Integrin alpha 3-subunit expression modulates alveolar epithelial cell monolayer formation

Richard L. Lubman, Xiao-Ling Zhang, Jie Zheng, Leah Ocampo, Melissa Z. Lopez, Srihari Veeraraghavan, Stephanie M. Zabski, Spencer I. Danto, and Zea Borok

Division of Pulmonary and Critical Care Medicine and Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California 90033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated expression of the alpha 3-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha 3-integrin subunit on AEC monolayer formation. alpha 3-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha 3- and beta 1-integrin subunits coprecipitated in immunoprecipitation experiments with alpha 3- and beta 1-subunit-specific antibodies, consistent with their association as the alpha 3beta 1-integrin receptor at the cell membrane. Treatment with blocking anti-alpha 3 monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha 3- and beta 1-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha 3beta 1-integrin receptor at the cell membrane. Blockade of the alpha 3-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha 3-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha 3beta 1-integrin receptor in AEC adhesion.

alveolar epithelium; beta 1-integrin; cell adhesion; extracellular matrix; transepithelial resistance


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE PULMONARY ALVEOLAR AIR SACS are lined with a continuous layer of epithelial cells across which gas exchange occurs. The alveolar epithelium is composed of alveolar type I (AT1) and type II (AT2) cells that interact with the extracellular matrix (ECM; basement membrane) via cell surface receptors. This interaction influences many alveolar epithelial cell (AEC) functions, including cell adhesion and spreading, and modulates the repair of the alveolar epithelial barrier after lung injury.

The integrins are a family of cell surface proteins that mediate cell adhesion and cell-cell interactions. Integrins are heterodimeric proteins that consist of one each of several distinct alpha - and beta -subunits that together determine the specificity of the molecule as a receptor for different ECM proteins. Several different integrin subunits have been identified in human and rat lungs and AECs, with particular subunits thought to play a role in modulation of AEC function in an ECM ligand-specific fashion (6, 7, 12, 19, 23, 24, 29, 38, 42, 46, 48). Current data also indicate that the expression of different integrin receptors in the lung, and in alveolar epithelium in particular, changes after lung injury or inflammation and after neoplastic transformation (1, 2, 14, 20, 26, 33, 35-37, 39, 43).

The alpha 3beta 1-integrin (VLA-3) is a receptor for several known ligands, including laminin, fibronectin, collagen type IV, epiligrin, and entactin/nidogen (2). It is present in many epithelia, and its expression is required for morphogenesis of both lung and kidney (27). The alpha 3-integrin subunit and its beta 1-subunit companion are expressed in normal human alveolar epithelium in both AT1 and AT2 cells, where it is presumed that they form functional receptors (46). The specific functions and substrates for the alpha 3beta 1-integrin in the lung and alveolar epithelia are largely unknown.

In this study, we investigated the expression and function of the alpha 3beta 1-integrin in AEC monolayers. In this model, primary cultured rat AT2 cells form confluent, electrically resistive monolayers of cells that gradually acquire AT1 cell phenotypic properties (4, 5, 9). Our results indicate that AECs in culture express alpha 3- and beta 1-integrins that form alpha 3beta 1-integrin heterodimers based on coprecipitation studies. Blockade of integrin binding with specific anti-integrin monoclonal antibodies (MAbs) results in decreased AEC adhesion and monolayer integrity, consistent with a role for this ECM receptor in the formation of an intact epithelial barrier in situ.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation and preparation of rat AEC monolayers. AT2 cells were isolated from adult male Sprague-Dawley rats by disaggregation with elastase (2.0-2.5 U/ml; Worthington Biochemical, Freehold, NJ), followed by differential adherence on IgG-coated bacteriological plates (16). The enriched AT2 cells were resuspended in a minimal defined serum-free medium (MDSF) consisting of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture in a 1:1 ratio (Sigma, St. Louis, MO) supplemented with 1.25 mg/ml of bovine serum albumin (BSA; Collaborative Research, Becton Dickinson, Franklin Lakes, NJ), 10 mM HEPES, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml of sodium penicillin G, and 100 µg/ml of streptomycin (5). Cells were plated onto tissue culture-treated polycarbonate (Nuclepore, Pleasanton, CA) filter cups (Transwell, Corning Costar, Cambridge, MA), 8-well chamber slides (Falcon, Becton Dickinson, Franklin Lakes, NJ), or 24-well tissue culture plastic dishes (Falcon, Becton Dickinson) at a density of 1.0 × 106 cells/cm2. Cultures were maintained in a humidified 5% CO2 incubator at 37°C. AT2 cell purity (>90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid (32). Cell viability (>90%) was measured by trypan blue dye exclusion.

Media were changed, thereby removing nonadherent cells, on the second day after plating. Monolayers were subsequently fed every other day. Cells were maintained in MDSF or in MDSF supplemented with either alpha 3-integrin blocking MAb CP11 (clone P1B5; Oncogene Research, Cambridge, MA) (8), alpha 3-integrin blocking MAb Ralph 3.2 (Santa Cruz Biotechnology, Santa Cruz, CA), or nonimmune mouse IgG1 at 0, 1, 2, or 5 µg/ml. The range of concentrations of blocking Ab used was chosen in accordance with the manufacturer's recommendations. Transepithelial resistance (TER) was measured with a rapid screening device (Millicell-ERS, Millipore, Bedford, MA) as previously described (9). RNA and protein were harvested from monolayers at daily intervals for Northern and Western blotting, respectively.

A549 cells. A549 cells (a human adenocarcinoma-derived cell line) were obtained from the American Type Culture Collection and cultured in medium containing DMEM supplemented with 10% fetal bovine serum, 100 U/ml of sodium penicillin G, and 100 µg/ml of streptomycin. Cells were plated at a density of 2 × 106/cm2 on 100-mm tissue culture plastic dishes (Falcon, Becton Dickinson), grown until confluent in a humidified 5% CO2 incubator at 37°C, and solubilized directly into immunoprecipitation lysis buffer (see Western analysis and coprecipitation studies) for immunoprecipitation studies.

Immunofluorescence. Two sets of fresh-frozen sections (4 µm) of adult rat lung were fixed in 100% methanol at -20°C, blocked with PBS-3% BSA (pH 7.4) to reduce nonspecific reactivity, reacted sequentially with anti-alpha 3-integrin subunit polyclonal antibody (PAb) 1920P (Chemicon, Temecula, CA) and FITC-labeled goat anti-rabbit secondary Ab, and postfixed with 3.7% Formalin. The labeled sections were then viewed by differential interference contrast (DIC) optics and immunofluorescence (IF) with an Olympus microscope equipped with epifluorescence optics set at ×300 magnification. Monolayers (day 7) grown on chamber slides maintained in MDSF were rinsed with cold PBS, fixed with 100% methanol at -20°C for 10 min, rinsed in PBS again, and treated with PBS-3% BSA. Monolayers were reacted in situ with anti-alpha 3-integrin subunit PAb 1920P. After extensive washing, the monolayers were incubated with FITC-labeled goat anti-rabbit secondary Ab and viewed by epifluorescence at ×600 magnification.

Western analysis and coprecipitation studies. SDS-PAGE was performed with the buffer system of Laemmli (30), and immunoblotting (Western blotting) was performed with procedures modified from Towbin et al. (44). For detection of integrin subunits, AEC monolayers were solubilized directly into 2% SDS sample buffer at 37°C for 15 min. Equal amounts of cell protein in sample buffer were resolved by SDS-PAGE under reducing conditions for the detection of beta 1-integrin subunit or under nonreducing conditions for the detection of alpha 3-integrin subunit as previously described (22). Proteins were electrophoretically blotted onto Immobilon-P (Millipore). The blots were blocked for 2 h with 5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris and 500 mM NaCl, pH 7.5, and then incubated with primary PAb 1920P for detection by immunoblot.

Blots were incubated with horseradish peroxidase-linked goat anti-rabbit or anti-mouse IgG conjugates for 1 h, and antigen-Ab complexes were visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL). The relative intensity of protein bands was quantified by densitometry. Protein concentrations were determined with the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA), and BSA was used as a standard. Prestained or biotin-labeled molecular weight standards (Bio-Rad) or Cruz Marker molecular weight standards (Santa Cruz Biotechnology) were used to determine the apparent molecular masses of the blotted proteins.

For coprecipitation studies, AECs were first immunoprecipitated with either anti-alpha 3-integrin subunit Ab (PAb 1920P, MAb CP11, or MAb Ralph 3.2) or anti-beta 1-integrin subunit (MAb 141720, Transduction Labs, Lexington, KY). Briefly, AEC monolayers were solubilized in immunoprecipitation lysis buffer [TBS (0.05 M Tris), pH 8.0, 1% NP-40, and 1% BSA] for 1 h on ice and then centrifuged at 10,000 g for 20 min to remove insoluble material. Before immunoprecipitation, the resulting supernatant was preincubated with both goat IgG-agarose and rabbit or mouse serum-agarose for 1 h at 4°C to reduce nonspecific binding to primary and secondary Abs. The preclarified supernatants were then incubated with either anti-alpha 3- or anti-beta 1-integrin Ab overnight at 4°C. After incubation with this primary Ab, the samples were incubated with secondary Ab (goat anti-rabbit or anti-mouse IgG) that had been conjugated to agarose beads for 1 h. After it was washed twice with lysis buffer, once with TBS at pH 8.0, and once with TBS at pH 6.0, the bound antigen was eluted from the goat anti-rabbit or anti-mouse agarose beads in 2% SDS sample buffer and processed for SDS-PAGE immunoblotting as described above. Either anti-alpha 3-integrin subunit PAb 1920P or anti-beta 1-integrin subunit MAb 141720 was used to blot the coprecipitated integrin subunits.

RNA isolation and Northern analysis. Total RNA was isolated from AEC monolayers by the acid guanidinium-phenol-chloroform method (10). Equal amounts of RNA (5-20 µg) were denatured with formaldehyde, size-fractionated by agarose gel electrophoresis under denaturing conditions, and transferred to nylon membranes (Hybond N+, Amersham Life Sciences, Cleveland, OH). RNA was immobilized by ultraviolet cross-linking. Blots were prehybridized for 2 h at 65°C in 1 M sodium phosphate buffer (pH 7), 7% SDS, and 1% BSA. Hybridization was performed for 16 h at 65°C in the same buffer. Blots were probed with an integrin subunit-specific oligonucleotide probe for the alpha 3-subunit (Biognostik, Chemicon, Temecula, CA). Probes were labeled with [alpha -32P]dCTP (Amersham) by the random-primer method with the use of a commercially available kit (Boehringer Mannheim, Indianapolis, IN). Blots were washed at high stringency (0.5× saline-sodium citrate; 75 mM NaCl and 7.5 mM sodium citrate, pH 7.0, with 0.1% SDS at 55°C) and visualized by autoradiography. Differences in RNA loading were normalized with a 24-mer oligonucleotide probe rRNA end labeled with [gamma -32P]ATP for 18S (31). Binding was detected by autoradiography and quantified by densitometry.

Cell number. AECs grown for 24 h in MDSF or MDSF supplemented with 5 µg/ml of alpha 3-integrin blocking MAb CP11, alpha 3-integrin blocking MAb Ralph 3.2, or nonimmune mouse IgG in 24-well tissue culture plastic plates were washed three times with ice-cold PBS, trypsinized (0.05% trypsin) for 30 s, and resuspended in PBS. The cell number was counted with a Coulter Counter (Coulter Electronics, Hialeah, FL).

Nuclear staining of AEC monolayers with propidium iodide. AEC monolayers grown in MDSF or MDSF supplemented with 5 µg/ml of anti-alpha 3 MAb CP11 were fixed on day 3 for IF (as described in Immunofluorescence). Fixed monolayers were stained with propidium iodide (Molecular Probes, Eugene, OR), a selective nuclear stain. Stained specimens were viewed by epifluorescence microscopy at ×200 magnification.

Scanning electron microscopy. AEC monolayers grown in MDSF or MDSF supplemented with 5 µg/ml of anti-alpha 3 MAb CP11 were fixed on day 3 with 2.5% glutaraldehyde and 1.5% OsO4, dehydrated, and critical-point dried. Specimens were viewed by scanning electron microscopy (SEM) at ×400 magnification.

Chemicals. Except where otherwise indicated, cell culture media and all other chemicals were purchased from Sigma and were of the highest commercial quality available.

Statistical analysis. Results are expressed as means ± SE. Significance of differences (P < 0.05) was determined by ANOVA.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Labeling of lung sections with anti-alpha 3 MAb. Figure 1 shows corresponding DIC and epifluorescence images indicating diffuse alveolar staining by the anti-alpha 3 PAb 1920P. The staining pattern, which is similar to that shown for other anti-alpha 3 Abs in human lung (46), is consistent with staining of both AT1 and AT2 cells in situ.


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Fig. 1.   Labeling of rat lung sections with anti-alpha 3 antibody (Ab). Corresponding differential interference contrast (DIC; A) and epifluorescence (B) images indicate diffuse alveolar staining by the anti-alpha 3 polyclonal antibody (PAb) 1920P (×300 magnification).

Immunofluorescence, Western blotting, and Northern blotting of alpha 3-integrin in AEC monolayers. AEC monolayers labeled with the anti-alpha 3 PAb 1920P showed staining on day 7 as illustrated in the epifluorescence image shown in Fig. 2A. The same Ab labels a single band corresponding to the expected molecular mass (145 kDa) of the alpha 3-integrin subunit on Western blot (Fig. 2B). AEC monolayers expressed progressively increasing amounts of alpha 3-integrin subunit protein as shown in this representative Western blot (Fig. 2C). Protein abundance increased maximally by day 6 to ~3.5 times that observed on day 1. Similarly, the 5-kb transcript corresponding to the expected size of the alpha 3-integrin subunit mRNA was present on Northern blot from days 1 to 8, with a relative increase in mRNA levels evident as a function of time in culture. alpha 3-Integrin subunit mRNA levels were maximal by day 6 at ~4 times those observed on day 1 (Fig. 2D).


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Fig. 2.   Immunofluorescence (IF), Western blotting, and Northern blotting of alpha 3-integrin in alveolar epithelial cell (AEC) monolayers. A: AEC monolayers stained with anti-alpha 3 PAb 1920P (day 4, ×600 magnification). B: single band corresponding to the predicted molecular mass (MW; 145 kDa) of the alpha 3-integrin subunit on Western blot labeled with the same Ab. C: AEC monolayers expressed progressively increasing amounts of alpha 3-integrin subunit protein on days 1, 3, 6, and as shown in representative Western blot and the corresponding quantitation; n = 3 experiments. * Significantly different from day 1. D: 5-kb transcript corresponding to the predicted size of the alpha 3-integrin subunit mRNA was detectable on Northern blot from days 1 to 8, with a relative increase in mRNA levels evident as a function of time in culture as indicated in the corresponding quantitation; n = 2 experiments. * Significantly different from day 1.

Coprecipitation of integrin alpha 3- and beta 1-subunits. As illustrated in Fig. 3A, immunoprecipitates of day 4 AEC lysates containing either anti-beta 1-integrin subunit MAb 141720 (lane 2) or anti-alpha 3-integrin subunit PAb 1920P (lane 3) were resolved by SDS-PAGE and immunoblotted with the anti-alpha 3-integrin subunit PAb. Positive 145-kDa bands are seen for both alpha 3 and beta 1 immunoprecipitates, indicating that both alpha 3 and beta 1 Abs either precipitate or coprecipitate the alpha 3-integrin subunit. Figure 3A, lane 1, shows day 8 AEC lysate blotted as a positive control, demonstrating immunodetection of the same 145-kDa alpha 3-integrin subunit in the alpha 3 and beta 1 immunoprecipitates as in total cell lysate. Immunoprecipitations with rat IgG (Figure 3A, lane 4) or in the absence of primary Ab (lane 5) were negative for alpha 3-integrin subunit.


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Fig. 3.   Coprecipitation of integrin alpha 3 (A)- and beta 1 (B)- subunits as shown by Western blot. A: AECs (day 4) immunoprecipitated (IP) with either anti-beta 1-integrin subunit monoclonal antibody (MAb) 141720 (lane 2) or anti-alpha 3-integrin subunit PAb 1920P (lane 3) were blotted with the anti-alpha 3-integrin subunit MAb. Positive 145-kDa bands are seen for both, indicating that the Abs either precipitate or coprecipitate the alpha 3-integrin subunit. Lane 1, AEC (day 8) lysate blotted as a positive control. Precipitations with rat IgG (lane 4) or in the absence of primary (1°) Ab (lane 5) were negative. B: AECs (day 4) immunoprecipitated with either the anti-beta 1-integrin subunit MAb (lane 1) or the anti-alpha 3-integrin subunit PAb (lane 2) and blotted with the anti-beta 1-integrin subunit MAb were positive for 120-kDa bands, indicating that both Abs precipitate or coprecipitate the beta 1-integrin subunit. Precipitations with rat IgG (lane 3) or in the absence of 1°Ab (lane 4) were negative. Lane 5, molecular mass markers (nos. on right).

As illustrated in Fig. 3B, day 4 AEC lysates immunoprecipitated with either the anti-beta 1-integrin subunit MAb (lane 1) or anti-alpha 3-integrin subunit PAb (lane 2), resolved by SDS-PAGE, and immunoblotted with the anti-beta 1-integrin subunit MAb were positive for 120-kDa bands, indicating that both alpha 3 and beta 1 Abs either precipitate or coprecipitate the beta 1-integrin subunit. Immunoprecipitations with rat IgG (Fig. 3B, lane 3) and in the absence of primary Ab (lane 4) were negative for beta 1-integrin subunit. Molecular mass standards are shown in Fig. 3B, lane 5. These results are consistent with the formation of heterodimeric alpha 3beta 1-integrin receptors in AECs.

Immunoprecipitation of alpha 3-integrin by anti-alpha 3-integrin blocking Abs. As shown in Fig. 4, lysates of A549 cells (lanes 1 and 3) and day 4 rat AEC lysates (lanes 2 and 4) were immunoprecipitated with either anti-alpha 3-integrin subunit MAb CP11 (lanes 1 and 2) or Ralph 3.2 (lanes 3 and 4), and the precipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-alpha 3-integrin subunit PAb 1920P. Positive 145-kDa bands are seen in Fig. 4, lanes 1 and 2, indicating that MAb CP11 (an anti-human integrin Ab) precipitates both human and rat alpha 3-integrin subunits. A positive 145-kDa band is also seen in Fig. 4, lane 4, but is absent in lane 3, indicating that MAb Ralph 3.2 (an anti-rat integrin Ab) precipitates rat, but not human, alpha 3-integrin subunits. These results indicate the potential for specific interaction between each anti-integrin blocking Ab and the rat alpha 3-integrin subunit in the functional studies described below.


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Fig. 4.   Immunoprecipitation of alpha 3-integrin by anti-alpha 3-integrin blocking Abs. Lysates of A549 cells (lanes 1 and 3) or day 4 rat AEC lysates (lanes 2 and 4) were immunoprecipitated with either anti-alpha 3-integrin subunit MAb CP11 (lanes 1 and 2) or Ralph 3.2 (lanes 3 and 4), and the precipitates were blotted with anti-alpha 3-integrin subunit PAb 1920P. Positive 145-kDa bands are seen in lanes 1 and 2, indicating that MAb CP11 precipitates both human and rat alpha 3-integrin subunits. A positive 145-kDa band is also seen in lane 4 but is absent in lane 3, indicating that MAb Ralph 3.2 (an anti-rat integrin Ab) precipitates only rat alpha 3-integrin subunits. +, Presence; -, absence.

Effect of anti-alpha 3 blocking MAb on AEC cell adhesion. AECs grown in 24-well plates in MDSF or MDSF supplemented with 5 µg/ml of either anti-alpha 3-integrin blocking Ab CP11 or Ralph 3.2 or mouse IgG1 were gently rinsed with PBS at 24 h, and adherent cells were trypsinized off the plates and counted with a Coulter Counter. As indicated in Fig. 5, the absolute number of adherent cells per well was no different for MDSF versus MDSF plus IgG1. In contrast, AECs grown in MDSF plus CP11 or Ralph 3.2 showed markedly reduced numbers of adherent cells per well. These results indicate that either anti-alpha 3-integrin blocking Ab is capable of reducing cell adhesion at 24 h.


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Fig. 5.   Effect of anti-alpha 3 blocking MAb on AEC adhesion. After a gentle rinse with PBS, AECs (n = 3 experiments) adherent at 24 h when grown in 24-well plates in minimal defined serum-free medium (MDSF) or MDSF supplemented with 5 µg/ml of either anti-alpha 3-integrin blocking Ab CP11 or Ralph 3.2 or mouse IgG1 were trypsinized off the plates and counted with a Coulter Counter. The absolute number of adherent cells per well is no different for MDSF vs. MDSF+IgG1, whereas AECs grown in MDSF+CP11 or +Ralph 3.2 showed markedly reduced numbers of adherent cells per well. * Significantly different cell number from MDSF alone.

Effect of anti-alpha 3 blocking MAb on AEC monolayer formation: TER. AEC monolayers grown in the absence of anti-alpha 3-integrin subunit blocking Ab formed confluent monolayers, with TER > 1,000 Omega  · cm2 on day 3, whereas AECs grown in the presence of 5 µg/ml of Ab CP11 developed no measurable TER after an equal amount of time in culture (Fig. 6). Intermediate concentrations of Ab (1 and 2 µg/ml) resulted in measurable but lower TER than no-Ab controls (data not shown). In separate experiments, AECs grown in the presence of 5 µg/ml of Ab Ralph 3.2 showed no measurable resistance on day 3, whereas TER was no different for monolayers treated with 5 µg/ml of mouse IgG of the same subclass as the anti-alpha 3 blocking Ab (2.86 ± 0.12 kOmega · cm2) compared with that for untreated monolayers (3.13 ± 0.19 kOmega · cm2). TER 1,000 Omega  · cm2 (indicating development of confluent, electrically tight monolayers) gradually develops on days 4-6 under all conditions studied.


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Fig. 6.   Effect of anti-alpha 3 blocking MAb on AEC monolayer formation: transepithelial resistance (TER). AEC monolayers grown in the absence of anti-alpha 3-integrin subunit blocking Ab form confluent monolayers with TER > 1,000 Omega  · cm2 on day 3, whereas AECs grown in the presence of Ab CP11 develop no measurable TER after an equal amount of time in culture. TER 1,000 Omega  · cm2 (indicating development of confluent, electrically resistive monolayers) gradually develops on days 4-6 under both conditions. * Significantly different from control monolayers on same culture day.

Effect of anti-alpha 3 blocking MAb on AEC monolayer formation: nuclear staining and SEM. As shown in Fig. 7A, fluorescence microscopy of fixed AEC monolayers (day 3) grown in the absence of anti-alpha 3 blocking Ab and stained with the nuclear stain propidium iodide showed uniform distribution of nuclei consistent with the development of confluence for monolayers. In contrast, monolayers grown in the presence of 5 µg/ml of anti-alpha 3 blocking Ab CP11 showed large areas of the filter without nuclei (i.e., without cells), probably due to the failure of cells to adhere to the substratum and/or cell sloughing before or during fixation (Fig. 7B). As shown in Fig. 8A, control monolayers on day 3 appeared confluent by SEM, whereas those grown in the presence of 5 µg/ml of anti-alpha 3 blocking Ab CP11 (Fig. 8B) showed areas of nonconfluence. Monolayers grown in the presence of blocking Ab appeared to show cells being sloughed off, leaving areas of bare filter.


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Fig. 7.   Effects of anti-alpha 3 blocking MAb on AEC monolayer formation: nuclear staining. A: fluorescence microscopy of fixed AEC monolayers (day 3) stained with the nuclear stain propidium iodide shows uniform distribution of nuclei, consistent with the development of confluence for monolayers grown in the absence of anti-alpha 3 blocking MAb CP11. B: in contrast, monolayers grown in the presence of 5 µg/ml of anti-alpha 3 blocking Ab show large areas of the filter without nuclei (i.e., without cells) due to the failure of cells to adhere to the substratum and/or to cell sloughing before or during fixation (×200 magnification).



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Fig. 8.   Effects of anti-alpha 3 blocking MAb on AEC monolayer formation: scanning electron microscopy (SEM). Untreated control monolayers (A) on day 3 appear confluent by SEM, whereas those grown in the presence of 5 µg/ml of anti-alpha 3 blocking MAb CP11 (B) show areas of nonconfluence. Monolayers grown in the presence of blocking Ab show cells appearing sloughed off, leaving behind bare filter (×400 magnification).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrated in this study that primary cultured AECs express both integrin alpha 3- and beta 1-subunits. Integrin alpha 3-subunit mRNA and protein are detectable from day 1 and increase with time in culture through day 8, coincident with the period during which the cells spread to form confluent monolayers and undergo transdifferentiation from AT2 cells toward the AT1 cell phenotype (4, 5). Integrin alpha 3- and beta 1-subunits can be reciprocally coprecipitated, consistent with their association as the alpha 3beta 1-integrin receptor at the cell membrane. AEC monolayer formation can be partially and transiently inhibited by anti-alpha 3-subunit Abs previously shown to have blocking activity as indicated by adhesion assays, bioelectric measurements, and morphological assessment. Taken together, these data demonstrate that cultured AECs express the alpha 3beta 1-integrin receptor, a mediator of epithelial cell adhesion, and indicate a role for this receptor in mediating AEC adhesion.

Integrin alpha 3-subunits have been previously identified in adult rat and human alveolar epithelia as well as in fetal lung and isolated fetal distal lung epithelial cells (6, 7, 12, 19, 23, 24, 29, 42, 46, 48). Staining for the integrin alpha 3-subunit is abundant in evolving alveolar walls during the canalicular stage of development (19-21 days gestation in the rat) where it colocalizes with laminin-5 (epiligrin), one of its ECM ligands (46). Antisera to both the integrin alpha 3-subunit and its only known companion, the integrin beta 1-subunit, have been shown to label adult human alveolar surfaces diffusely (46) in a pattern similar to that observed with the anti-integrin alpha 3-subunit PAb used in the current study (Fig. 1). Integrin alpha 3-subunit is also known to be expressed in some lung epithelium-derived cell lines (e.g., LM5 cells, an AT2-derived cell line), but it has not been previously identified in adult AECs grown in primary culture (29).

alpha 3-Integrin subunit mRNA is detectable in AECs as a 5-kb transcript by Northern blot. alpha 3-Integrin subunit protein is detectable in AECs by IF (Fig. 2A) and as a 145-kDa protein on Western blot (Fig. 2B). Both alpha 3-integrin subunit mRNA and protein are minimally detectable in freshly isolated AT2 cells (data not shown), whereas mRNA levels and protein abundance increase from day 1 through day 8 in culture, with the greatest increases occurring during the first few days after plating (Fig. 2, C and D). The most likely explanation for the relatively low amount of this integrin subunit after cell isolation may be that transcriptional downregulation and protein turnover of alpha 3-integrin occur rapidly in the absence of a continuous input of signal from an intact basement membrane. Conversely, attachment of cells and secretion of basement membrane components likely create positive feedback for further matrix secretion, integrin expression, and cell adhesion (13, 15, 25). Although little is known about the specific mechanisms by which alpha 3-integrin expression is regulated in any tissue, the recent description of the genomic organization of the human and mouse alpha 3-integrin subunit genes may facilitate further efforts in this direction (21, 45).

It is also possible that the quantitative differences in alpha 3-integrin subunit expression observed in the present study reflect some degree of differential expression between AT1 and AT2 cells. AT2 cells, especially those grown on permeable supports, have been shown to transdifferentiate toward the AT1 cell phenotype on the basis of morphology and expression of several distinct AT1 cell markers, with a time course resembling that found for the increase in alpha 3-integrin subunit expression (4, 5). AT1 and AT2 cells have been shown to reside on basement membranes of somewhat different composition, consistent with a role for different alpha 3beta 1-integrin receptor-matrix interactions in the transition from one differentiated phenotype to the other (40). Alternatively, alpha 3-integrin expression may be relatively greater in AT1 versus AT2 cells for the simple reason that AT1 cells have more basolateral surface in contact with basement membrane than AT2 cells and would need to express relatively more integrin (and other cell surface) receptor than their AT2 cell counterparts to maintain a similar membrane density of attachments. Notwithstanding the demonstration of the labeling of AT2 cell basolateral membranes by an anti-alpha 3-integrin subunit Ab in situ in at least one report (46), indicating that these cells express alpha 3-integrin to some degree, further studies will be necessary to distinguish among these possibilities.

alpha 3beta 1-Integrin receptors are expressed in a variety of epithelial cells, including keratinocytes, and in smooth muscle and connective tissue. Although many of the known integrin subunits can associate with multiple molecular partners, alpha 3-integrin subunits have only been shown to form heterodimers with beta 1-integrin subunits to form functioning integrin receptors. Conversely, beta 1-integrin subunits, which are expressed ubiquitously, can associate to form integrin receptors with at least 10 different alpha -subunits. In the current study, we have shown that the alpha 3-integrin subunit associates with its beta 1-integrin subunit partner in AECs by demonstrating reciprocal coprecipitation (Fig. 3, A and B). This does not preclude the association of beta 1-integrin subunits with other alpha -integrin subunits such as the alpha 5-integrin subunit shown to be present in AECs (38) or the currently less likely association of alpha 3-integrin subunits with other as yet unidentified beta -integrin subunits. The likely association of beta 1-integrin subunits with other alpha -integrin subunits is supported by the fact that relatively little alpha 3-integrin subunit appears to have been recovered by immunoprecipitation with an anti-beta 1-subunit Ab (Fig. 3A), whereas the anti-alpha 3- and anti-beta 1-subunit Abs precipitate similar amounts of beta 1-subunit protein (Fig. 3B). This result may be a consequence of the fact that most or all of the alpha 3-integrin subunit present in heterodimers is paired with a beta 1-integrin subunit, whereas a substantial fraction of the beta 1-integrin subunit present in heterodimers is paired with an alpha -integrin subunit other than alpha 3-integrin. The apparent lack of efficiency with which the beta 1-integrin subunit coprecipitates the alpha 3-integrin subunit may therefore be due, at least in part, to the fact that much of the alpha -integrin subunit coprecipitated by the anti-beta 1-subunit Ab is actually some other alpha -subunit isoform that remains undetected on the immunoblot.

The alpha 3beta 1-integrin receptor is best known as a mediator of cell-matrix attachment. In this study, decreased adhesion by AECs during monolayer formation in the presence of alpha 3-integrin subunit blocking Abs (shown to specifically associate with the alpha 3-integrin subunit in rat AECs by immunoprecipitation studies; Fig. 4) was demonstrated in several different ways. First, AECs cultured in the presence of anti-alpha 3-integrin subunit Ab with blocking activity show markedly fewer adherent cells at 24 h compared with those plated with IgG or no Ab (Fig. 5). Second, AT2 cells plated from day 0 in the presence of blocking Ab show a delay in the development of electrically resistive monolayers (i.e., development of TER > 1,000 Omega  · cm2) relative to untreated controls (Fig. 6). Although control monolayers develop high electrical resistance by day 3 as previously reported (9), treated monolayers fail to show any measurable TER at the same point. Third, epifluorescence images of propidium iodide-stained nuclei from day 3 monolayers show large patches of bare polycarbonate filter in treated versus untreated AECs (Fig. 7). These bare areas could result either from failure of cells to attach (and secrete appropriate ECM components) or from suboptimal adhesion to the ECM and/or adjacent AECs, with subsequent loss during fixation and processing. The large defects created in the monolayers provide ample explanation in either case for the lack of electrical resistance, which depends on AEC confluence and the formation of functional tight junctions between cells. Finally, SEM images of treated versus untreated monolayers on day 3 confirm the lack (or loss) of cell adhesion to the underlying substratum (Fig. 8). Although the precise mechanism by which cell attachment is impaired cannot be determined from these images, the sloughed appearance of some of the cells on this and other similar images (data not shown) suggests that a relative lack of adhesiveness rather than a complete inability to attach to the substratum may account for the delay in development of electrically resistive monolayers.

After day 3, the gradual development of TER > 1,000 Omega  · cm2 by AEC monolayers treated with the alpha 3-integrin subunit blocking Ab can be explained in several ways. As shown in Fig. 2, expression of the alpha 3-integrin subunit, and presumably of the alpha 3beta 1-integrin receptor, increases from day 1 through day 8. ECM components serving as integrin ligands are continuously secreted by the cells after attachment and promote further anchoring of the cells to the substratum (17). Therefore, part of the loss of effect of the blocking Ab may simply be due to the stoichiometric increase in alpha 3beta 1-integrin and ECM ligands relative to the amount of Ab present. Alternatively, expression of other integrin receptors capable of binding the same or additional matrix components may gradually assume the functions of alpha 3beta 1-integrin. Several other integrin receptors, including alpha 2beta 1, alpha 5beta 1 and alpha Vbeta 3, that collectively bind to a wide variety of matrix proteins have been described in adult AECs (23, 24, 38). The lack of effect of blocking Ab when it is added after the development of TER in untreated monolayers (data not shown) is compatible with either of these explanations and suggests that both may be operative.

Little information is currently available concerning specific functions of the alpha 3beta 1-integrin receptor in adult alveolar epithelium. Transforming growth factor-beta (TGF-beta ) is shown to be increased in alveolar lining fluid during inflammatory reactions of the lung and to be present in AECs of developing lungs and hyperplastic type II cells during repair. In 1997, Kim and Yamada (25) suggested that growth factors such as TGF-beta induce an increase in ECM and enhance the ability of the cells to respond to this increase. Surprisingly, Kumar et al. (29) demonstrated downregulation of alpha 3-integrin subunit expression in the AT2-derived cell line LM5 after treatment with TGF-beta 1, although expression of alpha 6- and beta 1-integrin subunits (which form an integrin receptor that binds laminin similar to alpha 3beta 1-integrin receptor) concurrently increased. Thus although growth factor-induced regulation of alpha 3-integrin could be shown to occur in this study, a specific role for this integrin in lung inflammation and repair could not. In contrast, the strong correlation between the loss of alpha 3-integrin expression and lung cell dedifferentiation, tumorigenesis, and metastasis suggests that the presence of this ECM receptor is at least one of many obligatory components of a nonmalignant phenotype in AECs (1, 2, 14, 20, 26, 33, 36, 43).

The adhesive function of the alpha 3beta 1-integrin receptor in alveolar epithelium may be important during development as well as for normal cell turnover and repair from injury in the adult. Several studies (6, 7, 12) have demonstrated the presence of alpha 3-integrin in fetal lung epithelium, including developing alveolar epithelium. Fetal distal lung epithelium expresses alpha 3-integrin subunit that has been shown to bind to laminin in vitro (7). alpha 3-Integrin-deficient mice survive to birth but die shortly thereafter due to severe defects in kidney and lung organ formation (27). The latter appears to be due to a relative failure of branching morphogenesis rather than from any abnormality of the alveolar epithelium, although perinatal mortality of the alpha 3-integrin-deficient mice precludes any evaluation of the role of this integrin in adult AEC biology in this model. Further insight into the role of the alpha 3beta 1-integrin receptor in alveolar epithelium during development and in the adult may be possible due to the availability of mice deficient in multiple integrin subunits and/or conditional knockouts that allow for normal lung development through the perinatal period.

In addition to its role as an adhesion molecule in AECs, alpha 3beta 1-integrin may serve a variety of cellular functions as it does in other cell types. alpha 3beta 1-Integrin has been shown to modulate the assembly of pericellular matrices via its effects on the rate of ECM component secretion (47), increase matrix turnover by activation of matrix metalloproteases and phagocytosis of the matrix (3, 11, 28), regulate epithelial cell surface polarity (34), and effect cell proliferation (18). Given its ability to activate multiple cellular signaling pathways (41), it is likely that the alpha 3beta 1-integrin has numerous effects on AEC biology that remain to be identified.

In summary, we have identified the alpha 3beta 1-integrin receptor in AECs grown in primary culture. Inhibition of integrin-ECM interaction with an Ab with blocking activity transiently reduced the ability of AECs to form confluent, electrically resistive monolayers, indicating a role in cell-substratum adhesion for this receptor. Further studies will be necessary to define the involvement of the alpha 3beta 1-integrin receptor in both fetal and adult alveolar epithelial development, in lung injury and repair, and in the pathogenesis of lung neoplasia.


    ACKNOWLEDGEMENTS

We thank Dr. Edward D. Crandall for thoughtful encouragement and support of this work. We note with appreciation the expert technical support of Martha Jean Foster and Susie Parra.


    FOOTNOTES

This work was supported in part by the American Heart Association and American Heart Association-Western States Affiliate; the American Lung Association; the Baxter Foundation; National Heart, Lung, and Blood Institute Research Grants HL-03609, HL-38578, HL-38621, HL-51928, and HL-62569; and the Hastings Foundation.

Address for reprint requests and other correspondence: R. L. Lubman, Division of Pulmonary and Critical Care Medicine, GNH 11900, Univ. of Southern California, 2025 Zonal Ave., Los Angeles, CA 90033 (E-mail: rlubman{at}hsc.usc.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 18 June 1999; accepted in final form 10 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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