RAPID COMMUNICATION
Laminin 2 attachment selects myofibroblasts from fetal mouse lung

Guillermo Flores-Delgado, Pablo Bringas, and David Warburton

Center for Craniofacial Molecular Biology, Department of Pediatric Surgery, and Developmental Biology Program, Children's Hospital Los Angeles Research Institute, University of Southern California Schools of Dentistry and Medicine, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Laminins (LNs) are extracellular matrix glycoproteins that are involved in cell adhesion, proliferation, and differentiation. So far, 11 LN variants (LN1 to LN11) have been described. In the lung, at least six LN variants have been identified. However, only the role of LN1 has been characterized to any extent. In this study, we hypothesized that the LN2 variant may play a role during lung development. We identified, by RT-PCR analysis, that the alpha 2-chain mRNA of LN2 is expressed during mouse lung development. LN2 adhesion assays were then performed with cells from fetal mouse lung primary cultures. Our results showed that a specific subpopulation of fetal lung cells that expressed vimentin, alpha -smooth muscle actin, and desmin attached onto LN2, whereas the cells that did not adhere to LN2 as well as the total cell population were able to adhere readily on fibronectin. Cell attachment onto LN2 was inhibited by EDTA. In addition, we demonstrated, by RT-PCR and Western analysis, that the LN2-adherent cells autoexpressed the alpha 2-chain of LN2. In the late pseudoglandular period, LN2 was localized by immunohistochemistry in the basement membrane of airways and blood vessels and around mesenchymal cells. We conclude that LN2 is expressed during lung development and that a specific subpopulation of fetal lung mesenchymal cells expressing a myofibroblastic phenotype can be selected by attachment to LN2 in primary culture. These findings lead us to speculate that LN2 may play a key role in the cell biology of myofibroblasts during lung development.

merosin; lung development; mesenchymal cells; cell adhesion

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

LAMININS (LNs) are a family of extracellular matrix (ECM) glycoproteins. They are composed of one central chain (alpha ) and two lateral chains (beta  and gamma ) that are linked by disulfide bonds to form a cross-shaped molecule. The LN family is composed of 11 variants (LN1 to LN11) (33), which result from the combination of different chain isoforms. To date, five alpha -, three beta -, and two gamma -chain isoforms have been characterized. Their expression and distribution is tissue specific.

In the lung, the identification of various chain isoforms of LN (alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, and gamma 1) suggests that at least six LN variants are present (2, 8, 12, 15, 35). The LN1 variant has been extensively characterized during lung development (9, 18, 20). It is involved in lung morphogenesis and lung epithelial cell polarization (26, 31). However, the role and importance of the other LN variants identified during lung development remain to be elucidated.

Specific domains localized in the different chain isoforms of LN1 play specific roles during lung development. The cross region of the alpha 1-chain of LN1 participates in epithelial-mesenchymal interaction. The globular region of the beta - and gamma -chains are involved in cell polarization. In addition, a fragment containing the carboxy-terminal region, obtained after degradation of LN1, participates in the in vitro formation of alveolar-like structures (22, 27-29).

The LN2 (alpha 2, beta 1, gamma 1) variant was originally described as being specific to muscle cells, the placenta, and peripheral nerves (10, 21). However, in recent studies (1, 4, 35), LN2 has also been identified in other tissues including the lung. Virtanen et al. (35) have recently identified the alpha 2-chain (molecular mass 300 kDa) of LN2 in both lung epithelial cells and smooth muscle cells during the pseudoglandular and canalicular stage of embryonic human gestation. In addition, in patients with severe chronic asthma, the alpha 2-chain isoform of LN2 was identified in the basement membrane of epithelial airways, suggesting its participation in remodeling of the ECM (1).

In the present study, we hypothesized that the LN2 variant may be expressed and play a role during fetal lung development. To test this hypothesis, we identified the expression of the alpha 2-chain isoform of LN2 during mouse lung development using RT-PCR analysis. We determined that a particular subpopulation of mesenchymal cells derived from primary cultures of embryonic day (ED) 16 mouse embryos attach selectively to LN2. This subpopulation of fetal lung cells expressed vimentin, alpha -smooth muscle actin, and desmin (VAD), cell markers characteristic of VAD-type myofibroblasts (6, 7, 13, 17). In addition, we show that this cell subpopulation autoexpressed LN2. Our data suggest that LN2 plays an important role during lung development by directing adherence of a specific mesenchymal cell population with a myofibroblastic phenotype.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. SeaKem agarose LE was purchased from Intermountain Scientific. RT-PCR kit was from Perkin-Elmer (Foster City, CA). Trizol, 100-bp DNA ladder, LN1, LN2, and fibronectin (FN) were obtained from GIBCO BRL (Gaithersburg, MD). Antibody against alpha 2-chain isoform was from Chemicon (Temecula, CA). Dulbecco's modified Eagle's medium (DMEM); fetal bovine serum; penicillin-streptomycin; Triton X-100; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue; MTT); and antibodies against keratin (clone PCK-26), vimentin (clone VIM-13), alpha -smooth muscle actin (clone 1A4), and desmin were purchased from Sigma (St. Louis, MO). The antibody directed against smooth muscle myosin heavy chain was kindly provided by Dr. M. Schwartz (Children's Hospital Los Angeles, CA).

RT-PCR reactions. For PCR amplification, total RNA was extracted from ED12 to ED18 mouse lung tissue or ED16 primary cell cultures according to the manufacturer's instruction (Trizol, GIBCO BRL). To synthesize cDNA, 1 µg of total RNA was incubated in 20 µl of reaction buffer (5 mM MgCl2, 50 mM KCl, and 10 mM Tris · HCl, pH 8.3) with 2.5 µM oligo(dT)16 primer and 2.5 U/µl of murine leukemia virus reverse transcriptase that was purchased as an RT-PCR kit (Perkin-Elmer). The RT reaction was carried out in one cycle at 42°C for 20 min and 99°C for 5 min. The PCR reaction was carried out in three steps as follows: 94°C for 90 s (1 cycle); 94°C for 35 s, 60°C for 35 s, and 72°C for 35 s (32 cycles); and 72°C for 6 min and 4°C for 5 min (1 cycle). In some experiments, the second step consisted of 22 cycles, but the results were similar. PCR analysis was performed with LN-alpha 1 primers: sense, 5'-AGTCCTTCAGCGCTCGTCCC-3'; antisense, 5'-GATCGACGCCGCTTGTTTCC-3'; size product 521 bp. LN-alpha 2 primers were sense 5'-CGACCGGATGCTGAAGGAAC-3' and antisense 5'-CCTCGGACATTGGTGGCAAC-3', size product 445 bp. The mouse beta -actin primers, used as controls, were previously described by Kaartinen et al. (16): sense, 5'-GTGGGCCGGTCTAGGCACCA-3'; antisense, 5'-GGTTGGCCTTAGGGTTCAGG-3'; size product 246 bp.

Primary cell cultures. Cells were isolated from ED16 mouse lungs as described by Schuger et al. (26), with some minor modifications. In brief, ED16 mice embryos were removed from the uteri under sterile conditions. The lungs were dissected out and placed in a petri dish containing 10 ml of Hanks' balanced salt solution with 100 U/ml of penicillin and 100 µg/ml of streptomycin. The lungs were cut into small pieces, and the medium was exchanged after two washes with Hanks' balanced salt solution containing 0.1% trypsin-EDTA and incubated at 37°C for 20-30 min. Isolated cells were filtered through a 100-µm nylon mesh. The cells were pelleted by centrifugation and resuspended in DMEM-10% fetal bovine serum containing 100 U/ml of penicillin and 100 µg/ml of streptomycin. The cells were plated in a 75-cm2 tissue culture flask and incubated for 1 h at 37°C. Nonattached cells were washed out. The attached cells were grown for 24 h at 37°C in a 5% CO2 atmosphere. Immunohistochemical analysis of these primary cell cultures revealed them to be principally mesenchymal-type cells containing <1% of keratin-positive cells (data not shown), a specific cell marker of epithelial cells.

Cell adhesion assays. Culture plates with 96 wells or 35-mm petri dishes were coated with different concentrations of LN1, LN2, and FN. The coated plates were incubated overnight at 4°C. The wells were saturated with 2 mg/ml of BSA for 2 h at room temperature. Fetal lung cells from primary cultures were washed with PBS, pH 7.5, containing 5 mM glucose and 2 mM EDTA. The cells were detached with 0.05% trypsin-EDTA and resuspended in DMEM containing 2 mg/ml of BSA and 1 mM MgCl2. The total cell suspensions were centrifuged and resuspended in DMEM. Aliquots containing 50,000 cells/50 µl DMEM were seeded in each well of the 96-well plates. In the experiments performed with the 35-mm petri dishes, aliquots of 200,000 cells/ml were seeded. Cells were incubated for 60 min at 37°C in a 5% CO2 atmosphere. Unattached cells were removed by aspiration. In some experiments, unattached cells were replated over FN- or LN2-coated dishes. Adherent cells were washed two times with DMEM and then incubated in DMEM containing 1 mg/ml of MTT for 60 min at 37°C in a 5% CO2 atmosphere to be quantified. Color absorbance derived from the metabolized MTT by viable cells was determined at 560 nm in an MRX microplate reader (Dynatech Laboratories). The number of cells adhered onto LN2 and LN1 was calculated from a standard curve prepared with cells attached onto FN-coated wells (1 µg FN/well) and incubated with MTT. The total number of cells plated onto FN adhered to this substrate. Experiments were performed in at least triplicate, and values are means ± SD. Viability of the cells in all experiments was checked by the exclusion of trypan blue, and only cell cultures containing >95% of viable cells were used.

Western analysis. Cell cultures were washed two times with PBS solution. The cells were incubated with 0.1% Triton X-100 for 10 min in the presence of a proteinase inhibitor cocktail. The cell extract was recovered and centrifuged at 10,000 g for 10 min. The supernatant was recovered, and SDS solution was added to a final concentration of 2%. Protein determination was performed with the bicinchoninic acid protein assay (Pierce, Rockford, IL). Equal amounts of cell extract proteins and commercially purified LN1 and LN2 were separated by SDS-polyacrylamide gel electrophoresis. Separated proteins were electrotransferred onto nitrocellulose paper. Blotted proteins were immunoreacted with the respective primary antibodies. Bands were revealed with the appropriate peroxidase-conjugated secondary antibody (Bio-Rad, Richmond, CA) and stained with a diaminobenzidine substrate kit (Pierce).

Immunohistochemical analysis. Cells and fetal lung tissue were fixed and processed for immunohistochemistry following the protocol indicated in the Histomouse SP kit (Zymed Laboratories). The primary antibodies used were anti-cytokeratin (1:500; Sigma), anti-alpha 2-chain isoform of LN2 (1:40; Chemicon), anti-vimentin (1:50; Sigma), anti-alpha -smooth muscle actin (1:1,000, Sigma), anti-desmin (1:300; Sigma), and anti-alpha -smooth muscle myosin heavy chain (1:300; provided by Dr. M. Schwartz). After subsequent washing of the samples in PBS, the secondary antibody (biotinylated antibody; Zymed Laboratories) was applied for 30 min. Finally, the antigen immunodetection was visualized after incubation of the streptavidin-peroxidase substrate. Tissues and cells were counterstained with hematoxylin and covered with a coverslip. The samples were examined and photographed with an Olympus BH2 microscope.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

alpha 2-Chain isoform of LN2 is expressed during fetal mouse lung development. To determine whether LN2 was expressed during mouse lung development, we identified the expression of the alpha 2-chain isoform of LN2 by RT-PCR analysis. We designed specific primers for the mouse alpha 2-chain isoform of LN2 and the alpha 1-chain isoform of LN1. We used published primers of beta -actin as a control. Total RNA extracted from ED12 to ED18 fetal lung tissue was reverse transcribed, and cDNA was used for the PCR reactions. As shown in Fig. 1, the amplified products of expected size for the alpha 2-chain (445 bp) and alpha 1-chain (521 bp) isoform of LN were identified during fetal mouse lung development from ED12 to ED18. No amplified products were observed in samples incubated without reverse transcriptase. The identification of the alpha 2- and alpha 1-chain products indicated that LN2 and LN1, respectively, are expressed during lung development.


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Fig. 1.   Detection of alpha 2-chain mRNA of laminin (LN) 2 from embryonic days 12 to 18 fetal mouse lung by RT-PCR. Total RNA from each indicated fetal mouse lung embryonic day was reverse transcribed. cDNA synthesized was amplified with alpha 1-chain, alpha 2-chain, or beta -actin primer. Amplified products of alpha 1-chain (alpha 1; top), alpha 2-chain (alpha 2; middle), or beta -actin (bottom) were then separated in 3% agarose gels and stained by ethidium bromide. A 100-bp DNA ladder was used as a size product reference. Nos. on left, molecular size of respective amplified product.

Fetal mouse lung cells attach differentially to LN2, LN1, and FN. To determine whether LN2 plays a biological role as an adhesive substrate for fetal lung cells, we performed a cell adhesion assay using commercially purified human LN2 as substrate, which has high homology with mouse LN2 (2). We compared its adhesive properties with LN1 and FN. Primary cultures of lung cells from ED16 mouse embryos were used in the cell adhesion assays. Cells were plated onto 96-well plates that had been previously coated with different concentrations of LN2 or LN1. They were incubated for 60 min at 37°C, and the unattached cells were removed. The cells that adhered onto LN2 or LN1 were then incubated with medium containing MTT to be quantified as described in METHODS. The cells that attached onto both substrates were observed to be uniformly distributed. In addition, we observed that cells attached onto LN2 were flattened, and some of these cells had many hairlike projections extending outward from the cytoplasm. In contrast, cells plated onto LN1 adhered but remained rounded, and only a few of them flattened out over the surface. As shown in Fig. 2, the maximal density of cells that adhered onto both substrates was reached at a concentration of 7.5 µg · ml-1 · well-1. The number of cells able to adhere onto LN1 or LN2 remained constant at concentrations > 10 µg/ml (data not shown). The number of cells attached onto LN2 was four times greater than those attached onto LN1. However, only one-third of the total fetal lung cell population was able to adhere to LN2. This observation was highly reproducible. A longer time of incubation did not increase the number of cells attached to LN2.


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Fig. 2.   Differential adhesion of fetal mouse lung cells plated onto 96-well plates coated with increasing concentrations of LN2 and LN1. Aliquots containing 50,000 cells/50 µl were seeded on coated wells and incubated for 60 min at 37°C. Unattached cells were removed by aspiration. Cells that adhered to matrix substrate were carefully washed two times and then incubated in DMEM containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl blue; MTT) for quantification. Number of cells was determined by color absorbance at 560 nm with a standard curve as described in METHODS. Values are means ± SE from 4 wells in a representative experiment that was performed at least 3 times (P < 0.005).

The viability of the LN1- and LN2-unattached cells was verified to be >96%, and these cells were replated onto FN. The LN1- and LN2-unattached cell populations were able to attach onto FN, and the cell morphologies were similar. In addition, we observed that the total fetal lung cell population was able to adhere and spread onto FN.

To better quantitate the cells adhering to LN2, we plated the cells onto 35-mm petri dishes coated with LN2 (30 µg/ml) or FN (30 µg/ml). The concentration of each substrate used to coat the petri dishes was determined to allow the maximal number of attached cells. We observed again that only a subpopulation of cells (32 ± 5%) was able to adhere to LN2, whereas the total population (94 ± 3%) adhered to FN (Fig. 3). We also replated the LN2-unattached cell population on LN2, and the cells were incubated for 2 h at 37°C. We observed that LN2-unattached cells were still not able to adhere to LN2. The attachment of fetal lung cells to LN2 was abrogated when cells were seeded in the presence of 4 mM EDTA.


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Fig. 3.   Differential adhesion of fetal mouse lung cells plated on LN2 or fibronectin (FN) substrate. Primary cell cultures from embryonic day 16 fetal mouse lung cells were plated onto 35-mm petri dishes previously coated with FN (30 µg/ml), LN2 (30 µg/ml), or BSA (2 mg/ml). Cells plated on LN2 were incubated in absence and presence of 4 mM EDTA. Cells were allowed to attach to the different substrates for 1 h at 37°C in a 5% CO2 atmosphere. Nonattached cells were removed, and attached cells were trypsinized and counted. Viability of cells was verified before and after each experiment. This experiment was performed at least 4 times. Values are means ± SE from a representative experiment in triplicate. Cells plated onto BSA or in presence of EDTA were unable to adhere.

LN2-attached subpopulation of fetal lung cells expresses a myofibroblastic phenotype. Some reports (3, 11) have indicated that mesenchymal cells attach preferentially to FN compared with LN1. We were therefore interested to determine whether the subpopulation of cells that were adhering to LN2 were epithelial or mesenchymal cells. The epithelial cell characterization was performed with a keratin antibody (clone PCK-26; Sigma) that recognizes keratin of high and low molecular weight in different species, including mouse epithelial cells. As illustrated in Fig. 4A, we identified that the cell population attached to LN2 did not contain epithelial cells. In contrast, the cells that adhered to LN2 expressed the mesenchymal cell marker vimentin (Fig. 4B), indicating that they were of mesenchymal origin. Furthermore, we found that ~90% of cells in this subpopulation of mesenchymal lung cells also expressed alpha -smooth muscle actin and desmin, specific markers of myofibroblasts (6, 7, 17). As illustrated in Fig. 4C, the immunostaining pattern with the desmin antibody showed a perinuclear and radial distribution. The presence of cytoplasmic filaments was observed in the cells immunostained with the alpha -smooth muscle actin antibody (Fig. 4D). To determine whether the cells in this subpopulation were myofibroblasts or smooth muscle cells, we used an antibody directed against alpha -smooth muscle myosin, a specific cell marker of smooth muscle cells (6, 13). As shown in Figs. 4E and 5, only a small number of cells (9 ± 4.8%) showed positive immunostaining with this antibody, indicating that the LN2-adherent cell population consisted principally of myofibroblastic cells. On the other hand, we observed that the population of cells unable to attach onto LN2 contained more cells with a smooth muscle phenotype (24 ± 5.2%) and fewer cells expressing the myofibroblastic phenotype (Fig. 5). Myofibroblastic cells expressing vimentin, alpha -smooth muscle actin, and desmin have been classified as VAD type (6, 7). The above observations led us to conclude that the LN2-adherent cell subpopulation of fetal mouse lung mesenchymal cells corresponds to VAD-type myofibroblasts. As shown in Fig. 5, both LN2-adherent cells and nonadherent cells expressed LN2.


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Fig. 4.   Phenotype characterization of fetal lung cell subpopulation that adhered to LN2. Cells attached to LN2 were trypsinized and then plated onto Labtek wells for immunostaining analysis as described in METHODS. Cells were immunostained with anti-keratin (A), anti-vimentin (B), anti-desmin (C), anti-alpha -smooth muscle actin (D), anti-alpha -smooth muscle myosin heavy chain (E), or anti-LN2 (F) antibody. A shows negative cell immunoreactivity and demonstrates absence of epithelial cells in subpopulation of fetal lung cells adhered to LN2. B demonstrates positive immunoreactivity of cells to vimentin, indicating mesenchymal cell origin. C and D show positive staining to desmin and alpha -smooth muscle actin, both cells markers of myofibroblastic phenotype. E: only a small number of cells show positive staining to alpha -smooth muscle myosin heavy chain. F shows positive reactivity of cells to LN2 antibody. Bar, 50 µm.


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Fig. 5.   Comparison of abundance of cells bearing myofibroblastic phenotype in cell subpopulation that adheres onto LN2 vs. nonadherent cells. LN2-adherent and nonadherent cells were seeded separately onto Labtek wells (2 × 103 cells/well). They were fixed and processed for immunohistochemistry in presence of desmin (DSM), alpha -smooth muscle actin (SMA), alpha -smooth muscle myosin heavy chain (SMM), and LN2 antibodies as described in METHODS. Cells that showed both positive and negative immunoreactivity to antibodies were quantified under the microscope. Values are means ± SE from a representative experiment performed at least 3 times. LN2-adherent cell population showed that majority of cells have a myofibroblastic DSM and alpha -SMA phenotype (solid bars) compared with nonadherent cell population (open bars). Only a small number of cells showed positive immunoreactivity to alpha -SMM, indicating that LN2-adherent cells have myofibroblastic VAD phenotype (see text). Both LN2-adherent and nonadherent cells showed positive immunoreactivity to LN2 antibody.

Fetal lung mesenchymal cells attached to LN2 also autoexpress LN2. To confirm whether the LN2-adherent subpopulation of mesenchymal autoexpressed LN2 and was not due to a contamination derived from the coated plates, we analyzed by RT-PCR assay the presence of LN2 mRNA from total mRNA extracted from the mesenchymal cell subpopulation adhered onto LN2. The results indicated that alpha 2-chain mRNA of LN2 was indeed present in this subpopulation but not the alpha 1-chain of LN1 (Fig. 6A). Furthermore, the expression of the alpha 2-chain isoform was verified by Western analysis. The subpopulation of cells adhered to LN2 was trypsinized and subcultured onto a cell culture petri dish without LN2. The cells were incubated overnight at 37°C in a 5% CO2 atmosphere. The cells were recovered and lysed for Western blot analysis as indicated in METHODS. We used a monoclonal antibody directed to a fragment of the human alpha 2-chain of LN2, which detected a polypeptide of 70 kDa in the cell extract under reducing conditions (Fig. 6B). The specificity of the antibody was confirmed by preincubation of the antibody with LN2. The preincubated antibody failed to detect the alpha 2-chain fragment both in the fetal lung cell culture extract and in the human LN2 (data not shown).


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Fig. 6.   Detection of LN2 in fetal lung cell subpopulation adhered to LN2. In A, total RNA obtained from LN2-adhered cell cultures was reverse transcribed. cDNA synthesized was amplified with beta -actin, alpha 2-chain, and alpha 1-chain primers. Amplified products of alpha 2-chain (lane 1), alpha 1-chain (lane 2), and beta -actin (lane 3) were separated in 3% agarose gels and stained by ethidium bromide. A 100-bp DNA ladder was used as a size product reference. Nos. on left, molecular size of respective amplified product. In B, identification of alpha 2-chain expression in LN2-adhered cell cultures by Western blot. LN2-adhered cell cultures were trypsinized and cultured onto a petri dish without LN2 for 24 h at 37°C in a 5% CO2 atmosphere. Cell extract from these cell cultures and purified LN2 were separated by 7.5% SDS-PAGE, electrotransferred to a membrane, and incubated with a monoclonal antibody directed to an 80-kDa fragment of alpha 2-chain of human LN2. A band at 80 kDA was detected in human LN2 (lane 1), and a 70-kDa band was visualized in fetal mouse lung cell extracts (lane 2).

LN2 is present in fetal lung tissue. The in vitro analysis of the role of LN2 led us to determine the in vivo distribution of LN2 in fetal lung tissue. We used ED16 lung tissue corresponding to the late pseudoglandular stage of mouse lung development. We used the monoclonal antibody against the alpha 2-chain of LN2 in immunohistochemical analysis. The immunolocalization of LN2 observed in the ED16 lung tissue showed a strong signal around the basement membrane of airways and blood vessels; this pattern matched with the tissue distribution of alpha -smooth muscle actin (data not shown). However, the LN2 antibody also showed immunoreactivity around some mesenchymal cells, although this staining was weaker than that observed around the basement membrane (Fig. 7).


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Fig. 7.   Identification of alpha 2-chain of LN2 in fetal lung tissue. Fetal mouse lung tissue corresponding to pseudoglandular stage (embryonic day 16) was processed for immunohistochemistry as described in METHODS. Expression of alpha 2-chain of LN2 was localized in basement membrane of airways (arrow) and around blood vessels and mesenchymal cells. Bar, 50 µm.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In the present report, we have shown that alpha 2-chain mRNA of LN2 is expressed in embryonic lung tissue, suggesting that LN2 may play a biological role during mouse lung development. The expression of LN2 was originally reported to be specific to the basement membrane of striated muscle, trophoblasts, and peripheral nerves (10, 21). However, our results presented here, together with recent data from other laboratories (4, 35), indicate that LN2 has a more general distribution. On the other hand, one report (33) has indicated that LN2, together with LN1, may also contribute in the assembly of the basement membrane, indicating that LN2 may actively participate in the structural organization of the ECM.

Our results obtained by PCR suggest that the pattern of expression of the alpha 2-chain during mouse lung development is different from that of the alpha 1-chain. This expression pattern was observed in all the experiments performed during the present work. In addition, our results indicated that LN2 is expressed by fetal lung cells from the beginning of lung development. Previous reports (10, 21) have shown that LN2 is expressed in earlier embryonic stages that precede lung development.

A recent study (35) also indicated a distinct expression pattern and distribution of the alpha 2-chain during human lung development. In the human study, the alpha 2-chain of LN2 was localized principally in the basement membrane of bronchial epithelial cells during the pseudoglandular stage of lung development, whereas in the canalicular stage, the alpha 2-chain was localized along peribronchial smooth muscle cells. These data suggest that LN2 may play a role during bronchial smooth muscle cell development in human embryonic lung.

A previous study (1) has further indicated that LN2 is expressed in adult patients with severe asthma but not in healthy patients, suggesting that LN2 may play a biological role in lung ECM remodeling. LN2 has also been implicated in congenital muscular dystrophy (36, 39); a partial mutation in the alpha 2-chain gene is a major cause of this disease (32). LN2 null mutant animals die at an early age. However, it is not known whether patients or animals with LN2-related disease have abnormal lung myofibroblasts.

In the present report, we determined for the first time a specific biological role for LN2 as a selective cell-adhesive substrate for a specific subpopulation of fetal lung mesenchymal cells. In addition, we demonstrated a differential pattern of cell adhesion for mesenchymal cells between LN2 and LN1. This study was performed with primary cell cultures derived from ED16 lungs. This embryonic day corresponds to the pseudoglandular stage in mouse lung development. The best yield of isolated mesenchymal cells that adhere to LN2 was obtained on ED16.

Our results further support the hypothesis that each LN variant may play a specific role during lung development. Both LN1 and LN2 contain the beta 1- and gamma 1-chains but differ in the alpha -chain. Therefore, the alpha -chain may contribute directly to the differences in adhesive properties. There is only 46% homology between mouse LN alpha 2- and alpha 1-chains (2). Several domains in the alpha 1-chain of LN1 with ascribed cell-adhesive properties, such as RGD and SIKVAV sequences, are not found in the alpha 2-chain (2). Differential attachment to LN2 in comparison to LN1 suggests the presence of specific receptors for the mouse alpha 2-chain in fetal lung mesenchymal cells.

LN1 (alpha 1, beta 1, gamma 1) was the first laminin purified and characterized (34). Its expression has been demonstrated to be crucial for the early progression of embryonic development. During lung development, LN1 is expressed principally by epithelial cells (35). In addition, LN1 promotes the organotypic rearrangement of isolated mesenchymal and epithelial cells from fetal mouse lung when cultured together (26, 31). In particular, the alpha 1-chain (400 kDa) of LN1 was observed to be widely distributed in pulmonary basal membranes of lung tissue during human fetal development (20, 35). In addition, the LN1 variant has been described to be a preferential substrate for epithelial cells (3). The subpopulation of cells that attach to LN2 expressed vimentin, a protein found in fibroblastic cells but not in keratin, a specific marker of epithelial cells. We also noted that a majority of these cells expressed alpha -smooth muscle actin and desmin, two intracytoplasmic proteins considered to be phenotypic markers of myofibroblasts and/or smooth muscle cells (6, 7, 13, 17). Thus our observations indicate for the first time that LN2 may be involved in the attachment of a specific subpopulation of fetal lung mesenchymal cells that bear a myofibroblastic phenotype.

In vivo, the concept of fibroblast heterogeneity is now well accepted. It is suggested that fibroblast populations are modulated during development to produce a heterogeneous phenotypic population in different organs (6, 7, 17, 24). Thus fibroblastic cells are relatively undifferentiated and can assume a particular phenotype according to physiological needs and/or microenviromental stimuli (24, 25). Cultured fibroblasts may also express different phenotypic features. When grown in vitro, subpopulations of fibroblasts derived from normal tissue acquire several phenotypic features of myofibroblasts. This observation has been indicated by different groups (6, 7, 24). Among the smooth muscle cell markers expressed by myofibroblastic populations, alpha -smooth muscle actin is the most common, followed by desmin (6). Smooth muscle myosin heavy chain expression is observed to a lesser extent, and it is principally attributed to be a specific marker of smooth muscle cells (6, 13). Our results showed that the LN2-adherent subpopulation consisted of ~90% of cells expressing alpha -smooth muscle actin and desmin, and only a small number of cells (9 ± 4.8%) were observed to express smooth muscle myosin heavy chain (Fig. 5). Myofibroblastic cells expressing vimentin, desmin, and alpha -smooth muscle actin have been classified as VAD type (6). Our results suggest that the subpopulation of LN2-adherent cells reported herein correspond principally to VAD-type myofibroblasts. Although we observed that almost all mesenchymal cells expressed LN2 (Fig. 5), the selective attachment of the subpopulation of myofibroblasts to LN2 suggests the presence of specific LN2 cell membrane receptors for this substrate in this particular cell subpopulation. In contrast, we observed that the cell population unable to attach to LN2 was, however, able to attach to FN, indicating that the latter cell population may not bear the specific receptors necessary to attach to LN2. Thus our results strongly support the existence of fibroblast heterogeneity during lung development and suggest that the LN2-adherent cell subpopulation may play an important role in pathological states such as asthma in which an elevated expression of LN2 and cell-matrix remodeling occurs (1).

The autoexpression of LN2 in the subpopulation of cells that adhered to LN2 was corroborated by RT-PCR and Western analysis. A monoclonal antibody to the alpha 2-chain detected a single 70-kDa band in the lung cell subpopulation that attached to LN2. This was somewhat lower than the molecular mass (80 kDa) observed with human alpha 2-chain. However, this difference has also been observed by another group (11) and may reflect species differences in the relative glycosylation of the respective molecules.

In a recent publication, Schuger et al. (30) indicated that an antibody directed against the alpha 1-chain of LN1 was able to modify the phenotype of smooth muscle cells in embryonic lung explants and decreased the expression of desmin. However, an antibody directed against the alpha 2-chain did not produce any effects. These results, together with our data, suggest that LN2 may principally play a biological role as a cell adhesion substrate for a specific subpopulation of mesenchymal cells. Preliminary studies in our laboratory indicate that growth factors, together with this LN2-cell interaction, may modulate the proliferation and migration of this cell subpopulation. In contrast, LN1 may be involved in controlling differentiated cell functions and play a role as a preferential substrate for epithelial cells.

Several integrin and non-integrin receptors have been described for LN1 and LN2 (11). In the lung, the integrin receptors alpha 3beta 1 and alpha 6beta 1 bind LN1 (3, 19). However, the integrin receptors present in fetal lung mesenchymal cells that bind LN2 are unknown. Integrin function is dependent on divalent cations (23), and the presence of chelating agents such as EDTA completely inhibits their function. Herein, we have demonstrated that the attachment of fetal lung mesenchymal cells to LN2 can be abrogated by the presence of EDTA, suggesting the involvement of an integrin receptor in the adhesion to LN2. A recent report (5) has indicated that LN1 and LN2 are able to bind both alpha 1beta 1- and alpha 2beta 1-integrins. However, the differential adhesion pattern of mesenchymal cells to LN1 versus LN2 suggests the involvement of another type of integrin. We are currently isolating and characterizing membrane receptors derived from lung mesenchymal cells that bind LN2.

The RGD sequence domain found in FN and some ECM glycoproteins interacts directly with integrins. In lung cells, the integrin alpha 5beta 1 is the principal receptor for FN (3). Our results show that only a subpopulation of fetal lung mesenchymal cells binds to LN2; meanwhile, the cells that did not attach to LN2 can bind to FN, further suggesting that specific receptors for LN2 may mediate selective adherence to LN2 versus FN.

Recent studies (14, 37) have identified the dystrophin-dystroglycan complex as a non-integrin receptor that binds LN2. However, the role of this receptor in the attachment of lung mesenchymal cells to LN2 remains to be characterized.

The presence of other variants of LN in the lung leads to the speculation that each variant may have a specific role during lung development, and this remains to be elucidated. The recent identification of the alpha 3-chain isoform of LN in lung (12) suggests that LN5, LN6, or LN7 may also be present (33). The latter chain isoform has been localized in the basement membrane of epithelial cells in human fetal lung (35). In addition, the expression of the beta 2-chain isoform that forms part of the variants LN3, LN4, and LN7 was demonstrated in alveolar type II cells during development of rabbit lung (9). Its distribution was localized in the basement membrane of airways and arterial blood vessels. The presence of the alpha 4-chain that forms part of LN8 has also been observed to be highly expressed in lung tissue (15). The LN2 immunostaining observed in fetal lung tissue agrees with previous observations, indicating that LN2 is expressed by mesenchymal cells.

To our knowledge, this is the first report indicating that LN2 is an adhesive substrate for fetal lung myofibroblasts. In the past, several studies (3, 38) have used either individual or mixed ECM proteins such as Matrigel or Gelfoam as cell substrates. However, the cell membrane receptors involved in the attachment to these substrates may differ from those used in the cell attachment to LN2.

In conclusion, we have shown that LN2 is present in a specific temporospatial distribution in embryonic mouse lung and is involved in the differential attachment of a subpopulation of fetal lung mesenchymal cells that express a myofibroblastic phenotype. We speculate that LN2-cell interaction may be involved in the migration, distribution, and proliferation of this LN2-adherent cell population during mouse lung development.

    ACKNOWLEDGEMENTS

We thank Pei Jia Chen and Norman W. Lautsch for technical advice and Valentino Santos for photographic assistance.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-44977 and HL-44060.

Address for reprint requests: D. Warburton, Center for Craniofacial Molecular Biology, School of Dentistry, Univ. of Southern California, 2250 Alcazar St., Los Angeles, CA 90033.

Received 10 October 1997; accepted in final form 29 May 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(3):L622-L630
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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