Expression of N-deacetylase/sulfotransferase
and 3-O-sulfotransferase in rat alveolar type II
cells
Zhong-Yuan
Li,
Kazunori
Hirayoshi, and
Yasuhiro
Suzuki
Department of Ultrastructural Research, Institute for Frontier
Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan
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ABSTRACT |
Basal laminae beneath alveolar type I cells are suggested to contain
highly sulfated heparan sulfate-containing proteoglycans (PGs), and
cultured type II cells accumulate highly sulfated matrices. To
characterize the regulation of PG synthesis during the transition from
type II cells to type I cells, we examined mRNA expression of
N-deacetylase/sulfotransferase (NST) and
3-O-sulfotransferase (3-OST), two enzymes specific for
heparan sulfate synthesis. We found that both freshly isolated and
cultured type II cells expressed NST and 3-OST as shown by in situ
hybridization. Expression of surfactant-associated protein A, B, and C
mRNAs, determined by semiquantitative PCR, decreased during culture.
Expression of type I cell marker T1
mRNA increased except in cells
cultured on an Engelbrecht-Holm-Swarm gel. Expression of NST was
dependent on cell density and matrix and was intense in conditions
where cells spread fully, whereas 3-OST expression was unchanged in the
conditions examined. The PG sulfation inhibitor sodium chlorate significantly inhibited cultured type II cell spreading, and this inhibition was reversed by sodium sulfate. These results suggest that
highly sulfated PGs modified by NST are necessary for the spreading of
cells during transdifferentiation of type II cells to mature type I cells.
heparin/heparan sulfate proteoglycan; cell spreading; sodium
chlorate; T1
; surfactant-associated proteins
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INTRODUCTION |
CELL-MATRIX
INTERACTION is important to cell morphology, differentiation, and
proliferation. Epithelial cells produce their own matrix made of
fibronectin, thrombospondin, and laminin and prepare appropriate
substrata for themselves (11). In the lung, it was shown
that components of the alveolar basement membrane (ABM) are highly
sulfated beneath type I cells (25, 38)
compared with those beneath type II cells, and the difference is mainly due to the presence of highly sulfated heparan sulfate (HS)
proteoglycans (PGs) as shown histochemically by differential enzyme
digestion (25, 38). The difference in
sulfation of extracellular matrix- and cell-associated PGs is important
because it may modify cell behavior, such as the response to growth
factors (26, 27).
Type II cells are thought to be a progenitor of type I cells
(2). Type II cells lose their phenotypic expression of
lamellar bodies and alkaline phosphatases and change into flattened
cells when they are cultured for several days on tissue culture plastic at low density. These cells then progressively assume the appearance of
type I cells (6) and come to express a marker for type I cells (4). Type II cells are known to biosynthesize a
variety of basement membrane-related components in short-term culture (2-10 days), including fibronectin (20), laminin
(20), type IV collagen (35), and entactin
(30), and some of them became highly sulfated after
prolonged culture as reported by Sannes et al. (28). The
above-mentioned results suggest that the regulation of heparin (H) and
HS (H/HS) synthesis is different between type II and type I cells.
To examine the changes in H/HSPG synthesis during the transition of
cultured type II cells into type I cells, we evaluated N-deacetylase/sulfotransferase (NST) and
3-O-sulfotransferase (3-OST) mRNA expression in type II
cells cultured in various conditions because only H/HS are specifically
sulfated at the O-3 and N positions of some disaccharides among all the
glycosaminoglycans. We also evaluated the expression of
surfactant-associated protein (SP) A, SP-B, and SP-C, i.e., type II
cell markers, and T1
, recently reported as a candidate marker gene
of type I cells to monitor the transition of these two types of cells.
In this study, we molecularly cloned the coding region of 3-OST cDNA
from the rat lung and demonstrated 1) its expression in type
II cells by in situ hybridization; 2) that NST and T1
mRNAs were variously upregulated during culture in different culture conditions, with the rapid disappearance of expression of SPs except in
cells cultured on Engelbrecht-Holm-Swarm (EHS) gels; and 3)
that sodium chlorate, a proteoglycan sulfation inhibitor, significantly
inhibited type II cell spreading in culture.
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MATERIALS AND METHODS |
Cloning and sequencing of rat 3-OST and NST cDNAs.
Pathogen-free adult male Wistar rats (Shimizu Laboratory Supplies,
Kyoto, Japan) weighing 200-250 g were anesthetized by
intraperitoneal injection of pentobarbital sodium (100 mg/kg body wt)
and killed by exsanguination from the abdominal aorta. Total RNA in the
lungs was extracted with TRIzol Reagent (Life Technologies, Grand
Island, NY). Two micrograms of total RNA were annealed with
oligo(dT)15 primer (Promega, Madison, WI) and reverse
transcribed with reverse transcriptase of Rous-associated virus 2 (TaKaRa Biomedicals, Shiga, Japan). The cDNA products were then
subjected to PCR with primers that have the same sequences as those of
the mouse, with the forward primer
5'-ggaattcatatgaccttgctgctcctgggtg-3' containing an
EcoR I restriction site and the reverse primer
5'-gctctagatcagtgccagtcgaatgttctg-3' containing an Xba I
restriction site. The PCR was carried out with a GeneAmp PCR System
9700 (PE Applied Biosystems, Foster City, CA) for 5 min at 95°C, 30 cycles of denaturing for 1 min at 95°C, annealing for 1.5 min at
65°C, and synthesis for 2 min at 72°C. The 3-OST RT-PCR fragment
was then cloned into the EcoR I-Xba I sites of
the vector pGEM4Z. This plasmid was designated pGEM4Z-3-OST and
transformed into DH-5
. The transformed DH-5
was plated on
modified Luria-Bertani gel (1% Bacto Tryptone, 0.5% Bacto-yeast
extract, 0.5% NaCl, 0.1% glucose, 1.5% agarose, and 0.01%
ampicillin) and selected with
X-galactosidase-isopropyl-
-D-thiogalactopyranoside. The pGEM4Z-NST clones were constructed and analyzed in the same manner except that the forward primer was
5'-aactgcagatgttcctgctgtttgtcttctgcc-3' with a Pst I
restriction site and the reverse primer was
5'-ggaattcctacagtctttcaagcccaggttcg-3' with an EcoR I
restriction site.
DNA sequencing of the inserts was carried out with the dye terminator
method with a Ready Reaction Kit (PE Applied Biosystems) in an ABI
PRISM 377 DNA Sequencer (PE Applied Biosystems) with the primers
described above. In the case of 3-OST, pUC/M13 primers (Promega) of the
pGEM4Z vector were also used to ascertain the sequences of the primer
and the ligation region. The nucleotide sequence of rat 3-OST reported
here has been submitted to GenBank with accession number AF177430.
Northern blot analysis.
Poly(A)+ mRNAs were prepared with Oligotex-dT30 (Super)
(TaKaRa Biomedicals). After denaturation, they were separated on a 1%
agarose gel containing 6% (vol/vol) formaldehyde, transferred onto a
positively charged nylon membrane (Boehringer Mannheim, Indianapolis,
IN), and then subjected to prehybridization for 2 h and
hybridization for 16 h at 42°C. Digoxigenin (Dig)-labeled double-stranded probes (936 bp) of 3-OST were prepared from the pGEM4Z-3-OST clone with a Dig DNA labeling and detection kit, and the
membrane was washed under contingent conditions according to the
instructions of the manufacturer (Boehringer Mannheim). The membrane
was detected with a Dig luminescent detection kit (Boehringer Mannheim)
and exposed to Kodak X-AR film (Eastman Kodak, Rochester, NY) for photography.
The size of 3-OST mRNA was estimated by comparison with the markers 28S
(~4.5-kb) and 18S (~2-kb) rRNAs.
Type II cell isolation and culture.
Rat alveolar type II cells were isolated with the method of Dobbs et
al. (9). The viability as assessed by trypan blue exclusion and purity as assessed by alkaline phosphatase staining (12) of isolated type II cells were >90%. To prepare
specimens of freshly isolated type II cells for in situ hybridization,
5 × 104 cells were spun down at 130 g for
1 min onto a poly-L-lysine-coated glass slide at 4°C. For
the preparation of cultured cells, the cells were plated at a density
of 2.5 × 104/cm2 on glass coverslips in
24-well cluster plates and cultured in Dulbecco's modified Eagle's
medium (DMEM) containing 10% FCS and 100 U/ml of penicillin G, 100 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B at 37°C
in a humidified incubator in a 5% CO2 atmosphere.
Nonadherent cells were removed after 16 h by washing with DMEM,
and the culture was continued for different time courses by changing
the medium every other day. On termination of the culture, the cells
were washed with DMEM and subjected to in situ hybridization.
In situ hybridization and immunocytochemistry.
Cell specimens were fixed in freshly prepared 4% paraformaldehyde in
phosphate-buffered saline (PBS; pH 7.2) for 10 min. The slides were
immersed in 3× PBS for 2 min to stop the fixation, washed two times
with 1× PBS for 2 min, and dehydrated sequentially in a series of 30, 60, 80, 95, and 100% ethanol solutions for 2 min each. After they had
completely dried, the slides were used immediately or kept at
70°C.
The DNA probes were prepared as described in Northern blot
analysis, and the ribonucleotide probes were prepared with a Dig RNA labeling kit (SP6/T7; Boehringer Mannheim). In situ hybridization was performed according to the instructions of the manufacturer. The
final color development was performed for 4 h for type II cells
and overnight for fibroblasts. To identify cells, double staining with
anti-SP-B (37) and anti-vimentin antibodies as well as
with wide-spectrum anti-keratin antibodies (DAKO, Carpinteria, CA)
(19) was performed. For double staining, the reagents were added to the specimen in the following order for binding of probes to
mRNA, alkaline phosphatase-labeled anti-Dig antibody binding to the
probe, incubation with mouse anti-SP-B or mouse anti-vimentin antibody,
incubation with a secondary antibody (goat anti-mouse IgG Alexa
568; Molecular Probes, Eugene, OR), and finally immersion into
color substrate solution for in situ hybridization. Before the final
reaction with substrate solution, photographs were taken under a
fluorescence microscope (IX70, Olympus Optical, Tokyo, Japan), and the
places were marked by scratching with a glass cutter. The same scopes
were again photographed after reaction with color substrate solution
for 3-OST or NST mRNA by in situ hybridization. For double staining of
vimentin and keratin, the cells were photographed on the same film by
changing fluorescence filters for the secondary antibodies (goat
anti-mouse IgG Alexa 568 and goat anti-rabbit IgG Alexa 488). In the
immunohistochemistry control study, the preimmune serum supplied by the
manufacturer was used instead of the primary antibodies (SP-B, keratin,
or vimentin).
Expression of differentiation markers and NST and 3-OST mRNAs in
cultured type II cells.
Type II cells (1 × 106) were plated at low (2 × 104 cells/cm2), medium (1 × 105 cells/cm2), and high (5 × 105 cells/cm2) densities. To prepare EHS gels,
0.3 ml of 5 mg/ml of Matrigel (Becton Dickinson Labware, Bedford, MA)
was added to 24-well plates. For the EHS-coated and collagen-coated
surfaces, 7 µg/cm2 of protein were applied to 100-mm
culture dishes. The cells were cultured as described in Type II
cell isolation and culture. Total RNA was extracted with TRIzol
Reagent and reverse transcribed to cDNA as described in Cloning
and sequencing of rat 3-OST and NST cDNAs. Changes in 3-OST and
NST mRNAs as well as in mRNAs of SP-A, SP-B, SP-C, T1
, syndecan-1,
and
-actin (internal standard) were examined by semiquantitative PCR
with the primers shown in Table 1.
In a preliminary experiment, the linearity of the PCR was examined in
NST, SP-A, and
-actin at 17, 19, 21, 23, 25, 27, and 29 cycles. All
of the 3 PCR products examined at 25 cycles were within the linear
range. The result was similar to that reported (17). cDNA
corresponding to 50 ng of total RNA was used for 3-OST, NST, T1
,
syndecan-1, and
-actin with 25 cycles, and 250 ng of total RNA were
used for SP-A, SP-B, and SP-C mRNAs with 30 cycles to determine
how long they remain by culturing. The total volume of the reaction
mixture was 25 µl. The PCR products (2 µl) were separated on a 2%
agarose gel in Tris-acetate-EDTA buffer containing 0.5 µg/ml
of ethidium bromide at 100 V for 40 min and photographed with an
electronic ultraviolet transilluminator connected to a
charge-coupled device video camera module. Three microliters (120 ng) of
/Hind III digest-
X174/Hae III digest Loading Quick DNA size marker (TOYOBO) were separated at the same time.
Photographs were taken, adjusting each marker band to the same density.
The density of each band was measured with a densitometer, and the peak
area was measured with an image analyzer (JIM-5000, JEOL, Tokyo,
Japan). The relative amount of each PCR product was normalized to the
amount of
-actin and plotted as relative fluorescence units.
-Actin was reported to be a suitable control in cultured type II
cells (1). Results are shown as means ± SE of
4-6 experiments.
Effect of sodium chlorate on cell spreading.
Sodium chlorate (0, 10, 20, or 40 mM) was added when type II cells were
seeded at a density of 2.5 × 103
cells/cm2 in 35-mm culture dishes. The cells were cultured
for 1, 2, or 3 days, and cell size was determined with an image
analyzer as described in Expression of differentiation markers
and NST and 3-OST mRNAs in cultured type II cells, with cultured
cells photographed under phase contrast with a video camera. The
specificity of sodium chlorate as an inhibitor of proteoglycan
sulfation was examined by culturing cells in the presence of 20 mM
sodium chlorate with and without 10 mM sodium sulfate. The sodium
concentration was adjusted to 150 mM.
After culture of 2 × 105 cells for 2 days in the
presence and absence of 20 mM sodium chlorate, the number of cells
recovered and cell viability were examined. The cells were harvested by incubating with 0.1% trypsin and 0.02% EDTA, and viability was determined by trypan blue exclusion.
The incorporation of 5-bromo-2'-deoxyuridine (BrdU) into cultured cells
was performed. After being cultured for 3 days with and without sodium
chlorate, the cells were further cultured in the presence of 10 µM
BrdU for 24 h. After staining of BrdU with anti-BrdU antibody
(Boehringer Mannheim), vimentin staining was performed to monitor the
percentage of type II cells.
All values are expressed as means ± SE. Differences between means
were evaluated by nonpaired Student's t-test with a
commercially available computer software package (StatView, Abacus
Concepts, Berkeley, CA).
 |
RESULTS |
Isolation and characterization of the cDNA and deduced amino
acid sequences of rat 3-OST.
Six independent pGEM4Z-3-OST clones were obtained and sequenced.
As shown in Fig. 1, in all clones, the
inserted fragment from the ATG start codon to the stop codon was 936 bp, containing a single open reading frame. The cDNA and amino acid
sequences showed 94.8 and 97.7% identity, respectively, to those of
the mouse (34). There were seven substitutions of amino
acid residues: His19, Glu33, Thr49,
Ser51, Leu190, Leu224, and
His294 in the mouse were changed to Pro, Gly, Ala, Thr,
Val, Phe, and Arg, respectively, in the rat. Some changes seem to have
no great effect on the protein structure: both Thr51 and
Ser51 are neutral, Leu190 and
Val190 are nonpolar, Leu244 and
Phe244 are nonpolar, and His294 and
Arg294 are basic amino acids in the mouse and rat,
respectively.

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Fig. 1.
Composite sequence in the coding region of rat
3-O-sulfotransferase (3-OST) cDNA and the deduced amino acid
sequence. Short thick underlines, difference from the deduced mouse
3-OST amino acid sequence reported in GenBank (accession no. AF019385)
(34); long thin underlines, primer sequences used in this
experiment that are the same as those of the mouse. *, Stop codon.
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Northern blot analysis demonstrated the 3-OST mRNA transcript to be
~1.8 kb (Fig. 2).

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Fig. 2.
Northern blot analysis of 3-OST from rat lung. Five
micrograms of poly(A)+ mRNA were electrophoresed on a
formaldehyde-agarose gel, transferred onto a positively charged nylon
membrane, and hybridized with 3-OST cDNA probe labeled with digoxigenin
(Dig). The membrane was then detected with a Dig luminescent detection
kit and exposed to Kodak X-AR film for photography. The 3-OST mRNA was
detected as a single band of ~1.8 kb. 18S and 28S, 18S (~2-kb) and
28S (~4.5-kb) rRNAs, respectively.
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NST sequence.
Five independent pGEM4Z-NST clones were sequenced in the same
manner as pGEM4Z-3-OST. We cloned a 531-bp fragment that encodes the NH2-terminal 177 amino acids from Phe21 to
Asp197. The DNA sequence of isolated cDNA clones was
identical to that reported (13; and data not shown).
Expression of 3-OST and NST mRNA in rat lung type II cells detected
by in situ hybridization.
Most of the freshly isolated cells were found to express 3-OST mRNA by
in situ hybridization. To ascertain that type II cells express 3-OST
message, double staining was performed. As shown in Fig.
3, nearly all of the freshly
isolated type II cells that were stained with monoclonal SP-B antibody
(A) expressed 3-OST mRNA (B). NST mRNA was also
expressed in these cells. As shown in Fig. 3, almost all the cells that
were stained with SP-B antibody (C) expressed NST mRNA
(D). There was a good overlay of NST mRNA and SP-B protein.
As shown in Fig. 3, insets, the vimentin-positive cells did
not express 3-OST mRNA (Fig. 3, A and B) or NST
(Fig. 3, C and D) mRNA. No signals were
detected with sense probes of 3-OST and NST or in the
immunohistochemistry control study (data not shown).

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Fig. 3.
Immunohistochemical analysis and in situ hybridization of
3-OST and N-deacetylase/sulfotransferase (NST) in freshly
isolated type II cells. A and C: type II cells
(red) stained with anti-surfactant-associated protein (SP) B antibody.
B and D: mRNAs of 3-OST and NST, respectively, in
type II cells detected by in situ hybridization with 3-OST and NST
probes, respectively. A and B and C
and D are each the same sample. Type II cells expressed
3-OST and NST mRNAs, but fibroblasts did not. Insets:
fibroblasts stained with anti-vimentin antibody immunohistochemically.
Bar: 40 µm in A-D; 80 µm in
insets.
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3-OST and NST mRNA expression in cultured type II cells.
As shown in Fig. 4, the shapes of
the type II cells, which were stained with anti-keratin antibody, and
the contaminating fibroblasts, which were stained with anti-vimentin
antibody, were distinct (B). 3-OST mRNA was expressed in
type II cells cultured for 3 days at low density, whereas fibroblasts
expressed little 3-OST mRNA message (Fig. 4D). The type II
cells strongly expressed NST mRNA; the fibroblasts less so (Fig.
4F). No signals were detected with the sense probes for
3-OST and NST or in the immunohistochemistry control study (data not
shown).

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Fig. 4.
Immunohistochemical analyses and in situ hybridization of
3-OST and NST in type II cells cultured for 3 days. A,
C, and E: type II cells under phase contrast.
B: type II cells stained with anti-keratin antibody (green)
and fibroblasts stained with anti-vimentin antibody (red). D
and F: mRNAs of 3-OST and NST, respectively, in type II
cells detected by in situ hybridization with 3-OST and NST probes,
respectively. A and B, C and
D, and E and F are each the same
sample. A, C, and E, arrowheads:
fibroblasts. Type II cells expressed 3-OST mRNA, whereas fibroblasts
(D) did not. Fibroblasts (E, arrowheads)
expressed less NST mRNA than type II cells (F). Bar, 40 µm.
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Effect of cell density and matrix substrate on the expression of
mRNA in cultured type II cells.
Figure 5 shows a representative
electrophoretic profile of RT-PCR products. Although T1
and NST
mRNAs were expressed at low levels in freshly isolated type II cells,
their expression increased remarkably in type II cells cultured on
plastic, with a concomitant decrease in SP-A mRNA expression. But in
cells cultured on EHS gels, there was no increase in the expression of
NST mRNA with a high expression level of SP-A mRNA.

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Fig. 5.
Representative electrophoretic pattern of RT-PCR products
of mRNA from type II cells cultured with different cell densities,
matrices, and culture times. Lane 1, freshly isolated type
II cells; lanes 2-4, cells cultured at low
cell density on plastic for 1, 3, and 6 days, respectively; lanes
5-7, cells cultured at medium cell density on
plastic for 1, 3, and 6 days, respectively; lanes
8-10, cells cultured at high cell density on
plastic for 1, 3, and 6 days, respectively; lanes
11-13, cells cultured at high cell density on
Engelbrecht-Holm-Swarm (EHS) gels for 1, 3, and 6 days, respectively.
SP-A, surfactant-associated protein (SP) A.
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As shown in Fig. 6, top, in
cells cultured on plastic, mRNA expression of SP-A, SP-B, and SP-C
decreased rapidly to nearly undetectable levels within 3 days. However,
there was a tendency for this decrease to be delayed according to the
cell density. T1
mRNA increased rapidly irrespective of cell density
(Fig. 6, bottom). Expression of NST mRNA increased rapidly
in cells cultured at low density but was delayed in cells cultured at
medium density. In cells cultured at high density, expression of NST mRNA was not induced early, although it gradually increased and reached
the same level as in cells cultured at low density by day
14. These results indicated that cell density affects expression of NST mRNA in cultured type II cells.

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Fig. 6.
Effect of cell density on the changes in expression of
mRNA from type II cells cultured on plastic. L, low cell density; M,
medium cell density; H, high cell density; Synd1, syndecan-1; RFU,
relative fluorescence units. RT-PCR products were semiquantitated.
Fluorometric analysis of amplicon fluorescence was normalized to
-actin and is plotted as means ± SE.
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Compared with that in cells cultured on plastic at low density, the
decrease in the expression of SP-A, SP-B, and SP-C mRNAs was slightly
delayed in cells cultured on EHS- or collagen-coated plates (Fig.
7, top). Moreover, in cells
cultured on EHS gels at high density, mRNA expression of SP-A, SP-B,
and SP-C first decreased on day 1, and then SP-A and SP-B
mRNAs gradually recovered to relatively high levels that were retained
even on day 9 of culture, although a complete recovery was
not achieved. Therefore, these results indicated that SP-A, SP-B, and
SP-C mRNA expression was maintained better at high cell density and on
the EHS gel surface than at low density and on the plastic surface.
This result is consistent with other reports that type II epithelial
cells cultured on EHS gels retain their morphology and differentiation better than those cultured on plastic (31-33). As
shown in Fig. 7, bottom, expression of T1
and NST mRNAs
was kept at a low level in cells cultured on EHS gels and at a
relatively low level in cells cultured on collagen-coated plates. In
the latter condition, cell spreading was not remarkable compared
with that on plastic (see Effects of cell density and
matrix substrate on cell spreading) because hydrated collagen
produced a thin gel at this concentration (7 µg/cm2).

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Fig. 7.
Effect of substrates on the changes in expression of mRNA
from cultured type II cells. HEg, high cell density on EHS gel; LEc,
low cell density on EHS-coated plate; LCc, low cell density on
collagen-coated plate. RT-PCR products were semiquantitated.
Fluorometric analysis of amplicon fluorescence was normalized to
-actin and is plotted as means ± SE.
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Expression of syndecan-1, a representative of cell membrane HSPGs, and
3-OST mRNAs was kept at almost the same level as that of freshly
isolated type II cells.
Effects of cell density and matrix substrate on cell spreading.
The behavior of type II cell spreading differed according to the cell
density and matrix substrate used in culture. As shown in Fig.
8, type II cells seeded on plastic at low
density were distributed mainly as single cells or groups of two cells
(A) and after 3 days spread out showing a large attenuated
cytoplasm (B). In medium- and high-density cultures, the
cells formed small and large aggregates, respectively, at 1 day of
culture (Fig. 8, C and E, respectively),
and their spreading was greatly inhibited by cell-cell contact
after 3 days when type II cells were nearly confluent (Fig. 8,
D and F). Aggregates of type II cells
with a three-dimensional nature formed on the EHS gel at high cell density after 3 days of culture. In this case, type II cells did not
spread (Fig. 8, G and H). Cells cultured on the
EHS-coated surface at low density showed some spreading after 1 day and
spread to the same extent as those cultured on plastic at low cell
density (Fig. 8, I and J). Type II cells cultured
on the collagen-coated surface at low density spread only slightly
(Fig. 8, K and L), although some spread well at
places where the coating appeared to be thinner (data not shown).

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Fig. 8.
Effect of cell density and matrix substrate on cell
spreading in cultured cells under phase contrast. A and
B: cells cultured on plastic at low cell density for 1 and 3 days, respectively. C and D: cells cultured on
plastic at medium density for 1 and 3 days, respectively. E
and F: cells cultured on plastic at high density for 1 and 3 days, respectively. G and H: cells cultured on
EHS gels at high density for 1 and 3 days, respectively. I
and J: cells cultured on EHS-coated surfaces at low density
for 1 and 3 days, respectively. K and L: cells
cultured on collagen-coated surfaces at low density for 1 and 3 days,
respectively. Bar, 40 µm.
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Effect of sodium chlorate on spreading of cultured type II cells.
As shown in Fig. 9, sodium chlorate
inhibited the spreading of type II cells cultured for 3 days in a
dose-dependent manner. Significant inhibition of type II cell spreading
was observed with 20 and 40 mM sodium chlorate at 2 days of culture,
and at all concentrations of sodium chlorate, highly significant
inhibition was observed at 3 days of culture compared with that in
control cultures. The specificity of sodium chlorate as an inhibitor of proteoglycan sulfation was examined by culturing the cells in the
presence of sodium chlorate with and without sodium sulfate. As shown
in Table 2, the inhibiting effect of
sodium chlorate on cell spreading was antagonized by sodium
sulfate (P < 0.0001 vs. control and sodium
chlorate-treated cultures). These results suggest that sulfation
of PGs is necessary for cell spreading, especially after 2 days
of culture.

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Fig. 9.
Effect of sodium chlorate on type II cell
spreading. The freshly isolated type II cells were exposed to different
concentrations of sodium chlorate for 1, 2, and 3 days. Cell size was
measured as described in MATERIALS AND METHODS. Each point
shows mean ± SE of 50 cells from a representative of 3 experiments with similar results. Significantly different from 0 mM
sodium chlorate group:  P < 0.0001; P < 0.05.
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To exclude the possibility that sodium chlorate is toxic to cultured
cells, we assessed cell viability and DNA synthesis in the presence of
sodium chlorate. After culture at medium cell density on plastic
for 2 days in the presence and absence of 20 mM sodium chlorate, the
total number of cells recovered was 0.96 × 105 ± 0.05 × 105 and 0.89 × 105 ± 0.02 × 105, respectively, and the viability of
cells was 95.8 ± 1.5 and 96 ± 0.12%, respectively
(n = 3 experiments; no significant difference between
them). We also measured the incorporation of BrdU as an indicator of
DNA synthesis. As shown in Table 3, cells
actively incorporated BrdU in the presence of sodium chlorate, although sodium chlorate showed a mild inhibition of DNA synthesis (~1.0% of
cells were labeled in sodium chlorate-treated cells compared with 2.2%
in control cells among the vimentin-negative cells; ~40% in sodium
chlorate-treated vimentin-positive cells compared with 47.8% in
control cells). Statistical analysis was not done because the number of
experiments was too small. Therefore, the ability of sodium chlorate to
inhibit cell spreading may not be due to its toxicity in this
experiment.
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DISCUSSION |
To investigate H/HS metabolism in the type II cells, we first
cloned two key enzymes for H/HS synthesis, 3-OST and NST. As far as we
know, this is the first study to demonstrate that pulmonary alveolar
type II cells in adult rat express 3-OST and NST mRNAs.
Although we do not know the exact sequences of the primer region
(around the start and stop codons) in the rat, the successful PCR
(successful annealing) suggests that they are identical in the rat and
the mouse. Major variation in the amino acid sequence occurred only in
the amino terminus (the carboxy-terminal 260 residues were conserved),
suggesting that the carboxy-terminal domain possesses sulfotransferase
activity as deduced by Shworak et al. (34). The rat 3-OST
mRNA expressed in the lung was ~1.8 kb, similar to that of the mouse
(1.7 kb), but we did not find the 2.3- and 3.3-kb bands that were
expressed in the rat endothelial cell line (RFPEC) (34).
This may indicate that the 3-OST genes are regulated in a tissue- or
cell type-specific fashion or that the transcripts may differ by
alternative splicing because the latter has been shown in the mouse
(34).
We examined the changes in the expression of NST and 3-OST together
with the changes in type II cell markers (SP-A, SP-B, and SP-C) and a
type I cell marker (T1
) in various culture conditions. We found that
at least two factors regulate the expression of these protein mRNAs:
cell density and matrix. The expression of type II cell markers was
rapidly lost in all conditions except in culture on EHS gels. The
present results are consistent with other reports (6,
31), although the precise mechanism of the effects of the
EHS gel on the cultured cells is unknown. Considering that at a higher
density of cells the disappearance of mRNA of SPs was slightly delayed,
cell-cell interaction or inhibition of spreading of cells seems to be
important in the regulation of expression of SPs. Cell form is
important for keeping the type II cell phenotype as suggested by
Shannon et al. (32).
T1
was reported to be a marker gene expressed solely in the alveolar
type I epithelial cell of the adult lung (22,
39), and type II cells in monolayer cultures rapidly
upregulate expression of T1
mRNA and protein (10,
22). The present results further support the fact that
T1
may be a good indicator of the transdifferentiation of type II
cells into type I cells because no increase in T1
mRNA expression
was observed in cells cultured on EHS gels. In the present study,
contrary to the expression of SPs, the expression of T1
was not
dependent on cell density because cells cultured at high density on
plastic also exhibited a rapid increase in T1
mRNA, and this was not
suppressed when the cells became confluent at 3 days of culture. The
expression of T1
may depend more on matrix conditions than on
cell-cell interaction because the collagen-coated plates delayed the
increase in mRNA, whereas the EHS coating did not suppress the
expression in low-density cultures.
NST expression was also regulated by two factors, matrix and cell
density. Cell density had a greater effect on mRNA expression than on
matrix because a rapid increase in mRNA was observed in low cell
density cultures but not in high-density cultures. In the latter
condition, NST mRNA expression was kept low during 6 days of culture,
and it took ~2 wk to reach the same level of expression as in low
cell density cultures. NST is one of the specific enzymes for synthesis
of H/HSPGs. N-sulfation is the first step of sulfation in H/HS
synthesis and is followed by sulfation at the O-2 and O-6 positions
and, finally, at the O-3 position (24). HS is important
for various cellular activities (15, 40);
however, the regulatory mechanism of its expression has not been
thoroughly studied. Sannes et al. (28) reported that type
II cells synthesize more highly sulfated basement membrane components
in a 21-day culture than in a 7-day culture. The present results in low
cell density cultures (which are almost the same density as theirs)
show that type II cells respond to synthesize NST mRNA within 1 day of
starting the culture and coincide with the notion that type I cells may
actively biosynthesize or maintain the heavily sulfated ABM.
We also examined 3-OST expression during culture. However, its
expression did not change remarkably under the conditions examined, although a slight increase was seen in cultures at low cell density. It
is reported that type II cells and A549 cells derived from lung cancer
synthesize anticoagulant PGs (3, 36). The
present result that 3-OST mRNA is expressed in type II cells supports the hypothesis that the heparin-like PGs are synthesized by type II
cells because sulfation at the O-3 position is necessary in the active
form of heparin, which binds to antithrombin. We speculate that the
highly sulfated ABM beneath type I cells is mainly N-sulfated rather
than 3-O-sulfated.
As a comparison with the increase in the expression of NST mRNA, we
also investigated the mRNA expression of syndecan-1, a core protein of
one of the main cell-bound HSPGs (41). In most cells,
syndecan synthesis is mainly regulated at the level of gene
transcription (5). Therefore, the level of syndecan-1 mRNA
generally represents the protein level. In this experiment, syndecan-1
core protein mRNA was generally kept at the same level as in control
cells throughout the culture period. Although we have yet to determine
the actual degree of sulfation in this protein, it is suspected that
cultured type II cells synthesize highly sulfated syndecans from an
early stage of culture, considering the different ratio of mRNA levels
of NST to syndecan. Syndecan is reported to be important as a
coreceptor for growth factors (23) and is also reported to
induce cell spreading in transfected Raji cells
(14). Reich-Slotky et al. (21) reported that
the binding of keratinocyte growth factor and acidic fibroblast growth factor to receptors is modified by sulfation of cell-associated HS.
Therefore, it is speculated that the response to some growth factors in
type II cells may be altered in cultured cells by further sulfation of
syndecan molecules.
We found that the inhibition of cell spreading was induced by sodium
chlorate treatment and reversed by the addition of sodium sulfate.
Sodium chlorate-treated cells actively incorporated BrdU, although the
percentage of labeled cells was slightly reduced by sodium chlorate
treatment. Reportedly, sodium chlorate, which reduces adenosine
3'-phosphate 5'-phosphosulfate utilized for sulfation, inhibited
sulfate incorporation into HSPGs in HeLa cells (18),
smooth muscle cells (29), human breast cancer cell lines
MCF-7 and MDA-MB-231 (8), and kidney epithelia
(7), generally in a dose-dependant manner ranging from 10 to 60 mM. When exposed to 10 mM sodium chlorate,
[3H]thymidine incorporation into cultured smooth muscle
cells decreased by 66%, whereas [35S]sulfate
incorporation into cell-associated HS proteoglycans was reduced by 90%
(29). SR91 cell viability was not affected with 50 mM
sodium chlorate as assessed by trypan blue exclusion (16).
In the range of lower concentrations used here (10-20 mM), sodium
chlorate seemed not so toxic to type II cells because we did not find a
significant decrease in the number of viable cells recovered when
cultured at a concentration of 20 mM for 2 days.
The biological meaning of these findings in in vivo lungs is not known
at present, but we speculate that NST expression is important in the
recovery from lung injury. On injury, type I cells will be lost first
because they are more susceptible to injury than type II cells
(2), and during the wound healing process, type II cells
proliferate and spread to cover naked ABM, which may be facilitated by
upregulation of NST. Further studies are ongoing to prove this
hypothesis by preparing antibodies against NST, 3-OST, and syndecan-1.
In summary, we examined mRNA expression of NST and 3-OST, two enzymes
specific for HS synthesis, to characterize the regulation of PG
synthesis during the transition from type II cells to type I cells in
various culture conditions. Cultured type II cells lost their marker
protein SP-A, SP-B, and SP-C expression irrespective of culture
conditions except for culture on EHS gels. NST and type I cell marker
T1
expression rapidly increased during culture, but NST expression
was dependent on cell density, whereas T1
expression was regulated
by matrix. Type II cells cultured on EHS gels rarely expressed NST or
T1
. 3-OST expression was generally kept at a low level, and
syndecan-1, a representative PG, was not upregulated by culturing
either. Upregulated expression of NST coincided with cell spreading,
and the PG sulfation inhibitor sodium chlorate reversibly inhibited
cultured type II cell spreading. These results suggest that highly
sulfated PGs modified by NST are intimately involved in cell spreading
during transdifferentiation from type II cells to type I cells.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
Y. Suzuki, Dept. of Ultrastructural Research, Institute for Frontier Medical Sciences, Kyoto Univ., Sakyo-ku, Kyoto 606, Japan (E-mail: suzuki{at}frontier.kyoto-u.ac.jp).
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.
Received 30 November 1999; accepted in final form 22 March 2000.
 |
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