1 Division of Pulmonary Medicine, Feinberg School of Medicine, Northwestern University, Morton 4-616, 303 East Chicago Avenue, Chicago, IL 60611, USA
2 Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Morton 4-616, 303 East Chicago Avenue, Chicago, IL 60611, USA
3 Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, Department of Physiology and Biophysics, University of Iowa, 400 Eckstein Medical Research Building, Iowa City, IA 52242, USA
* Author for correspondence (e-mail: j-jones3{at}northwestern.edu)
Accepted 22 March 2005
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Summary |
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Key words: Matrix adhesion, Matrix receptors, Stretching
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Introduction |
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Although it has been established that cells sense external forces, in part, through mechanosensitive channels and growth-factor-receptor circuitry, there are considerable data that indicate that certain mechanical signals are transduced via matrix cell-surface receptors, including the integrins (Blount, 2003; Chen et al., 1999
; Hamill and Martinac, 2001
; Hynes, 1992
; Hynes, 2002
; Ingber, 2003
; Tschumperlin et al., 2004
; Wang et al., 1993
). However, whether matrix receptors and their ligands, including the laminins and laminin-binding proteins, mediate such mechanical-signal transduction by converting mechanical signals into chemical signals in the cytoplasm of a cell is not clear (Edwards, 2001
; Liu et al., 1999
; Nilius and Droogmans, 2001
).
The composition of the matrix to which AECs adhere in the intact lung has been studied by several groups. Specifically, several laminin subunits have been identified in the matrix-rich basement membrane of AECs in vivo (Coraux et al., 2002; Mizushima et al., 1998
; Pierce et al., 1998
; Virtanen et al., 1996
). This basement membrane is covered by a sheet of cells composed mainly of type-I (AT1, alveolar type-I pneumocytes) cells (Crapo et al., 1982
). The squamous AT1 cells line
95% of the alveolar surface area and are susceptible to oxidant stress (Crapo et al., 1982
). They share the basement membrane with type-II (AT2, pneumocyte type II) cells, which are cuboidal and cover less than 5% of the alveolar surface (Crapo et al., 1982
). The type-II cells produce surfactant, proliferate and differentiate into type I cells (Crapo et al., 1982
; Fehrenbach, 2001
; Uhal, 1997
). In the basement membrane of the lung, subunits of laminin show precise, developmentally regulated patterns of expression (Coraux et al., 2002
; Mizushima et al., 1998
; Pierce et al., 1998
; Virtanen et al., 1996
). For example, laminin
1 expression is restricted to early human lung morphogenesis, whereas the laminin
5 is continuous from early lung development to adult life (Pierce et al., 1998
; Virtanen et al., 1996
). Moreover, the
3 laminin subunit is expressed in the basement membrane of the alveoli of adult rat lung, where it localizes with ß1,
1 and ß2 subunits. Indeed, some authors have speculated that alveolar cells might express at least five laminin isoforms (Pierce et al., 1998
). In addition, several matrix-receptor complexes have been identified in the developing and adult lungs. In particular, the
2,
3 and
6 integrin subunits are found in developing alveolar tissue and in adult alveoli, and might serve in AEC adherence to laminins in the basement membrane (Virtanen et al., 1996
). The ß4 integrin appears to be restricted to the bronchi, where it assembles into hemidesmosome structures that tether bronchial airway epithelial cells to laminin 5 in the basement membrane (Michelson et al., 2000
).
In this study, we used primary rat AECs to assay the molecular underpinning of cellular response to physical force. In particular, we tested the hypothesis that matrix molecules secreted by lung epithelial cells are crucial molecular links in the process of `converting' a mechanical stress in the form of stretching into a cytoplasmic signal. We present the first analyses of the function and organization of laminin 6 secreted by epithelial cells and we provide data that implicate dystroglycan as a component of a complex that transduces mechanical signals. Moreover, our results reveal that a novel matrix fibrillar complex transduces mechanical signals that regulate the activity of p42/p44 mitogen-activated-protein kinase (MAPK) in rat AECs. Our data and conclusions contrast with those of others, who have stated that mechanotransduction does not require `force-dependent biochemical processes within the cell or cell membrane' (Tschumperlin et al., 2004).
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Materials and Methods |
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Antibodies and other reagents
Polyclonal rabbit antisera against p42/p44 MAPK and activated (phosphorylated) p42/p44 MAPK were purchased from Cell Signaling Technology (Beverly, MA). The mouse monoclonal antibodies CM6 and 5C5 against the laminin 3 subunit were described previously (Baker et al., 1996
; Langhofer et al., 1993
). Mouse monoclonal antibodies against the
2 and ß3 subunits of rat laminin 5 were originally provided by Desmos (San Diego, CA). A rabbit antiserum against the ß1 laminin subunit was obtained from P. Yurchenco (Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ). Mouse monoclonal antibody (mAb) against the
1 laminin subunit (mAb 1920) was obtained from Chemicon International (Temecula, CA). A goat polyclonal antiserum against the
5 laminin subunit (G20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A rabbit antiserum against nidogen was obtained from Calbiochem (San Diego, CA). A hamster mAb (Ha2/5) that inhibits the function of rat ß1 integrin was purchased from BD Pharmingen (Franklin Lakes, NJ). A function-inhibitory rat antibody against
3 integrin (Ralph 3.1), a mouse mAb against the ß2 laminin subunit and mouse mAb against perlecan were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Mouse IgM IIH6 against
-dystroglycan has been described elsewhere (Henry et al., 2001
). Mouse mAb 8D5 against ß-dystroglycan was purchased from Novocastra Labs (Newcastle, UK). Mouse IgM Ab1 against actin was purchased from Calbiochem. Control IgGs and IgMs, and fluorescein, rhodamine and horseradish-peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). For imaging of unfixed matrix proteins under stretching, we labeled the matrix with the antibody 5C5 followed by Alexa-Fluor-488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR).
The GRGDSP peptide was purchased from Invitrogen (Carlsbad, CA) and added to the medium of the AECs at a concentration of 100 µg ml1 in serum-free medium for 24 hours before stretching.
short hairpin RNA protocol
A set of single-stranded oligonucleotides encoding the dystroglycan target short hairpin RNA (shRNA) and its complement were synthesized by Invitrogen (Carlsbad, CA).
2719Forward:
CACCGCTCATTGCTGGAATCATTGCCGAAGCAATGATTCCAGCAATGAGC
2719Reverse:
AAAAGCTCATTGCTGGAATCATTGCTTCGGCAATGATTCCAGCAATGAGC
The sequence was selected using an algorithm provided by Invitrogen. The oligonucleotide pair was annealed, ligated into the pENTR/U6 vector and used to transform competent Escherichia coli cells following an established protocol (Invitrogen). Plasmid DNA was isolated from the kanamycin-resistant colonies and sequenced. The pENTRY/U6 construct was used in a recombination reaction with the adenoviral vector pAD/BLOCK-iT-DEST (Invitrogen). The resulting shRNA adenoviral vector was linearized with PacI and transfected into 293A cells using Lipofectamine following Invitrogen protocols. In addition, an adenoviral vector containing shRNA targeting human (but not rat) lamin A/C (supplied by Invitrogen) was processed the same way and used as a negative control for the adenoviral infection. Approximately 12 days after transfection, the adenovirus-containing 293A cells were harvested and lysed to prepare a crude viral stock. The resultant viral stock was amplified and the viral concentration determined. 2 days after plating, AECs were infected with the virus at a multiplicity of infection of 1:10. The following day, the medium was replaced and assays were performed 24 hours later.
Cyclic stretching
AECs were cultured for 3 days. After serum starvation for 24 hours, the cells were subjected to equibiaxial stretching on day 4 using the Cyclic Strain Unit FX-3000 (Flexcell International, Hillsborough, NC). This unit consists of a controlled vacuum unit and a base plate to hold the culture plates. A vacuum was cyclically applied to elastomer-bottomed six-well culture dishes using the Flexercell device with loading stations in place to impose equibiaxial stretching at 30 cycles per minute and a stretching-relaxation ratio of 1:1, resulting in a 10% linear elongation of the membrane as measured microscopically. This is the same regimen used previously (Correa-Meyer et al., 2002). Cells were harvested at 10 minutes, after the initiation of cyclic stretching.
SDS-PAGE and immunoblotting
AECs were solubilized in sample buffer consisting of 8 M urea, 1% sodium dodecyl sulfate in 10 mM Tris-HCl, pH 6.8, 15% ß-mercaptoethanol (Laemmli, 1970). AEC matrix was prepared according to a previously published procedure (Langhofer et al., 1993
). The matrix proteins were collected from the culture dish by solubilization in the above sample buffer. Proteins were separated by SDS-PAGE, transferred to nitrocellulose and processed for immunoblotting as previously described (Harlow and Lane, 1988
; Klatte et al., 1989
; Laemmli, 1970
). Immunoblots were scanned and quantified using Molecular Analyst (BioRad, Richmond, CA).
Immunofluorescence microscopy
Cells on glass coverslips were extracted for 2 minutes at 20°C in acetone. Cells maintained on elastomer surfaces in six-well plates were fixed in 3.7% formaldehyde for 5 minutes, extracted in 0.5% Triton X-100 in PBS at 4°C for 8 minutes in situ and then washed thoroughly in PBS. Subsequently, the elastomer surface with attached cells was excised from the six-well plate and processed as if it were a coverslip. Appropriate single primary antibodies or mixtures were overlaid onto the cells and the preparations were incubated at 37°C for 60 minutes. The cells on coverslips or elastomer were washed in three changes of PBS and then overlaid with secondary antibodies, placed at 37°C for an additional 60 minutes, washed extensively and then mounted on slides (cells on coverslips) or covered with a glass coverslip (cells on elastomer membrane). All preparations were viewed on a Zeiss laser-scanning confocal microscope (LSM 510) (Carl Zeiss, Thornwood, NY). Microscope images were exported as TIF files and figures were generated using Adobe Photoshop software.
Image analyses of 3 laminin under stretching
AECs were cultured on elastomer membranes (Flexcell International) for 4 days. AEC matrix was prepared according to a previously published procedure and then incubated at 37°C for 60 minutes in an unfixed state using mAb 5C5 against the 3 laminin subunit (Langhofer et al., 1993
). After thorough washing in PBS, the membrane was incubated in Alexa-Fluor-488-conjugated goat anti-mouse IgG for an additional 60 minutes at 37°C. The membrane was mounted in a StageFlexer system (Flexcell International) on the stage of a Leica DMRXA2 microscope (Leica Microsystems, Wetzlar, Germany) coupled to a Hamamatsu Orca-ER digital camera (Hamamatsu, Bridgewater, NJ). This allowed the fibers to be viewed while they were undergoing cyclic stretching. The membranes were stretched as described above and images captured using OpenLab in both stretched and unstretched conformations. Measurements of fiber length and areas between fibers were generated by identifying fiducial points in the images, which were subsequently analyzed using the MetaMorph Imaging System (Universal Imaging, Molecular Devices, Downingtown, PA) by examining the same networks under stretching and non-stretching conditions. Significant differences between experimental conditions were analysed using a two-way analysis of variance (ANOVA). When the ANOVA indicated a significant difference between groups, individual differences were explored with two-tailed paired Student's t-tests using the Bonferroni correction for multiple comparisons. A significant difference was prospectively identified as P<0.05.
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Results |
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In support of the immunofluorescence data, western blotting confirms that primary AECs express a distinct subset of laminin subunits. Antibodies against 3, ß1, ß2 and
1 laminin recognize proteins of 190 kDa and 160 kDa, 200 kDa, 190 kDa, and 200 kDa (respectively) in immunoblots of AEC extracts, whereas antibodies against other laminin subunits including
2 and ß3 show no obvious reactivity against the same protein preparation (Fig. 4). The latter antibodies, however, show strong reactivity with extracts derived from the rat bladder-cell line 804G, which is known to secrete a laminin-5-rich matrix (Baker et al., 1996
; Langhofer et al., 1993
) (Fig. 4A). It should be realized that we detect ß2 laminin subunit in extracts of 804G cells even though this protein is not deposited into the matrix that 804G cells secrete onto their culture support (J. C. R. Jones et al., unpublished).
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Matrix and matrix-receptor involvement in the transduction of mechanical signals
A major goal of our study was to investigate whether mechanotransduction in AECs might involve matrix-receptor complexes (Chen et al., 1999; Ingber, 2003
; Meyer et al., 2000
; Pavalko et al., 1998
; Shyy and Chien, 2002
). Indeed, we hypothesized that the fibrous nature of the matrix of AECs might facilitate the transduction of stretching stimuli to adherent cells if it were to deform upon mechanical stimulation. To evaluate this possibility experimentally, we first subjected native AEC matrix to an equibiaxial stretch regimen (a linear 10% elongation at a frequency of 30 cycles per minute, with a stretching:relaxation ratio of 1:1), which Correa-Meyer et al. have previously shown elicits specific signaling events in AECs (Correa-Meyer et al., 2002
). We chose to stimulate the matrix of AECs mechanically at 4 days after plating because, by this time, the AECs have established the same extensive fibrillar matrix on elastomer membranes that we observe when the cells are plated onto glass coverslips (compare Figs 1, 2, 3 with Fig. 5).
3-Laminin subunits in unfixed, native AEC matrix were immunolabeled. The labeled preparation was visualized before stretching, during the stretching regimen mentioned previously and following stretching in liquid medium. An overlay of three images of the labeled matrix fibers under these distinct conditions is shown (Fig. 6A).
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We next derived measurements of the changes in area enclosed by the fibers (A/A0; where A is the area during stretching and A0 is the area before stretching) and the length of the fibers (L/L0; where L is the fiber length during stretching and L0 is the fiber length before stretching) in the matrix of AECs (Fig. 6B). Both fiber area and fiber length are increased significantly during stretching, and return more or less to the baseline after cessation of stretching.
A tightly adherent matrix undergoing equibiaxial stretch should exhibit isotropic expansion. To determine whether AEC matrix does so, we used the formula
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When AECs are subjected to the stretching regimen mentioned above, p42/p44 MAPK undergoes activation within 15 minutes (Correa-Meyer et al., 2002). Hence, we were able to use this endpoint, in combination with a collection of function-blocking antibodies to define which particular matrix components play a role in stretching-induced signaling in AECs. AECs were washed with serum-free medium and then incubated in the same medium with antibody or control immunoglobulin for 24 hours before applying the regimen of cyclic stretching. Extracts of cells were processed for immunoblotting using antibodies that recognize activated p42/p44 MAPK, and then reprobed with antibodies against total p42/p44 MAPK. We show pairs of images of one representative immunoblot and quantification of at least three trials of each experimental treatment (Fig. 7).
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Our data showing expression of dystroglycan by AECs suggest that dystroglycan might function as a receptor for the perlecan/laminin-6 complexes (Fig. 4C). This hypothesis is supported by the observation that the -dystroglycan function-perturbing antibody IIH6 effectively inhibits stretching-induced MAPK activation in AECs by about 30% (Michele and Campbell, 2003
) (Fig. 7A,B). To confirm this result, we infected AECs 2 days after plating with adenovirus encoding shRNA to knockdown specifically the production of dystroglycan (Fig. 7C) (Elbashir et al., 2002
). Knockdown of dystroglycan is >90% in AECs 2 days after infection (Fig. 7C). At the same time, we then assayed dystroglycan-shRNA and control-shRNA-expressing cells for MAPK activity following stretch (Fig. 7B). MAPK activation is inhibited by at least 50% in AECs in which production of dystroglycan is knocked down compared with untreated cells and cells expressing control shRNA (Fig. 7B).
It should be realized that AECs show no obvious morphological perturbation, including detachment or rounding, following treatment with any of the function-perturbing antibodies we used in these studies, or following treatment with adenovirus (not shown). Furthermore, laminin-6-fiber organization is not perturbed by our antibody or viral regimens. Moreover, the same approximate amount of total cell protein is collected from cultures of untreated cells and cells treated with antibodies or adenovirus at the conclusion of the stretch regimen (not shown). Together, these results indicate that no obvious loss or death of cells is caused by antibody incubation or viral infection.
Irrelevant IgG and IgM treatment has no impact on activation of MAPK in AECs subject to stretching (not shown). We have performed several other control studies to assess the specificity of matrix molecule involvement in transducing mechanical signals in the AECs. For example, AECs 3 days after plating were incubated with an excess of RGD-containing peptide in serum-free medium and then, 24 hours later, the treated cells were subjected to stretch (Fig. 7B). This peptide is a competitive inhibitor of the receptor-binding site on matrix molecules including fibronectin and vitronectin for several ß1- and ß3-subunit-containing integrin-family members (Hynes, 1992). The RGD-containing peptide does not inhibit stretching-induced p42/p44 MAPK activation in AECs, indicating that RGD-binding integrins, including
5ß1 and
vß3, are not involved in mediating signaling in the stretched cells. Rather, our results suggest roles for laminin-6, the proteoglycan perlecan and dystroglycan in mediating stretching-induced signaling in AECs.
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Discussion |
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Laminin heterotrimers assemble into polymeric matrix arrays in distinct ways in different cultured cell types. For example, in the rat epithelial cell line 804G, laminin 5 assembles into a "cat's-paw" array, whereas the same molecule assembles in cloud-like arrays, arcs and circles in the matrix of keratinocytes (deHart et al., 2003; Langhofer et al., 1993
). By contrast, laminin 1 assembles as a reticular network on the surface of muscle cells (Colognato et al., 1999
), and the organizational state of laminin 6 in AEC matrix resembles this. Exactly how laminin 6 assembles into cable-like structural arrays remains to be determined. However, our time-course studies suggest that perlecan is laid down in a fibrillar organization before laminin 6. Indeed, we speculate that AECs first assemble fibers of perlecan whose distribution subsequently determines the arrangement of laminin 6. Perlecan is certainly capable of doing so via an interaction with nidogen, which cross-links the immunoglobulin-like domain 3 of perlecan with an epidermal-growth-factor-like repeat within the laminin
1 subunit (Kvansakul et al., 2001
; Mayer et al., 1993
; Ries et al., 2001
). In support of this, we show that nidogen codistributes with perlecan and the subunits of laminin 6 in the matrix of AECs.
The fibrous nature of perlecan/nidogen/laminin-6-rich complexes in the matrix of AECs led us to ask whether these fibers provide the structural means for cells to resist and/or respond to mechanical stresses. Indeed, by visualizing labeled fibers, we have shown that the fibers adhere to the matrix at sites sufficiently close to one another to result in isotropic expansion during stretching. Hence, the ability of fibers rich in laminin 6 to respond to stretching by undergoing a precise deformation makes them ideal transducers of force from the outside to the inside of cells, initiating signaling cascades that, in turn, trigger specific physiological responses (Gillespie and Walker, 2001). To test this hypothesis, we used activation of the p42/p44 MAPK pathway as our endpoint. This important cytoprotective signal pathway is maximally activated within 15 minutes of the initiation of cyclic stretching in AECs (Correa-Meyer et al., 2002
). Using an antibody that we have previously shown to bind to and inhibit the function of the
3 laminin subunit, we have provided evidence that laminin 6 is one link in the molecular pathway that transmits an outside-in mechanical signal (Baker et al., 1996
). It should be realized that this pathway is distinct from the recently detailed growth-factor-receptor circuitry involved in mechanical signaling in bronchial airway cells (Tschumperlin et al., 2004
).
We initially assumed that laminin 6 would transduce a signal via its interaction with an integrin at the surface of AECs, because the role of integrins in signal transduction is well established (Howe et al., 1998; Hynes, 1992
; Hynes, 2002
). There are several potential integrin receptors for laminins containing the
3 laminin subunit, including the integrin heterodimers
6ß4,
3ß1 and
6ß1 (Carter et al., 1991
; Jones et al., 1998
). Our primary AECs do not produce the ß4 integrin subunit, as assessed by immunoblotting and immunofluorescence using a polyclonal antibody against the ß4 integrin subunit that recognizes ß4 integrin in rat bladder cells (not shown). By contrast, rat AECs produce ß1-containing integrin heterodimers. Thus, we focused on testing the hypothesis that ß1-subunit-containing integrin heterodimers mediate the signal initiated by stretching that impacts MAPK activation in AECs. We did so using an antibody that inhibits rat ß1-integrin function. However, function-perturbing antibodies against both ß1 and
3 integrins, as well as the RGD peptide, which inhibit the activity of a range of integrin receptors, fail to block stretching-induced p42/p44 MAPK activation in AECs (Hynes, 1992
; Hynes, 2002
).
If integrins are not involved in mediating mechanical signaling in AECs, what senses matrix deflections at the surface of these cells? A candidate receptor for mediating laminin-6 signaling is dystroglycan. Dystroglycan is best known as a component of the dystrophin-glycoprotein complex, whose absence or abnormality leads to muscular dystrophies and several types of cardiomyopathy (Michele and Campbell, 2003). This complex plays a structural role in muscle by forming a mechanically strong link between the sarcolemma and actin, but whether it transduces signals is controversial (Michele and Campbell, 2003
; Rybakova et al., 2000
; Spence et al., 2004
). When bound to laminin, dystroglycan is reported to inhibit MAPK activation (Ferletta et al., 2003
). In epithelial cells, the functions of dystroglycan are not clear, although it is believed to be primarily a structural protein involved in basement-membrane assembly, growth control, cytoskeleton organization, development of cell polarity and epithelial morphogenesis (Durbeej et al., 2001
; Muschler et al., 2002
).
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In summary, analyses of AECs and their response to stretching support a major role for dystroglycan in mediating integrin-independent, matrix-driven mechanotransduction in AECs. Our studies hold implications not only for the ways epithelial cells sense and respond to mechanical stimulation in general but also for the pathophysiology of certain lung diseases. Specifically, AECs undergo stretching when the lung is mechanically ventilated with high lung volumes. This might be injurious, leading to the failure of the epithelial sheet that covers the lung surface (Liu et al., 1999). Our results suggest that mechanically induced MAPK activation functions as a protective mechanism to ameliorate potential damage caused by overdistention of AECs. There is precedent for this because activation of MAPK pathways is known to promote the survival of several cell types, most notably in the nervous and immune systems (Bonni et al., 1999
; Holmström et al., 2000
). Defining the precise molecular pathway(s) by which AECs respond to mechanical stretching and elicit survival signals might lead to the design of strategies to protect the alveolar epithelium against injury induced by positive-pressure ventilation (Matthay et al., 2003
; Sznajder, 2001
).
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Acknowledgments |
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