1 Department of Pathology, Lung development
is a complex process in which epithelial-mesenchymal interactions play
a key role. A conserved secretory apparatus, the soluble
N-ethylmaleimide-sensitive factor
attachment protein receptor (SNARE) complex, is essential for
exocytosis in many cell types. Syntaxins, located on the terminal
plasma membrane (T-SNAREs), are a critical component of the
secretosomal complex involved in vesicular docking, fusion, and
exocytosis. We analyzed syntaxin 1A mRNA and protein in fetal rat lung
ontogeny, demonstrating peak expression on about day
19 of embryonic development, immediately preceding type
II pneumocyte differentiation. Syntaxin 1A is predominantly expressed
by lipofibroblasts, which are required for bombesin-like
peptide-induced surfactant phospholipid synthesis (choline uptake) by
isolated type II cells. In organ cultures, anti-syntaxin 1A antibody
HPC-1 blocks choline uptake both at baseline and when induced by
bombesin-like peptide or dexamethasone. HPC-1 also promotes thymidine
uptake in parallel in a dose-dependent fashion. These observations
indicate a potential role for syntaxin 1A during fetal lung
development, possibly through involvement in secretion of mesenchymal
cell-derived factors that induce terminal type II cell differentiation.
lung development; bombesin; dexamethasone; monoclonal antibody; surfactant phospholipids
LUNG DEVELOPMENT in the embryonic rat begins on
approximately embryonic day 11 (ED11)
as a ventral foregut outpouching. Subsequent lung development can be
divided into embryonic (days
12-15),
pseudoglandular (days
15-19),
canalicular (days
19-20),
saccular (day 20 to shortly after
birth), and alveolar (postnatal) stages. During these stages, the lung
bud undergoes repetitive branching, with eventual formation and
maturation of the gas-exchanging alveolar acinar units. Specialized epithelial cells, in particular the type II pneumocyte, differentiate, and surfactant production ensues (40).
Lung branching morphogenesis and epithelial cytodifferentiation require
that epithelial cells interact with surrounding mesenchymal cells
and/or basement membrane components. The expression of genes encoding
extracellular matrix, growth factors, and growth factor receptors has
been shown to be developmentally regulated in the lungs of rats and
mice (53, 56). Many growth factors produced by epithelial cells in the
developing lung can promote type II cell differentiation in a
mesenchymal cell-dependent fashion (19, 26, 33, 42, 49, 52). These
factors include epidermal growth factor (45, 50, 63), parathyroid
hormone-related protein (32), basic fibroblast growth factor (FGF)
(22), and platelet-derived growth factor (23). Other epithelial
cell-derived growth factors such as FGF10, bone morphogenetic
protein-4, epidermal growth factor, and bombesin-like peptide (BLP)
have also been implicated in the induction of branching morphogenesis
(5, 6, 29, 42). Pulmonary mesenchymal cells can signal the developing
epithelium through the secretion of extracellular matrix components
such as laminin, tenascin, and nidogen (51, 53, 67) or alternatively
via secreted growth factors such as acidic FGF and keratinocyte growth
factor (15). Recent work (4, 20) has implicated the secreted
mesenchymal factor sonic hedgehog and members of the
Gli gene family in pulmonary
embryogenesis. It appears that glucocorticoids can induce type II
pneumocyte differentiation and surfactant production via a mesenchymal
cell intermediary (54), potentially via a secreted substance derived from developing fibroblasts termed "fibroblast-pneumonocyte
factor" (48), the identity of which remains elusive.
In eukaryotic cells, regulated secretion occurs as a complex series of
steps beginning with exocytic vesicle formation and leading to docking
and fusion of the secretory vesicle with the plasma membrane (64). A
conserved molecular apparatus, known as the soluble
N-ethylmaleimide-sensitive factor
(NSF) attachment protein receptor (SNARE) complex, is highly conserved
in a wide variety of species including yeast, plants, fruit flies,
nematodes, invertebrates, and mammals (16, 27, 38). This complex is involved in the docking and fusion steps of the vesicular transport and
membrane trafficking pathways of several cell types. The primary components of this machinery have been identified, and their molecular interactions have been examined. In the process of vesicle docking and
fusion, a core complex is formed between the vesicular SNAREs, vesicle-associated membrane protein (VAMP), and synaptotagmin located
on the vesicular membrane surface and the terminal SNAREs, syntaxin,
and synapse-associated protein-25 (SNAP-25) located at the plasma
membrane. The exocytosis core complex then recruits soluble factors
such as NSF and soluble NSF attachment proteins ( Syntaxins are terminal SNAREs that have been defined primarily in terms
of their role in cellular secretion (7). As plasma membrane proteins,
syntaxins form part of the secretosomal docking complex involved in
exocytosis and were originally described in neurons as regulators of
synaptosomal trafficking. In mammals, syntaxin (Stx) 1A was originally
identified in a limited analysis as being neural or neuroendocrine
specific (7) but has since been reported in malignant colonic
epithelial cells as well (8). Other syntaxins have been known to be
widely distributed in a variety of tissues including neurons, endocrine
cells, and exocrine cells, in which they are also involved in regulated
exocytosis (7, 18, 37, 44).
Syntaxins have been implicated in developmental processes. Stx1 is
required for cellularization of
Drosophila embryos (11), and a
syntaxin homolog in Arabidopsis is
required for proper seedling development (38). Hirai et al. (24) found
that murine lung buds cultured with an antibody to epimorphin
demonstrated impaired morphogenesis, but the mechanism underlying this
observation has not been clarified. Subsequently, epimorphin was found
to be identical to Stx2 (7).
Here we examine the expression and function of Stx1A during late fetal
and early postnatal lung development in the rat. Initially, we
anticipated that Stx1A would be localized only to developing neurons
and possibly to neuroendocrine cells in view of its known specific
immunolocalization to the enteric nervous system in the developing rat
gut (10). Consistent with previous observations (7, 18, 44), we did
observe some Stx1A expression in pulmonary nerve fibers, epithelial
cells, and neuroendocrine cells. Surprisingly, however, most of the
Stx1A mRNA and protein is associated with a mesenchymal cell population
that is lipid rich and transiently expressed. We demonstrate that BLP
promotes the differentiation of isolated fetal mesenchymal cells into
lipofibroblasts and that the effect of BLP on isolated type II
pneumocyte differentiation requires the presence of these
lipofibroblasts. Finally, the anti-Stx1A antibody HPC-1 completely
blocks the effect of BLP on type II cell differentiation in fetal lung
organ cultures while it promotes cell proliferation. These observations
support a role for Stx1A in mediating lipofibroblast secretory
processes that contribute to terminal differentiation of the pulmonary epithelium.
Materials. Collagenase A
(~0.85 U/mg) and Protease Inhibitors Complete were
obtained from Boehringer Mannheim (Indianapolis, IN). Dulbecco's
modified Eagle's medium (DMEM) and Hank's balanced salt solution
(HBSS) were purchased from GIBCO BRL (Life Technologies, Gaithersburg,
MD); filter-sterilized fetal calf serum (FCS) was obtained from Atlanta
Biologicals (Norcross, GA). Anti-Stx1A antibody (HPC-1), MOPC21, and
rat IgG were obtained from Sigma (St. Louis, MO). Bovine serum albumin
(BSA) was supplied by Bio-Rad (Hercules, CA).
Primary fetal cell culture. Pregnant
Sprague-Dawley rats were killed on day
19 of gestation. The fetal lungs were obtained, and the
surrounding tissues (heart, thymus, extrapulmonary airways, and large
blood vessels) were discarded. The lungs were minced into 0.1-mm cubes
with a McIlwaine tissue chopper. The minced explants were placed into a
sterile culture flask containing 15 ml of HBSS (~1 ml/lung) and
collagenase (0.2 mg/ml). The flask was sealed and gently agitated at
37°C for 30 min, then an equal volume of ice-cold HBSS was added
and the entire mixture was filtered through two layers of sterile gauze
into sterile glass centrifuge tubes (Kimax). The cells were then
sedimented by centrifugation at ~20
g for 4 min, and the cell pellet was
washed again in HBSS.
The mixed fetal pulmonary cells were resuspended in warm serum-free
DMEM (~1 ml/lung) and plated onto 100-mm Corning tissue culture
plates (10 ml/plate) for incubation. Some of the minced lung explants
were placed directly into tissue culture plates and grown in DMEM with
10% (vol/vol) FCS.
Isolation of fetal pulmonary
fibroblasts. The plates containing a mixture of fetal
pulmonary cells were incubated for 20 min at 37°C in humidified 5%
CO2. After incubation, the
supernatant and nonadherent cells were removed for the preparation of
epithelial cells enriched for type II pneumocytes (see
Isolation of pulmonary epithelial
cells). Fresh DMEM with 10% (vol/vol)
FCS was added to the plates containing the adherent pulmonary fibroblasts.
Isolation of pulmonary epithelial
cells. Falcon bacteriological plates were coated with 5 ml of a solution of rat IgG (0.5 mg/ml) suspended in 50 mM Tris (pH
8.8) for 3-6 h at room temperature. The plates were washed three
times with HBSS and once with serum-free DMEM.
Plates containing mixed fetal pulmonary cells were incubated for 20 min
at 37°C in humidified 5% CO2.
After this, the nonadherent cells were harvested by gently swirling the
plates and transferred to IgG-coated plates, which were incubated at
37°C for 1 h under the same conditions. The supernatant containing
the nonadherent type II cells was then removed and placed on fresh
tissue culture plates, with FCS added to a final concentration of 10%
(vol/vol).
RT-PCR and Southern hybridization
analysis. Total RNA was prepared from whole lungs and
cell cultures by homogenization as previously described (13) with
TriReagent purchased from Molecular Research Center (Cincinnati, OH).
Single-stranded cDNA was prepared with total lung RNA (2 µg), Moloney
murine leukemia virus RT and random hexamers as described elsewhere
(55). Parallel reactions were carried out on each sample without adding
RT, hereafter designated RT As an internal control for the amplification, rat 18S RNA was amplified
from the same original volume of the RT reaction. A 568-bp DNA product
was amplified with 18S.F (5'-TAG CTC TTT CTC GAT TCC GTG
G-3') as a forward primer and 18S.R (5'-ATG ATC CTT CCG CAG
GTT CAC-3') as a reverse primer. PCR was carried out as above for
21 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 120 s.
To control for possible DNA contamination, 5% of each RT The PCR products were run on an 0.8% agarose gel, transferred by
capillary blotting to a nitrocellulose filter, and hybridized to a
specific 32P-labeled
oligonucleotide probe internal to the amplified sequence as follows:
Stx1A, Stx1A.2F (5'-CCC GAT GAG AAG ACC AAG GAG-3'); syntaxin 2 (epimorphin), EPI.2F (5'-TGG ATG CGG CCT TTG CTT
CGC-3'); GRP, GRP.2F (5'-CTT CTT CCC AGC GGA TGT
AT-3'); and DDC, DDC.2F (5'-TTT TTG GCT GGA AGA GCT GGG
G-3'). All filters were washed several ( Protein analysis. Protein lysates were
prepared with a functional protein lysis buffer composed of 20 mM HEPES
(pH 7.4), 2 mM EGTA, 50 mM For Western blotting, 40 µg of protein lysate were subjected to
SDS-PAGE on a 12% polyacrylamide gel and then electroblotted onto a nitrocellulose membrane. Transfer was assessed with Ponceau S
staining (Bio-Rad). The nitrocellulose filters were reacted first with
a monoclonal mouse anti-rat Stx1A antibody (HPC-1) at a 1:4,000
dilution followed by a biotinylated horse anti-mouse IgG at a 1:1,000
dilution. The filters were then treated with avidin and biotin
conjugated to horseradish peroxidase (ABC Reagent, Vector Laboratories)
and visualized by chemiluminscence with the HRPL kit (National
Diagnostics, Atlanta, GA).
Immunohistochemical and histochemical
analyses. For immunoperoxidase studies, lungs harvested
from ED19 rat fetuses were fixed for 4 h in 4% paraformaldehyde and
placed in 30% sucrose in PBS overnight before being embedded in
optimum cutting temperature compound and frozen on
powdered dry ice. Three-micrometer frozen sections were prepared, and
immunoperoxidase analyses were carried out with a mouse monoclonal
anti-Stx1A antibody (HPC-1) at a 1:50 dilution and the avidin-biotin
complex immunoperoxidase technique with diaminobenzidine and
biotinylated tyramide (tyramide system amplification) as a substrate as
previously described (60). In addition, serial 3-µm sections were
used for immunostaining for the neural or neuroendocrine markers
calcitonin gene-related peptide and protein gene product (PGP) 9.5, the
general epithelial marker keratin, and the mesenchymal markers desmin
and vimentin as previously described (21, 58, 59).
For detection of lipids in tissue sections, frozen sections of lungs
harvested from ED19 rat fetuses were stained for lipids with 0.5% oil
red O in 60% isopropanol for 15 min, with pre- and posttreatment 60%
isopropanol washes (14). Serial frozen sections were used for
immunoperoxidase analyses for Stx1A as described above.
Lung organ culture. Fetal lung was
harvested on ED18 at 4°C in Waymouth medium supplemented with 5%
FCS. The tissue was chopped into 0.5-mm cubes and cultured in six-well
plates with the same medium with and without added BLP or other agents.
Cultures were grown for 72 h at 37°C in 5%
CO2 on a rocking platform at 3 oscillations/min (57). Tissue viability was checked by histological
analyses, satisfactory baseline incorporation of
[3H]choline and
[3H]thymidine, intact
18S and 28S RNA bands on ethidium gels, and good positive control
responses with dexamethasone (Dex) and GRP after 72 h.
Determination of [3H]DNA,
[3H]disaturated
phosphatidylcholine, DNA, and protein content.
After 68 h of culture, tissue was incubated with
[3H]thymidine (4 µCi/ml) or
[3H]choline (16 µCi/ml; NEN-Dupont, Boston, MA) for 4 h at 37°C in 5%
CO2-air on a rocking platform
before being harvested and analyzed for
[3H]DNA, DNA content,
[3H]disaturated
phosphatidylcholine, and protein content.
[3H]thymidine
incorporation into acid-precipitable counts was carried out as
previously described (57). DNA was assayed after trichloroacetic acid precipitation with the method of Burton (12). For
determination of the rate of surfactant phospholipid synthesis,
lipids were extracted from cell homogenates with chloroform-methanol,
and [3H]choline
incorporation into phosphatidylcholine was determined as
previously described (61). Protein was determined with the method of
Bradford (9), with BSA as the standard. Experimental values were
normalized by defining the mean of the control groups as baseline and
expressing the values as percent changes above or below this.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-SNAPs) to form the mature 20S fusion
complex. Disruption of the mature complex occurs through NSF-mediated
ATP hydrolysis.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
reactions. Five percent of the RT
reaction volume was then used for PCR. A 652-bp DNA fragment coding for
Stx1A was amplified by PCR with Stx1A.F (5'-GAA AAC GTG GAG GAG
GTG AAG-3') as the forward primer and Stx1A.R (5'-ATC TTC
TTC CTG CGT GCC TTG-3') as the reverse primer. In addition, an
825-bp DNA fragment coding for Stx2 [epimorphin (EPI)] was
amplified with EPI.F (5'-TGG AGA CAC TGC TGT CGT CAT-3') as
the forward primer and EPI.R (5'-TCA TTT GCC AAC CGA CAA
GCC-3') as the reverse primer. Rat syntaxin sequences were taken
from Genbank accession numbers U35039 through U35047. As neural or
neuroendocrine markers, rat gastrin-releasing peptide (GRP) and Dopa
decarboxylase (DDC) were also amplified from certain RT reaction
products. A 283-bp DNA fragment coding for GRP was amplified by PCR
with RGRP.F (5'-ACT GGG CTG TAG GAC ACT T-3') as the
forward primer and RGRP.R (5'-GAG AAC CTG GAG CAG AGA GTC TAC CAA
CTT-3') as the reverse primer. A 501-bp DNA fragment coding for
DDC was amplified with DDC.F (5'-TTC TTC GCT TAC TTC CCC ACG
G-3') as the forward primer and DDC.R (5'-GGG TGA CAA CCA
CGA AGA AAG G-3') as the reverse primer. All PCRs were carried
out with the Hot-Start protocol for 30 cycles at 94°C for 30 s,
57°C for 30 s, and 72°C for 120 s.
reaction was subjected to PCR under the same conditions as the RT reactions. The primers used for Stx1A and Stx2 (epimorphin) span multiple introns so that genomic DNA clearly would yield a larger band
than the predicted size of cDNA amplification. None of our negative
control "RT
" reactions yielded RT-PCR products.
3) times under
high-stringency conditions (5× saline-sodium citrate at
60-65°C) and exposed to autoradiography film with intensifying screens.
-glycerophosphate, 1% Triton X-100, 10%
glycerol, 1 mM dithiothreitol, and 1× Protease Inhibitor Complete
as previously described (28). All protein lysates were assayed to
determine their protein content with the Bio-Rad assay with a BSA standard.
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RESULTS |
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Syntaxin mRNA and protein analyses.
RT-PCR analyses of rat fetal lungs during development demonstrated
elevated Stx1A mRNA levels between ED16 and ED19, with lower levels
from ED20 through postnatal development. These are semiquantitative
RT-PCR analyses comparing the relative levels of mRNAs normalized to
the relative levels of 18S RNA. Each lane represents a pool of mRNA
from at least 20 fetal lungs (lungs from 2 litters with 10-14
fetuses/litter), so that each blot demonstrates averaged data from
numerous fetal lungs. RT-PCR was carried out three times (with two
separate pools of RNA), consistently with the same result, that is,
that peak expression occurs at ED19. For comparison, mRNAs encoding
additional components of a potential secretosomal complex were also
analyzed, including Stx2 (epimorphin), Rab26, and Munc18; all mRNAs
were most highly expressed at ED19 (Fig.
1,
A and
C, and data not shown). Levels of 18S rRNA were near equivalence in all lanes. All RT-PCRs were
carried out with conditions such that positive control RNA (normal rat
brain in the present study) was in the linear range of detection (65).
Western blotting of protein lysates from the same fetal lungs
demonstrated elevated Stx1A protein levels between ED17 and ED20,
peaking at approximately ED18-19 (Fig. 1,
B and
D). These Western analyses were
carried out twice with two separate pools of protein lysates, with at
least 20 fetal lungs at each time point, with the same result, i.e.,
that peak Stx1A protein levels occur at ED18-19. It should be
noted that the Western analyses were developed with chemiluminescence
in which detection was semiquantitiative rather than absolutely
quantitative due to the rapid extinction of the substrate over time.
|
RT-PCR analysis of primary cell cultures from fetal rat lungs on ED19
(Fig. 2A)
demonstrated expression of the Stx1A gene in the mixed cell population
before enrichment (lane 1), >95% fetal fibroblasts (lane 2), >95%
distal lung epithelial cells enriched for type II pneumocytes
(lane 3), and intact whole lung explants (lane 4). Stx1A mRNA levels
in the fibroblast cultures were consistently over threefold greater
than those in type II cell cultures (Fig.
2C). In contrast, there was no
significant difference in Stx2 mRNA expression in fibroblasts versus
epithelial cells (Fig. 2, A and
C). 18S rRNA was
expressed at similar levels in both mesenchymal and epithelial cell
populations. Western blotting of protein lysates from the same primary
cultures demonstrated detectable Stx1A protein in all of the primary
cell cultures, with higher levels of Stx1A in the mesenchymal cell
cultures (Fig. 2B,
lane 2, and
C), consistent with the results of
RNA analyses.
|
We carried out immunostaining of these same cell populations grown on glass coverslips to verify that the fibroblast cultures were composed of >95% vimentin-positive and <5% Clara cell 10-kDa secretory protein (CC10) and surfactant protein C, keratin-positive cells (200 cells counted for each of 2 experiments). Conversely, the lung epithelial cultures enriched for type II pneumocytes were composed of >95% keratin-positive cells (approximately two-thirds surfactant protein C positive and one-third CC10 positive) and <5% vimentin-positive cells (200 cells counted for each of 2 experiments; data not shown). Over half of the cells in the fibroblast cultures were strongly Stx1A positive, whereas <20% of cells in the type II cell-enriched cultures were weakly Stx1A positive, with additional rare strongly Stx1A-positive cells having multiple dendritic processes, consistent with neuroendocrine cells (data not shown).
To further assess each of the primary cultures for the presence of neuroendocrine or neuronal cells, we carried out RT-PCR for DDC and GRP. The mesenchymal cell cultures demonstrated a complete absence of DDC and GRP mRNAs on ethidium gels, with only trace amounts in the type II cell-enriched populations. Southern blotting of the RT-PCR products demonstrated barely detectable levels of DDC and GRP mRNAs in the fibroblast cultures and low levels in the type II cell cultures relative to the cell mixtures and intact cell explants (data not shown).
Immunolocalization studies. Using the
Stx1A-specific antibody HPC-1 (3), we carried out definitive
immunolocalization studies in intact ED19 fetal lung frozen sections
(Figs.
3-5).
In the conducting airways, Stx1A was present at high levels in
occasional clusters of neuroendocrine cells, representing <5% of the
airway epithelial cells (Fig. 3A,
between red arrows). Stx1A was also present at high levels in nerve
fibers in the submucosa and muscularis (Fig. 3A, purple arrows). In serial
sections, the neuroendocrine cells immunostained for PGP9.5 (Fig.
3B, between red arrows)
and calcitonin gene-related peptide (Fig.
3C, between red arrows). The nerve fibers immunostained for both Stx1A (Fig.
3A) and PGP9.5 (Fig. 3B, purple arrows). An
additional serial section immunostained in parallel with PGP9.5
antiserum that had been preabsorbed with PGP9.5 (21) is devoid of
immunostaining (Fig. 3D). There was weak immunostaining of the nonneuroendocrine epithelial cells for both
Stx1A (Fig. 3A) and PGP9.5 (Fig.
3B,
left side of
left red arrow),
consistent with our previous published results in human fetal lungs
(21).
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Light-microscopic analysis of cross sections of ED19 rat lung demonstrated the most prevalent Stx1A staining in undifferentiated mesenchymal cells, with the highest levels surrounding developing blood vessels (Fig. 4B, blue arrow) and moderate levels occurring around the conducting airways (Fig. 4B, AW) and in the interstitium of the primitive alveoli (Fig. 4B, alv, white arrows). This staining pattern markedly differs from that of keratin immunostaining in an immediately serial section (Fig. 4A) in which the epithelium of the conducting airways was strongly positive and the developing alveolar epithelium was weakly positive, whereas mesenchymal cells were negative. Most of the Stx1A-positive undifferentiated mesenchymal cells are also positive for vimentin (Fig. 4C) and desmin (data not shown). However, endothelial cells and the desmin-positive differentiated smooth muscle cells surrounding the airways and blood vessels were not Stx1A positive.
This distribution of loose mesenchymal cells positive for Stx1A is reminiscent of the localization pattern of lipocytes, or lipid fibroblasts, in the developing lung. To determine whether the Stx1A-positive cells might indeed be the same population of mesenchymal cells, we carried out oil red O staining for lipids in parallel with HPC-1 immunostaining (Fig. 5A). The most intense HPC-1 immunostaining was again surrounding developing blood vessels (Fig. 5A, bv, thin arrows), with only moderate HPC-1 immunostaining around developing proximal conducting airways (Fig. 5A, Prox. AW, dashed-line arrow) and in the interstitium of the primitive alveoli (Fig. 5A, alv, thick arrows). The same population of mesenchymal cells contains abundant intracellular lipid, staining as red intracellular droplets against a background with dark blue-purple hematoxylin counterstain (Fig. 5B).
Type II cell differentiation and cell proliferation in
lung explants. To determine whether the effect of BLP
on type II cell differentiation requires the presence of cocultured
fibroblasts, similar to Dex (54), we analyzed
[3H]choline uptake
into saturated phosphatidylcholine as the rate-limiting step in
surfactant phospholipid synthesis. As shown in Fig.
6A, there
was no BLP-induced increase in choline uptake in ED18 type II cell
cultures in the absence of added mesenchymal cells, whereas there was
over a 50% increase in choline uptake in the presence of ED18 fetal
fibroblasts (P < 0.001).
Furthermore, BLP induced a significant increase in
[3H]triglyceride
uptake directly into ED18 fetal fibroblasts (62), suggesting that
triglyceride uptake by fibroblasts is hormonally inducible by BLP,
similar to Dex (Fig. 6B) (46).
|
We then evaluated the potential role of Stx1A in mediating the effects of optimal concentrations of BLP and Dex on type II cell differentiation and DNA synthesis in ED18 whole lung organ cultures. We carried out five experiments analyzing inhibition of function in fetal lung organ cultures, comparing the well-characterized anti-Stx1A blocking monoclonal antibody (murine IgG1) HPC-1 (7, 66) to the irrelevant murine IgG1 MOPC-21. HPC-1 completely blocked the effect of BLP (1 nM) on [3H]choline uptake and reduced the effect of Dex to less than half of control values as shown in Fig. 6C (both P < 0.001 comparing HPC-1 with MOPC). In parallel cultures, [3H]thymidine incorporation was stimulated by HPC-1 (Fig. 6D), augmenting the small but significant BLP effect on cell proliferation (P < 0.03) and reducing the growth inhibitory effect of Dex (P < 0.005).
HPC-1 also blocks baseline choline incorporation and augments baseline [3H]thymidine incorporation in a dose-dependent fashion (Fig. 6E), indicating a significant role for Stx1A in lung automaturation and growth cessation.
The HPC-1 antigen was originally determined to be extracellular in location (2, 25). However, a more recent analysis (7) has demonstrated that the HPC-1 antigen may be cytoplasmically oriented. Regardless of its orientation, we do observe highly reproducible effects of HPC-1 on thymidine incorporation and choline incorporation in the fetal samples. The developing cells in fetal lung explants are well recognized to take up antisense oligodeoxynucleotides in culture experiments in the absence of any agent to enhance cellular permeability, and antisense oligodeoxynucleotides can clearly lead to functional and molecular alterations in the explant system (34, 36, 43, 63). The developing cells apparently actively endocytose or pinocytose the culture medium, which we believe to be the case in our experiments. To unequivocably test this hypothesis, we have carried out immunoperoxidase analyses using biotinylated horse anti-mouse IgG (IgG fraction) versus biotinylated normal horse IgG at the same protein concentration as the primary antisera on sections of cultured ED19 rat lungs exposed to medium alone, HPC-1, or the irrelevant isotype-matched murine IgG1 MOPC as our negative control (which was used in all of our HPC-1 functional experiments). These immunohistochemical analyses clearly demonstrate murine IgG (both HPC-1 and MOPC) within the cytoplasm of numerous mesenchymal cells in the rat fetal lung explants (data not shown), indicating that the monoclonal antibodies are taken up by the cells in culture and retained in sufficient quantities for visualization in tissue sections. There was no immunostaining observed with normal horse IgG instead of horse anti-mouse IgG with the exception of occasional alveolar macrophages. Sections of the same lungs incubated without IgG added to the culture medium did not immunostain with horse anti-mouse IgG.
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DISCUSSION |
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The present study demonstrates the expression of mRNAs encoding multiple synaptosomal proteins in rat fetal lungs, including Stx1A, Stx2 (epimorphin), Rab26, and Munc18, all of which peak at ED19, immediately before type II cell differentiation and the onset of surfactant synthesis. We have explored the distribution and function of Stx1A in depth because this molecule is most highly expressed in mesenchymal cells that did not express detectable markers of neuroendocrine or neuronal differentiation by RT-PCR or immunostaining.
There are at least three potential mechanisms for Stx1A-mediated effects on cell differentiation and growth in fetal lungs both at baseline and induced by Dex and BLP. First, Stx1A could be acting as a critical relay molecule, regulating the secretion of other mesenchymal cell-derived molecules similar to its role in regulated exocytosis from neurons and neuroendocrine cells (30). Second, Stx1A could regulate the topographical translocation of membrane-bound molecules from the interior of vesicles to the external surface of the plasma membrane and thus could alter the availability of receptors on the cell surface. Such a role for exocytosis core complex proteins has been implicated for receptors such as GLUT-4 in pancreatic cells as well as for transporters such as aquaporin in renal collecting duct cells (39, 41). Third, Stx1A, or a fragment of Stx1A, could be secreted by fetal mesenchymal cells to function directly as developmental signaling molecules (31, 47), although it is probably unlikely that it directly interacts with the target cell. We cannot rule out the possibility that HPC-1-mediated inhibition of growth factor secretion by neuroendocrine cells or other epithelial cells might be contributing to the dose-dependent decline in baseline choline uptake in fetal lung organ cultures.
A previous study (17) indicated that Stx1A is not expressed in mature adipocytes. The mesenchyme is known to play an important role in specifying epithelial cell fates. The expression of Stx1A in a transient population of lipid-rich fibroblasts is of particular interest because this syntaxin was previously demonstrated to be expressed only by neurons and neuroendocrine cells in adult mammals, the only exception being one report of two malignant colonic epithelial cells (8). The peak of Stx1A mRNA and protein expression on ED19 coincides with the peak expression of several developmental signaling molecules (53), including the receptor for GRP, the major known pulmonary BLP (1).
A significant role for Stx1A in rat pulmonary development is indicated by the observed effects of HPC-1 on type II cell differentiation and cell proliferation in fetal lung organ cultures. The ability of HPC-1 to block Dex- and BLP-induced type II cell differentiation supports a role for secretory fibroblasts in these processes (35). The dose-dependent increase in new DNA synthesis and the parallel decrease in new surfactant production suggest that Stx1A can indirectly regulate both cell proliferation and type II pneumocyte differentiation. A role for syntaxins in development is not without precedent. Cellular proliferation has been demonstrated to be regulated by syntaxin 1 in Drosophila embryos (11). A syntaxin homolog in Arabidopsis is required for proper seedling development (38). Hirai et al. and Oka and Hirai demonstrated that syntaxin 2 (epimorphin) could play a role in early lung morphogenesis (24) and induce human endothelial cells in culture to form tubular structures resembling capillaries (47).
In summary, the present study demonstrates Stx1A gene expression is developmentally regulated in fetal lung mesenchymal cells and that Stx1A can, in turn, regulate both cell differentiation and proliferation in a dose-dependent fashion, probably as a key component of the secretory complex mediating fibroblast exocytosis in fetal lungs.
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ACKNOWLEDGEMENTS |
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Present address of B. H. Brimhall: Dept. of Pathology, Univ. of Colorado School of Medicine, Campus Box A022, 4200 E. Ninth Ave., Denver, CO 80262.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. E. Sunday, Dept. of Pathology, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 (E-mail: sunday{at}a1.tch.harvard.edu).
Received 29 October 1998; accepted in final form 9 April 1999.
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