Differential regulation of centrin genes during ciliogenesis in human tracheal epithelial cells

Michel LeDizet, James C. Beck, and Walter E. Finkbeiner

Cardiovascular Research Institute and Department of Pathology, University of California, San Francisco, California 94143-0566

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Centrins are small calcium-binding proteins found in a variety of cell types, often in association with microtubule-organizing centers. Here we present results regarding the expression of centrins during the in vitro differentiation of human tracheal epithelial cells. When grown at an air-liquid interface, these cells differentiate into mucus-secreting cells or undergo ciliogenesis. In immunofluorescence and immunoelectron microscopy experiments, an anti-centrin antibody stained exclusively the basal bodies of the ciliated cells. There was no staining over the axonemes or the striated rootlets. Northern blots and RT-PCR analysis of the three known human centrin genes showed that these genes have distinct patterns of expression during the growth and differentiation of human tracheal epithelial cells. Centrin-1 is never transcribed. Centrin-2 mRNA is present at all times, and its concentration increases when ciliogenesis occurs. Centrin-3 mRNA is found at a constant level throughout the entire process. This differential regulation suggests that centrins are not interchangeable but instead have unique functions.

cellular differentiation; air-liquid interface; respiratory epithelium

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CENTRINS ARE SMALL calcium-binding proteins that belong to the superfamily of calmodulin and yeast CDC31. Originally identified in Tetraselmis (31), centrins have since been found in a wide variety of protozoa, plants, and animals (reviewed in Ref. 20). Immunoreactive centrin has been detected in many different cellular structures, suggesting that centrins may have several distinct functions. Many centrin-containing structures are microtubule-organizing centers or are attached to such centers: centrioles or basal bodies, pericentriolar material, mitotic spindle poles (with or without centrioles), striated contractile fibers extending from one basal body toward another ("distal fibers") or toward the nucleus ("striated rootlets"; also known as "flagellar rootlets") (reviewed in Ref. 20). In addition, centrin is also encountered in the transition zone, linking a basal body to the axoneme (32) as a subunit of Chlamydomonas inner dynein arms (17, 29), and in soluble form (28).

In view of the variety of structures containing centrin, it is not surprising that cells may harbor multiple centrin isoforms. Paoletti et al. (28) showed that 2-dimensional gel electrophoresis can resolve 10 species of immunoreactive centrins in human cells. A critical issue is to determine whether individual centrin isoforms have a specific location within the cell and, more importantly, specific functions.

Many centrin-containing structures are found exclusively in ciliated cells. This prompted us to determine whether ciliogenesis was accompanied by the appearance of specific centrin species. We chose to study centrin expression during the in vitro differentiation of human tracheal epithelial (HTE) cells (43). In this model system of ciliogenesis, tracheal epithelial cells are isolated from autopsy specimens and grown on a collagen layer at an air-liquid interface. The cells rapidly multiply to form a multilayered cell sheet composed of nondifferentiated cells. Subsequently, 60-80% of the apical cells become ciliated. Such a system is well suited to our purpose because most structures expected to contain centrins are encountered. Therefore, a large number of centrin isoforms may be synthesized. Moreover, the time at which centrin isoforms appear during the differentiation process may give us information regarding their function.

Antibodies specific for individual centrin isoforms are not generally available. For this reason, we studied centrin gene expression at the mRNA level. Three human centrin genes have been identified to date: centrin-1 was isolated from a testis cDNA library (6), whereas centrin-2 (originally named caltractin) was found in libraries derived from umbilical vein endothelial mRNA and from T-cell lymphoblastic leukemia mRNA (19). Centrin-3 was recently identified among cDNAs derived from a T-lymphoblastic cell line (24). The centrin-1 and -2 proteins are 84% identical. Centrin-3 is more closely related to yeast CDC31 and is 54% identical to both centrin-1 and centrin-2 (24).

We found that the three centrin genes have very different expression patterns during the differentiation of human tracheal epithelial cells. Centrin-1 is never expressed. Centrin-2 mRNA is always present, and its concentration increases as ciliated cells appear. Centrin-3 mRNA is found at a constant concentration throughout the differentiation process. These results suggest that the three genes encode proteins with distinct functions. Furthermore, centrin-2 is the isoform most likely to be found in cilia-associated structures.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Isolation and Culture

Tracheae were obtained from patients without chronic airway disease up to 24 h postmortem. HTE cells were isolated as previously described (7, 43) and suspended in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mix containing 2% low-protein serum replacement (LPSR-1, Sigma, St. Louis, MO) supplemented with penicillin (105 U/l), streptomycin (100 mg/l), gentamicin (50 mg/l), and amphotericin B (2.5 mg/l). Cell counts and estimates of viability were made with trypan blue and a hemocytometer. Cells were plated onto 12-mm Transwell inserts (0.4-µm pore size; Corning Costar, Cambridge, MA) containing Vitrogen 100 collagen (Celtrix Laboratories, Palo Alto, CA) at a density of 1.67 × 106 live cells/well. The following day, the cultures were rinsed with phosphate-buffered saline (PBS). Throughout the culture period, we maintained the cells in air-liquid interface cultures with medium (1 ml) only on the basal side of the filters. Immersed cultures were grown with medium on both the basal and apical sides of the filters (1 and 0.7 ml, respectively). All cultures were maintained at 37°C in a humidified atmosphere of 95% air-5% CO2.

Morphology

Light microscopy. Support membranes with the attached collagen layer and cell sheet were cut out of the culture inserts and frozen in TissueTek optimum cutting temperature compound (Sakura Finetek). Ten-micrometer sections were cut on a cryostat (Leica CryoCut 1800), transferred to charged glass slides, and stained with toluidine blue or hematoxylin and eosin.

Scanning electron microscopy. Cultures were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, for 1 h at room temperature, followed by two 10-min washes with 0.1 M sodium cacodylate, pH 7.4. Samples were then postfixed at room temperature with 1% osmium tetroxide in 0.1 M sodium cacodylate, pH 7.4, followed by two 10-min washes with 0.1 M sodium cacodylate. The samples were then dehydrated with a graded series of ethanol washes and infiltrated with Peldri II (Ted Pella, Redding, CA). The last change of Peldri II was cooled to below 23°C, and the samples were maintained under vacuum until all Peldri II evaporated. Samples were subsequently sputter coated with an Anatach Hummer X with a gold-palladium alloy and then placed onto scanning electron microscope specimen mounts. The specimens were viewed with a JEOL JSM 840 scanning electron microscope.

Immunocytochemistry

To identify and localize centrin, we used the anti-centrin monoclonal antibody 20H5 (35) generously provided by Dr. Jeffrey L. Salisbury (Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN). This antibody recognizes the proteins encoded by all three human centrin genes (24). The antibody was used at a 1:200 dilution.

Light-microscopic immunocytochemistry. Cultured cells were dissociated in a solution of saline-trypsin-versene (0.05% trypsin and 0.02% EDTA in 0.9% NaCl, wt /vol). Isolated cells were spun onto glass slides with a cytocentrifuge. Indirect immunofluorescence was performed as previously described (8). Briefly, the antibody was diluted in PBS containing 2% normal goat serum-0.6% Triton X-100 and applied to the cells for 2 h at room temperature. Next, the slides were rinsed three times for 5 min with PBS containing 1% normal goat serum-0.3% Triton X-100. The slides were incubated (30 min at room temperature) with goat anti-mouse IgG-fluorescein isothiocyanate diluted 1:40 in PBS. After a final rinse with PBS, they were covered with 1,4-diazabicyclo(2.2.2)octane (DABCO) solution and glass coverslips before examination with a fluorescence microscope.

Electron-microscopic immunocytochemistry. HTE cells were cultured as described in Cell Isolation and Culture on Millicell HA filters (Millipore). The filters were washed two times with PBS and fixed for 60 min with 3% freshly depolymerized paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2. We cut the filters out of their insert and processed them in glass vials to avoid subsequent cross-reaction between the plastic insert and the LR White resin (London Resin, Basingstoke, UK) (25). After three 10-min washes in 0.1 M sodium phosphate buffer (pH 7.2), the cells were dehydrated with a graded series of ethanol washes up to 100% and then slowly infiltrated with increasing concentrations of LR White resin (hard grade) in 100% ethanol up to 100% resin. Next, filters were cut into smaller pieces and placed in oven-dried gelatin capsules that were then filled with fresh resin. Polymerization of the resin was performed at 50°C for 24-48 h to preserve antigenicity (25). Filters were cross-sectioned to pale gold or silver thickness (80-100 nm) and picked up onto formvar-coated or carbon-stabilized 100-mesh nickel grids.

Immunocytochemistry was performed as previously described (43). At each step, the grids were floated section side down on drops of the solutions. Residual free aldehydes remaining from the fixation were inactivated by incubating the grids on drops of 0.1 M glycine in PBS for 30 min (12). Nonspecific immunoreactive binding sites were then blocked with a solution of PBS containing 1% fish gelatin, 1% globulin-free bovine serum albumin (BSA), and 0.02% sodium azide, pH 7.2, for 30 min (3). The grids were subsequently incubated for 60 min with the anti-centrin monoclonal antibody (diluted in PBS-1% BSA-0.02% sodium azide, pH 7.2). Grids incubated in SP 2/O myeloma culture supernatant were included as negative controls. The grids were washed three times for 10 min each in PBS and two times for 10 min each in the PBS-1% BSA-0.02% sodium azide (pH 7.2). To prevent aggregation of the secondary antibody, the sections were brought to pH 8.2 with a 10-min incubation on drops of PBS-1% BSA (globulin free)-0.02% sodium azide (pH 8.2) (37). Then the sections were incubated in 10-nm gold-conjugated goat anti-mouse IgG (Biocell Gold Conjugates, Ted Pella) diluted 1:75 with PBS-1% BSA-0.02% sodium azide (pH 8.2) for 60 min. The grids were then washed six times for 5 min each in a solution of PBS-0.1% BSA (globulin free)-0.02% sodium azide (pH 7.2) and three times for 5 min in PBS (pH 7.2) and dip washed with double-distilled water. The grids were then poststained with saturated aqueous uranyl acetate before they were viewed in the electron microscope.

Molecular Biology Techniques

Genomic DNA isolation. Genomic DNA was isolated from fresh human blood with a DNA isolation kit (Boehringer Mannheim) or with standard procedures.

RNA isolation. Total cellular RNA from the cultured cells was prepared by homogenizing the entire content of a culture insert into 2 ml of TRIzol Reagent (Life Technologies). Yields were 20-50 µg/culture insert. RNA from fresh (uncultured) cells was prepared by homogenizing strips of native tracheal epithelium (obtained 16 h or less postmortem) in TRIzol Reagent.

cDNA synthesis. RNA (2 µg) was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) as recommended by the manufacturer. Genomic DNA contaminating the total RNA was hydrolyzed with RNase-free DNase (0.5 unit of enzyme, 15 min, 37°C; Promega) in RT buffer before the initial denaturation step. The final reaction volume was 25 µl.

Gene-specific DNA probes. The 3'-untranslated region of the centrin-1 gene was amplified from genomic DNA with the primers C1-770 and C1-alpha 1088 (see Table 1). The PCR product was purified from an agarose gel with GeneClean (Bio101), digested with EcoR I and Xho I, and cloned into pBluescript SK(+) (Stratagene). The nucleotide sequence of the subcloned fragment was determined to confirm the identity of the probe.

                              
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Table 1.   PCR primers used in this study

To subclone the 3'-untranslated region of the centrin-2 gene, RNA isolated from HTE cells grown at an air-liquid interface for 21 days was reverse transcribed with Anch1(dT) as a primer (see Table 1). An aliquot of the cDNAs was then amplified with the primers Anch1 and C2-586. The PCR product was subcloned as above.

Northern blot hybridization. At each time point, total RNA was isolated as described in RNA isolation. Polyadenylated RNA was selected by oligo(dT) cellulose chromatography with a rapid mRNA purification kit (Amresco), separated on a formaldehyde-containing agarose gel, and transferred to a GeneScreen membrane (NEN Research Products) with standard procedures. Each lane contained the polyadenylated RNA obtained from five culture inserts (100-150 µg of total RNA). For one experiment (see Fig. 7A), each lane contained 20 µg of total RNA. To produce radiolabeled probes, the inserts of the plasmids described in Gene-specific DNA probes were purified and used in random-primed labeling reactions. The hybridization was performed for 1 h at 68°C in QuickHyb solution (Stratagene). The blots were washed to a final stringency in 0.1× saline-sodium citrate (1× saline-sodium citrate is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). Detection of the bound probe was performed by autoradiography or with a Molecular Dynamics phosphorimager.

PCR amplification of centrin sequences. The sequences of human centrin-1, centrin-2, and centrin-3 cDNAs were retrieved from GenBank (accession nos. U03270, X72964, and Y12473, respectively). The coding regions span nucleotides 49-564 in centrin-1, 48-563 in centrin-2, and 1-504 in centrin-3. We chose regions of greatest heterogeneity to design gene-specific primers. Table 1 contains the sequences of the primers used and their position in the cDNA sequences. We performed PCR reactions in a volume of 30 µl with GeneAmp PCR system 2400 (Perkin-Elmer, Emeryville, CA). Reaction conditions were as recommended by the supplier of the Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). The template consisted of either 1 µl of cDNA synthesis reaction or 0.2 µg of human genomic DNA. After an initial denaturation step of 4 min at 94°C, the samples were subjected to 35 cycles (or less; see Semiquantitative RT-PCR assay for centrin mRNAs) of amplification (20 s at 94°C, 30 s at 60°C, and 60 s at 72°C). Forty cycles of amplification were performed to detect centrin-1 cDNA. When genomic DNA was amplified, we performed 35 cycles with the following parameters: 30 s at 94°C, 30 s at 55°C, and 2 min at 72°C. Some of the amplifications were performed with a Perkin-Elmer DNA thermal cycler 480; for these experiments, the durations of the denaturation and hybridization steps were 45 and 90 s, respectively.

After amplification, aliquots of the PCR reactions (typically 6 µl) were analyzed by electrophoresis on 1× Tris-borate-EDTA agarose gels containing 0.4 µg/ml of ethidium bromide. The gels were photographed under ultraviolet transillumination.

Semiquantitative RT-PCR assay for centrin mRNAs. To compare the abundance of centrin-2 and centrin-3 mRNAs in different samples, we introduced several modifications to our basic protocol. 1) The amount of specific product in a PCR reaction typically increases with the number of cycles until a plateau phase is reached. We found that the plateau phase was reached after 33 cycles for both centrin-2-specific and centrin-3-specific reactions. We therefore only performed 30 cycles of amplification so as to remain within the phase of exponential accumulation of PCR product. 2) We used high initial concentrations of nucleotides (200 µM each) and primers (0.5 µM each) to ensure that they did not become limiting. 3) We lowered the influence of tube-to-tube variation in the PCR product yield by performing each amplification in duplicate, using, if possible, cDNAs synthesized in independent RT reactions. This precaution may also correct possible unequal efficiencies in the RT of individual mRNAs. The data presented here represent the average of two or more independent PCR amplifications. The individual data points are usually within 10% (or less) of the average value. 4) Gel images were recorded and quantitated with an AlphaImager digital-imaging system (Alpha Innotech, San Leandro, CA). The volume of PCR analyzed and the exposure times were chosen to remain within the range of linear response of the charge-coupled device camera. Control experiments showed that, under our conditions, the signal recorded by the imaging system is directly proportional to the amount of DNA being visualized (data not shown).

RT-PCR amplification of glyceraldehyde-3-phosphate dehydrogenase mRNA. Table 1 shows the sequences of primers GAP-811 and GAP-alpha 1198 that are based on the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA sequence (GenBank accession no. M33197). The template consisted of 1 µl of cDNA synthesis reaction. After an initial denaturation step of 4 min at 94°C, the samples were subjected to 24 cycles of amplification (30 s at 94°C, 30 s at 65°C, and 45 s at 72°C). The plateau phase of amplification was reached after 28 cycles. Quantitation of the specific PCR product was performed as described for the centrin transcripts.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Morphological Differentiation of HTE Cells

Yamaya et al. (43) previously described the basic culture conditions allowing growth and differentiation of HTE cells. For this study, we wanted to correlate levels of centrin gene expression with cellular differentiation and, in particular, with ciliogenesis. We therefore monitored the appearance of cilia as a function of time in culture and isolated RNA from the cells at various time points.

In a typical experiment, isolated epithelial cells obtained from a single trachea were seeded in 50-200 culture inserts containing a layer of collagen. They were then grown at an air-liquid interface as described in MATERIALS AND METHODS. After various lengths of time in culture (4-21 days), RNA was prepared from two to five culture inserts while cells from other inserts were fixed and examined microscopically. Because differentiation appears to occur synchronously in all cultures, the cells from which RNA was isolated can be expected to be identical to those that were examined microscopically.

After the cells are plated, they rapidly multiply and form a thin multilayered pseudoepithelium consisting of undifferentiated cells. Figure 1A shows the luminal surface of cells grown for 3 days at an air-liquid interface. No ciliated cells are present. Over the next few days, the cell sheet becomes progressively thicker. Figure 1B shows the luminal surface of a cell sheet where the first ciliated cells are visible together with secretory cells. The length of time preceding the onset of ciliogenesis in the cultured cells varies from trachea to trachea and is typically 13-15 days. Over the 3-4 days after the appearance of the first cilia, the number of ciliated cells increases dramatically. After 18-21 days in culture, transmission electron microscopy shows that the pseudostratified cell sheet is composed of a range of cell types, with taller columnar cells at the luminal surface. Transmission electron micrographs of similar cell sheets have been previously published (43). The columnar cells at the luminal surface are either ciliated epithelial cells or secretory goblet cells. Figure 1C shows that ciliated cells account for 60-80% of the apical surface in fully differentiated cell sheets.


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Fig. 1.   Scanning electron micrographs of cultured human tracheal epithelial cells. Cells were grown at an air-liquid interface (A-C) or immersed (D). A: day 3. No ciliated cells are present. B: day 12. Scattered ciliated cells are present. C: day 20. Luminal surface of cell sheet is composed predominantly of ciliated cells. D: day 20. No ciliogenesis occurs if cells are grown immersed. Bar, 10 µm.

The presence of an air-liquid interface is critical to the appearance of ciliated cells. Figure 1D shows the luminal surface of cells grown immersed for 21 days. No ciliated cells are visible.

Immunolocalization of Centrin

We used the monoclonal antibody 20H5 raised against Chlamydomonas centrin (35) to determine the location of centrin in cultured HTE cells. This antibody recognizes the proteins encoded by all three human centrin genes (24). Immunoperoxidase labeling of cells grown at an air-liquid interface showed staining limited to the apical portion of the ciliated luminal cells (data not shown). No staining was detected in cells grown immersed.

Using immunofluorescence microscopy, we observed an intense punctate staining directly underneath the apical membrane of the ciliated cells (Fig. 2A), a distribution consistent with labeling of the basal bodies. The staining was punctate and in a narrow plane of focus, indicating that axonemes and striated fibers were not stained.


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Fig. 2.   Immunocytochemical detection of centrin. A: immunofluorescence microscopy of ciliated cells obtained by trypsinization and cytocentrifugation of cells grown at an air-liquid interface. Staining is located in apical cytoplasm in a punctate pattern corresponding to distribution of basal bodies. Bar, 20 µm. B: immunogold electron microscopy of a ciliated cell from an air-liquid interface culture. Gold particles are located primarily within basal bodies and transition zones. Ax, axoneme; Bb, basal body; Sf, striated fiber; Tz, transition zone. Bar, 1 µm. C: immunogold electron microscopy of a ciliated cell from native tracheal epithelium shows an identical distribution of centrin. Bar, 1 µm.

Immunoelectron microscopy confirmed that staining is limited to the basal bodies; although the staining was sparse, gold particles were clearly concentrated over the basal bodies and in the transition zone between basal body and axoneme (Fig. 2B). A similar staining pattern was observed in native tracheal epithelial cells (Fig. 2C). There was no staining on the striated fibers that are clearly visible in Fig. 2B. A small number (5) of gold grains can be seen over the axonemes in Fig. 2C. However, their concentration is clearly much lower than that over the basal bodies and transition zones, and they probably represent background staining.

The Centrin-1 Gene Is Not Transcribed in Cultured HTE Cells

We then analyzed the expression of each of the three known human centrin genes to determine which are transcribed during growth and differentiation of HTE cells. Expression of the centrin-1 gene was first analyzed through Northern blot hybridization experiments. To limit cross-reactions with other related centrin genes, we used the 3'-untranslated region of the centrin-1 gene (nucleotides 770 through 1088) as a probe. This probe was obtained by PCR amplification of genomic DNA (see MATERIALS AND METHODS). No signal was detected in Northern blot experiments with RNA isolated from HTE cells cultured at an air-liquid interface or immersed (data not shown). However, the same radiolabeled probe readily detected a single sequence in human DNA in Southern blot experiments. These experiments indicate that, in cultured HTE cells, centrin-1 mRNA is found at a very low concentration, if at all.

We sought to detect centrin-1 transcripts using the more sensitive RT-PCR technique. RNA was prepared from cultured HTE cells and reverse transcribed. cDNA aliquots were then amplified with the primers C1-88 and C1-alpha 587 (see Table 1). Amplification of genomic DNA with these primers yields a 500-bp product (Fig. 3, lane a), the identity of which was confirmed by digestion with the restriction endonucleases Bgl II and Stu I. This is the size expected from amplification of a cDNA, making it impossible to distinguish genuine amplification of cDNAs from amplification of genomic DNA contaminating the RNA preparation. It was therefore essential to treat the RNA with RNase-free DNase before RT. Figure 3, lanes b and c, shows aliquots of PCR reactions performed with template cDNAs derived from HTE cells grown for 21 days either immersed or at an air-liquid interface. No specific product is visible. It should be noted that 40 cycles of amplification were performed, which should allow the detection of even minute amounts of cDNA. In further experiments, no product was obtained by amplification of cDNAs derived from HTE cells cultured for shorter lengths of time (12, 14, and 16 days, immersed and air-liquid interface cultures). We also failed to obtain any PCR amplification product when other centrin-1-specific primers were used (data not shown). These results show that the centrin-1 gene is not transcribed in cultured HTE cells.


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Fig. 3.   PCR amplification of centrin sequences. Primers used were specific for centrin-1 (lanes a-d) or centrin-2 (lanes e-h) genes. Template consisted of genomic DNA (lanes a and e), cDNAs derived from human tracheal epithelium (HTE) cells grown immersed (lanes b and f), cDNAs derived from HTE cells grown at an air-liquid interface (lanes c and g), and cDNAs derived from human testes (lanes d and h). An aliquot of PCR reactions was analyzed by agarose gel electrophoresis in presence of ethidium bromide. Nos. at left, position of DNA molecular-weight markers.

Figure 3, lane d, shows that a PCR product was obtained when human testis cDNA was used as a template. No product was obtained if the enzyme was omitted from the RT reaction, proving that this product is truly derived from a cDNA. This result confirms that the centrin-1 gene is transcribed in human testes (from which its cDNA was isolated) (6).

Centrin-1 Is Not Expressed in Human Tracheal Epithelium

The absence of centrin-1 transcripts could be an artifact of cell culture. We therefore sought to detect centrin-1 mRNA in uncultured human tracheal epithelium. RNA was purified from strips of tracheal epithelium obtained 16 h or less postmortem and reverse transcribed. Forty cycles of amplification with the primers C1-88 and C1-alpha 587 failed to yield any detectable product (Fig. 4, lane a). As a positive control, we used the same reaction mix to amplify 200 ng of genomic DNA. The expected 500-bp product was observed after 34 cycles of amplification (Fig. 4, lane b). Therefore, we conclude that the centrin-1 gene is not transcribed in native, uncultured tracheal epithelial cells.


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Fig. 4.   RT-PCR detection of centrin sequences in fresh (uncultured) human tracheal cells. Primers used were specific for centrin-1 (lanes a and b), centrin-2 (lane c), and centrin-3 (lane d). Samples amplified were genomic human DNA (lane b) and cDNAs derived from native tracheal epithelium (lanes a, c, and d). No. of amplification cycles performed was 40 (lane a), 34 (lane b), 32 (lane c), and 38 (lane d). Nos. at left, position of DNA size markers (in bp).

Centrin-1 Is an Intronless Gene

The result of the amplification of cDNAs with the primers C1-88 and C1-alpha 587 had shown that most of the centrin-1 coding sequence was not interrupted by introns. We sought to determine whether this was true of the rest of the centrin-1 cDNA sequence.

The sequence, position, and orientation of the centrin-1-specific primers used are given in Table 1. In all of our experiments, the size of the PCR products obtained after amplification of genomic DNA was identical to that expected from the centrin-1 cDNA sequence; as we mentioned before, amplification with the primers C1-88 and C1-alpha 587 yielded a 500-bp product (Fig. 3, lane a), amplification with the primers C1-88 and C1-alpha 1088 yielded a 1,000-bp product, and, finally, amplification with the primers C1-770 and C1-alpha 1088 yielded a 300-bp product (data not shown). The identities of the PCR products were confirmed by determining their nucleotide sequence or by diagnostic digestions with restriction enzymes. We conclude from these experiments that most of the centrin-1 gene is devoid of introns, although we cannot rule out the presence of an intron upstream of nucleotide 88 or downstream of nucleotide 1088.

The Centrin-2 Gene Is Actively Transcribed in Cultured HTE Cells, Testes, and Native Tracheal Epithelium

RT-PCR experiments performed in parallel with those described in The Centrin-1 Gene Is Not Transcribed in Cultured HTE Cells showed that the centrin-2 gene is transcribed in cultured HTE cells. Figure 3 (lanes e-h) shows the PCR products obtained with the primers C2-88 and C2-alpha 604. Amplification of genomic DNA yielded a 2-kb product, indicating that one or more introns interrupt the centrin-2 coding sequence (Fig. 4, lane e). A 500-bp product was obtained after amplification of cDNAs derived from HTE cells grown for 21 days immersed (Fig. 3, lane f) or at an air-liquid interface (Fig. 3, lane g), as well as after amplification of testis cDNAs (Fig. 3, lane h). The identity of this PCR product was confirmed by digesting it with the enzymes Hind III and Sty I and observing fragments of the expected sizes. A centrin-2-specific PCR product could also be obtained after 32 cycles of amplification of cDNAs derived from native human tracheal epithelium (Fig. 4, lane c).

Comparison of Fig. 3, lanes f and g, suggested that there may be more centrin-2 mRNA in HTE cells grown at an air-liquid interface than in HTE cells grown immersed. We therefore sought to quantitate the levels of centrin-2 gene expression during the growth and differentiation of HTE cells.

We first studied the expression of the centrin-2 gene through Northern blot experiments. To avoid cross-reactions with other centrin genes, we used the 3'-untranslated region of the centrin-2 cDNA as a probe (see MATERIALS AND METHODS). This probe was radiolabeled and applied to a blot of polyadenylated RNA isolated from HTE cells grown at an air-liquid interface for various lengths of time. Each lane contains polyadenylated RNA prepared from five culture wells (~100-150 µg of total RNA). As expected, the probe detected a 1.2-kb mRNA. A typical autoradiogram is shown in Fig. 5A. The radioactivity bound to this mRNA was quantified with a phosphorimager and is recorded underneath each band. To account for differences in the amount of RNA loaded in each lane, the centrin-2 probe was removed and a new probe specific for the "housekeeping" gene GAPDH was applied. The GAPDH gene is usually assumed to be transcribed at a constant level in cells. The 1.4-kb mRNA detected is shown in Fig. 5B. At each time point, the intensity of the centrin-2 signal was divided by the corresponding GAPDH signal to yield an estimate of the centrin-2 mRNA abundance in the original sample. The number obtained for day 12 cultures was chosen as a reference, and all other numbers are expressed as a percentage of this number.


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Fig. 5.   Quantitation of centrin-2 transcripts by Northern blot hybridization. RNA was isolated from HTE cells grown at an air-liquid interface for 12, 15, 18, or 21 days. Each gel lane contains polyadenylated RNA prepared from 5 culture inserts (100-150 µg of total RNA). A: portion of an autoradiogram showing binding of a centrin-2-specific probe to a 1.2-kb mRNA. Nos. at bottom, amount of radioactivity bound. B: portion of an autoradiogram showing binding of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe to a 1.4-kb mRNA. Nos. at bottom, amount of radioactivity bound. C: centrin-2-specific signal was normalized relative to GAPDH signal and is expressed as a percentage of signal obtained from day 12 cells (see text for details).

Figure 5C shows the centrin-2 mRNA abundance after 12, 15, 18, and 21 days of culture at an air-liquid interface. The concentration of centrin-2 mRNA after 15, 18, and 21 days in culture at an air-liquid interface is approximately double the concentration seen at day 12. Similar results were obtained with cells from three different tracheae, although the exact timing of the increase in mRNA concentration varied from experiment to experiment.

We then sought to independently confirm these results using a semiquantitative method based on RT-PCR. We took several precautions (see MATERIALS AND METHODS) to ensure that the amount of PCR product synthesized is proportional to the concentration of centrin-2 mRNA in the sample.

RNA was prepared from HTE cells grown at an air-liquid interface for various lengths of time. Two micrograms of RNA were reverse transcribed, and a cDNA aliquot was amplified with the primers C2-88 and C2-alpha 604. The mRNA level observed at the earliest time point was fixed at 1.0, and all other data are expressed as multiples of this level. Figure 6 shows the result of two experiments with cells obtained from two different tracheae. Over time, the centrin-2 mRNA concentration increases 2.5-fold to reach a maximum after 17-19 days in culture.


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Fig. 6.   Quantitation of centrin-2 and centrin-3 transcripts by RT-PCR amplification. Centrin-2 (triangle ) and centrin-3 (bullet ) mRNAs were detected by RT-PCR with total RNA isolated from HTE cells grown at an air-liquid interface for various lengths of time. Amount of specific PCR product is expressed as a multiple of the amount obtained at the earliest time point. A and B: results of the same experiment performed with cells of 2 different tracheae. Each data point represents average of 2 or 3 independent PCR amplifications. Most individual data points were within 10% of the average.

Comparison of Fig. 6, A and B, shows that the magnitude of the change in centrin-2 concentration is essentially the same (2.5-fold). However, the timing of this change varies; in the experiment shown in Fig. 6A, the centrin-2 mRNA concentration did not increase markedly until days 15-17. In contrast, in the experiment shown in Fig. 7B, the centrin-2 mRNA concentration increased as early as day 9 and reached a maximum after 17 days in culture. The length of time in culture before the change in centrin-2 mRNA concentration and the appearance of ciliated cells varies from trachea to trachea. The experiment shown in Fig. 6A is more typical than that shown in Fig. 6B.

Microscopic examination of frozen sections showed that the accumulation of centrin-2 mRNA coincides with the appearance of ciliated cells; for instance, in the experiment shown in Fig. 6A, the first ciliated cells appeared after 15 days in culture. The number of ciliated cells increased after 17 days and was maximal after 19 and 21 days in culture.

The Centrin-3 Gene Is Expressed at a Constant Level in Cultured HTE Cells

Centrin-3 is the most recently isolated member of the human centrin family. The 3'-untranslated region is not included in the published sequence (24), making it difficult to design a gene-specific probe to use in Northern blot hybridizations. Instead, we chose to determine the level of centrin-3 expression using the RT-PCR method described in MATERIALS AND METHODS. HTE cDNAs were amplified with the primers C3-1 and C3-alpha 503 (see Table 1). The 500-bp PCR product obtained was digested with the restriction enzyme Hind III to yield fragments of 350 and 150 bp, confirming its identity. No product was obtained if reverse transcriptase was omitted from the cDNA synthesis reaction (data not shown).

We quantitated the amount of PCR product derived from HTE cells grown for various lengths of time at an air-liquid interface and normalized the results as above. Figure 6 shows that the concentration of centrin-3 mRNA does not change more than 50% in comparison with that observed after 4 days in culture. In particular, the centrin-3 mRNA concentration does not systematically increase over time.

Centrin-3 transcripts were also detected in RNA derived from native human tracheal epithelium (Fig. 4, lane d). However a minimum of 35 cycles of amplification had to be performed to observe a specific PCR product. In contrast, 25 cycles of amplification were sufficient to detect a specific product from cultured HTE cell RNA. Therefore, centrin-3 is expressed in native epithelium, although at a much lower level than in cultured HTE cells.

Centrin-2 and -3 Are Expressed at a Low Constant Level in HTE Cells Grown Immersed

We previously mentioned that ciliogenesis does not occur in cells grown immersed. If the change in centrin-2 mRNA concentration we observed in air-liquid interface cultures is indeed linked to ciliogenesis, it should not take place if the cells are grown in immersion.

To test this hypothesis, HTE cells from one trachea were grown either at an air-liquid interface or in immersion. RNA was purified after 7 or 14 days and analyzed by Northern blot with a probe specific for centrin-2 transcripts. Figure 7 A shows that, after 7 days in culture, the centrin-2 mRNA concentration was approximately identical in immersed cells and in cells grown at an air-liquid interface. Between 7 and 14 days of culture, the centrin-2 mRNA concentration increased as expected in cells grown at an air-liquid interface. In contrast, this concentration did not change in immersed cells. This result reinforces our conclusion that the change in centrin-2 mRNA concentration that we observed is truly linked to the occurrence of ciliogenesis.


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Fig. 7.   Comparison of centrin gene expression levels in HTE cells grown immersed or at an air-liquid interface. A: Northern blot analysis of centrin-2 mRNA abundance in HTE cells. Cells were grown either at an air-liquid interface (Air) or immersed (Imm) for 7 (d7) or 14 (d14) days. Each lane contains 20 µg of total RNA. B: centrin-2 (C2), centrin-3 (C3), and GAPDH transcripts were detected by RT-PCR as described in text. RNA was prepared before onset of ciliogenesis (Early; days 4-6) or after appearance of ciliated cells (Late; days 12-18). In each case, we calculated ratio of amount of PCR product obtained from cells grown at an air-liquid interface to that obtained from immersed cells. Data are averages ± SE from 2 (Early) or 4 (Late) different tracheae.

We then sought to generalize this observation using our semiquantitative RT-PCR assay. Using cells derived from several different tracheae, we determined mRNA concentrations for centrin-2, centrin-3, and GAPDH. We then expressed our results as the ratio of the concentration seen in cells grown at an air-liquid interface to that in cells grown immersed (air-to-immersed ratio; Fig. 7B). At early time points (4-6 days in culture), we found that the concentrations of centrin-2 and centrin-3 mRNAs were identical in both types of culture (ratios of 1.08 and 0.94, respectively). These numbers are the averages of the results of two experiments with cells derived from two different tracheae. We then analyzed RNA derived from four independent experiments where the cells were grown for longer times (12-18 days) so that ciliogenesis could occur. We found that the average concentration of centrin-2 mRNA was 2.45 times higher in cells grown at an air-liquid interface than in immersed cells (n = 4; SE = 0.25). In contrast, the concentration of the GAPDH mRNA was identical in both types of cells (air-to-immersed ratio = 0.96; n = 3; SE = 0.08). The concentration of centrin-3 mRNA was slightly higher in cells grown at an air-liquid interface (air-to-immersed ratio = 1.65; n = 4; SE = 0.26).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Centrins are Ca2+-binding proteins found in a variety of subcellular structures, often in association with microtubule-organizing centers. Many centrin-containing structures are found exclusively in ciliated cells, suggesting that centrin gene expression may be a useful marker in the study of ciliogenesis. For this reason, we sought to characterize the expression patterns of human centrin genes during ciliogenesis.

In Vitro Ciliogenesis in HTE Cells

The culture system we used in this study has been previously partially characterized (43). Growth of HTE cells at an air-liquid interface results in the formation of a multilayered pseudoepithelium. The apical surface of the cell sheet consists of ciliated cells (60-80% of the area) and mucus-producing goblet cells. The mechanism by which the air-liquid interface improves the differentiation of HTE cells is not known.

Very similar culture systems have been previously described for cells obtained from other organisms (1, 4, 5, 8, 15, 16, 18, 27, 39, 40). One important quantitative feature of our system is the abundance of ciliated cells; the area covered by ciliated cells in our system is much greater than that reported in cultures of rat cells (where it represents 5-15% of the area) (4). This feature certainly facilitates the detection of changes in gene expression linked to ciliogenesis.

Cells derived from different tracheae become ciliated after a variable length of time. The most typical value is 13-15 days until the appearance of ciliated cells. However, this value can occasionally be as early as 11 days or as late as 17 days. This variability is reflected in the timing of gene expression changes as illustrated in Fig. 6. Because these are primary cultures, this variability may be linked to the health of the epithelium before isolation of the cells.

Distribution of Centrin in Ciliated Cells

Our immunostaining data show centrin to be largely confined to the basal bodies of ciliated cells. Centrin has been shown before to be a component of basal bodies in many species (20). The absence of centrin outside the basal bodies is somewhat surprising. No staining was seen over the striated rootlets that are clearly visible in electron micrographs (Fig. 2). In contrast, centrin is an important component of striated fibers in algae (see below). Absence of anti-centrin staining in striated rootlets has been previously noted in chick and mouse tracheal ciliated cells (20) and mammalian photoreceptor cells (41). This may indicate that in mammals centrin is not an obligatory component of these structures. Alternatively, centrin may be present but in a conformation preventing access by the antibody. However, the monoclonal antibody we used (20H5) stains striated rootlets in invertebrates (35). A third possibility is that the centrin isoform present in these structures is not recognized by the antibody we used. However, we think that this is unlikely because the 20H5 antibody bound 10 protein isoforms resolved by 2-dimensional gel electrophoresis in immunoblotting experiments (28). Moreover, it recognized all three bacterially produced centrin gene products (24).

The absence of centrin in the striated rootlets of vertebrate cilia may be functionally significant. Centrin is the major component of the striated rootlets of Tetraselmis (31). In several species of algae, including Chlamydomonas, Platymonas, Spermatozopsis, and Tetraselmis, these fibers were shown to contract in a Ca2+-dependent manner (23, 30, 33-36). The analysis of the Chlamydomonas centrin mutants (vfl2 and others) supported the hypothesis that centrin, a Ca2+-binding protein, mediated this contraction (38). Conceivably, a protein other than centrin mediates contraction of the striated rootlets in vertebrate ciliated epithelial cells.

Recent data (10) obtained in the human oviduct showed that the striated rootlets are much longer early in the ciliogenesis process than they are in mature ciliated cells. It is conceivable that centrin may be found in these longer fibers but may be absent from the "remnants" seen in older cells.

The 20H5 antibody also failed to stain the ciliary axoneme. In contrast, the same antibody stains Chlamydomonas axonemes, where centrin was shown to be a light chain associated with one type of inner dynein arms (17, 29), as well as the axonemes of the protist Holomastigotoides (21). Levy et al. (20), using a different anti-centrin antibody, found no staining in the axonemes of chick and mouse tracheal epithelia. The polypeptide composition of inner dynein arms has been studied in two vertebrate species, the pig and the Antarctic rockfish (11, 14). However, the published electrophoretograms do not include the region of the gel where centrin would be found. Therefore, the presence or absence of centrin in vertebrate inner dynein arms remains an open question.

Comparison of Gene Expression Levels by RT-PCR

To follow centrin gene expression during differentiation, we needed to compare the abundance of mRNAs in cells grown for various lengths of time. The first technique we used was Northern blot hybridization, followed by a quantitation of the signal bound specifically. However, we found that this technique was not well suited to our goals. It requires large amounts of RNA, and it does not lend itself well to determining expression levels of many different genes with the same RNA samples.

For these reasons, we developed a semiquantitative method based on RT-PCR to independently confirm the results of Northern blot analysis. Both RT and PCR amplification have the potential to give nonquantitative results. As described in MATERIALS AND METHODS, we took several precautions to ensure that our results are as quantitative as possible. Our task was made easier by the fact that we did not need to determine absolute abundances of mRNAs but merely needed to compare mRNA concentrations between similarly prepared RNA samples.

Three main lines of evidence indicate that our RT-PCR method yields accurate comparisons of mRNA abundances from sample to sample. 1) First, the conclusions regarding centrin-2 expression that were reached by this method were identical qualitatively and quantitatively to those reached through the Northern blot experiments. This agreement between the two methods was also noted for other genes involved in ciliogenesis (LeDizet and Finkbeiner, unpublished data). 2) The results obtained were very reproducible when duplicate amplifications of the same RNA samples were performed and when the experiments were duplicated with cells obtained from a different trachea. 3) Finally, we found that different centrin genes have different patterns of expression. Therefore, we can rule out the possibility that the variations in the amounts of RT-PCR products are due to the presence of inhibitory components in some of the RNA samples studied, to RNA degradation, or to errors in RNA quantitation. We are therefore confident that the amount of RT-PCR product synthesized under our conditions represents a reasonable estimate of mRNA abundance.

Centrin-1

Ciliated cells harbor several specific cytoskeletal structures known or suspected to contain centrin. For this reason, we hypothesized that ciliated cells might contain specific centrin isoforms, possibly expressed from a specific gene. At the outset of this work, we expected to find centrin-1 expressed in ciliated cells; the centrin-1 cDNA was isolated from human testes (6), an organ where active ciliogenesis takes place. A later abstract (42) reported centrin-1 expression in mammalian ciliated sensory cells, which contain a highly modified ciliary axoneme.

We found, however, that the centrin-1 gene is never transcribed in HTE cells, either native or cultured. It is therefore clear that ciliogenesis can occur in the absence of centrin-1. Furthermore, with the possible exception of ciliated sensory cells, centrin-1 expression appears restricted to testes; we searched the expressed sequence tag (EST) section of GenBank for ESTs derived from the centrin-1 gene. Only two human ESTs were found, and both originated in testis cDNA libraries (see Table 2).

                              
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Table 2.   Human ESTs derived from known centrin genes

One possible explanation of these results is that the centrin-1 protein is needed exclusively in testes. However, the absence of introns in the centrin-1 sequence raises the intriguing possibility that it represents a processed pseudogene. As such, an mRNA derived from it might not be translatable. This possibility could be tested with the development of antibodies specific for centrin-1.

Centrin-2

In contrast to centrin-1, the centrin-2 gene appears to be ubiquitously expressed. ESTs derived from the centrin-2 gene were identified on the basis of their high homology (>95%) to the centrin-2 cDNA sequence. To date, 29 ESTs derived from the centrin-2 gene can be found in the GenBank database, and they originate from many different organs and cell types (see Table 2). This suggests that centrin-2 is present in structures found in all cell types, such as centrosomes.

In cultured HTE cells, the centrin-2 mRNA is always detectable. Its abundance increases approximately twofold after 15-19 days of growth at an air-liquid interface, which coincides with the appearance of ciliated cells. As expected, this concentration increase does not take place in cells grown immersed, which never undergo ciliogenesis. Furthermore, our data indicate that centrin, as detected by immunostaining, is concentrated in the basal bodies of ciliated cells. Taken together, these results strongly suggest that centrin-2 is present in the basal bodies of ciliated cells.

It should be pointed out that the centrin-2 mRNA abundance was determined in RNA samples prepared from the entire multilayered pseudoepithelium. Ciliogenesis only occurs in the topmost layer of cells. Therefore, although 60-80% of the cells in the top layer acquire cilia, they represent a much smaller proportion of the total number of cells in the culture. Presumably, centrin-2 gene induction only occurs in the ciliated cells and not in the cells of the lower layers. As a result, the twofold increase in overall centrin-2 mRNA abundance represents a much larger increase within the ciliated cells.

The mechanism by which the concentration of centrin-2 mRNA increases is not known. An increase in the levels of mRNAs encoding ciliary proteins was shown to accompany ciliogenesis in many different organisms (2, 9, 13, 22, 26). In Chlamydomonas, this increase was shown to be the consequence both of increased gene transcription and of a selective mRNA stabilization (13).

Centrin-3

Centrin-3 is the most recently discovered member of the human centrin gene family (24). The predicted centrin-3 protein is only 54% identical to that of centrin-1 or centrin-2, suggesting a markedly different function. We identified 27 ESTs derived from the centrin-3 gene (see Table 2). These ESTs originate from a variety of tissues and cell types, although their distribution appears less wide than that of centrin-2-derived ESTs. Centrin-3 mRNA appears abundant in cultured HTE cells, where 25 cycles of PCR amplification were sufficient to detect a specific product. Thirty-five cycles of PCR amplification were sufficient to detect a specific product from RNA isolated from native tracheal epithelial cells. These results contrast with those of Middendorp et al. (24). These authors reported a very low centrin-3 mRNA abundance in a human lymphoblastic cell line where two rounds of PCR amplification with nested primers were necessary to obtain 500 ng of PCR product. Centrin-3 gene expression may therefore be very variable in different cell types.

We found that the centrin-3 mRNA concentration varied on either side of a constant value during the growth and differentiation of HTE cells. There did not seem to be a systematic trend to these variations. In particular, the centrin-3 mRNA abundance did not consistently increase over time as was the case with centrin-2 mRNA. Although we cannot be certain that the concentration of centrin-3 protein mirrors that of its mRNA, this result would suggest that centrin-3 is not found in cilia and cilia-associated structures. This conclusion is supported by the finding that centrin-3 mRNA is only slightly lower in cells grown immersed compared with cells grown at an air-liquid interface. The much lower abundance in centrin-3 mRNA observed in native, uncultured epithelial cells is harder to explain but may be significant.

In conclusion, we have shown that the genes encoding the three known human centrin genes are regulated independently during the growth and differentiation of HTE cells. In the absence of antibodies specific for individual centrin isoforms, one can only presume that changes in mRNA levels are reflected in the abundance of the corresponding proteins. However, our finding that the three centrin genes have markedly different expression patterns suggests that centrin isoforms are not interchangeable and that they each fulfill unique functions. Furthermore, the centrin-2 mRNA abundance closely mirrors the fraction of ciliated cells within the pseudoepithelium and may therefore constitute a useful marker for the study of this differentiation process.

    ACKNOWLEDGEMENTS

We express our gratitude to Susan Y. Chun, Rebecca Siegel-Wasserman, and Lorna T. Zlock for outstanding technical help; to Margaret Mayes for instruction in histological techniques; to Zac Cande, Gianni Piperno, Joel Rosenbaum, and Tim Stearns for useful, encouraging, and stimulating discussions; and to Amie Franklin and Gianni Piperno for critical readings of the manuscript before publication.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-42368 and Multidisciplinary Training Program in Lung Disease Grant T32-HL-07185.

Present addresses: M. LeDizet, Dept. of Medical Pathology, Univ. of California, Davis Medical Center, 2315 Stockton Blvd, Sacramento, CA 95817; J. C. Beck, Alza Corporation, 950 Page Mill Rd., Palo Alto, CA 94303.

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.

Address for reprint requests and present address of W. E. Finkbeiner: Dept. of Medical Pathology, Univ. of California, Davis Medical Center, 2315 Stockton Blvd., Sacramento, CA 95817.

Received 3 February 1998; accepted in final form 15 September 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(6):L1145-L1156
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