Laminin alpha -chain expression and basement membrane formation by MLE-15 respiratory epithelial cells

Nguyet M. Nguyen1, Yushi Bai2, Katsumi Mochitate2, and Robert M. Senior1

1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110; and 2 Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Basement membranes have a critical role in alveolar structure and function. Alveolar type II cells make basement membrane constituents, including laminin, but relatively little is known about the production of basement membrane proteins by murine alveolar type II cells and a convenient system is not available to study basement membrane production by murine alveolar type II cells. To facilitate study of basement membrane production, with particular focus on laminin chains, we examined transformed murine distal respiratory epithelial cells (MLE-15), which have many structural and biochemical features of alveolar type II cells. We found that MLE-15 cells produce laminin-alpha 5, a trace amount of laminin-alpha 3, laminins-beta 1 and -gamma 1, type IV collagen, and perlecan. Transforming growth factor-beta 1 significantly induces expression of laminin-alpha 1. When grown on a fibroblast-embedded collagen gel, MLE-15 cells assemble a basement membrane-like layer containing laminin-alpha 5. These findings indicate that MLE-15 cells will be useful in modeling basement membrane production and assembly by alveolar type II cells.

murine alveolar type II cells; transforming growth factor-beta ; extracellular matrix


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BASEMENT MEMBRANES ARE THIN sheets of extracellular matrix that serve as a structural support for cells. They also act as a selective barrier to various solutes and affect cell properties, including proliferation, differentiation, adhesion, and migration (for review, see Ref. 6). The major components of basement membranes are laminins, type IV collagens, entactins (nidogens), and perlecan. Basement membrane composition varies between tissues and even in the same tissue at different stages of development. Laminins contribute to the diversity of basement membranes. Laminins are a family of heterotrimeric glycoproteins in which each member contains an alpha -, beta -, and gamma -chain. To date, five alpha -, three beta -, and three gamma -chains forming 15 laminin isoforms have been described. Laminin-gamma 3 is the most recently described (23) laminin chain and is the only laminin chain that is not found in basement membranes. In the current model, basement membrane assembly occurs via linking of a type IV collagen network with a laminin network by entactin (41).

In the lung, alveolar type I and alveolar type II cells rest on a continuous basement membrane; however, the composition of the basement membrane under both cell types is not the same. Differences exist in the location of basement membrane anionic sites beneath alveolar type I and type II cells (34). Subsets of alveolar type II cells have localized interruptions or discontinuities of the basement membrane where cytoplasmic processes extend and contact or closely approximate interstitial fibroblasts and extracellular matrix, whereas the basement membrane beneath type I cells does not (5).

The alveolar epithelial basement membrane is complex in composition and undergoes changes during development. Recent studies (29, 30) highlight the variability of laminin alpha -chain expression in the lung. Laminin-alpha 1 and -alpha 2 are present in fetal lungs whereas laminin-alpha 3, -alpha 4, and -alpha 5 are present in both fetal and adult lungs (29). Laminin-alpha 1 and -alpha 5 are colocalized, but laminin-alpha 1 expression is restricted to the first trimester whereas laminin-alpha 5 expression is present at the end of the first trimester and continues throughout adulthood (30). All three laminin beta -chains and laminin-gamma 1 and -gamma 2 are found in both fetal and adult lungs (10, 11, 37). Little is known about the effects of injury and disease on the expression of laminin chains in the lung.

Alveolar type II cells in culture produce basement membrane components, including type IV collagen, laminin, perlecan, and entactin-1 (8, 15, 31, 36). Although alveolar type II cells can be isolated for study of production of basement membrane, obtaining them is labor intensive and time consuming. Additionally, harvest and culture of alveolar type II cells can be complicated by contamination with fibroblasts and differentiation into alveolar type I cells with time. To circumvent these problems, cell lines are widely used. The human alveolar epithelial cell line A549 and the rat alveolar epithelial cell line SV40-T2 have been used to study basement membrane production, but no mouse alveolar epithelial cell line has been identified for similar studies (15, 22, 31). With availability of knockout/transgenic mouse models, mice are utilized extensively in studies of cell and molecular biology and a mouse cell line would be valuable.

MLE-15 cells are an immortalized cell line obtained from lung tumors of transgenic mice containing the simian virus 40 (SV40) large T antigen under the transcriptional control of the human surfactant protein C (SP-C) promoter (21). MLE-15 cells have many characteristics of alveolar type II cells, including polygonal epithelial cell morphology, microvilli, cytoplasmic multivesicular bodies, multilamellar inclusion bodies, expression of SP-A, SP-B, and SP-C, and secretion of phospholipids (39). MLE-15 cells have been used to examine distal respiratory epithelial cell functions such as Fas-dependent apoptosis, chemokine response to silica, transcription of thyroid transcription factor-1, and protein expression in response to hypertonic stress, but their ability to produce extracellular matrix has not been determined (1, 20, 40, 42). We have found that MLE-15 cells constitutively make several laminin chains, including laminin alpha 5, as well as other components of basement membrane, and assemble these components into a subepithelial basement membrane-like structure when grown in the presence of fibroblasts.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and cell culture. MLE-15 cells were obtained from Dr. Jeffrey Whitsett (University of Cincinnati) and grown in HITES medium. HITES medium is RPMI 1640 (GIBCO-BRL, Gaithersburg, MD) supplemented with 2% fetal bovine serum (GIBCO-BRL), 100 U/ml penicillin, 100 µg/ml streptomycin, 1% insulin-transferrin-sodium selenite, 5 µg/ml transferrin, 10 nM hydrocortisone, 10 nM beta -estradiol, 2 mM glutamine, and 2 mM HEPES. For studies involving transforming growth factor-beta 1 (TGF-beta 1), confluent cultures were serum deprived for 24 h, and then TGF-beta 1 (R&D Systems, Minneapolis, MN) was added to a final concentration of 10 ng/ml for 24 h. Before fixation for immunohistochemistry and in situ hybridization studies, cells were treated with 10 mg/ml brefeldin A for 3 h to prevent secretion of extracellular proteins.

SV40-T2 cells (a gift from Dr. Jerome Brody, Boston University) were grown in DMEM with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 10% FCS. Unless otherwise noted, all reagents were obtained from Sigma Chemical (St. Louis, MO).

Culture of MLE-15 cells on collagen I gel and fibroblast-embedded collagen I gel. MLE-15 cells were cultured on fibroblast-embedded collagen I gel (Fgel) to investigate the influences on basement membrane formation. Fibroblasts were prepared from adult male Jcl/Fischer 344 rats (Japan Cle, Tokyo, Japan), as described previously (15). First, Fgel was prepared by mixing fibroblasts (1.8 × 105 cells) with 0.72 ml of 1 mg/ml neutralized type I collagen solution (acid-extracted type I collagen from bovine dermis; Koken, Tokyo, Japan) in DMEM, pH 7.2, casting the cell suspension on a cell culture insert with a polyethylene terephthalate (PET) membrane (Becton Dickinson Labware, Franklin Lakes, NJ) and allowing it to gel in a 5% CO2 incubator for 1 h. After polymerization, fibroblasts were supplied with DMEM containing 10% FCS, 0.2 mM ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Tokyo, Japan), and 10 mM HEPES, pH 7.2, and cultured for 3 days. Next, MLE-15 cells (8 × 105 cells) grown in HITES medium were seeded on the Fgel (MLE-15 Fgel) or on 0.72 ml of 1.5 mg/ml type I collagen gel (MLE-15 gel) and cultured for 2 wk in an equal volume mixture of DMEM and RPMI 1640 supplemented with 2% FCS, 100 mM glutamine, 0.2 mM ascorbic acid 2-phosphate, and 10 mM HEPES, pH 7.2. MLE-15 cells were hyperproliferative in HITES medium and formed multilayers in both MLE-15 Fgel and MLE-15 gel. Accordingly, insulin-transferrin, sodium-selenite, hydrocortisone, and beta -estradiol were excluded, and equal mixtures of fibroblast and MLE-15 cell medium (without HITES) were used for MLE-15 gel and MLE-15 Fgel cultures.

Northern blot analysis. Total RNA was isolated from MLE-15 cells with the ToTally RNA kit (Ambion, Austin, TX). For Northern hybridization, 10 µg of total RNA were denatured in 50% formamide, 1 M formaldehyde, and 50 ng/µl ethidium bromide at 68°C for 5 min and then separated by electrophoresis through a 1% agarose gel containing 1 M formaldehyde. RNA was passively transferred to charged Hybond N+ nylon membrane (Amersham, Arlington Heights, IL), fixed by treatment with 50 mM NaOH for 5 min, and hybridized. cDNA probes were radiolabeled by random priming with [32P]dCTP. mRNAs were detected using probes made from nt 5494-5892 of laminin-alpha 1 cDNA (35) and nt 658-990 of laminin-alpha 5 cDNA (28). For glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (pRGAPDH13), a 1.3-kb rat cDNA was used as previously described (36). After hybridization, membranes were washed and exposed to Kodak AR X-ray film at -70°C for 1-4 days with an intensifying screen.

RT-PCR. RNA (1 µg) was added to each RT reaction mixture including 20 pmol of each primer. RT was performed using random hexamers by incubating at 25°C for 10 min, 42°C for 30 min, and 94°C for 5 min. PCR was performed using specific mouse primers for laminin-alpha 1 to -alpha 5 and GAPDH as shown in Table 1. All primers were obtained from Life Technologies (Grand Island, NY). All PCR reactions were denatured at 94°C and extended at 72°C, and the annealing temperatures and number of cycles varied as given in Table 1. Primers for GAPDH were amplified in the same reaction mixture as laminin alpha -chains but were added after 6-10 cycles, depending on the laminin alpha -chain under investigation. PCR products were separated in 1% agarose gels, transferred onto BrightStar-Plus nylon membranes (Ambion, Austin, TX), and hybridized with 32P-labeled specific oligonucleotide probes in standard saline citrate (SSC), 1% SDS, 1× Denhardt's solution, and 50 µg/ml denatured salmon sperm DNA at 42°C overnight. Membranes were washed in 6× SSC and 0.1% SDS for 15 min at room temperature and then for 15 min at 52°C. Quantification of PCR products was performed using the Bio-Rad molecular imager system (GS-525; Hercules, CA) and expressed as the ratio of laminin to GAPDH.

                              
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Table 1.   Primers and probes for PCR and Southern analysis

In situ hybridization. 35S-labeled riboprobes were prepared from linearized cDNA templates for in situ hybridization of MLE-15 cells as described previously (25). MLE-15 cells were cultured on glass slides until near confluence, serum deprived for 1 day, treated with TGF-beta for 1 day, and fixed in methanol. The methods used for in situ hybridization were as described in detail previously (36). Negative controls hybridized with 35S-labeled sense probes were performed simultaneously with the corresponding antisense samples.

Antibodies. Recombinant domain G3-G4 of mouse laminin-alpha 5 (a gift from Dr. Ulla Wewer, University of Copenhagen) (12) was expressed in the PET vector system (Invitrogen, Carlsbad, CA) and used to generate a rabbit polyclonal antibody. Rat monoclonal antibody to mouse laminin-alpha 1 was a gift from Dr. Dale Abrahamson (University of Kansas). Rabbit polyclonal antibody to perlecan recognizing the core protein of heparan sulfate proteoglycan was a gift from Dr. K. Kimata (Aichi Medical University). Rabbit polyclonal antibody to mouse type IV collagen, rat monoclonal antibody to mouse laminin-1 (MAb1975, which recognizes the pepsin-digested fragment of laminin-1, P1), and rat monoclonal antibody to mouse entactin (MAb1946, which recognizes a recombinant region of the G1/link domain) used in immunofluorescence studies were from Chemicon International (Temecula, CA). Rabbit polyclonal antibody to type IV collagen used in immunoprecipitation was from Collaborative Biomedical Products (Becton-Dickinson Labware, Bedford, MA). Rabbit polyclonal antibody to recombinant entactin was raised as previously described (36). Fluorescein-conjugated goat antibodies to rabbit IgG F(ab')2 and rat IgG F(ab')2 were from Rockland (Gilbertsville, PA).

Immunohistochemistry. MLE 15 cells were cultured in two-well glass chambers (NUNC, Fisher Scientific, PA), treated with TGF-beta 1 as described in Cells and cell culture, and fixed in methanol. Immunohistochemical analysis of MLE-15 cells with polyclonal laminin-alpha 5 and monoclonal laminin-alpha 1 antibodies was performed with the diaminobenzidine Envision kit (DAKO, Carpinteria, CA) and the Vectastain Elite avidin-biotin complex kit (Vector Laboratories, Burlingame, CA), respectively.

MLE-15 Fgel and MLE-15 gel were fixed in 4% cold paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing 0.2 M sucrose for 1 h, cryoprotected in 20% sucrose, and frozen in TissueTek OCT compound (Miles, Elkhart, IN). Transverse cryosections (5 µm) were blocked with 1-5% normal goat serum, incubated with specific primary antibodies, and then treated with FITC-conjugated secondary antibodies. Control sections were treated with normal serum instead of the primary antibodies and run parallel with sections exposed to primary antibodies. Coverslips were mounted in 50% glycerol in PBS. Representative fields were photographed with a Leica DMRBE confocal fluorescence microscope (Buffalo, NY).

Tissue processing for transmission electron microscopy. The fixatives and dyes required for electron microscopy were obtained from TAAB (Berkshire, UK), except for Quetol resin, which was obtained from Nissin (Tokyo, Japan). Tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) containing 0.2 M sucrose and 0.1% tannic acid and then postfixed with 1% osmium tetraoxide. The tissues were dehydrated through a series of graded ethanol, replaced with n-butyl glycidyl ether, and embedded in Quetol resin. After ultrathin sectioning, specimens were stained with lead citrate and uranyl acetate and examined with a JEOL JEM-2000FX microscope.

Metabolic labeling and immunoprecipitation. Cells were seeded into six-well culture plates (Costar, Cambridge, MA) at 2 × 106 cells/well with complete medium for 2 days. In studies involving TGF-beta 1, the TGF-beta 1-treated wells were serum deprived for 24 h before TGF-beta 1 treatment; control wells were maintained in serum-free medium as described above during the same time period. Cells were washed with methionine-free RPMI 40 medium, after which fresh medium with all additives including FCS (dialyzed to remove methionine) was added, followed by 50 µCi of [35S]methionine (ICN Biomedical, Costa Mesa, CA). Cultures were returned to a 5% CO2 incubator for 24 h, after which the conditioned medium was collected. To obtain secreted matrices, we treated cells as described previously by Rannels et al. (32). Briefly, cells were exposed to 1 ml of 0.25 M NH4OH, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM EDTA, and wells were washed with PBS-EDTA (1 mM). DNA and nuclear debris were removed by treatment with 1 ml of 50 mM Tris, pH 8.0, 1 M NaCl, 1 mM PMSF, and 1 mM EDTA for 15 min at 4°C, and wells were washed with PBS-EDTA (1 mM). STRIPA (500 µl) (150 mM NaCl, 5 mM EDTA, 5 mM urea, 1 mM PMSF, 1% deoxycholate, 1% SDS, 1% Triton X-100, 1% Nonidet P-40, and 50 mM Tris, pH 8.0) was added, and wells were scraped and the contents collected. Medium and secreted matrices were stored at -80°C.

Conditioned medium or scraped secreted matrices were mixed with specific antibody or preimmune serum, incubated for 1 h at 37°C, and then incubated overnight at 4°C. Before incubation with perlecan antibody, the sample for perlecan immunoprecipitation was pretreated with heparitinase and protease inhibitors as previously described (14). The immune complexes were separated by the addition of protein A-Sepharose (Zymed Laboratories, San Francisco, CA) and incubation for 3 h at room temperature. The pellets were washed, resuspended in electrophoresis buffer, incubated at 60°C for 15 min, and microcentrifuged, and the supernatants were transferred to new tubes. beta -mercaptoethanol was added to a final concentration of 2%, after which samples were boiled for 5 min and subjected to PAGE in 6% gels. Gels were incubated for 30 min at room temperature in Amplify (Amersham), rinsed with water, dried, and exposed to autoradiographic film for 7-21 days at -70°C.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of laminin alpha -chain mRNAs. RT-PCR for laminin alpha 1- to alpha 5-chain mRNAs in MLE-15 cells revealed high expression of laminin-alpha 5 and slight expression of laminin-alpha 3 but virtually no expression of laminin-alpha 1, -alpha 2, and -alpha 4 at baseline (Fig. 1). Northern blot analysis confirmed the expression of laminin-alpha 5 mRNA in MLE-15 cells (Fig. 2A) with mouse placenta as a positive control. Likewise, in situ hybridization (Fig. 2B) and immunohistochemical staining (Fig. 2, C and D) for laminin-alpha 5 revealed strong expression and production in MLE-15 cells. Metabolic labeling and immunoprecipitation of MLE-15-conditioned medium showed production and secretion of laminin alpha 5-chain as well as laminin beta 1/gamma 1-chains (Fig. 3).


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Fig. 1.   RT-PCR of laminin alpha -chains from MLE-15 cell RNA. RNA was extracted from MLE-15 cells, and RT-PCR was performed with specific laminin alpha -chain primers concurrently with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers as an internal control. Signal intensity for laminin alpha -chains was normalized to signal intensity for GAPDH and expressed as the ratio of laminin alpha -chain mRNA to GAPDH mRNA; n = 3 for laminin-alpha 1 and -alpha 5 and n = 2 for laminin-alpha 2, -alpha 3, and -alpha 4. Laminin-alpha 5 is the prominent alpha -chain produced by MLE-15 cells. Laminin-alpha 3 production is slight whereas the production of other laminin alpha -chains is minimal.



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Fig. 2.   Laminin-alpha 5 mRNA and protein in MLE-15 cells. A: autoradiogram of Northern blot analysis for laminin-alpha 5 (Lam alpha 5) and GAPDH mRNAs in MLE-15 cells. Mouse placental mRNA was used as positive control. B: darkfield view of cultured MLE-15 cells hybridized in situ for laminin-alpha 5 mRNA. Positive signal is visible as silver grains surrounding nuclei. C: immunohistochemical detection of laminin-alpha 5 is seen as brown cytoplasmic staining of variable intensity among most MLE-15 cells. D: no staining is seen with preimmune serum. Original magnification: B, ×100; C and D, ×200.



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Fig. 3.   Production of laminin-alpha 5 by MLE-15 cells in culture. The culture medium of MLE-15 cells was replaced with methionine-deficient medium containing [35S]methionine as described in MATERIALS AND METHODS. After 24 h, the culture medium was harvested and immunoprecipitated using polyclonal anti-laminin-alpha 5 serum (I) or preimmune serum (PI). Shown is a fluorogram of an SDS-6% PAGE, with the location of laminin-alpha 5 and the laminin-beta 1/gamma 1 complex indicated.

Effect of TGF-beta 1 on laminin alpha -chain expression. Because TGF-beta 1 promotes the production of several basement membrane proteins (14, 18, 24), semiquantitative RT-PCR for laminin-alpha 1 to -alpha 5 was performed on MLE-15 cell mRNA after the addition of TGF-beta 1. To lower the basal constitutive expression of laminin-alpha 5, we serum deprived the cells for 24 h before the addition of TGF-beta 1. Figure 4 shows that laminin-alpha 1 production was induced eightfold with TGF-beta 1, an effect that was confirmed by in situ hybridization (Fig. 5, A and B) and immunohistochemistry (Fig. 5, D and E). In comparison, TGF-beta 1 had only a minimal effect on laminin-alpha 5 mRNA expression as was the case for laminin-alpha 2, -alpha 3, and -alpha 4 mRNA (data not shown). The optimum exposure time for the maximal effect of TGF-beta 1 on laminin alpha -chain expression was determined to be 18-24 h (data not shown).


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Fig. 4.   Effect of transforming growth factor-beta 1 (TGF-beta 1) on laminin-alpha 1 and -alpha 5 mRNA. RNA was extracted from MLE-15 cells treated with 10 ng/ml TGF-beta . RT-PCR was performed with specific primers for laminin-alpha 1 and -alpha 5 and GAPDH. Results are presented as the ratio of laminin alpha -chain to GAPDH mRNA expression. Laminin-alpha 1 expression in MLE-15 cells increased significantly with TGF-beta 1, whereas laminin-alpha 5 expression was increased, but not significantly, by the addition of TGF-beta 1. The experiments were done in triplicate.



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Fig. 5.   TGF-beta 1 induces expression of laminin-alpha 1 mRNA and protein in MLE-15 cells. MLE-15 cells grown on glass slides for 2 days were treated with 10 ng/ml TGF-beta 1 for 24 h and fixed for in situ hybridization or immunohistochemical staining. A: in the absence of TGF-beta 1, laminin-alpha 1 signal is similar to background by in situ hybridization. B: with TGF-beta 1 treatment, laminin-alpha 1 signal is strong and surrounds cell nuclei. With immunohistochemistry, laminin-alpha 1 is barely detectable without TGF-beta 1 (D), but positive staining is present in nearly all cells treated with TGF-beta 1 (E). As a negative control for immunohistochemical staining, rat serum instead of laminin-alpha 1 antibody was used on cells treated with TGF-beta (C). Original magnification: A and B, ×100; C-E, ×200.

Basement membrane protein production. In addition to production of laminin alpha 5- and beta 1/gamma 1-chains, metabolic labeling and immunoprecipitation of MLE-15 cell-conditioned medium also demonstrated production of type IV collagen and perlecan but not entactin (Fig. 6). Immunoprecipitation of the insoluble material deposited under MLE-15 cells was negative for laminin-alpha 5 or -beta 1/gamma 1, type IV collagen, and entactin (data not shown), indicating that although MLE-15 cells make some components of basement membrane, they do not assemble a basement membrane when grown on plastic.


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Fig. 6.   Production of basement membrane proteins by MLE-15 cells. Metabolic labeling and immunoprecipitation of MLE-15 cell medium with anti-sera for major components of basement membrane. MLE-15 cell medium showed immunoreactive bands with antibodies against type IV collagen (Col IV; lane 3) and perlecan (Perl; lane 5) but not entactin (Ent; lane 4). SV40-T2 cell medium was used as a positive control for entactin immunoprecipitation (lane 1). No bands were seen with preimmune serum (lane 2).

Basement membrane formation. In previous studies, basement membrane formation was observed (15) when rat type II epithelial cells (SV40-T2) were grown on Fgel but not on a collagen I gel. Because MLE-15 cells did not form a basement membrane when cultured on plastic, we examined basement membrane formation when MLE-15 cells were grown synchronously for 2 wk on collagen gels (MLE-15 gel) and Fgel (MLE-15 Fgel) by immunofluorescence and transmission electron microscopy. Similar to rat SV40-T2 cells, MLE-15 cells exhibited a cuboidal morphology on collagen gel (MLE-15 gel) and a squamous appearance when grown on fibroblast-embedded collagen gel (MLE-15 Fgel) (15). In MLE-15 gel, only faint, spotty immunofluorescence for laminin, type IV collagen, and perlecan was present (Fig. 7, A, C, and E). However, in MLE-15 Fgel, distinct, partially continuous linear deposits of fluorescence for laminin, type IV collagen, and perlecan were seen at the interface between MLE-15 cells and Fgel (Fig. 7, B, D, and F). Because MLE-15 cells do not produce entactin, we did not detect entactin when MLE-15 cells were grown on a collagen gel without fibroblasts (Fig. 7G). However, integration of entactin was observed in the MLE-15 Fgel culture when detected with the polyclonal antibody against mouse entactin (Fig. 7H) but not with the rat monoclonal antibody (MAb1946) against mouse entactin (data not shown). This suggests that the integrated entactin was derived from the embedded rat fibroblasts since the polyclonal antibody is crossreactive with rat entactin (36).


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Fig. 7.   Immunofluorescent staining of basement membrane constituents in MLE-15 cells seeded on type I collagen gel (MLE-15 gel) or rat pulmonary fibroblast-embedded type I collagen gel (MLE-15 Fgel) and cultured for 2 wk. Integration of laminin (LN-1; A and B), type IV collagen (C and D), perlecan (E and F), and entactin (G and H) was examined in transverse sections of MLE-15 gel (A, C, E, and G) and MLE-15 Fgel (B, D, F, and H). The arrowheads and arrows indicate apical surfaces of MLE-15 cells and deposits of basement membrane components beneath MLE-15 cells, respectively. In MLE-15 gel, the deposits are diffuse and dotted. In MLE-15 Fgel, deposits are enhanced and linear, but discontinuous. A-H are all at the same magnification. Bars: 20 µm.

To identify the laminin alpha -chains integrated into the basement membrane-like material, we examined MLE-15 Fgel by immunohistochemistry. Intermittent positive staining was observed for laminin-alpha 5 at the interface between MLE-15 cells and Fgel (Fig. 8). This is consistent with the profile of laminin alpha -chains produced by MLE-15 cells.


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Fig. 8.   Immunofluorescent staining of laminin-alpha 5 in MLE-15 Fgel. MLE-15 cells were cultured on Fgel for 2 wk. Immunolocalization of laminin-alpha 5 was examined in transverse sections. A discontinuous fluorescent band of laminin-alpha 5 was observed at the interface between MLE-15 cells and Fgel. The arrowheads and arrows indicate apical surfaces of MLE-15 cells and deposits of basement membrane components beneath MLE-15 cells, respectively. Bar: 20 µm.

These findings by immunofluorescence microscopy were supported by transmission electron microscopy. Virtually no electron-dense deposits corresponding to basement membrane were observed beneath MLE-15 cells in MLE-15 gel (Fig. 9A). However, in MLE-15 Fgel, short lengths of lamina densa, corresponding to basement membrane, were present beneath the basal surface of MLE-15 cells (Fig. 9B).


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Fig. 9.   Transmission electron micrograph of extracellular matrix structure beneath MLE-15 cells in MLE-15 gel and MLE-15 Fgel. A: in MLE-15 gel, electron-dense deposits were rarely observed beneath the basal surface. B: in MLE-15 Fgel, short lengths of lamina densa (arrows) consistent with basement membrane were observed along with areas lacking lamina densa (arrowheads). A and B are at the same magnification. Bar: 1 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

As a surrogate for normal murine type II alveolar epithelial cells, MLE-15 cells have been used to study surfactant and phospholipid production (19), chemokine expression (1, 2), and induction of water channel proteins (20). MLE-15 cells have been transfected with various genes (7, 40) and infected with retrovirus-mediated HSV-tk gene (42). However, the production of basement membrane proteins by MLE-15 cells has not been investigated, although it is well known that alveolar epithelial cells make these proteins. Considering the current interest in murine cell and molecular biology, the availability of a murine respiratory epithelial cell that produces basement membrane proteins could prove quite useful.

Different isoforms of collagen, proteoglycans, and laminins give basement membranes heterogeneity and functional specificity. In this study, we concentrated on laminins. Although 15 laminins have been described, the chain with the most variability in the laminin trimer is the alpha -chain. In the normal lung, laminin-alpha 1 and -alpha 2 are seen only in fetal development while laminin-alpha 3, -alpha 4, and -alpha 5 are seen in both fetal and adult stages (29, 30). Mesenchymal cells produce laminin-alpha 2 and -alpha 4, epithelial cells produce laminin-alpha 3 and -alpha 5, and both epithelial and mesenchymal cells produce laminin-alpha 1 (13). The MLE-15 cell line is an immortalized cell line derived from alveolar type II epithelial cells of lung tumors generated in adult mice. Accordingly, production of laminin-alpha 3 and -alpha 5 was expected, but not production of mesenchymal laminin-alpha 2 and -alpha 4. Consistent with this, we found that MLE-15 cells express laminin-alpha 5 and -alpha 3 but do not express laminin-alpha 2 and -alpha 4. Laminin-alpha 5 expression was most abundant with lesser expression of laminin-alpha 3. Because production of laminin-alpha 1 is normally restricted to early fetal lungs, we did not expect to find laminin-alpha 1 production and this was the result with MLE-15 cells at baseline.

TGF-beta increases production of many extracellular matrix proteins, including the basement membrane proteins type IV collagen (18), laminin-beta 2 (24), perlecan, and entactin (14). Virtually every mammalian cell produces and has receptors for TGF-beta , and TGF-beta levels are increased in inflammation and fibrosis (4). We found that MLE-15 cells exposed to TGF-beta 1 increase expression and production of laminin alpha -chains, similar to human and rat type II cell lines (14). A new observation is that TGF-beta 1 induces production of laminin-alpha 1. Laminin-alpha 1 is the most extensively studied laminin alpha -chain and is present in the lung only during early fetal development (29, 38). It is somewhat surprising that an adult-derived cell line could be induced to make laminin-alpha 1. This may occur in MLE-15 cells because it is a transformed cell line. Alternatively, because TGF-beta is increased in many injury states and TGF-beta leads to increased production of extracellular matrix, it is possible that adult alveolar epithelial cells might produce some "fetal proteins" as part of the response to injury.

The substrata on which alveolar type II cells are grown affects their phenotype and function (for review, see Ref. 9). With complex extracellular matrices such as human amniotic membrane, type II cell morphology is cuboidal when grown on the basement membrane side and attenuated when grown on the stromal side (27). When cultured on individual matrix substrates such as laminin-1 or type I collagen, type II cell morphology is cuboidal, whereas with fibronectin and type IV collagen, the morphology is attenuated. We examined laminin alpha 1- and alpha 5-chain expression by MLE-15 cells grown on plastic alone and on different matrix substrates such as mouse laminin-1 derived from the Engelbreth-Holm-Swarm (EHS) tumor, type IV collagen, and pepsin-digested laminin-10/11 (formed by the trimers alpha 5/beta 1/gamma 1 and alpha 5/beta 2/gamma 1, respectively). We did not find differences in cell morphology or expression of laminin alpha 1- or alpha 5-chains so MLE-15 cells do not appear to be as responsive to the extracellular matrix as primary type II cells.

We found that MLE-15 cells produce laminin, type IV collagen, and perlecan, but they do not independently assemble a basement membrane. MLE-15 cells do not produce entactin, the molecule that links laminin with type IV collagen. However, in the presence of fibroblasts, a basement membrane-like material containing entactin is deposited underneath the MLE-15 cells. Because lung mesenchymal cells are the main source of lung entactin, the entactin seen was probably produced by fibroblasts and recruited to the basement membrane by the MLE-15 cell. Alternatively, fibroblasts may secrete a factor(s) that induces entactin expression by MLE-15 cells. However, this explanation is unlikely since the rat monoclonal antibody to mouse entactin failed to detect deposits of entactin even in the presence of fibroblasts. Rat transformed alveolar type II cells (SV40-T2) produce laminin, type IV collagen, perlecan, and a small amount of entactin. However, like MLE-15 cells, they are unable to form a basement membrane unless grown in the presence of fibroblasts or Matrigel (an extracellular matrix derived from EHS tumor with basement membrane that contains laminin-1, growth hormones, and cytokines) (15, 16) even though they make entactin. These data support the concept that basement membrane assembly requires more than the presence of the components of basement membrane but rather interaction between epithelial cells and fibroblasts.

In summary, previous studies (1, 2, 7, 19, 20, 40, 42) have found MLE-15 cells useful as a surrogate to investigate different aspects of alveolar type II cell function. The present study extends these earlier observations by revealing that MLE-15 cells spontaneously produce basement membrane proteins, specifically laminins containing alpha 5- and alpha 3-chains, type IV collagen, and perlecan, and that MLE-15 cells can interact with fibroblasts to form a subepithelial basement membrane-like structure. Accordingly, MLE-15 cells may be useful for studies of basement membrane production, assembly, and function by murine alveolar type II cells.


    ACKNOWLEDGEMENTS

We thank Dr. Jeffrey Whitsett for providing MLE-15 cells and G. L. Griffin and M. S. Mudd for technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-29594; the Alan A. and Edith L. Wolff Charitable Trust; and a Research Fellowship from the American Lung Association (N. M. Nguyen).

For questions and comments pertaining to the microscopy studies, contact K. Mochitate (E-mail: mochitat{at}nies.go.jp).

Address for reprint requests and other correspondence: R. M. Senior, Dept. of Medicine, Barnes-Jewish Hospital, North Campus, 216 South Kingshighway, St. Louis, MO 63110 (E-mail: seniorr{at}msnotes.wustl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 14, 2001;10.1152/ajplung.00379.2001

Received 24 September 2001; accepted in final form 1 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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