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Published ahead of print on February 14, 2003, doi:10.1165/rcmb.2002-0196OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 98-105, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0196OC

Identification of Three Genes of Known Function Expressed by Alveolar Epithelial Type I Cells

Renli Qiao, Beiyun Zhou, Janice M. Liebler, Xian Li, Edward D. Crandall and Zea Borok

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

Address correspondence to: Renli Qiao, M.D., Ph.D., Division of Pulmonary and Critical Care Medicine, University of Southern California, IRD 602, 2020 Zonal Avenue, Los Angeles, CA 90033. E-mail: rqiao{at}usc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To identify genes of known function expressed by type I (AT1) cells, changes in gene expression during transdifferentiation of alveolar epithelial cells (AEC) in primary culture from type II (AT2) to type I–like cell phenotype were evaluated. Total RNA from AEC on Day 0 or Day 8 was hybridized to a rat microarray for screening. Eight upregulated genes on Day 8 were selected for further investigation. Northern analysis confirmed upregulation of three of these genes, PAI-1, P2X4, and P15INK4B. The corresponding proteins were evaluated in cultured AEC and results correlated with expression in AT1 cells. In AEC monolayers, all three proteins increased between Day 1 and Day 8. In mixed populations of freshly isolated rat lung cells, concurrent labeling with the AT1 cell-specific antibody, VIIIB2, localized these proteins to AT1 cells. In whole lung, all three proteins were detected in alveolar epithelium in a location consistent with expression in AT1 cells. Identification of novel AT1 cell genes of known function suggests an active role for AT1 cells in alveolar homeostasis. Furthermore, expression of these gene products in AT1-like cells, in freshly isolated AT1 cells, and AT1 cells in whole lung indicates that AT1-like cells reflect many of the properties of AT1 cells in situ.

Abbreviations: alveolar epithelial cells, AEC • aquaporin-5, AQP5 • fluorescein isothiocyanate, FITC • immunofluorescence microscopy, IFM • phosphate-buffered saline, PBS • reverse transcriptase–polymerase chain reaction, RT-PCR • sodium dodecyl sulfate, SDS • saline sodium citrate, SSC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The alveolar epithelium is comprised of two morphologically distinct cell types: cuboidal type II (AT2) cells containing lamellar bodies and located at the corners of the alveolar spaces, and large, flattened type I (AT1) cells with expansive cytoplasmic processes that cover the majority of the gas exchange surface of the lung (1, 2). AT2 cells have been extensively characterized with respect to their roles in surfactant production and active ion transport (3, 4). In the adult, AT2 cells are also believed to serve as the progenitors for replacement of both AT2 and AT1 cells during normal maintenance and repair of the alveolar epithelium following injury (5, 6).

Despite the fact that AT1 cells cover the majority of the internal surface area of the lung and play a presumptive role in gas exchange, their functional properties are less well characterized. With recent improvements in techniques for purification of AT1 cells and immunostaining, evidence is accumulating to support an active role for AT1 cells in aspects of lung function besides gas exchange (7, 8). For example, recent studies in isolated AT1 cells and AT1 cells in situ demonstrate that AT1 cells express functional Na transport proteins, including Na+,K+-ATPase, and thereby may contribute to fluid clearance from alveolar spaces (7).

Due to difficulty in isolating AT1 cells of high yield and purity, AT2 cells grown in culture for several days have been used as a model with which to study AT1-like cell properties. Over a period of 3–4 d, isolated rat AT2 cells gradually lose their characteristic hallmarks, and change morphologically to resemble AT1 cells (9, 10, 11). Concurrently they acquire a number of phenotypic markers specific for AT1 cells in situ, including aquaporin-5 (AQP5) and T1{alpha}/RTI40 (12, 13), as well as reactivity with the AT1 cell-specific monoclonal antibody (Ab) VIIIB2 (11), suggesting that these cells are transdifferentiating toward a type I cell-like phenotype (AT1-like cells) resembling the situation in vivo.

Although useful for identification of the AT1 cell phenotype, available AT1 cell markers have provided limited insight into AT1 cell functions. Identification of additional AT1 cell genes would be helpful in defining potentially novel AT1 cell functions. To identify candidate type I cell genes of known function, we performed a single microarray analysis to screen for genes upregulated during transdifferentiation of AT2 cells in primary culture toward an AT1 cell-like phenotype. We selected eight apparently upregulated genes for further validation (Table 1), based on their known functions and potential relevance to AT1 cell biology. Upregulation of expression in AT1-like cells with time in culture was confirmed by Northern and Western analyses and immunofluorescence microscopy (IFM). Three genes (plasminogen activator inhibitor [PAI-1], P2X purinoceptor 4 [P2X4], and cyclin-dependent kinase 4 inhibitor 2B [P15INK4B]) were identified that are upregulated in AT1-like cells. Expression of all three upregulated proteins in both isolated AT1 cells and AT1 cells in situ confirms the usefulness of this approach for identification of additional type I cell genes.


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  		TABLE 1 Eight genes studied by Northern/Western analysis

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Isolation and Culture
AT2 cells were isolated from the lungs of adult male Sprague-Dawley rats (150–200 g) by disaggregation with elastase (2.0–2.5 U/ml; Worthington Biochemical, Lakewood, NJ), followed by panning on IgG-coated bacteriologic plates as previously described (9, 14). Enriched AT2 cells were plated in defined serum-free medium onto tissue culture–treated polycarbonate (Nuclepore) filter cups (Transwell; Corning-Costar, Cambridge, MA) at a density of 1 x 106 per cm2 and grown to confluent high-resistance monolayers (9, 10). Media were changed on the second day after plating and every other day thereafter. Cells were maintained in a 5% CO2 incubator at 37°C. AT2 cell purity (> 85%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid. Cell viability (> 95%) was measured by trypan blue dye exclusion.

Mixed alveolar cell populations that included AT1 cells were isolated using several modifications of previously described methods for AT2 and AT1 cell isolation (7–9, 14). Briefly, lungs from adult male Sprague-Dawley rats (250–300 g) were perfused via the pulmonary artery with RPMI-1640 containing 25 mM HEPES (solution A). Lungs were lavaged with phosphate-buffered saline (PBS; pH 7.2) containing 5 mM each of EDTA and EGTA, and then filled with 10 ml of solution B (solution A with 5% dextran) containing elastase at 8 U/ml (Worthington) and collagenase (0.1%; Worthington) at 37°C for a total of 40 min. Lung tissue was dissected away from large airways and chopped in solution B containing 20% fetal bovine serum and DNase (3 mg/ml; Sigma, St. Louis, MO). Lung fragments were agitated by end-over-end rotation and filtered sequentially through 100- and 40-µm filters (BD Labware, San Jose, CA) and 20-µm Nitex mesh (Tetko, Elmsford, NY). Cytocentrifuged preparations of these mixed alveolar cell populations were processed for IFM.

RNA Extraction
Total cellular RNA was extracted from freshly isolated AT2 cells, from AEC at different times in culture, and from whole rat lung by the acid phenol-guanidinium-chloroform method of Chomczynski and Sacchi (15).

cDNA Array Hybridization
cDNA array analysis was performed with an Atlas rat 1.2 expression array according to the manufacturer's manual (BD Biosciences Clontech, Palo Alto, CA; http://www.clontech.com/techinfo/manuals/PDF/PT3140-1.pdf). Briefly, RNA samples harvested from freshly isolated AT2 cells adhered for 1 h to remove macrophages, and from AEC maintained in culture for 8 d, were reverse transcribed using the cDNA Synthesis Primer Mix and [{alpha}32P]dATP and hybridized to two separate arrays. Each array was spotted with ~ 10 ng of sequence-verified gene-specific cDNA fragments representing 1,185 known genes, of which 9 encoded housekeeping genes. The arrays were prehybridized in ExpressHyb Solution (BD Biosciences Clontech) containing 100 µg/ml heat-denatured salmon sperm DNA at 68°C for 30 min. Radioactively labeled probe mix was heat-denatured and then added directly to the prehybridization solution to attain a final probe concentration of 6 x 106 counts/min. Arrays were hybridized overnight at 68°C. After hybridization, membranes were washed three times using 2x saline sodium citrate (SSC; 75 mM NaCl, 7.5 mM sodium citrate, pH 7.0) with 1% sodium dodecyl sulfate (SDS) at 68°C for 30 min, followed by washing in 0.1% SSC–0.5% SDS at 68°C. Membranes were then rinsed with 2x SSC for 5 min by continuous agitation at room temperature and exposed to a Storm 860 phosphorimager (Amersham Biosciences, Arlington, IL) for 3 d. Data were analyzed using AtlasImage (BD Biosciences Clontech) to determine both ratio and difference values between the two arrays after global normalization to the averaged intensities of all nine housekeeping genes.

Reverse Transcriptase–Polymerase Chain Reaction
One microgram of rat lung RNA was reverse transcribed with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) after treatment with DNase I (DNA-free; Ambion, Austin, TX) to remove residual DNA. cDNA synthesis was at 42°C for 50 min. DNA was amplified with pfu polymerase (Stratagene, Kingsport, TN) for 30–35 cycles using gene-specific primer pairs for eight genes that were upregulated by cDNA array on Day 8. Sequences of forward (F) and reverse (R) primers and annealing temperatures for each of the eight upregulated genes are shown in Table 1. PCR products were cloned into TOPO cloning vector (Invitrogen) and inserts verified by restriction enzyme mapping and sequencing. The inserts were then used to generate DNA probes for Northern analysis.

Northern Analysis
Aliquots of RNA (10 µg) extracted from freshly isolated AT2 cells or from AEC grown on filters for 1 or 8 d were denatured with formaldehyde, size-fractionated by agarose gel electrophoresis under denaturing conditions, and transferred to nylon membranes (Hybond N+; Amersham-Pharmacia Biotech, Buckinghamshire, UK). Membranes were visualized under ultraviolet light to detect 28S and 18S rRNA to ensure equal loading. RNA was immobilized by ultraviolet crosslinking. Blots were prehybridized for 2 h in Church and Gilbert buffer (1 M Na phosphate buffer, pH 7, 7% SDS, and 1% bovine serum albumin). Hybridization was performed for 16 h at 65°C in the same buffer. Probes were labeled with [{alpha}32P]dCTP (Amersham) by the random primer method using a commercially available kit (Roche Molecular Biochemicals, Indianapolis, IN). Blots were washed at high stringency and signals were detected with a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Antibodies
Polyclonal rabbit anti-rat AQP5 and polyclonal rabbit anti-rat P2X4 antibody (Ab) and blocking peptide were from Chemicon (Temecula, CA). Synthetic rat PAI-1 peptide and polyclonal rabbit anti-rat PAI-1 Ab were from American Diagnostics (Greenwich, CT). Polyclonal rabbit anti-P15 Ab with blocking peptide were from Research Diagnostics (Flanders, NJ). VIIIB2 is a murine monoclonal Ab to rat AT1 cells generated in our laboratory (11). It recognizes an epitope in the apical membrane of AT1 cells that is not detectable in other rat lung or non-lung cells. MF20, a monoclonal Ab to chicken skeletal muscle myosin heavy chain, was a gift from Don Fischman (Cornell University) and was used as a negative control for VIIIB2 in double-labeling experiments. HRP-conjugated anti-rabbit Ab for Western blotting was from Promega (Madison, WI). For IFM in isolated cells and AEC, signal was amplified with biotinylated anti-rabbit IgG followed by fluorescein isothiocyanate (FITC)- or Texas Red-conjugated avidin (Vector, Burlingame, CA). Anti-mouse secondary Abs conjugated to FITC or Texas Red used for IFM were from Jackson ImmunoResearch Laboratory (West Grove, PA).

Western Blotting
SDS-PAGE was performed using the buffer system of Laemmli (16) and immunoblotting was performed using procedures modified from Towbin and coworkers (17). AEC grown on polycarbonate filters for 1 or 8 d were rinsed with cold PBS (pH 7.2) and then solubilized directly into SDS sample buffer (2% SDS, 10% glycerol, 10% ß-mercaptoethanol, pH 6.8). Protein concentrations were measured using the DC Protein Analysis System (BioRAD, Hercules, CA). Equal amounts of cell protein in sample buffer were resolved by SDS-PAGE and electrophoretically blotted onto Immobilon-P membranes (Millipore, Bedford, MA). Membranes were blocked in 5% nonfat dry milk and incubated with primary Abs (1:200 for anti-P2X4 and PAI-1, 1:100 for anti-P15, and 1:1,000 for anti-AQP5) at 4°C overnight. Blots were washed with TBS-T (20 mM Tris-7.5, 0.5 M NaCl, 0.01% Tween-20), and incubated with horseradish peroxidase–conjugated secondary Ab for 45 min at room temperature. Antigen–Ab complexes were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ).

IFM
All samples for IFM were fixed with 4% paraformaldehyde. For P15 staining, the cells were permeabilized with Tween-20 for 10 min. Concentrations for each of the primary Abs were: anti–PAI-1 1:100, anti-P2X4 1:100, and anti-P15 1:50. After immunologic reaction (see below), slides were mounted in Vectashield antifade mounting medium with 4',6-diamidino-2-phenylindole (DAPI, blue) or propidium iodide (PI, red) for nuclear staining (Vector). Slides were viewed with an Olympus BX60 microscope equipped with epifluorescence optics (Olympus, Melville, NY). Images were captured separately in monochrome with filters for DAPI, FITC, or rhodamine isothiocyanate using a cooled charge-coupled device camera (Magnafire; Olympus) or with a confocal microscope (Nikon Eclipse, TE 300; Nikon, Melville, NY) equipped with a Nikon PCM2000 laser scanner and SimplePCI Imaging System 4.0 (Compix, Cranberry Township, PA). For co-localization, monochrome images of the same field were pseudocolored and superimposed on each other. Images were imported into Adobe Photoshop (Adobe Systems, Mountain View, CA) as TIFF files.

IFM in AEC Monolayers
After incubation with CAS block (Zymed, South San Francisco, CA) for 30 min at room temperature, monolayers were incubated at 4°C overnight with primary Abs in CAS, followed by washing with TBS-T. Monolayers were incubated with biotinylated secondary Abs at 1:250 in CAS at room temperature for 45 min, followed by washing with TBS-T. Finally, monolayers were incubated with FITC-conjugated avidin at 1:400 for 45 min, followed by washing in TBS-T. Rabbit IgG at the same concentrations as the primary Abs was used as a negative control.

IFM in Freshly Isolated Lung Cells
Cytocentrifuged preparations of mixed alveolar cell populations were processed for IFM following fixation in 4% paraformaldehyde. Slides were blocked with CAS for 45 min at room temperature and incubated in primary rabbit Abs, followed by washing with TBS-T. Slides were then incubated with biotinylated secondary anti-rabbit Abs at 1:400. VIIIB2 was mixed with the secondary anti-rabbit Abs for identification of AT1 cells. After washing, slides were incubated with a mixture of FITC-conjugated avidin at a final concentration of 1:400 and Texas Red–conjugated anti-mouse Ab at a final concentration of 1:200. Rabbit IgG and MF20 were substituted for rabbit polyclonal Abs and mouse monoclonal Ab VIIIB2, respectively, as negative controls.

IFM of Rat Lung Sections
Normal rat lung was inflated and fixed in 4% paraformaldehyde following perfusion with PBS to remove red blood cells, and airway lavage to remove macrophages. Lungs were embedded in paraffin and 4-µm-thick sections were cut. After deparaffinization and rehydration, the slides underwent microwave antigen retrieval (Antigen Unmasking Solution; Vector). The lungs were processed with Catalyzed Signal Amplification Kit according to the manufacturer's protocol (DAKO, Carpinteria, CA), except in the final step FITC-conjugated avidin replaced alkaline phosphatase–conjugated avidin for signal detection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Microarray Analysis
We detected changes in the expression of 156 genes in AT1-like cells following transdifferentiation of AT2 cells. Among the genes that were apparently upregulated in AEC on Day 8, eight with potential functional relevance to AT1 cell biology were selected for validation and further characterization (Table 1). Because microarray was performed only once for use as a screening tool, the full microarray analysis is not included here.

Reverse Transcriptase–Polymerase Chain Reaction and Northern Analysis of Upregulated Genes in AEC
To validate array results by Northern analysis, we generated cDNA probes by reverse transcriptase–polymerase chain reaction (RT-PCR) using RNA samples from whole rat lung as template. Using the gene-specific primer pairs listed in Table 1, we successfully generated cDNA for seven of the eight selected genes. Despite its presence on the array, CBLP was not detected by RT-PCR in whole lung RNA, indicating that it is not expressed in significant amounts in lung. Sequence of the amplified fragments was identical with the published sequences for these genes.

Although KCHNL and ST3 were detected by RT-PCR in whole lung and the identities of the cloned inserts were confirmed by DNA sequencing, neither KCHNL nor ST3 mRNA were detected using these inserts as probes in either AT2 or AT1-like cells by Northern blotting (data not shown). Therefore, in alveolar epithelial cells, ST3 and KCHNL mRNAs appear not to be expressed in significant amounts. Although both BMP4 and ERK1 were detectable by Northern blot in mRNA derived from AEC, changes in expression could not be confirmed by Northern blot. Levels of expression were similar in AT1-like cells compared with AT2 cells (data not shown), indicating that expression of these two genes is not changed during the process of transdifferentiation.

Northern blotting confirmed upregulation of mRNA levels of PAI-1, P2X4, and P15 in AT1-like cells (Day 8) compared with freshly isolated AT2 cells (Day 0) or AEC on Day 1 (Figure 1). PAI-1 mRNA was not detected in AT2 cells or on Day 1 but was present as a single 3.5-kb band in AT1-like cells. P2X4 and P15 mRNA were both detected as single bands of 1.4 and 1.3 kb, respectively, and the density of the bands increased markedly in AT1-like cells compared with AT2 cells and AEC on Day 1.



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  		Figure 1. Northern analysis of upregulated genes in AEC. These representative Northern blots demonstrate an increase in mRNA for PAI-1, P2X4 and P15INK4B in AT1-like cells (Day 8) compared with freshly isolated AT2 cells (Day 0) and AEC on Day 1.

 
Western Analysis
To confirm that upregulation of the three genes correlated with increased expression of the corresponding proteins, protein samples from AEC on early and late days in culture were analyzed by Western blot. PAI-1, P2X4, and P15 proteins were detected in AECs with higher levels of expression in AT1-like cells compared with AEC on Day 1. The same protein samples were also analyzed by Western blotting for the AT1 cell marker AQP5. AQP5 expression was detected only in AEC on Day 8 in culture, confirming that AT2 cells had transdifferentiated toward the AT1-like phenotype between Day 1 and Day 8 as previously reported (7, 12) (Figure 2).



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  		Figure 2. Western analysis of upregulated proteins in AEC. Representative Western blots demonstrate an increase in PAI-1, P2X4, and P15INK4B protein in AEC on Day 8 (AT1-like cells) compared with AEC on Day 1. PAI-1 is detected as a doublet at 45–50 kD and its expression is higher in AT1-like cells than on Day 1 (PAI-1). Synthetic rat PAI-1 protein is shown on the right as positive control. P2X4 is detected as a protein core (45 kD) and a glycosylated form (~ 66 kD), both of which are hardly visible in Day 1 but increase markedly in AT1-like cells (P2X4). P15 is a single band at 15 kD, and its expression increased in AT1-like cells compared with AEC on Day 1 (P15). The type I cell phenotypic marker AQP5 (27 kD) is detected only in AEC on Day 8, consistent with transdifferentiation of AEC from AT2 to AT1 cell-like phenotype.

 
PAI-1 was expressed as a doublet of ~ 40–50 kD, consistent with previous reports of PAI-1 expression in AEC (18, 19). Representative Western blot (n = 3) demonstrates that low levels of PAI-1 protein were present on Day 1 but increased markedly in AT1-like cells on Day 8 (Figure 2, PAI-1). A positive control using synthesized PAI-1 protein confirmed the size of the PAI-1 protein bands. When PAI-1 Ab was premixed with excessive synthetic PAI-1 peptide, both bands were competed out (data not shown).

P2X4 proteins consist of a 45-kD peptide core and a 65-kD glycosylated form, both of which have been reported in brain and endothelial cells (20, 21). Representative Western blot (n = 3) demonstrates minimal P2X4 expression on Day 1, with clear bands of appropriate sizes in AT1-like cells on Day 8 (Figure 2, P2X4). Both bands were competed out by pre-incubation of Ab with 5-fold excess blocking peptide (data not shown) demonstrating Ab specificity.

P15 was detected as a single 15-kD band. P15 was detected in AEC on Day 1 but, as shown in this representative Western blot (n = 3), levels were much higher in AT1-like cells than AT2 cells (Figure 2, P15). When the primary antibody was premixed with 5-fold excess blocking peptide, this band was competed out (data not shown), demonstrating Ab specificity.

Localization by IFM in AEC Monolayers
Because PAI-1, P2X4, and P15 were all upregulated on Day 8 by Western blot, we undertook localization of all three proteins by IFM in AEC on early and late days in monolayer culture. Using this approach, all three proteins were present in much higher levels in AT1-like cells on Day 8 compared with Day 1 (Figure 3). Due to its intracellular distribution, PAI-1 was detected with a confocal microscope as a punctate cytoplasmic signal (Figure 3, upper panel). P2X4 was more homogenously distributed, consistent with surface expression of a receptor protein (Figure 3, middle panel). P15 protein was preferentially distributed in a perinuclear location (Figure 3, lower panel). Staining patterns were homogenously distributed throughout the monolayer, indicating high purity of cells and homogeneous upregulation of the proteins of interest within the monolayers.



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  		Figure 3. Immunofluorescence microscopy of AEC monolayers grown on polycarbonate filters. Cells were stained with biotinylated Abs for PAI-1 (upper panels), P2X4 (middle panels), and P15 (lower panels) in AEC on Day 1 and Day 8 (AT1-like cells) and detected with FITC-conjugated avidin (green). Nuclei are labeled with propidium iodide (red). All three proteins are markedly upregulated in AEC on Day 8 compared with Day 1. Negative controls using rabbit IgG are not reactive (data not shown). Each image is representative of three separate experiments. Original magnification: x400.

 
Localization of PAI-1, P2X4, and P15 by IFM in Freshly Isolated AT1 Cells
To confirm that observations in AT1-like cell monolayers were representative of AT1 cells in vivo, results in AEC monolayers were correlated with findings in freshly isolated AT1 cells within mixed alveolar cell populations. Alveolar cell populations isolated as described above comprise a crude mixture of macrophages, AT1, and AT2 cells of approximately equal numbers (7). To localize each of the three target proteins in an AT1 phenotype–specific manner, we labeled cells concurrently with Abs for each of the three proteins and with the AT1 cell-specific monoclonal Ab, VIIIB2. As shown in Figure 4, representative double-label IFM images demonstrate co-localization of the AT1 cell marker VIIIB2 (green), with PAI-1, P2X4, and P15 (red).



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  		Figure 4. Immunofluorescence microscopy of freshly isolated rat lung cells. AT1 cells are identified by the AT1 cell-specific mAb VIIIB2 (green). Cells were concurrently labeled with primary Abs (red) against either PAI-1 (upper panels), P2X4 (middle panels), or P15 (lower panels). Nuclei are labeled with DAPI (blue). Each of the three target proteins co-localizes with VIIIB2 staining, consistent with expression in AT1 cells. Each image is representative of three separate experiments. Original magnification: x400.

 
Localization of PAI-1, P2X4, and P15 by IFM in Whole Lung
IFM was undertaken to confirm expression of PAI-1, P2X4, and P15 in whole lung. As shown in Figure 5, all three proteins were detected in alveolar epithelium in a location consistent with expression in AT1 cells. No reactivity was seen with substitution of rabbit IgG for the primary Abs. High power views of the same lung sections are shown as insets for each condition (Figure 5).



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  		Figure 5. Immunofluorescence microscopy in whole lung sections. Abs to either PAI-1, P2X4, or P15 are reactive (green) with cells in the alveolar septae, in a location consistent with expression in AT1 cells. Nuclei are labeled with propidium iodide (red). Rabbit IgG (negative control) showed only faint autofluorescence. Each image is representative of three separate experiments. Original magnification: x400. Higher power (x1,000) views are shown as insets.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used cDNA array analysis to screen for expression of genes with known function that were upregulated in AEC in primary culture that had transdifferentiated toward an AT1 cell phenotype to identify biological properties of AT1 cells. Eight upregulated genes identified in AT1-like cells on day 8 (Table 1) were selected for validation by other methods and for further exploration based on their known functions or their potential involvement in processes relevant to AT1 cell biology (e.g., ion transport (P2X4, KChNL), signal transduction (ERK1, CBLP, BMP4), cell cycle progression (P15INK4B), matrix remodeling (ST3) and lung injury (PAI-1)) (22–28). Upregulation of three of these genes and their corresponding proteins, PAI-1, P2X4, and P15INK4B, was confirmed by Northern and Western analyses in AEC in primary culture on Day 8 compared with freshly isolated AT2 cells or AEC on Day 1. Concurrent labeling of cytocentrifuged preparations using an AT1 cell-specific marker, VIIIB2, and specific Abs to each of these proteins confirmed their expression in freshly isolated AT1 cells. All three proteins were also detected in alveolar epithelium in situ in a location consistent with expression in AT1 cells. Identification in AT1 cells of three genes of known function suggests an active role for AT1 cells in alveolar homeostasis, commensurate with the large proportion of the alveolar surface that they cover.

Investigation of the functions of AT1 cells in the lung and their potential contribution to alveolar homeostasis has been limited by the inability to routinely isolate AT1 cells of high yield and/or purity and maintain them in culture. Studies in AT1-like cell monolayers derived from AT2 cells after several days in primary culture have provided useful insights into AT1 cell properties. The notion that AT2 cells undergo transdifferentiation toward an AT1-like cell phenotype with time in culture has gained increased acceptance with the demonstration that AT1-like cells express all the known phenotypic markers associated with AT1 cells in situ (9, 11, 13, 14). AT1 cells have also recently been shown to express functional Na transporters (7, 8), confirming previous studies in AT1-like cell monolayers. In the current study, we compared patterns of gene expression in freshly isolated AT2 and AT1-like cells after several days in culture to identify novel AT1 cell-associated genes of known function. Culture conditions were identical to those used in our previous studies in which AT2 cells were shown to progress toward an AT1-like cell phenotype (9, 11). Transdifferentiation toward an AT1-like cell phenotype was confirmed by detection of AQP5 expression by Western blot only in AEC on Day 8, consistent with our previous studies (9, 11). Genes identified initially in AT1-like cells were also identified subsequently in AT1 cells. Using this approach, we identified three different genes of known function that are expressed by AT1 cells. In addition to providing new phenotypic markers for AT1 cells, these findings suggest potential biological functions for AT1 cells. More detailed kinetic analyses of changes in gene expression during the process of transdifferentiation in vitro should prove useful to investigate the hierarchy of regulatory pathways that govern the transition from AT2 to AT1 cell phenotype in vivo.

PAI-1
PAI-1 is a member of the family of serine protease inhibitors (29). It is the major inhibitor of plasminogen activators in plasma and also within the alveolar space, rapidly inactivating both tissue-type and urokinase-type plasminogen activator through formation of enzymatically inactive complexes (29). Suppression of fibrinolytic activity within the alveolar spaces has been demonstrated in a number of acute and chronic inflammatory lung disorders, including the acute respiratory distress syndrome and idiopathic pulmonary fibrosis (30–32) and in animal models of lung injury (24, 33). PAI-1 is a critical determinant of net fibrinolytic activity within the alveolar space, with local increases in PAI-1 promoting fibrotic repair by inhibiting fibrin degradation (31). Consistent with this, transgenic mice that are deficient in PAI-1 are protected from both bleomycin and hyperoxic lung injury (24, 33), whereas mice that overexpress a PAI-1 transgene develop more extensive fibrosis after bleomycin-induced lung injury (33). Although alveolar macrophages have been considered the major cellular source of PAI-1 within the alveolar space (22, 23), AEC have also been shown to synthesize and secrete active PAI-1 in vitro (18, 19). Gross and colleagues evaluated PAI-1 production by AEC in culture, and demonstrated an increase in PAI-1 activity over time (18). Although it was suggested from that study that AEC may serve as an additional source of PAI-1 within the alveolar space in vivo, further localization and confirmation of expression in AT1 cells was not undertaken.

An increase in PAI-1 mRNA in AT1-like cells compared with AT2 cells was demonstrated by Northern blotting using a gene-specific probe. Parallel changes were noted in PAI-1 protein in AT1-like cells using a specific Ab to PAI-1. PAI-1 protein was also localized to isolated AT1 cells, and was detected in alveolar septae in whole lung in a location consistent with expression in AT1 cells. Upregulation of both PAI-1 mRNA and protein in AEC over time in culture are consistent with regulation at a transcriptional level, as has been demonstrated in other systems (29). This differs slightly from the results of Gross and coworkers, in which the increase in PAI-1 activity over time in culture was not accompanied by an increase in mRNA levels (18). Some of the differences from the previous study may be explained by differences in culture conditions used, because in the current study AEC were maintained in serum-free conditions on polycarbonate filters. Given the large surface area of the lung that is lined by AT1 cells, their contribution to PAI-1 activity within the alveolar space is likely significant and suggests an important role for AT1 cells in regulation of fibrinolytic activity on the alveolar surface under normal conditions and following injury.

P2X4
Purinergic receptors are cell surface molecules that mediate the physiologic effects of purine nucleotides (21). P2X receptors are ATP-gated ion channels that act as Ca2+-permeable, nonselective cation channels, whereas P2Y receptors are traditional G protein–coupled receptors that mobilize Ca2+ from intracellular stores (20, 21, 34). P2X receptors have a wide tissue distribution, and multiple P2XR channel isoforms, including P2X4, have been identified in airway epithelial cell primary cultures as well as cell lines derived from both airway and alveolar epithelium (20, 34). P2Y receptors have been identified in AT2 cells, where they are involved in ATP-triggered surfactant secretion (3, 27). However, no data were available on whether purinergic receptors are also present in AT1 cells.

Upregulation of P2X4 in AT1-like cells was demonstrated by Northern analysis using a gene-specific probe. Using a specific Ab to P2X4, P2X4 protein was confirmed to be upregulated during transdifferentiation of AT2 cells toward an AT1-like phenotype, and was also present in freshly isolated AT1 cells and within the alveolar septae in whole lung sections. The functional role of these receptors in AT1 cells remains to be determined, but the recent demonstration that intracellular Ca++ release initiated in AT1 cells can be transmitted to AT2 cells via gap junctions suggests the possibility that signals transduced via P2X receptors in AT1 cells may be similarly transmitted to adjacent AT2 cells (35).

P15INK4B
P15INK4B belongs to the INK4 proteins which specifically inhibit the catalytic subunits of cyclin-dependent kinase 4 (CDK4). When quiescent cells enter the cell cycle, genes encoding D-type cyclins are induced in response to mitogenic signals, and the cyclins assemble with their catalytic partners, CDK4 and CDK6, as cells progress through G1. Thus, the effect of P15INK4B is to arrest cells in early G1 by preventing cyclin D binding (28). In this regard, it has been suggested that TGF-ß–mediated cell cycle arrest may be mediated by induction of P15INK4B transcription and subsequent inhibition of CDK4 and CDK6 kinases (36).

AT2 cells serve as the progenitors for AT1 cells, following lung injury and during normal cell turnover. In contrast to AT2 cells, AT1 cells are incapable of division. Although AT1 cells have been shown to be able to revert to the AT2 cell phenotype under certain culture conditions (9, 12), this has not yet been demonstrated in vivo. The majority of AT2 cells are normally maintained in a quiescent state, and the shift toward a proliferative phenotype (e.g., in response to growth factors [e.g., KGF] and extracellular matrix signals) is mediated by induction of various cyclins and CDKs and suppression of CDK inhibitors (CDKI) (37, 38). The processes that regulate the transition from a cell with proliferative potential (AT2 cell) to a possibly more quiescent phenotype (AT1 cell) are poorly understood (39). Relative levels of expression of cyclins and CDKs on the one hand, and CDKIs on the other, during transition from the AT2 to AT1 cell phenotype has not previously been evaluated. In this study, marked increases of P15 mRNA during transdifferentiation from AT2 to an AT1-like cell phenotype were demonstrated by Northern and Western blotting in AEC monolayers. Expression of P15 protein was also demonstrated in freshly isolated AT1 cells and AT1 cells in situ. These findings suggest that upregulation of P15INK4B during the transition from AT2 to AT1 cell phenotype in vitro and in vivo may contribute to a loss of proliferative capacity of AT1 cells.

In summary, by screening for differences in gene expression between freshly isolated AT2 cells and AEC in primary culture, we have identified three proteins of known function that are expressed in AT1-like cells in a phenotype-specific manner. Furthermore, we have confirmed that all three proteins are also expressed in freshly isolated AT1 cells and AT1 cells in situ, suggesting potentially novel functions for AT1 cells. These findings demonstrate that useful insights regarding AT1 cell properties can be derived from analyses of AT1-like cells in culture. Furthermore, identification of several AT1 cell genes of known function suggests an active role for AT1 cells in alveolar homeostasis.


   Acknowledgments
 
The authors note with appreciation the expert technical assistance of Zerlinde Balverde, Martha Jean Foster, Susie Parra, and Juan Ramon Alvarez. They acknowledge the assistance of Dr. Brad Scherer, BD Clontech Laboratories, for help with microarray analysis. This work was supported by the National Institutes of Health (HL38578, HL38621, HL38658, HL62569, HL64365, and HL72231), American Lung Association of California, American Heart Association (Grant in Aid 9950442N), and the Hastings Foundation. E.D.C. is Hastings Professor of Medicine and Kenneth T. Norris Jr. Chair of Medicine.

Received in original form September 29, 2002

Received in final form February 3, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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