MAP kinase, which increases by 5 min and peaks by 10 min, whereas total p44 protein decreases by 10 min of strain. IHC confirmed increased phosphorylation of p42/44 MAP kinase within 10 min of the strain stimulus along the area of maximal strain (the periphery; Fig. 6, C and D). No statistically significant increase in MAP kinase phosphorylation was seen in the central area of the well where strain is not as great, suggesting a direct effect of strain on MAP kinase activation (data not shown). There is also a suggestion of upregulation of total MAP kinase stimulated by 30 min of strain, but the difference is not statistically significant (Fig. 6A). The processing of samples for IHC resulted in a small number of some cells being washed off the plate, with cytospin documenting that these cells had similar straining to the cells that remained plated (data not shown). The presence of immunoreactive cells under the control conditions was not unexpected because these cells are proliferating even under control conditions.


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Fig. 6.   H441 cells exposed to 20% elongation for 60 cycles/min demonstrated increased p42/44 mitogen-activated protein (MAP) kinase phosphorylation by Western blot analysis utilizing a 10% polyacrylamide gel (A; densitometry of Western blots in B) by as well as immunohistochemical staining (C) by 5-10 min, returning to baseline by 24 h. A: I, membrane probed for phosphorylated p42/44 MAP kinases. II, membrane stripped and reprobed for total p42/44 MAP kinases. MAP kinase 42, 40 ng of bacterially expressed, fully phosphorylated 42-kDa MAP kinase. C: immunohistochemistry (IHC) at periphery of well. H441 cells were unstrained (I) and strained (II) for 10 min. Arrows show positive staining (original magnification ×10, insets ×20; n = 3). D: quantification of immunohistochemistry. Values are no. of positively stained cells in strained sample expressed as percent of unstrained control sample. * P < 0.05 compared with unstrained control sample.

AP-1, a transcription factor that signals the mitogenic response to a variety of proliferative stimuli, translocates from the cytoplasm to the nucleus after activation, binding to promoter regions of regulatory genes (19). Nuclear extract of strained cells demonstrated a shift of AP-1 complex on polyacrylamide gel (Fig. 7A), and IHC demonstrated increased phosphorylation of the serine 63 residue of the c-jun subunit of AP-1 (Fig. 7, B and C). Supershifts were performed with c-fos rabbit polyclonal IgG antibody (Santa Cruz) but did not result in further shifting. The lack of supershifting could be explained by activation of a different AP-1 moiety than the antibody used to supershift identified so the IHC was performed to confirm the AP-1 activation.


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Fig. 7.   H441 cells exposed to 20% elongation for 60 cycles/min demonstrated a 2-fold increase in activating protein (AP)-1 translocation into the nucleus at 1 h by electrophoresis mobility shift assay (A) as well as increased phosphorylation of the serine-63 residue on c-jun by immunocytochemistry at the periphery (B and C). HELA, AP-1 control; shift, nuclear extract incubated with 32P-labeled AP-1 oligonucleotide; cold, incubated with 32P-labeled and excess nonlabeled AP-1 oligonucleotides; NS, incubated with nonspecific competitor (AP-2). B: IHC at periphery of well. H441 cells were unstrained (I) and strained (II) for 60 min. Arrows, positive staining (original magnification ×10, inset ×20; n = 3). C: quantification of immunohistochemistry. * P < 0.05 compared with unstrained control cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanical strain has been found to induce many responses in a variety of different systems. It has been found to regulate a variety of vasoactive molecules such as tissue plasminogen activator, nitric oxide, and prostacyclin in endothelial cells (16, 31). It induces growth in vascular endothelium, cardiac muscle, bone, skeletal muscle, and aortic endothelium (41). Strain also regulates extracellular matrix molecule formation and morphological development (22, 38). Postpneumonectomy lung growth is well described in animal models as well as in postoperative pneumonectomy patients (4, 35). It also has been shown that strain induces proliferation in fetal rat lung cells in three-dimensional strain (28) and in the human diploid lung fibroblast cell line IMR-90 using a similar two-dimensional strain model (3). Work by Liu et al. (28) failed to demonstrate increased proliferation of isolated fetal rat lung pulmonary epithelial cells in a two-dimensional strain model, which may be related to different plating properties of cell lines vs. primary cell isolates or the presence of nonepithelial cell types in a primary cell isolate. Subsequent studies by Xu and co-workers (45) demonstrated an increase in fetal pulmonary epithelial cell number in response to strain.

Although it is difficult to identify cellular and molecular responses to mechanical strain in a complex in vivo system, an in vitro model provides the opportunity to focus on molecular mechanics of strain-related effects in individual cell types. The effect of strain on pulmonary epithelial cells and the cell signaling mechanisms involved in strain-induced growth are poorly understood. The current work demonstrates the ability of pulmonary epithelial cells to proliferate in response to mechanical strain. The findings that this proliferation occurred along the area of maximal strain but not along the central area, which is exposed to less strain in this model, suggests that the mechanism of strain-induced proliferation is a direct effect and does not exclusively involve soluble molecules that may have been released by the strained cells because all the cells in a well were incubated in the same medium.

To minimize adverse mechanical strain responses, including disregulated proliferation, and maximize beneficial reparative effects, the mechanisms leading to proliferation need to be identified. This work supports the hypothesis that strain induces proliferation in pulmonary epithelial cells. The hypotheses that strain-induced signaling occurs within seconds to minutes of initiating strain and that tonic strain also induces proliferation were supported.

A number of cellular signaling cascades have been identified as having important roles in cellular proliferation, a phenomenon observed after strain in other cell types, as previously discussed. These are initiated by a variety of mechanisms including alterations in Ca2+ influx, phosphorylation of tyrosine kinase receptors, activation of adenylate cyclase, guanylate cyclase, phospholipases A2 and C, and stimulation of a variety of other non-tyrosine kinase receptors that result in a complex intracellular cascade of events (13, 17, 18, 43). Which, if any, of these is involved in strain-induced proliferation in the lung is not well known. The current data implicate phosphorylation of three tyrosine residues with proteins of ~125, 97, and 14 kDa in the cellular response to strain. The high molecular mass phosphorylated tyrosine protein was hypothesized to be p125FAK, an intracellular signaling molecule activated by beta 1-integrin-dependent cell adhesion (25). Integrins are transmembrane glycoproteins known to transduce extracellular mechanical signals into the cell. It has been shown that human osteosarcoma cells exposed to mechanical strain exhibit altered expression of beta 1-integrin within 24 h of strain application (5). Work by Wang et al. (42) and Nebe et al. (30) has shown that beta 1-integrin transduces mechanical strain signals in endothelial cells and a hepatocyte cell line, respectively. Immunoprecipitation and Western blotting did not support the hypothesis that the high molecular mass phosphotyrosine was p125FAK.

AP-1 is likely to be a signaling molecule in pulmonary epithelial cell response to strain for a number of reasons. AP-1 is important in early lung development, injury, and apoptosis (29). DNA binding activity of AP-1 is increased in response to strain in endothelial cells (24). The expression of c-fos, one member of the AP-1 complex, increases transiently in the lung at birth when ex utero breathing is initiated (20) and is also expressed during postpneumonectomy lung growth (39). Binding sites for AP-1 have been identified in the promoters for transforming growth factor (21) and vascular endothelial growth factor (9), both of which are expressed during lung growth and development. The current work supports the involvement of AP-1 as a secondary signaling molecule activated in response to mechanical strain. Which genes are regulated by this activation are not yet known. Blocking antisense oligonucleotides for platelet-derived growth factor (PDGF) have been found to inhibit strain-induced proliferation in fetal rat lung cells, implicating PDGF in mechanical strain-induced growth (26). Mechanical strain has been found to increase fibronectin synthesis in bladder smooth muscle cells (2). Fetal rabbit type II cells have been shown to exhibit an increase in phosphatidylcholine synthesis in response to strain (34). Northern blot analysis of RNA extracted from cells after 24-72 h of cyclic mechanical strain showed no difference compared with unstrained cells in message abundance of PDGF, fibronectin, SP-A, or SP-B with or without dexamethasone pretreatment to induce SP-B message (unpublished observations).

To determine whether phosphorylation of the three proteins tyrosine phosphorylated in response to strain is necessary or sufficient to initiate the signaling cascade leading to proliferation in mechanically strained pulmonary epithelial cells, a tyrosine kinase inhibitor was used. Genistein, which preferentially inhibits ATP binding to a tyrosine kinase rather than to serine or threonine kinases, was used to determine whether phosphorylation of the observed proteins is critical to the strain-induced proliferative response. Blocking phosphorylation of tyrosine proteins inhibits the proliferative response to strain.

In summary, cyclic as well as tonic mechanical strain induces proliferation of pulmonary epithelial cells. In addition, mechanical strain-induced signaling is initiated within seconds of the strain response and utilizes tyrosine kinases, whereas activation of FAK via tyrosine phosphorylation does not appear to have a role in the strain response. AP-1 and p42/44 MAP kinases are also involved as secondary messengers of mechanical strain, although their role in the proliferative response requires further study. Tyrosine phosphorylation has been shown to be necessary for strain-induced proliferation.


    ACKNOWLEDGEMENTS

We thank John McBride and Christine Miller for the help with the in vitro strain model study design, Richard Watkins for assistance with imaging, and Bruce Holm and Michael O'Reilly for a thoughtful review of the manuscript.


    FOOTNOTES

This work has been supported in part by an American Lung Association Grant and National Heart, Lung, and Blood Institute Grant HL-03910.

Address for reprint requests and other correspondence: P. R. Chess, Univ. of Rochester, Box 651, 601 Elmwood Ave, Rochester, NY 14642 (E-mail: patricia_chess{at}urmc.rochester.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. §1734 solely to indicate this fact.

Received 16 April 1999; accepted in final form 27 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adkins, WK, Hernandez LA, Coker PJ, Buchanan B, and Parker JC. Age effects susceptibility to pulmonary barotrauma in rabbits. Crit Care Med 19: 390-393, 1991[ISI][Medline].

2.   Baskin, L, Howard PS, and Macarak E. Effect of physical forces on bladder smooth muscle and urothelium. J Urol 150: 601-607, 1993[ISI][Medline].

3.   Bishop, JE, Mitchell JJ, Absher PM, Baldor L, Geller HA, Woodcock-Mitchell J, Hamblin MJ, Vacek P, and Low RB. Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity. Am J Physiol Lung Cell Mol Physiol 265: L126-L133, 1993.

4.   Cagle, PT, and Thurlbeck WM. Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 138: 1314-1326, 1988[ISI][Medline].

5.   Carvalho, RS, Scott JE, and Yen EH. The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cells. Arch Oral Biol 40: 257-264, 1995[ISI][Medline].

6.   Chess, PR, Ryan RM, and Finkelstein JN. Tyrosine kinase activity is necessary for growth factor-stimulated rabbit type II pneumocyte proliferation. Pediatr Res 36: 481-486, 1994[Abstract].

7.   Chess, PR, Ryan RM, and Finkelstein JN. H441 pulmonary epithelial cell mitogenic effects and signaling pathways in response to HGF and TGF a. Exp Lung Res 24: 27-39, 1998[ISI][Medline].

8.   Evans, MJ, Cabral LJ, Stephens RJ, and Freeman G. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp Mol Pathol 22: 142-150, 1975[ISI][Medline].

9.   Ferrara, N, Houck K, Jakeman L, and Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 13: 18-32, 1992[ISI][Medline].

10.   Gerstmann, DR, deLemos RA, Coalson JJ, Clark RH, Wiswell TE, Winter DC, Kuehl TJ, Meredith KS, and Null DM, Jr. Influence of ventilatory technique on pulmonary baroinjury in baboons with hyaline membrane disease. Pediatr Pulmonol 5: 82-91, 1988[ISI][Medline].

11.   Gil, J, Bachofen H, Gehr P, and Weibel ER. Alveolar volume-surface area relation in air- and saline-filled lungs fixed by vascular perfusion. J Appl Physiol 47: 990-1001, 1979[Abstract/Free Full Text].

12.   Gilbert, JA, Weinhold PS, Banes AJ, Link GW, and Jones GL. Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro. J Biomech 27: 1169-1177, 1994[ISI][Medline].

13.   Han, O, Sumpio BE, and Basson MD. Mechanical strain rapidly redistributes tyrosine phosphorylated proteins in human intestinal Caco-2 cells. Biochem Biophys Res Commun 250: 668-673, 1998[ISI][Medline].

14.   Hanks, SK, and Polte TR. Signaling through focal adhesion kinase. Bioessays 19: 137-145, 1997[ISI][Medline].

15.   Hernandez, LA, Peevy KJ, Moise AA, and Parker JC. Chest wall restriction limits high airway pressure-induced lung injury in young rabbits. J Appl Physiol 66: 2364-2368, 1989[Abstract/Free Full Text].

16.   Iba, T, and Sumpio BE. Tissue plasminogen activator expression in endothelial cells exposed to cyclic strain in vitro. Cell Transplant 1: 43-50, 1992[Medline].

17.   Ingram, AJ, Ly H, Thai K, Kang M, and Scholey JW. Activation of mesangial cell signaling cascades in response to mechanical strain. Kidney Int 55: 476-485, 1999[ISI][Medline].

18.   Johnson, GL, and Vaillancourt RR. Sequential protein kinase reactions controlling cell growth and differentiation. Curr Opin Cell Biol 6: 230-238, 1994[ISI][Medline].

19.   Karin, M, Liu Z, and Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 9: 240-246, 1997[ISI][Medline].

20.   Kasik, JW, Wan YJ, and Ozato K. A burst of c-fos gene expression in the mouse occurs at birth. Mol Cell Biol 7: 3349-3352, 1987[ISI][Medline].

21.   Kim, SJ, Angel P, Lafyatis R, Hattori K, Kim KY, Sporn MB, Karin M, and Roberts AB. Autoinduction of transforming growth factor beta 1 is mediated by the AP-1 complex. Mol Cell Biol 10: 1492-1497, 1990[ISI][Medline].

22.   Korver, TH, van de Stadt RJ, Kiljan E, van Kampen GP, and van der Korst JK. Effects of loading on the synthesis of proteoglycans in different layers of anatomically intact articular cartilage in vitro. J Rheumatol 19: 905-912, 1992[ISI][Medline].

23.   Kozma, LM, Rossomando AJ, and Weber MJ. Comparison of three methods for detecting tyrosine-phosphorylated proteins. Methods Enzymol 201: 28-43, 1991[ISI][Medline].

24.   Lan, Q, Mercurius KO, and Davies PF. Stimulation of transcription factors NF kappa B and AP1 in endothelial cells subjected to shear stress. Biochem Biophys Res Commun 201: 950-956, 1994[ISI][Medline].

25.   Lin, TH, Rosales C, Mondal K, Bolen JB, Haskill S, and Juliano RL. Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells. A possible signaling role for the Syk tyrosine kinase. J Biol Chem 270: 16189-16197, 1995[Abstract/Free Full Text].

26.   Liu, M, Liu J, Buch S, Tanswell AK, and Post M. Antisense oligonucleotides for PDGF-B and its receptor inhibit mechanical strain-induced fetal lung cell growth. Am J Physiol Lung Cell Mol Physiol 269: L178-L184, 1995[Abstract/Free Full Text].

27.   Liu, M, Skinner SJ, Xu J, Han RN, Tanswell AK, and Post M. Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol Lung Cell Mol Physiol 263: L376-L383, 1992[Abstract/Free Full Text].

28.   Liu, M, Xu J, Souza P, Tanswell B, Tanswell AK, and Post M. The effect of mechanical strain on fetal rat lung cell proliferation: comparison of two- and three-dimensional culture systems. In Vitro Cell Dev Biol Anim 31: 858-866, 1995[ISI][Medline].

29.   Molinar-Rode, R, Smeyne RJ, Curran T, and Morgan JI. Regulation of proto-oncogene expression in adult and developing lungs. Mol Cell Biol 13: 3213-3220, 1993[Abstract].

30.   Nebe, B, Rychly J, Knopp A, and Bohn W. Mechanical induction of beta 1-integrin-mediated calcium signaling in a hepatocyte cell line. Exp Cell Res 218: 479-484, 1995[ISI][Medline].

31.   Oluwole, BO, Du W, Mills I, and Sumpio BE. Gene regulation by mechanical forces. Endothelium 5: 85-93, 1997[Medline].

32.   Pryhuber, GS, Ma OR, Clark JC, Hull WM, Fink I, and Whitsett JA. Phorbol ester inhibits surfactant protein SP-A and SP-B expression. J Biol Chem 265: 20822-20828, 1990[Abstract/Free Full Text].

33.   Roberts, AB, and Mitchell J. Pulmonary hypoplasia and fetal breathing in preterm premature rupture of membranes. Early Hum Dev 41: 27-37, 1995[ISI][Medline].

34.   Scott, JE, Yang SY, Stanik E, and Anderson JE. Influence of strain on [3H]thymidine incorporation, surfactant-related phospholipid synthesis, and cAMP levels in fetal type II alveolar cells. Am J Respir Cell Mol Biol 8: 258-265, 1993[ISI][Medline].

35.   Sekhon, HS, and Thurlbeck WM. A comparative study of postpneumonectomy compensatory lung response in growing male and female rats. J Appl Physiol 73: 446-451, 1992[ISI][Medline].

36.   Sinkin, RA, Sanders RS, Horowitz S, Finkelstein JN, and Lomonaco MB. Cell-specific expression of fibronectin in adult and developing rabbit lung. Pediatr Res 37: 189-195, 1995[Abstract].

37.   Skinner, SJ, Somervell CE, and Olson DM. The effects of mechanical stretching on fetal rat lung cell prostacyclin production. Prostaglandins 43: 413-433, 1992[Medline].

38.   Sottiurai, VS, Kollros P, Glagov S, Zarins CK, and Mathews MB. Morphologic alteration of cultured arterial smooth muscle cells by cyclic stretching. J Surg Res 35: 490-497, 1983[ISI][Medline].

39.   Thet, LA, Allen PL, and Tang YL. Spatial-temporal patterns of c-fos proto-oncogene and type I collagen gene expression during compensatory lung growth (Abstract). Am Rev Respir Dis 143: A520, 1991.

40.   Tsuno, K, Prato P, and Kolobow T. Acute lung injury from mechanical ventilation at moderately high airway pressures. J Appl Physiol 69: 956-961, 1990[Abstract/Free Full Text].

41.   Vandenburgh, HH. Mechanical forces and their second messengers in stimulating cell growth in vitro. Am J Physiol Regulatory Integrative Comp Physiol 262: R350-R355, 1992[Abstract/Free Full Text].

42.   Wang, N, Butler JP, and Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260: 1124-1127, 1993[ISI][Medline].

43.   Wasilenko, WJ. Phosphoinositides and cell growth. Adv Exp Med Biol 321: 147-151, 1992[Medline].

44.   Wirtz, HR, and Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 250: 1266-1269, 1990[ISI][Medline].

45.   Xu, J, Liu MY, Tanswell AK, and Post M. Mesenchymal determination of mechanical strain-induced fetal lung cell proliferation. Am J Physiol Lung Cell Mol Physiol 275: L545-L550, 1998[Abstract/Free Full Text].

46.   Zhu, X, and Assoian RK. Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell 6: 273-282, 1995[Abstract]. (Corrigenda. Mol Biol Cell 7: June 1996, p. 1001.)


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