Sulfation of extracellular matrices modifies growth factor
effects on type II cells on laminin substrata
Philip L.
Sannes,
Jody
Khosla,
Cheng-Ming
Li, and
Ines
Pagan
Department of Anatomy, Physiological Sciences, and Radiology,
College of Veterinary Medicine, North Carolina State University,
Raleigh, North Carolina 27606
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ABSTRACT |
The alveolar basement membrane contains a
variety of extracellular matrix (ECM) molecules, including laminin and
sulfated glycosaminoglycans of proteoglycans. These mixtures exist
within microdomains of differing levels of sulfate, which may
specifically interact to be key determinants of the known capacity of
the type II cell to respond to certain growth factors. Isolated type II cells were exposed to either acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), or keratinocyte growth factor
(KGF; FGF-7) on culture wells precoated with laminin alone or in
combination with chondroitin sulfate (CS), high-molecular-weight heparin, or their desulfated forms. Desulfated heparin significantly elevated FGF-1- and FGF-2-stimulated DNA synthesis, whereas desulfated CS and N-desulfated heparin elevated FGF-7-stimulated DNA synthesis by
type II cells on laminin substrata. When FGF-1 was mixed into the
various test matrix substrata, DNA synthesis was significantly increased in all cases. These results demonstrated that decreased levels of sulfate in ECM substrata act to upregulate responses to
heparin-binding growth factors by alveolar epithelial cells on laminin
substrata.
sulfated proteoglycans; basement membrane; acidic fibroblast growth
factor; basic fibroblast growth factor; keratinocyte growth factor
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INTRODUCTION |
LAMININ IS AN ESTABLISHED COMPONENT of the alveolar
basement membrane (ABM) (7) and is biosynthesized by type II cells in
vitro (21). These cells also actively synthesize a variety of other
extracellular matrix (ECM) molecules as well, including fibronectin,
type IV collagen, heparan sulfate proteoglycan, chondroitin sulfate
(CS)-dermatan sulfate proteoglycan, glycosaminoglycans (GAGs)
associated with a number of uncharacterized proteoglycans (for review,
see Ref. 5), and entactin (30). Those known to be a part of the ABM are
laminin (7), heparan sulfate proteoglycan (Perlecan) (25), CS, CS
proteoglycan (Bamacan), entactin (25), and type IV collagen (2). The
relationships between the alveolar epithelial cells and their
underlying ABMs are important ones and are likely to influence all
their various functions (1, 3, 31-33).
Laminin appears to play a prominent role in maintaining cellular
phenotype and functional character as evidenced by type II cell
behavior in vitro on purified laminin surfaces and laminin-enriched matrix substrata such as are found in Engelbreth-Holm-Swarm (EHS) sarcoma matrix (22). Importantly, laminin has also been shown to
inhibit incorporation of thymidine into cellular DNA in
serum-containing cultures compared with uncoated surfaces (22). The
latter study also showed that EHS matrix (<60% laminin) was even
more inhibitory to thymidine incorporation. The reason for this
"added" inhibition was not clear but could be related to the
basic complex nature of ABM matrix structure (7, 23, 36). As visualized
immunocytochemically, laminin appears homogeneous and symmetrically
distributed within the ABM (7), which differs markedly from the
heterogeneous and asymmetrical distribution of sulfated macromolecules
found there. Specifically, the ABM microdomains beneath type II cells are distinctly less sulfated than the same region adjacent to type I
cells (8, 11, 23, 35). These anionic sites were shown to be chiefly
associated with heparan sulfates (36), which raised the question of
their functional significance with regard to type I and type II cells.
Recent evidence (26) has indicated that CS and heparin can reduce the
capacity of the type II cell to incorporate thymidine in response to
heparin-binding growth factors in vitro, whereas their chemically
desulfated forms restore this capacity. These data would seem to
support the notion that sulfate per se can modulate type II cell
responses to select growth factors (23), but, given the complex
structure of the ABM in situ, it is likely that the interactions
between soluble or bound growth factors, ECMs, and cell surfaces are
multiple in nature. A key question therefore becomes, how do molecules
such as laminin and associated (bound) sulfated proteoglycans (29),
which have the tendency to inhibit DNA synthesis, behave in combination
with specific growth factors that promote DNA synthesis in alveolar type II cells? The aim of this study was to determine whether laminin
added to collagen substrata in combination with native or desulfated CS
or heparin modified isolated rat type II cell responses to
heparin-binding growth factors.
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MATERIALS AND METHODS |
Cell preparation. Type II pneumocytes
were isolated from 200- to 240-g Fischer CDF (F-344) rats (Charles
River Laboratories, Wilmington, MA) by the panning method of Dobbs et
al. (4). Briefly, animals were anesthetized by intraperitoneal
injection of Nembutal containing heparin and perfused via the hepatic
portal vein with HEPES-saline solution until the lungs were free of
blood. The lungs were then dissected free and lavaged via the trachea with a HEPES-saline solution containing glucose and EDTA. The lungs
were then continuously insufflated via the trachea with 4.3 U of
elastase (Worthington Biochemicals, Freehold, NJ; Boehringer Mannheim
Biochemicals, Indianapolis, IN) at 5 cmH2O for 20 min, and the reaction
was stopped with fetal bovine serum. The lungs were minced
and filtered sequentially through two layers of 165-µm silk mesh
followed by four layers of 42-µm silk mesh, and the recovered cells
were centrifuged and panned on IgG-coated bacteriological plastic
culture dishes for 1 h at 37°C in a 10%
CO2
incubator. Adherent cells were discarded, and
nonadherent cells were examined with a modified Papanicolaou stain and
judged to be 90-95% type II cells as evidenced by lamellar
body-like structures and cell size. These cells were 80-95%
viable as defined by trypan blue exclusion.
Matrix substrata preparation.
Twenty-four-well tissue culture plates (Becton Dickinson, Lincoln Park,
NJ) were first coated with 6 µg/cm2 of type I collagen
(Sigma, St. Louis, MO), and the coating was allowed to dry at room
temperature overnight in a laminar flow hood. This treatment was
designed to optimize adherence of the ECM molecules to the culture
surface. Optimal ECM concentration was established in a previous study
(26), so the focus in these experiments was on the impact each specific
ECM combination had on the growth factor conditions. Designated
chambers were then coated with 16 µg/cm2 of laminin (Collaborative
Research Products, Bedford, MA) either alone or in combination with 35 µg/cm2 of CS [type A;
major repeating disaccharide,
-glucuronic
acid-[1
3]-N-acetyl-
-galactosamine-4-sulfate-[1
4], 70% pure (remaining is chondroitin 6-sulfate); Sigma],
high-molecular-weight heparin (13,500-15,000 mol wt; Calbiochem,
La Jolla, CA), or their specifically desulfated forms (see
below). In selected experiments, 100 ng/ml of
either acidic fibroblast growth factor (FGF-1) or basic fibroblast
growth factor (FGF-2) were mixed with the matrix substrata preparations
(final concentration of 15 ng/cm2). ECMs were evaporated to
dryness in a laminar flow hood, covered, and stored at 3°C until
use. In separate experiments, laminin was added to ECM substrata at
concentrations that varied from 0.1 to 1,000 µg/ml (0.016-160
µg/cm2).
The CS and high-molecular-weight heparin were subjected to solvolytic
desulfation with dimethyl sulfoxide (18). The treated and nontreated
samples were dialyzed against deionized water, and the content of
covalently bound sulfate was measured with a Dionex conductivity
detector (17) after hydrolysis in 6 N HCl vapor at 109°C for 6 h
and separation on an anion-exchange column (Dionex, Atlanta, GA). A
sample without hydrolysis served as the blank to correct for free
sulfate. Only ECM components that were >85% desulfated were used in
testing type II cell responses. Specifically, N-desulfated
high-molecular-weight heparin was purchased from Sigma.
Cell culture. Isolated cells were
seeded at 1 × 103/mm3
on the various substrata in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum (Life Technologies, Gaithersburg,
MD) overnight in a 10% CO2
incubator at 37°C. In selected experiments, 100 ng/ml of FGF-1 or
FGF-2 were mixed with matrix substrata beforehand or were added to the
medium at seeding. Optimal growth factor concentrations
had been established (15, 20) and confirmed in our preparations (26).
Attached cells (>85%) were washed with serum-free hormonally defined
(SFHD) medium (12) and then incubated with the same medium with or
without one of the following growth factors: 100 ng/ml of FGF-1
(bovine, Collaborative Research Products), 100 ng/ml of FGF-2 (bovine,
Collaborative Research Products), 10 ng/ml of keratinocyte growth
factor (KGF; FGF-7; R&D Systems, Minneapolis, MN), 10 ng/ml of
transforming growth factor (TGF)-
(R&D Systems), or 10 ng/ml of
TGF-
(R&D Systems). The latter two were utilized as growth factor
controls, and serum-free medium without growth factor added served as
an additional control. The levels chosen for each were
designed to optimize cell responses and were fixed so as to focus on
variations in response to the ECM substrata outlined above. To the SFHD
medium with or without growth factors, 1 µCi/ml of
[3H]thymidine (final
concentration) was added, and cells were cultured for 48 h. Cell
lysates were then processed and counted in a scintillation counter
according to standard methods (22). The remaining volume was used for
determining the protein content of each sample. Treatment groups were
organized and analyzed in quadruplicate. Experimental protocols were
repeated a minimum of seven times. In parallel preparations, some cells
were fixed with either 1% paraformaldehyde or acid alcohol (70%
ethanol and 1% HCl) for histochemical localization of type II
cell-specific antigens (26).
Data analysis. Means ± SE for the
disintegrations per minute per microgram of protein were developed for
the various treatment groups (in quadruplicate for each experiment).
Different treatment groups within experiments were compared with
laminin control substrata by analysis of variance (ANOVA, E-Z Stat,
Trinity Software, Campton, NH) within experiments.
P values < 0.05 were considered
significant.
Data are also expressed as a percentage of control value (laminin
treatment alone for each growth factor treatment) so that
The
significance of a percent value above or below 100% (control) was
determined by the P value (<0.05)
established by the raw data. The results presented in graphic form were
representative experiments that were consistent with or closely
resembled the outcomes of at least five or more repeats of the same
protocol.
Cell adhesion assay. To ensure that
the starting cell numbers for the various treatments were comparable,
we evaluated adhesion according to standard methods (34). After the
24-h seeding period in serum-containing medium, cells on the various
matrices were washed with PBS. Attached cells were then either treated
with additional medium-growth factor combinations or terminated. After an additional 48 h, attached cells in the medium-growth factor combinations were terminated by fixation with 3.7% Formalin for 8 min
at room temperature and stained with 1% toluidine blue in PBS for 2 h
at room temperature. Cells were then washed three times, air-dried, and
solubilized with 2% SDS for 30 min at 37°C. Absorbance of the
solubilized cells was measured in a spectrophotometer at 650 nm.
Nonadherent cells were similarly quantitated after centrifugation and
identical treatment.
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RESULTS |
For the short duration of these experiments (72 h total), no changes in
phenotype were detected for the isolated type II cells under any of the
various treatments as defined by alkaline phosphatase and surfactant
protein A histochemistry (26).
Isolated type II cells seeded on laminin plus desulfated heparin
exposed to FGF-1 showed a significant increase (232% of control values) in incorporation of thymidine compared with laminin alone (Fig.
1). Untreated heparin caused
a modest reduction in thymidine uptake that was not significant
compared with laminin controls. Similarly, laminin plus desulfated
heparin substrata and FGF-2 stimulated a significant increase (256% of
control values) in thymidine incorporation by type II cells (Fig.
2), with the modest increases in other
groups not significant. Cells exposed to FGF-7 showed significant
increases in thymidine uptake on substrata containing either laminin
plus desulfated CS (174% of control values) or laminin plus
N-desulfated heparin (190% of control values) (Fig.
3); those containing laminin
plus heparin showed decreased thymidine uptake, but the decrease was
not significant.

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Fig. 1.
Thymidine uptake by isolated type II cells exposed to 100 ng/ml of
fibroblast growth factor (FGF)-1 for 48 h is significantly elevated on
laminin + desulfated heparin substrate (232% of laminin control
value). MW, molecular weight. Data are means ± SE of quadruplicate
wells in 1 experiment, results of which were confirmed in at least 5 other independent experiments. ANOVA was performed on raw data
(dpm/µg protein). * Significant difference in groups compared
specifically with laminin control substrate,
P < 0.05.
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Fig. 2.
Thymidine uptake by isolated type II cells exposed to 100 ng/ml of
FGF-2 for 48 h is significantly increased on laminin + desulfated
heparin substrate (256% of laminin control value). Data are means ± SE of quadruplicate wells in 1 experiment, results of which were
confirmed in at least 5 other independent experiments. ANOVA was
performed on raw data (dpm/µg protein). * Significant
difference in groups compared specifically with laminin control
substrate, P < 0.05.
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Fig. 3.
Thymidine uptake by isolated type II cells exposed to 10 ng/ml of
keratinocyte growth factor (KGF; FGF-7) for 48 h is significantly
increased on laminin + desulfated chondroitin sulfate (174%) and on
laminin + N-desulfated heparin (190%). Data are means ± SE of
quadruplicate wells in 1 experiment, results of which were confirmed in
at least 5 other independent experiments. ANOVA was performed on raw
data (dpm/µg protein). * Significant difference in groups
compared specifically with laminin control substrate,
P < 0.05.
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For comparison, TGF-
was used as a non-heparin-binding growth
factor, with unexpected results. Type II cell responses to TGF-
on
the substrata used in this study were all stimulatory above laminin
alone. Specifically, laminin plus CS, laminin plus desulfated CS, and
laminin plus N-desulfated heparin stimulated dramatic increases in
thymidine uptake (561, 456, and 437% of laminin controls,
respectively; Fig. 4). Thymidine
incorporation results from experiments with cells exposed to either
serum-free medium without growth factors or TGF-
(additional growth
factor control) were extremely low, and comparisons between groups were not significant (data not shown).

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Fig. 4.
Thymidine uptake by isolated type II cells exposed to 10 ng/ml of
transforming growth factor (TGF)- for 48 h is significantly
increased on laminin + chondroitin sulfate (561%), laminin + desulfated chondroitin sulfate (456%), and laminin + N-desulfated
heparin (437%). Data are means ± SE of quadruplicate wells in 1 experiment, results of which were confirmed in at least 5 other
independent experiments. ANOVA was performed on raw data (dpm/µg
protein). * Significant difference in groups compared
specifically with laminin control substrate,
P < 0.05.
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Figure 5 compares the raw data of the
thymidine incorporation by type II cells exposed to the various growth
factor and matrix substrata conditions. Although all
growth factor concentrations used were considered optimal for thymidine
incorporation by published standards, the responses on the substrata
used were highly varied. For instance, laminin alone and laminin plus
heparin exposures resulted in the lowest thymidine incorporation with
all growth factors, with the notable exception of TGF-
. Responses to
FGF-1, FGF-7, and TGF-
were generally high, with the former two
highest on desulfated matrices and lowest on sulfated matrices. FGF-2 followed a pattern similar to the other heparin-binding growth factors
but at a generally lower level of incorporation. TGF-
was surprising
with its very low response to laminin alone but with highly elevated
responses to all matrix substrata combinations with laminin regardless
of sulfation.

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Fig. 5.
Thymidine uptake by type II cells exposed to 4 different growth factors
while on various extracellular matrix substrata. CS, chondroitin
sulfate; DCS, desulfated chondroitin sulfate; H, heparin; DH,
desulfated heparin; NDH, N-desulfated heparin; TGF- , transforming
growth factor- . Data are direct comparison of a variety of
treatments employed. Growth factor concentrations used were considered
optimal for thymidine incorporation, but responses on substrata used
were highly varied. Laminin alone and laminin-heparin exposures
resulted in lowest thymidine incorporation with all growth factors,
with notable exception of TGF- . Responses to FGF-1, FGF-7, and
TGF- were generally high, with former two highest on desulfated
matrices and lowest on sulfated. FGF-2 followed pattern similar to the
other heparin-binding growth factors but at a generally lower level of
incorporation. TGF- was surprising with its very low response to
laminin alone but with highly elevated responses to all matrix
substrata combinations with laminin regardless of sulfation.
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Type II cells were exposed to different concentrations of laminin
substrata alone in the presence of 10 ng/ml of KGF to determine whether
there was a dose relationship with its effects on thymidine incorporation (Fig. 6). A
slight elevation over collagen alone was observed at 0.016 µg/cm2 (0.1 µg/ml) of laminin,
followed by a steady decrease in thymidine incorporation toward 160 µg/cm2. Laminin is typically
used as a substrate in tissue culture between 0.16 and 16 µg/cm2 (the latter used in the
present experiments). These levels are generally considered to yield
optimal cell attachment with acceptable levels of growth.

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Fig. 6.
Thymidine uptake by isolated type II cells exposed to 10 ng/ml of KGF
on different concentrations of laminin substrata (0-1,000 µg/ml
or 0-160 µg/cm2). Over
the range of laminin concentrations tested, thymidine incorporation
decreased as a function of increasing laminin concentration.
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The impact of the timing and mode of growth factor addition on
thymidine uptake by type II cells was tested by adding either FGF-1 or
FGF-2 to the seeding medium or to the preformed culture substrata. The
results were particularly profound for FGF-1, which stimulated
150-190% increases in thymidine uptake over cells for which FGF-1
was mixed with laminin alone (Fig. 7).
Unlike the results seen in Fig. 1, in which FGF-1 was added only after
24 h of seeding in conventional serum-containing medium (no additional growth factors added), all matrix substrata were significantly stimulatory of thymidine uptake regardless of sulfation. This changed
somewhat when FGF-1 was added to the medium instead of to the matrix,
which resulted in significant elevations in thymidine uptake only in
the desulfated CS and N-desulfated heparin groups (165 and 150% of
laminin controls), although all were elevated. The effects were such
that the typical reduction in thymidine uptake expected from sulfated
matrix substrata was lost. The impact of this approach was less
profound for FGF-2, with a significant increase observed only in the
desulfated CS group (530% of laminin control value; Fig.
8). When these growth factors were mixed
with collagen only (no laminin or sulfated ECMs), significant elevation in thymidine uptake only occurred with FGF-1 and FGF-2 when added to
the medium (Fig. 9), a result that was the
reverse of that seen with laminin mixtures.

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Fig. 7.
FGF-1 increased thymidine uptake compared with FGF-1 (100 ng/ml) mixed
with laminin alone on all substrata (50-190%).
Data are means ± SE of quadruplicate wells in
individual experiments, results of which were confirmed in at least 5 other independent experiments. When FGF-1 was added to media
(+media/seed) instead of matrix (+matrix/seed), significant elevations
in thymidine uptake appear only in laminin + DCS and laminin + N-DH
groups (165 and 150% of laminin control). * Significant
difference in groups compared with laminin control substrate,
P < 0.05.
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Fig. 8.
FGF-2 (100 ng/ml) gave a significant increase only in
DCS group (530% of laminin control).
Data are means ± SE of quadruplicate wells in
individual experiments, results of which were confirmed in at least 5 other independent experiments. * Significant difference in groups
compared with laminin control substrate,
P < 0.05.
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Fig. 9.
Thymidine uptake (expressed as percent untreated
control) by isolated rat type II cells in response to 100 ng/ml of
either FGF-1 or FGF-2 added either to extracellular matrix (w/ECM)
substrata before seeding or to media (w/media) at the time of seeding
on substrata containing collagen alone. * Significant differences
are seen when FGFs are added to media but not to matrix.
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To attempt to define the influence the various treatments had on
adhesion, we exposed the cells to standard serum-containing medium on
the various ECM substrata, followed by growth factor addition at 24 h.
At 24 h, laminin plus desulfated heparin was the only matrix to give
modest but significant enhancement of adherence (137% of laminin
alone). The addition of FGF-1 enhanced adherence between the 24- and
48-h cultures on laminin plus CS and laminin plus desulfated CS,
whereas FGF-7 depressed adherence on laminin plus heparin and laminin
plus N-desulfated heparin (Fig. 10). The
other growth factors tested did not influence the adherence of type II
cells on the various substrata in a significant fashion.

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Fig. 10.
Effects of either FGF-1 or FGF-7 on adherence of rat
type II cells to selected substrata mixed with laminin. Significantly
increased adherence of type II cells at 24 h in serum-containing medium
is seen only with laminin + DH. After 24 h, addition of FGF-1 resulted
in elevated adherence of type II cells on laminin + CS and laminin + desulfated CS, but addition of FGF-7 reduced adherence on laminin +H
and laminin + NDH. * Significant difference in groups compared
with laminin control substrate, P < 0.05.
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DISCUSSION |
Laminin has previously been shown to support type II cell attachment
and maintenance of phenotype, including cuboidal shape (22). In
addition to being an important point of attachment for epithelial
cells, specific domains of the laminin molecule act to structurally
link or bind other components of the ABM, including heparan sulfate
proteoglycans (14) and entactin (6). These structural relationships are
well established, but their biological consequences may be much greater
than presently appreciated. In other biological systems, for instance,
FGF-2 is a survival factor for embryonal carcinoma cells cultured on
plastic but becomes mitogenic for the same cells when cultured on
laminin substrata (28). Laminin has also been shown to directly affect
gene expression as demonstrated for the
-casein gene in mammary
epithelium in the presence of lactogenic hormones (35). These data
could be relevant to the current study, which was undertaken to
determine whether the capacity of type II cells to incorporate
thymidine into DNA in response to selected growth factors was affected
by the presence of laminin alone or in combination with selected ECMs
with varying levels of sulfate.
Previous studies have demonstrated that the heparin-binding growth
factors FGF-1, FGF-2, and FGF-7 (KGF) stimulate isolated type II cell
incorporation of thymidine into DNA (15, 16, 20), and FGF-1 and FGF-2
have been immunolocalized in the type II cell and ABM, respectively
(24). The effects of FGF-1 and FGF-2 on bromodeoxyuridine (BrdU)
incorporation by isolated type II cells were shown to be inhibited by
sulfated CS and low- and high-molecular-weight heparins, and this
phenomenon was reversed by their chemical desulfation (26). In the
latter study, in which the "base" substratum was collagen type I,
laminin was shown to modestly support BrdU incorporation by type II
cells exposed to FGF-1 and FGF-2. Under the conditions used in the
present study, type II cell incorporation of thymidine into DNA was not
supported by laminin alone in the presence of most heparin-binding
growth factors tested (FGF-1, FGF-2, and FGF-7), and higher
concentrations tended to be even less supportive (see Fig. 6). This
would seem to support previous observations on the effects of laminin
on type II cell DNA synthesis (22). In the present study, desulfated high-molecular-weight heparin combined with laminin gave significant increases in thymidine incorporation compared with laminin substrata alone in the presence of FGF-1 or FGF-2, whereas desulfated CS and
N-desulfated heparin significantly enhanced FGF-7-stimulated DNA
synthesis. These modulatory effects support a previous report (26) that
indicated that desulfated CS supported BrdU incorporation into DNA when
combined with type I collagen in the presence of FGF-2. Taken together,
these observations support the notion that the undersulfated
microdomains of adult ABMs beneath type II cells (8, 11, 23, 36) could
act to support or promote responses to growth factors.
The aim of the present study was to develop an artificial combination
of ECM components within substrata that mimicked the in vivo state of
the ABM by including laminin with mixtures (high and low sulfate
content) used in a previous study (26). Clearly, the data indicate that
a lack of support of laminin for DNA synthesis (22) is dramatically
overcome by selected desulfated GAGs in the presence of certain FGFs.
The importance of sulfated molecules in the activity of FGF-1 and FGF-2
is well established (10, 19). FGF-1 and FGF-2 bind to cell surfaces via
a dual-receptor system consisting of a low-affinity binding site that
is a heparan sulfate and a high-affinity binding site (FGF receptor)
that is linked to tyrosine kinase (10, 13). Successful activation of
the FGF ligand by the low-affinity binding site is crucial for binding
the high-affinity site and appears to be related to sulfation (9, 10).
This dependency has been shown to be different between FGF-1 and FGF-2,
with the former requiring a high content of 6-O-sulfate groups and the
latter a fully N-sulfated decasaccharide enriched in 2-O- and
6-O-sulfated disaccharide units (10). Similarly, Guimond et al. (9)
prevented FGF-2 activation and 3T3 fibroblast mitogenesis by selective
2-O- and 6-O-desulfation of heparin. It would be expected that the
molecular constructs on the FGF ligand that interact with these
low-affinity "activating" sites on the cell surface would be
probable targets for competitive inhibition by CS and
high-molecular-weight heparin used in the present study and as
suggested previously (26). As noted above, however, these molecules
were not inhibitory in the mixtures used here, whereas their desulfated
forms were actually stimulatory. Although the mechanism(s) of this
action is not clear from the data, it is possible that the reduced
sulfate environment or the sugar sequences within the desulfated
molecules themselves act in concert with cell-surface heparan sulfate
proteoglycans (low-affinity receptor) to facilitate the necessary
ligand-receptor interaction critical for subsequent signaling events.
The fact that the sulfated forms of these GAGs were not as obviously
inhibitory of DNA synthesis as previously described (26) could be
explained by the possibility that the inherently "retarding
effects" of laminin were maximal and could not be enhanced further
by additional factors in the mixture. The role of laminin, if any, in
modulating heparin-binding growth factor activity at the cell surface
appears to be altered in a profound way by desulfated GAGs. These data,
at the very least, demonstrate that complex mixtures of ECMs in the
ABM, with their varying levels of sulfate, function in concert with one another, perhaps in sometimes subtle ways, to modulate type II cell
responses to growth factors.
The rather dramatic, stimulatory effects of TGF-
on the various
laminin-GAG substrata add another dimension to the discussion, although
a somewhat puzzling one. Its effects on type II cell DNA synthesis were
expected to be more modest, but this was not the case. The reason for
the enhancing effects of the GAGs on TGF-
effects is not obvious
from these experiments but, as suggested above, could reflect the
complexity of receptor-ligand interactions at cell surfaces and their
impact on signaling and other downstream events linked with DNA
synthesis and proliferative events. These observations could not be
readily explained and deserve further study.
It was of interest that the addition of FGF-1 to the preexisting
substrata or the medium during seeding overwhelmed its more selective
enhancement of thymidine incorporation by type II cells on laminin plus
desulfated heparin. In fact, all ECM substrata showed increased
thymidine incorporation compared with laminin alone. This could be
explained by the possibility that FGF-1 can gain access sooner to or
compete more effectively for its appropriate receptor complex by mixing
with the substrata. By comparison, when mixed with the medium in
soluble form, it was stimulatory in the laminin plus desulfated CS and
laminin plus N-desulfated heparin groups. FGF-2 was only stimulatory
when added in soluble form to the medium with the laminin plus
desulfated CS. This effectively supports an earlier study (26) using
BrdU incorporation as a measure of DNA synthesis.
Two important points arise from the adhesion studies. First, laminin
plus desulfated heparin enhanced adherence of type II cells, which
could have had an effect on the outcome of the thymidine incorporation
results for FGF-1 and FGF-2 by ostensibly increasing the number of
potential cells that could respond to the growth factors. Conversely,
FGF-7 in the presence of laminin plus N-desulfated heparin had a low
adherence (Fig. 9) yet high thymidine uptake (Fig. 3). These potential
problems in interpretation are minimized by the normalization of
thymidine incorporation with total protein.
An earlier report (26) presented data that support the notion that the
heavily sulfated regions of the ABM, such as those found adjacent to
type I cells, would be expected to retard or otherwise inhibit
epithelial responses to heparin-binding growth factors. In combination
with laminin, which characteristically does not support DNA synthesis
by type II cells (22), these effects would predictably be even greater.
This could explain the enhanced inhibitory effect of EHS matrix on
thymidine incorporation by type II cells in the original study by
Rannels et al. (22). The present study supports the converse of this
corollary, wherein regions of low sulfate within the ABM, like those
found beneath type II cells (8, 11, 23, 36), would be expected to
promote or at least be less inhibitory to potential growth factor
responses to heparin-binding growth factors (26), thus providing a
"window" of controlled growth factor responsiveness within the
alveolus. In further support of this paradigm are recent data
demonstrating that type II cells maintained in culture for periods
exceeding 7 days tend to biosynthetically add higher levels of sulfate
to nearly all of ECM components, including a novel sulfated laminin, compared with a rather modest level of sulfation within the first 7 days (27). Cells maintained in this manner would be considered more
"type I cell-like," thus suggesting that type I cells may actively biosynthesize/maintain the heavily sulfated ABM with which
they are known to be associated in vivo (8, 11, 23, 36). Such a
biosynthetic scheme could act as a further mechanism to help maintain
stable cell populations under normal conditions by allowing certain
cell types of common lineage to divide as necessary while preventing
excessive responsiveness to growth factors within a confined anatomic
region.
 |
ACKNOWLEDGEMENTS |
The authors are indebted to the support and helpful discussions
with Dr. Pi-Wan Cheng of the University of Nebraska Medical Center.
 |
FOOTNOTES |
These studies were supported by National Heart, Lung, and Blood
Institute Grant HL-44497 and a grant from the State of North Carolina.
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: P. L. Sannes, Dept. of Anatomy,
Physiological Sciences, and Radiology, College of Veterinary Medicine,
North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606.
Received 6 March 1998; accepted in final form 12 June 1998.
 |
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