* Department of Pathology and Laboratory Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201; Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455;
and § Connective Tissue Department, Institute for Experimental Medicine, Erlangen 91054, Germany
Laminins, the main components of basement
membranes, are heterotrimers consisting of ,
, and
polypeptide chains linked together by disulfide bonds.
Laminins-1 and -2 are both composed of
1 and
1
chains and differ from each other on their
chain, which is
1 and
2 for laminin-1 and -2, respectively.
The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial-mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin
1 chain. Synthesis of laminin
1 chain however returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical, since laminin
1 chain is not detected in
monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which
heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein
secretion, indicated both epithelial and mesenchymal
cells synthesize laminin
1 chain upon heterotypic cell-
cell contact. In a set of functional studies, embryonic
lung explants were cultured in the presence of monoclonal antibodies to laminin
1,
2, and
/
chains.
Lung explants exposed to monoclonal antibodies to laminin
1 chain exhibited alterations in peribronchial
cell shape and decreased smooth muscle development,
as indicated by low levels of smooth muscle
actin and
desmin. Taken together, our studies suggest that laminin
1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway
smooth muscle development.
BASEMENT membranes (BMs)1 are specialized, sheet-like extracellular matrices that divide tissues into
compartments. The BMs function as a dynamic
structure in morphogenesis, cell differentiation, and maintenance of the mature cellular structural and functional phenotypes (for review see Kleinman et al., 1993 Laminins (LMs) are the major constituents of BMs. This
complex family of extracellular matrix proteins plays important roles in cell adhesion, growth, morphology, and
migration (Timpl and Brown, 1994 The first LM reported, isolated from the Engelbreth
Holm-Shawm (EHS) tumor (Timpl, 1979), is now referred
to as LM-1(Burgeson et al., 1994 LM-1 plays important roles in lung development, more
specifically in the processes of branching morphogenesis
(Schuger et al., 1990a More recently, other LMs have been identified in the
developing lung, including LM-2 (Virtanen et al., 1996 Mouse lung development begins on day nine of gestation (Ten Have-Opbroek, 1981; Theiler, 1989 Here we present evidence suggesting that epithelial-
mesenchymal interaction is required for the synthesis of
the LM Antibodies
A polyclonal antibody against murine EHS LM was generated as previously described (Palm and Furcht, 1983 Generation of Epithelial and Mesenchymal
Monocultures and Cocultures
CD-1 strain (Charles River) mice were mated, and the day of finding a
vaginal plug was designated as day zero of embryonic development. Lungs
were removed at day 15 of gestation, minced, and placed in PBS containing 0.3% trypsin and 0.1% EDTA for 10 min at 37°C. A single cell suspension was obtained by forcing cell aggregates and pieces of tissue through a
micropipet several times. The cells were then filtered through a 100-µm-pore
mesh and resuspended in minimal essential medium (MEM; GIBCO BRL,
Gaithersburg, MD) with nonessential amino acids (GIBCO BRL), 0.29 mg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 mg/ml
amphotericin B, and 10% fetal bovine serum (Irvine Scientific, Santa
Ana, CA). Monocultures were generated by differential plating as previously described (Schuger et al., 1993 The cultures were established in 24-well plates (GIBCO BRL) or on
the upper surface of polycarbonate membrane inserts (Millipore, Bedford, MA). The polycarbonate membrane does not support cell attachment, and under these conditions the cells cluster together at the center of
the insert, allowing maximal cell-cell interaction. Approximately 3 to 6 × 105 cells were added to each well or insert. The cells were cultured in complete medium for 2 to 72 h. In some experiments involving epithelial-mesenchymal cocultures, the two cell populations were separated again by differential plating at the end of the coculture period. All of the cultures
employed for these studies were at passages 0 to 2.
Treatment of Epithelial-Mesenchymal Cocultures
with Brefeldin A
In these studies, organotypic epithelial-mesenchymal cocultures were exposed to the fungal metabolite brefeldin A (Sigma Chemical Co., St.
Louis, MO), an inhibitor of protein secretion at the pre-Golgi compartment (Klausner et al., 1992 Functional Studies on Lung Organ Cultures
Embryos were collected at day 12 of gestation. Their lungs were then dissected and the lower right lobes were cultured at the air-medium interface
on the upper surface of polycarbonate membrane inserts, in a serum free-defined medium, BGJb (GIBCO BRL; Schuger et al., 1996 Metabolic Labeling, Immunoprecipitation,
and SDS-PAGE
Epithelial and mesenchymal monocultures and cocultures (~2 × 107 cells
each) were incubated for 30 min in methionine-free medium supplemented with 1 mCi/ml of [35S]methionine (NEN Dupont, Boston, MA).
After incubation, the cells were washed, cultured in cold medium for an
additional 30 min, and then lysed in SDS sample buffer (0.0625 M Tris-HCL, pH 6.8, 10% glycerol, 2% SDS, and 2.5% 2 Western Blotting
Cell monocultures, cocultures, and lung organ cultures were lysed by boiling for 10 min in SDS sample buffer under reducing conditions for LM
and under nonreducing conditions for smooth muscle Immunohistochemistry
Immunolocalization of LM ELISA
Epithelial-mesenchymal cocultures and monocultures were washed and
incubated for 3 h in serum-free MEM containing 0.02% BSA (MEM-BSA). The culture fluids were then collected and added to quadruplicate
wells of a 96-well plate (Falcon Plastics, Oxnard, CA) in aliquots of 0.1 ml/
well. MEM-BSA served as a negative control. Serial dilutions of LM-1
were added to the assay plates to serve as a positive standard. After a 4-h
incubation, the fluids were removed and the ELISA was performed as described (Varani et al., 1985 LM-1 and LM-2 Are Expressed by the Mouse
Fetal Lung
Immunoblots using a polyclonal antibody against EHS
LM (Fig. 1, x-LM) that recognizes both
LM Monocultures of lung epithelial and mesenchymal cells as
well as homotypic cocultures (epithelial cells added to an
established epithelial monoculture, E/E; or mesenchymal
cells added to an established mesenchymal monoculture,
M/M) synthesized LM
Immunohistochemical studies showed that LM
Immunoprecipitation with a monoclonal antibody to the
LM
Epithelial-mesenchymal cocultures separated by a filter
did not synthesize LM
LM
LM Immunohistochemical studies showed no intracellular LM
Functional Studies Showed Alterations in
Peribronchial Cell Shape in Lung Explants Exposed
to Anti-LM Day 12 lung explants were cultured for 3 d in the presence
of monoclonal antibodies to LM chains Microscopic evaluation of hematoxylin- and eosin-stained
sections showed well preserved epithelial and mesenchymal cellular architecture with scattered mitotic figures in
both tissue compartments. A normal histological pattern
was seen in the mesenchyme of lung explants exposed to
antibodies against LM
To assess the magnitude of this effect, longitudinal sections of each explant including the primary and secondary
bronchi were projected on a television screen, and the
number of mesenchymal cells with elongated (polarized)
nuclei and with round (unpolarized) nuclei was determined. Although the main bronchi were surrounded by
two to three concentric layers of elongated mesenchymal
cells, only the cells directly apposed to the BM were counted.
By using this semi-quantitative method, we found a statistically significant difference in the number of elongated
versus round peribronchial cells in the explants exposed to
50 or 100 µg/ml of anti-LM
Functional Studies Suggested That LM To study the development of bronchial smooth muscle, we
used antibodies against two different smooth muscle-specific proteins, smooth muscle
Epithelial-Mesenchymal Contact Induces Synthesis of
LM Heterotypic cell interactions are long known to be essential for morphogenesis and cell differentiation, however,
the molecular mechanisms underlying these processes are
poorly understood. Most developing organs, including the
lung, are composed of two main cell populations, the epithelium and the mesenchyme, separated by a BM. Among
the BM constituents, LM-1 is known to play a significant role in morphogenic epithelial-mesenchymal interactions
(for review see Ekblom, 1996 LM Homotypic cocultures, epithelial-mesenchymal cocultures
separated by a filter, or monocultures exposed to conditioned media, did not produce LM The need for cellular contact was noticed in a variety of
cell-cell interactions (Reichmann et al., 1989 The simplest mechanism of cell-cell interaction leading
to LM Unfortunately, very little is known about the mechanisms regulating LM expression. Retinoic acid has been
shown to stimulate LM-1 synthesis by embryonic cells
(Carlin et al., 1983 LM There has been some uncertainty in the literature as to
what cells synthesize LM Ekblom and coworkers observed that the development
of epithelial cell polarity coincides with the expression of
LM Our studies as well as others (Virtanen et al., 1996 LM Lung explants cultured in the presence of antibodies
against LM Interestingly, two previous studies reported the differentiation of mesenchymal cells into smooth muscle upon
prolonged contact with the epithelium (Cunha et al., 1992 Most mesenchymal cells in the developing lung have a
round configuration and obviously do not express smooth
muscle markers. Interestingly, stretching forces cause undifferentiated mesenchymal cells to express smooth muscle-specific proteins (Yang, Y., and L. Schuger, unpublished observations). Based on this observation, we propose
that in the lung explants exposed to anti-LM On the other hand, LM-1 has a direct effect on muscle
cell shape, unrelated to stretching (Öcalan et al., 1988 In summary, we have shown that while LMs ).
). LMs consist of three
subunit polypeptide chains, classified as
,
, and
chains.
These are linked together by disulfide bonds and associate
into a cruciform tertiary structure by a triple-helical coiled
coil producing a long, rigid rod-like structure and two to
three shorter arms (for review see Engel, 1992
). The different LM chains share partial homology, particularly in
the globular and rod-like domains containing EGF-like repeats (Engel, 1992
) and domains participating in the
coiled-coil region (Iivanainen et al., 1995
; Miner et al., 1995
).
Additionally, the
chains contain a large COOH-terminal
globular domain with five internal repeat motifs that have
been identified as major sites for integrin binding (Hall et
al., 1990
; Kramer et al., 1990
; Elices et al., 1991
) and heparin binding (Skubitz et al., 1988
).
) and is composed of
1
(400 kD),
1 (210 kD), and
1 (200 kD) chains. LM-1 is
the earliest extracellular matrix molecule produced in
mouse embryogenesis (Wu et al., 1983
), and during development it can be detected in epithelial BMs of most organs, including the lung (Wan et al., 1983; Klein et al.,
1990
; Schuger et al., 1992
). The presence of LM
1,
1,
and
1 mRNA in the mouse lung epithelial and mesenchymal compartments (Schuger et al., 1992
; Thomas and Toziadek, 1994; Lallemand et al., 1995
) suggests that both
cell populations contribute to its production. However,
other studies have shown an exclusively epithelial cell origin
(Klein et al., 1990
). LM-1 deposits exclusively in epithelial BMs. Vascular BMs and the rest of the extracellular matrix do not contain LM-1 (Klein et al., 1990
; Schuger et al.,
1992
; Thomas and Toziadek, 1994; Lallemand et al., 1995
;
Virtanen et al., 1996
).
, 1991
), BM assembly (Schuger et al.,
1995
, 1996
), epithelial cell adhesion (Matter and Laurie,
1994
; Schuger et al., 1995
), and epithelial cell polarization
(Schuger et al., 1990b
, 1995
, 1996
). In addition, our recent
studies showed that LM-1 polymerization at the epithelial-mesenchymal interface is required for normal arrangement and polarization of bronchial smooth muscle
cells (Schuger, L., P. Yurchenco, and Y. Yang, manuscript
submitted for publication).
).
However, their functional activity and possible role in
morphogenesis remain to be elucidated. LM-2, formerly
referred to as merosin (Engvall, 1994
), is the main LM in
striated muscle and differs from LM-1 only in its
chain
(
2,
1,
1).
) and culminates in the neonatal period giving rise to a complex pattern of branched airways and alveoli (Ten Have-Opbroek,
1981). Cytological markers indicative of smooth muscle
differentiation, such as smooth muscle
actin, desmin,
and myosin start to appear in mesenchymal cells surrounding the trachea and main bronchi by days 11 to 12, progressing in a proximal to distal fashion (Roman and McDonald, 1992
; Yang, Y., and L. Schuger, unpublished
observations). LM-1 deposition in the developing lung, although restricted to the epithelial-mesenchymal interface,
coincides with the areas of bronchial smooth muscle development. Smooth muscle cells are not detected in the lung vasculature until late development (Roman and McDonald,
1992
; Yang, Y., and L. Schuger, unpublished observations).
1 chain, while the LM
2 chain is constitutively
synthesized by both cell types. Furthermore, monoclonal
antibodies to LM
1 chain but not to LM
2 or
/
chains
altered peribronchial mesenchymal cell shape and inhibited smooth muscle development in lung organ cultures. These results suggest that the LM
1 chain synthesis is
stimulated by epithelial-mesenchymal contact and may
play a role in airway smooth muscle development.
Materials and Methods
). The antibody reacts with LM-1
but not fibronectin or type IV collagen (McCarthy et al., 1983
). The antibody was further purified on an LM-1 affinity column (McCarthy and
Furcht, 1984
). Monoclonal antibodies to LM
1 (referred to as AL-4) and
1/
1 chains (referred to as AL-3) were generated by the immunization of
male LOU/MNCr rats against murine EHS LM. The preparation, purification, and characterization of these antibodies has been previously described (Skubitz et al., 1987
, 1988
). Normal rat IgG was purchased from
Cappel (Malvern, PA). A monoclonal antibody to the LM
2 chain (4H8-2)
was generated, purified, and characterized as previously described (Schuler
and Sorokin, 1995
). This antibody does not recognize other LM chains (Schuler and Sorokin, 1995
). A rabbit polyclonal antibody to low and high
molecular weight cytokeratins was purchased from Dako (Carpinteria,
CA). A mouse monoclonal antibody to smooth muscle
actin was obtained from Boehringer Mannheim (Indianapolis, IN). This antibody has
been shown to immunoreact with mouse smooth muscle
actin (Roman
and McDonald, 1992
). A mouse monoclonal antibody to desmin was purchased from Dako. EHS LM was purchased from Collaborative Biomedical
(Boston, MA).
). Culture samples were immunostained with an anti-keratin antibody to identify epithelial cells. Only mesenchymal monocultures with <1% keratin-positive cells and epithelial monocultures with 10% or less mesenchymal cell contamination were used to carry out experiments. Homotypic and heterotypic cocultures were generated by plating mixed lung cell populations directly isolated
from fetal lungs, by plating together different numbers of epithelial and/or
mesenchymal cells obtained from monocultures, or by adding different
numbers of one cell type to an established monoculture of the same or the
other cell type.
), to determine what cell type synthesizes LM
1 chain. Organotypic cocultures were generated by plating mixed lung
cell populations isolated from fetal lungs at high densities (1-5 × 106 cells/
ml; Schuger et al., 1993
). When plated at high density, the lung epithelial
cells form spheroid clusters and cysts and the mesenchymal cells grow
around them as a monolayer (Schuger et al., 1993
, 1995
). 2-d-old organotypic cocultures were washed and incubated for 3 h in complete medium
supplemented with 0, 1, 5, and 10 µg/ml of brefeldin A. At the end of the
culture period the cultures were washed and immunostained as described
below.
). The lung explants were cultured for 3 d in the presence of monoclonal antibodies to
LM
1 chain (monoclonal antibody AL-4),
2 chain (monoclonal antibody 4H8-2), LM
1/
1 chain (monoclonal antibody AL-3), control IgG,
or no treatment. Previous studies demonstrated that these antibodies penetrate into the lung explant and bind to the epithelial BM (Schuger et al.,
1991
). The immunoglobulins were added to the organ cultures at concentrations of 10, 50, and 100 µg/ml at the beginning of the experiment. These
concentrations were not toxic to the cells as indicated by a cytotoxicity assay based on Cr51 release (Schuger et al., 1989; data not shown). Since the
embryonic lungs are transparent, the number of terminal airway buds was daily determined as an indicator of branching morphogenesis. After 3 d in
culture, the explants were lysed or formalin fixed, and paraffin embedded.
5-µm-thick sections were cut from the latter and stained with hematoxylin-eosin for light microscopy evaluation. These experiments were done in
triplicate wells (1 explant/well) and were repeated seven times.
-mercaptoethanol; all
from BioRad, Richmond, CA). LM-1 was precipitated from the lysates
with 10 µg/ml of monoclonal antibody AL-4 (against LM
1 chain) and
protein A-Sepharose (Sigma Chemical Co.) as previously described
(Schuger et al., 1992
). The immunoprecipitates were eluted and fractionated in 4% polyacrylamide gels under reducing conditions. Radioactive bands were visualized by exposing the dried gels to X-ray film (Kodak
XAR-2; Eastman Kodak Co., Rochester, NY).
-actin and desmin.
Cell supernatants were diluted 1:2 in 2× SDS sample buffer and boiled
under reducing conditions. 30 µg of sample was resolved in a 4% acrylamide gel for LM studies and in a 15% gel for smooth muscle
actin and
desmin studies. The samples were transferred to nitrocellulose membranes (BioRad) according to the method of Towbin et al. (1979)
and
blocked with 5% dry nonfat milk in TBS-T (20 mM Tris base, 137 mM sodium chloride, 0.05% Tween-20, pH 7.6; all from BioRad). The membranes were then blotted for 1 h with a 1:100 dilution of polyclonal antibodies to EHS LM that recognize LM
1 and
2 chains or monoclonal
antibodies to LM
1 chain (AL-4), LM
2 chain (4H8-2), smooth muscle
actin, and desmin. This was followed by another hour of incubation with a 1:3,000 dilution of the secondary antibody, goat anti-rat, anti-rabbit, or
anti-mouse IgG depending on the primary antibody. The bands were detected by chemiluminescence using a commercial kit (Amersham Life Science, Arlington Heights, IL) and following the manufacturer's instructions.
1 chain was examined on organotypic cocultures and on embryonic lungs. Occasionally, double immunostaining (immufluorescence followed by immunoperoxidase) was done for LM-1 and
cytokeratins combined. Cocultures and 5-µm-thick lung frozen sections
were fixed for 5 min in absolute alcohol, exposed to 5% normal goat serum followed by treatment with a 1:50 dilution of monoclonal antibody to
LM
1 chain (AL-4) for 45 min at room temperature. The sections were
then washed in PBS and exposed to a 1:50 dilution of the secondary antibody (FITC-conjugated goat anti-rat IgG for LM-1; Cappel) for 30 min at
room temperature. To identify epithelial cells, some of the LM-1-stained
cocultures were then immunostained with anti-cytokeratin antibodies using a commercial peroxidase-anti-peroxidase kit (Dako) and following the manufacturer's instructions.
) using a monoclonal antibody to the LM
1
chain (AL-4).
Results
1 and
2 chains,
a monoclonal antibody to LM
1 chain (Fig. 1, x-LM
1),
and a monoclonal antibody to LM
2 chain (Fig. 1, x-LM
2) demonstrated the presence of LM
1,
2,
1, and
1
chains in the mouse developing lung. We noticed that
1
and
1 chains frequently migrated with similar Mr producing a single band in minigels. However, the two bands were resolved in larger gels.
Fig. 1.
Immunoblots demonstrating the presence of LM 1,
2,
1, and
1 chains in the developing lung (day 15 of gestation).
A polyclonal antibody against EHS LM that recognizes
1,
2,
1, and
1 chains (x-LM), a monoclonal antibody to the LM
1
chain (AL-4, x-LM
1), and a monoclonal antibody to LM
2
chain (4H8-2, x-LM
2) were used in these studies. LM-1 from
the EHS tumor and embryonic heart (rich in LM-2) served as
controls. Immunoblots with normal rabbit IgG or rat IgG did not
detect any protein bands.
[View Larger Version of this Image (42K GIF file)]
1 Chain is Induced by
Epithelial-Mesenchymal Interaction
2,
1, and
1 but not
1 chain
(Fig. 2). Epithelial-mesenchymal cocultures synthesized
LM
1 chain in addition to LM
2,
1, and
1 chains (Fig.
2). There were, however, differences in the amount of LM
1 chain synthesized by the various heterotypic coculture
systems. Epithelial-mesenchymal cocultures established
by plating epithelial and mesenchymal cell populations directly after trypsinization of lungs (passage 0) synthesized
the highest levels of LM
1 chain (Fig. 3, column 1), whereas
cocultures established by adding one cell type on top of a
heterotypic cell monolayer synthesized the lowest levels
(Fig. 3, columns 3 and 4). Cocultures generated by plating
a combination of epithelial and mesenchymal cells obtained from monocultures at a ratio of 1:3 to 1:5 synthesized intermediate levels of LM
1 chain (Fig. 3, column
2). In addition, these studies indicated that these cells may
require at least 4 h of coculture for LM
1 chain to be synthesized, since LM
1 chains were not detected after 2 to 4 h
of coculture by ELISA (not shown).
Fig. 2.
LM 1,
2,
1, and
1 chains produced by epithelial-
epithelial (E/E), mesenchymal-mesenchymal (M/M), and epithelial-mesenchymal (E/M) cocultures. A polyclonal antibody
against EHS LM that recognizes
1,
2,
1, and
1 chains was
used for immunobloting. LM
1 chain is observed only in epithelial-mesenchymal cocultures. As controls, the first lane contained
LM-1 from the EHS tumor and the second lane contained lung
extract. Immunoblots with normal rabbit IgG or rat IgG did not
detect any protein bands.
[View Larger Version of this Image (47K GIF file)]
Fig. 3.
ELISA showing different levels of LM 1 chain synthesis in different cocultures. (Column 1) Epithelial-mesenchymal
coculture established from mixed cell populations directly isolated from the lung. (Column 2) Epithelial-mesenchymal coculture established with cells from monocultures mixed in a 1:3 epithelial/mesenchymal ratio. (Column 3) Epithelial-mesenchymal
coculture established by adding epithelial cells to a mesenchymal
monolayer (both confluent at the time of determining LM-1 production). (Column 4) Epithelial-mesenchymal coculture established by adding mesenchymal cells to an epithelial monolayer
(both confluent at the time of determining LM-1 production). The bars represent SD. The means and SD are based on quadruplicate examples in a single experiment. These were repeated
three times with similar results.
[View Larger Version of this Image (60K GIF file)]
1 chain
was deposited exclusively at the epithelial-mesenchymal
interface (Fig. 4). No significant amount of LM
1 chain
was detected intracellularly in either cell type. The restricted epithelial-mesenchymal localization of LM
1 chain
was also observed in the developing lung. In day 12 lung,
LM-1 was found in the BM along the bronchial tree (Fig. 4, inset) where more sustained epithelial-mesenchymal
contact occurred, but not at the tips of the growing bronchi, where new epithelial-mesenchymal contacts are continuously established.
Fig. 4.
Epithelial-mesenchymal organotypic coculture double-stained with anti-keratin antibodies by immunoperoxidase (a)
and with anti-LM 1 chain antibody by immunofluorescence (b).
The epithelial cells are recognized because they form round clusters and stain light brown with anti-keratin antibodies (not clearly
appreciated in a black and white photograph). LM-1 is exclusively deposited at the epithelial-mesenchymal interface (arrows). (Inset) Day 12 lung stained with anti-LM
1 chain antibody shows LM-1 deposition restricted to the basement membrane
alongside the bronchial tree. Bar: (a and b) 20 µm; (inset) 0.2 mm.
[View Larger Version of this Image (118K GIF file)]
1 chain after metabolic radiolabeling confirmed the
synthesis of LM
1 chain by epithelial-mesenchymal cocultures but not by monocultures (Fig. 5). These studies
also showed that LM
1 chain is linked to
1 and
1 chains
forming full LM-1 molecules (Fig. 5).
Fig. 5.
Immunoprecipitation of 35S-metabolically labeled LM-1
synthesized by epithelial (E) and mesenchymal (M) monocultures and epithelial-mesenchymal cocultures (E/M) using a monoclonal antibody to the LM 1 chain. Only epithelial-mesenchymal cocultures produced detectable levels of LM-1.
[View Larger Version of this Image (58K GIF file)]
1 chain, however
2,
1, and
1
expression remained constant (Fig. 6). No Lm
1 chain was
produced by culturing monolayers in epithelial-, mesenchymal-, or epithelial-mesenchymal-conditioned medium
(not shown).
Fig. 6.
Immunoblots using a polyclonal antibody to LM-1 that
recognizes 1,
2,
1, and
1 chains. No LM
1 chain was detected in epithelial-epithelial (E/E) or mesenchymal-mesenchymal (M/M) cocultures, whereas LM
1 chain was seen in epithelial-mesenchymal (E/M) cocultures. LM
1 chain was not
detected in the absence of epithelial-mesenchymal contact (monocultures separated by a filter), whereas
2,
1, and
1 chains remained constant. The first lane is LM-1 from the EHS tumor,
used as control.
[View Larger Version of this Image (43K GIF file)]
1 chain synthesis seems to be directly proportional
to the extent of epithelial-mesenchymal contact. This was
suggested by studies in which increasing numbers of mesenchymal cells were added to prestablished (24-h-old) epithelial monocultures (Fig. 7). These ratios were selected
based on the normal epithelial/mesenchymal ratio in mixed
lung cell populations obtained from day 15 embryonic lungs.
Fig. 7.
Addition of mesenchymal cells to epithelial monocultures stimulates LM 1 chain synthesis. (M
) No mesenchymal
cells; (M+) 1:2 epithelial/mesenchymal cell ratio; (M++) 1:4 epithelial/mesenchymal cell ratio. The first lane is LM-1 from the
EHS tumor, used as control. The inset shows staining of the nitrocellulose membrane with 0.2% amido black after immunobloting to visualize the amount of protein loaded per lane.
[View Larger Version of this Image (63K GIF file)]
1 Chain Is Synthesized by Epithelial and
Mesenchymal Cells
1 chain in epithelial-mesenchymal cocultures (Fig. 4).
Additionally, we could not determine by immunoblotting
or immunoprecipitation which cell population synthesized
LM
1 chain, because this chain disappeared very rapidly
after interruption of epithelial-mesenchymal contact. However, we occasionally noticed a faint band corresponding to LM
1 chain in both epithelial and mesenchymal cell lysates right after isolation from the lung or from heterotypic cocultures (not shown). To identify the origin of LM
1 chain, we exposed organotypic cocultures to brefeldin
A, a fungal metabolite that inhibits protein secretion
(Klausner et al., 1992
). In organotypic cocultures, the epithelial cells form cysts and the mesenchymal cells surround them as a monolayer (Schuger et al., 1993
, 1995
); therefore, it is possible to determine what cell type accumulates LM
1 chain. Immunolocalization studies in brefeldin A-treated cultures indicated that LM
1 chain accumulates in both the epithelial cells and in the mesenchymal
cells surrounding them (Fig. 8).
Fig. 8.
Epithelial-mesenchymal organotypic cocultures exposed to 0 (a), 5 (b),
and 10 (c) µg/ml of brefeldin
A for 3 h, followed by immunostaining with anti-LM 1
chain antibody. e, Epithelial
cell; m, mesenchymal cells. In
untreated cocultures (a), LM
1 chain accumulates at the
epithelial-mesenchymal interface (arrow). In brefeldin
A-treated cocultures (b and
c), LM
1 chain can be detected in the epithelial cells
as well as in the mesenchymal cells surrounding them
(b, arrows). Bar, 20 µm.
[View Larger Version of this Image (40K GIF file)]
1 Antibody
1,
2, and
1/
1,
normal mouse IgG, or no immunoglobulin. Since embryonic lungs are transparent, the airway branching activity
was daily monitored by counting the number of terminal
airway buds per explant and looking for changes in airway
tree architecture or lumen caliber. All of the explants presented a similar number of terminal buds regardless of
treatment (32 ± 8) and showed no changes in airway architecture (not shown).
2 or LM
1/
1 chains or to normal
mouse IgG. In these explants the peribronchial mesenchymal cells were elongated in shape (polarized) and arranged
concentrically to the airway (Fig. 9, a, d, and e), whereas
the rest of the mesenchymal cells were round in shape (unpolarized). However, in the lung explants exposed to 50 or
100 µg/ml of monoclonal antibody to LM
1 chain, the
mesenchymal cells were uniformly round (unpolarized), regardless of their proximity to the bronchial tree (Fig. 9
b). The effect that mAb AL-4 had on mesenchymal cell
morphology was corrected by preincubation of AL-4 with
LM-1 (Fig. 9 c).
Fig. 9.
Photomicrographs
of a main bronchus including
peribronchial mesenchymal
cells in lung explants cultured for 3 d in the presence of 100 µg/ml of normal
mouse IgG (a), anti-LM 1
chain antibody (b), anti-LM
1 chain antibody preincubated with 200 µg/ml of LM-1
(c), anti-LM
2 antibody
(d), and anti-LM
1/
1 antibody (e). Note that in the
control (a) the peribronchial
mesenchymal cells (m) are
polarized (elongated and oriented concentrically to the
bronchus), whereas the peribronchial mesenchymal cells in explants exposed to anti-LM
1 chain antibody (b)
are round. This effect was
corrected by preincubation
of the antibody with LM-1
(c) and was not observed in
explants exposed to the other antibodies (d and e). The epithelial cells (e) showed no
morphological alterations. Bar,
20 µm.
[View Larger Version of this Image (59K GIF file)]
1 chain antibody compared to
the other mAbs (Fig. 10). Notice the high percentage of
unpolarized peribronchial mesenchymal cells in the lung
explants exposed to anti-LM
1 chain antibody (P < 0.005, with Student's t test) and how this is corrected with preincubation of the antibody with LM-1. No significant differences were found in the total number of mesenchymal
cells among the explants, including those treated with anti-LM
1 chain (350 ± 45).
Fig. 10.
Histogram showing the percentage of polarized (elongated) and unpolarized (round) peribronchial cells in day 12 embryonic lung explants exposed for 3 d to various anti-LM antibodies and control immunoglobulin. The number of polarized
(elongated) and unpolarized (round) peribronchial mesenchymal
cells was determined on histological sections of the explants.
These were cut longitudinally to and including the full main bronchus. Only the cells closest to the BM of the main bronchus and
its first order branches were quantitated. The bars represent SD.
The means and SD are based on five examples of each treatment.
[View Larger Version of this Image (38K GIF file)]
1 May Play a
Role in Bronchial Smooth Muscle Development
actin and desmin. Desmin
is an intermediate filament expressed in all muscle tissue
and, when absent, results in smooth muscle hypoplasia and
degeneration (Milner et al., 1996
). These antibodies recognized the two proteins in immunoblots of mouse adult gut
(rich in smooth muscle) and fetal lung tissue lysates (Fig.
11). Lung explant cultured for 3 d in the presence of a
monoclonal antibody against LM
1 chain showed a decrease in smooth muscle
actin and desmin proportional
to the concentration of antibody (Fig. 12 a). No change in
smooth muscle
actin and desmin expression was observed in lung explants cultured for 3 d in the presence of
monoclonal antibodies to LM
2 chain or to LM
1/
1
chains compared with controls (Fig. 12 b).
Fig. 11.
Immunoblots using monoclonal antibodies
to smooth muscle actin and
desmin on mouse adult gut
(rich in smooth muscle) and fetal lung tissue lysates, the
latter on day 15 of gestation. A single band migrating at an
Mr of ~42,000, consistent
with smooth muscle
actin,
and a single band migrating
at an Mr of ~52,000, consistent with desmin, are visualized in both adult gut and fetal lung.
[View Larger Version of this Image (76K GIF file)]
Fig. 12.
Detection of smooth muscle actin (SM-actin) and
desmin in lung explants cultured for 3 d in the presence of monoclonal antibodies against LM chains or control IgG. There is a decrease in smooth muscle
actin and desmin proportional to the
concentration of anti-
1 chain antibody added to the lung organ
cultures (a). The inset shows a portion of the nitrocellulose membrane stained with 0.2% amido black after immunobloting to visualize the amount of protein loaded per lane. No change in
smooth muscle
actin or desmin synthesis is observed in lung explants cultured for 3 d in the presence of a mAb to LM
1/
1
chains, a monoclonal antibody to LM-
2 chain, or normal mouse
IgG (b).
[View Larger Version of this Image (35K GIF file)]
Discussion
1 Chain
). In this study, using a combination of monocultures and cocultures isolated from
mouse embryonic lungs, we show that epithelial-mesenchymal interaction induces the synthesis of the LM
1
chain.
1 chain is not the first extracellular matrix protein
found to be modulated by epithelial-mesenchymal interactions. In a previous study using tissue recombinants,
Vanio et al. (1989)
showed that heterotypic interaction between epithelium and mesenchyme stimulates the synthesis of syndecan and tenascin during mouse tooth development. A similar mechanism seems to regulate the expression of certain cytokines in the skin (Reusch et al., 1991
; Smola
et al., 1993
) and in the developing tooth (Mitsiadis et al., 1995
).
1 chain, indicating
that LM
1 chain synthesis requires heterotypic cell-cell
contact. This kind of close cell-cell interaction does not
normally occur in the adult but is characteristic of developing organs. In the lung, epithelial and mesenchymal cells are in direct contact at the ends of the bronchial tree, where new airway buds are being formed (Grant et al., 1983
;
Jaskoll and Slavkin, 1984
). Deposition of LM
1 chain begins at these sites, where it is barely detected, and gradually accumulates alongside the bronchial tree, where more
prolonged heterotypic cell-cell contact has time to occur.
; Reusch et
al., 1991
; Yaeger, et al., 1991; Flaumenhaft et al., 1993
;
Smola et al., 1993
) and underscores the importance of juxtacrine or short range paracrine signals. Such mechanisms
have been proposed or demonstrated in many heterotypic
cell-cell interactions, including the activation of TGF-
in
smooth muscle cell-fibroblast coculture (Flaumenhaft et
al., 1993
), the transactivation of TGF-
precursor by adjacent cells (Anklesaria et al., 1990
), and the production of
casein by mammary epithelial cells upon coculture with
mammary mesenchymal cells (Reichmann et al., 1989
).
1 chain synthesis is that a molecule, or molecules,
produced by one cell type induces the synthesis of LM
1
by the other. For example, PDGF-A is produced by lung
epithelial cells and exerts its effect on the mesenchymal
cells surrounding them (Boström et al., 1996
). Similarly,
scatter factor is produced by mesenchymal cells but acts
on epithelial cells (for review see Rosen et al., 1994
). Alternatively, cell-cell interaction could involve a regulatory factor with autocrine and paracrine control over LM
1
expression. In such a case, LM
1 chain could be induced
in both cell types. Obviously, mediator roles could be played
by extracellular matrix components and/or their receptors,
instead of cytokines.
) and has been implicated as the mediator of epithelial-mesenchymal interactions in the tooth
(Mitsiadis et al., 1995
). However, although retinoic acid
seems to play a role in lung morphogenesis (Schuger et al., 1993
), it should be stressed that it does not affect LM-1
synthesis in lung organ cultures, monocultures, or cocultures (Mitra, R., and L. Schuger, unpublished observations).
1 Chain Is Synthesized by Epithelial and
Mesenchymal Cells
1 chain. Using in situ hybridization, Ekblom et al. (1990) described LM
1 chain mRNA
in epithelial cells only; however, we detected LM
1 chain
mRNA in both the epithelium and the mesenchyme
(Schuger et al., 1992
). More recently, LM
1 chain mRNA
has been reported in the epithelium and in mesenchymal
cells close to it (Thomas and Dziadek, 1994
). In the current study, we could not determine by immunobloting or
immunoprecipitation which cell type synthesizes the LM
1 chain. Furthermore, since its immunodetection was restricted to the epithelial-mesenchymal interface, immunohistochemistry was not contributory. The origin of LM
1
chain was however elucidated by exposing cocultures to brefeldin A, a fungal metabolite that blocks protein secretion (Klausner et al., 1992
). These studies showed that in
epithelial-mesenchymal cocultures exposed to brefeldin
A, LM
1 chain accumulated in both the epithelial and
mesenchymal cells, confirming its origin in both cell types.
1 chain in the developing kidney (Klein et al., 1988;
Ekblom et al., 1990). Based on experimental studies, the
authors proposed that one of the functions of LM
1 chain
is to induce epithelial cell polarization (Klein et al., 1988).
Our studies did not test the role of LM
1 chain in epithelial cell polarization but suggested that polarized cells produce more LM
1 chain than their unpolarized counterparts. For example, cocultures established in Millipore
inserts, which allow epithelial cells to form spheroids and
cysts surrounded by concentric layers of mesenchymal
cells, synthesized higher levels of LM
1 chain than surface-anchored bilayer cocultures (as shown in Fig. 3).
) confirmed that the developing lung produces LM
2 chain.
Immunoprecipitation studies indicated that LM
2 chain
is secreted by the cells as part of LM-2 (
2,
1,
1; He, L.,
and L. Schuger, unpublished results). In contrast to LM
1
chain, LM
2 chain is not regulated by epithelial-mesenchymal contact and may serve different functions than
LM-1. So far, the LM
2 chain has been shown to be essential for myotube stability (Vachon, 1996) in skeletal
muscle, however its role in smooth muscle biology remains
to be elucidated. Noteworthy, LM-2 deficiency is the underlying defect in several muscular dystrophies in which
respiratory problems lead to early death (Mendell et al.,
1995
). However, at the present time, it is unclear whether these are primary to the lung or secondary to poor ventilation due to muscular dysfunction.
1 Chain May Be Involved in Bronchial Smooth
Muscle Development
1 chain exhibited alterations in peribronchial
mesenchymal cell shape and synthesized lower levels of
smooth muscle
actin and desmin. The anti-LM
1 chain
antibody used in these studies reacts with the COOH-terminal G domain of the LM
1 chain, a major adhesion site
for several cell types, including myofibroblasts (Engvall,
1994
). Therefore, it is likely that the antibody perturbed mesenchymal cell adhesion to LM-1 and this in turn resulted in abnormal smooth muscle cell phenotype.
;
Duluc et al., 1994
). However neither study explored possible mechanisms underlying this process. In light of our results, it may be possible that LM
1 chain was induced by
the epithelial-mesenchymal contact, facilitating the differentiation of mesenchymal cells into smooth muscle.
1 antibody, the decreased peribronchial cell attachment to BM LM-1
prevented them from stretching in response to bronchial
intraluminal pressure. The deficient stretching capability
then resulted in a defective smooth muscle phenotype.
),
and that per se may explain our findings. Furthermore,
LM-1 plays roles in proliferation, migration, and differentiation of myoblasts (Goodman et al., 1989
; von der Mark
and Öcalan, 1989; Kroll et al., 1994
; Vachon et al., 1996
).
Therefore, LM
1 chain may influence smooth muscle differentiation via different mechanisms.
2,
1, and
1 chains are steadily produced by embryonic lung epithelial and mesenchymal cells, the synthesis of LM
1 chain is
controlled by epithelial-mesenchymal interactions. Our
studies further suggest that LM
1 chain may play a role in
the development of bronchial smooth muscle, perhaps by
controlling peribronchial mesenchymal cell attachment,
shape, and ability to respond to stretching forces. Additional studies focused on the biological activities of LM-1
on smooth muscle cell differentiation will be required to
elucidate the mechanism whereby LM-1 affects bronchial
smooth muscle development.
Received for publication 6 May 1997 and in revised form 6 August 1997.
Address all correspondence to Lucia Schuger, Wayne State University School of Medicine, Department of Pathology, Gordon H. Scott Hall of Basic Medical Sciences, 540 East Canfield Street, Detroit, MI 48201. Tel.: (313) 577-5651. Fax: (313) 577-0057.This work has been supported by National Institutes of Health grants HL48730-01 (L. Schuger) and CA60658 (A.P.N. Skubitz).
BM, basement membrane; LM, laminin.
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