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Am J Physiol Lung Cell Mol Physiol 279: L292-L301, 2000;
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Vol. 279, Issue 2, L292-L301, August 2000

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 T1alpha 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; T1alpha ; surfactant-associated proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 T1alpha , 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 T1alpha 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-5alpha . The transformed DH-5alpha 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-beta -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, T1alpha , syndecan-1, and beta -actin (internal standard) were examined by semiquantitative PCR with the primers shown in Table 1.

                              
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  		Table 1.   PCR primers used

In a preliminary experiment, the linearity of the PCR was examined in NST, SP-A, and beta -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, T1alpha , syndecan-1, and beta -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 lambda /Hind III digest-phi 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 beta -actin and plotted as relative fluorescence units. beta -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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.

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.

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 T1alpha 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.

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. T1alpha 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 beta -actin and is plotted as means ± SE.

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 T1alpha 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 beta -actin and is plotted as means ± SE.

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.

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: star star P < 0.0001; star  P < 0.05.


                              
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  		Table 2.   Reversibility of sodium chlorate in the inhibition of cell spreading

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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  		Table 3.   Effects of sodium chlorate on BrdU incorporation into cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (T1alpha ) 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).

T1alpha 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 T1alpha mRNA and protein (10, 22). The present results further support the fact that T1alpha may be a good indicator of the transdifferentiation of type II cells into type I cells because no increase in T1alpha mRNA expression was observed in cells cultured on EHS gels. In the present study, contrary to the expression of SPs, the expression of T1alpha was not dependent on cell density because cells cultured at high density on plastic also exhibited a rapid increase in T1alpha mRNA, and this was not suppressed when the cells became confluent at 3 days of culture. The expression of T1alpha 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 T1alpha expression rapidly increased during culture, but NST expression was dependent on cell density, whereas T1alpha expression was regulated by matrix. Type II cells cultured on EHS gels rarely expressed NST or T1alpha . 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 279(2):L292-L301
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