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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 345-352, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0002OC

Heparin Inhibits DNA Synthesis and Gene Expression in Alveolar Type II Cells

Cheng-ming Li, Donna Newman, Jody Khosla and Philip L. Sannes

Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Address correspondence to: Philip L. Sannes, Ph.D., Dept. of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Hillsborough Street, Raleigh, NC 27606. E-mail: philip_sannes{at}ncsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Responses of isolated type II alveolar cells to fibroblast growth factors (FGF) have been shown to be sensitive to the level of sulfation in extracellular matrix (ECM) substrata. These observations may reflect the specific in situ distribution and level of sulfation of ECM within the alveolar basement membranes (ABM) associated with type II cells. The goal of this study was to determine if the model sulfated ECM heparin modified DNA synthesis and gene expression by type II cells in a concentration dependent-manner. Isolated rat type II cells were exposed to different concentrations of heparin (0.005–500 µg/ml) in serum-free medium for 1–3 d with or without FGF-1 or FGF-2. The effects of heparin were examined by [3H]thymidine incorporation into DNA, total cell protein, cell number, and selected gene expression. Results indicated that heparin inhibited [3H]thymidine uptake in a concentration-dependent manner. Total protein, cell number, and FGF-2 protein expression and mRNA of FGF-1, -2, and FGF receptor-2 detected by reverse transcriptase–polymerase chain reaction were decreased by heparin. These results demonstrate that sulfated molecules in the ABM may play important regulatory role(s) in selected type II cell activities during normal cell homeostasis, turnover, and repair after lung injury.

Abbreviations: alveolar basement membrane, ABM • Dulbecco's modified Eagle's medium, DMEM • extracellular matrix, ECM • fibroblast growth factor, FGF • heparan sulfate, HS • phosphate-buffered saline, PBS • proteoglycan, PG • serum-free, SF • surfactant protein, SP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Normal lung function depends on an intact alveolar epithelium, which is composed of two morphologically distinct cell types. Type I alveolar epithelial cells are squamous cells that cover > 90% of the alveolar surface and function primarily in alveolar gas exchange and fluid balance. Type II alveolar cells are cuboidal cells that cover the remaining alveolar surface and are responsible for pulmonary surfactant secretion and metabolism. Their capacity to divide and differentiate is vital for maintenance of stable cell populations under homeostatic conditions and restoration of the integrity of the alveolar epithelium following injury (1, 2). The mechanisms which regulate proliferation and differentiation of type II cells in vivo are beginning to be better understood as involving numerous growth-stimulatory/inhibitory factors and some non–growth-factor molecules, such as hormones and extracellular matrix (ECM) components. To date, hepatocyte growth factor, transforming growth factor-{alpha} and -ß, epidermal growth factor, and members of the fibroblast growth factor family of cytokines (FGF-1, FGF-2, and FGF-7), have been found to modulate DNA synthesis, gene expression, and surfactant protein (SP) synthesis by type II cells (3–7). FGF-1 has been immunolocalized in type II cells in situ and FGF-2 in the alveolar basement membrane (8). Gene expression of FGF-2 and FGF receptors in isolated type II cells has also been documented (9).

Proteoglycans (PGs) and their polysaccharide chains, glycosaminoglycans, are components of the ECM and pericellular matrix adjacent to the external surface of cell membranes. GAGs from the ECM can regulate cell growth by their interaction with cell membrane proteins and by their ability to bind with some growth factors (10, 11). Heparan sulfates (HS) are known to bind, stabilize, and protect FGFs from proteolytic degradation (12), but also to release them either by selective enzymatic release (i.e., heparinase, plasmin, metalloproteases) or to heparinoid molecules with greater affinities for FGFs (12) or to binding factors (13). A number of studies have indicated that heparin and HSPGs bind to airway smooth muscle cells and strongly inhibit their growth in vitro (14, 15). The saccharide sequences and the number and type of sulfate groups are believed to play important roles in determining the mechanism(s) of action of heparin and HSs (4, 16–20).

Microdomains within the ABM beneath type II cells are known to have a more random distribution and decreased level of sulfation than that beneath type I cells, where the sulfation is more concentrated immediately adjacent to its basilar border (21–23). These cytochemically identifiable sites have been shown to represent HS (23). More importantly, the differences in sulfation within ABM sites between type I and II cells have been proposed to be potential determinant(s) of their functional differences and roles in maintenance of alveolar epithelium, especially as it pertains to regulation of growth control and differentiation (19, 20, 22). The purpose of the present study was to determine whether or not the model ECM heparin alters DNA synthesis and gene and protein expression of selected FGFs and their receptors. The results could help define the biologic role(s) for local sulfated ECMs in modulating/regulating type II cell responses to certain growth factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Preparation
Rat type II pneumocytes were isolated from pathogen-free 200–250-g Fisher CDF rats (Charles River Laboratory, Wilmington, MA) following the procedure of Dobbs and coworkers (24) with minor modifications (20). Isolated cells were suspended with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µg/ml gentamicin for 24 h.

Cell Culture and Treatment
Except experiments in which effects of cell density were evaluated, isolated type II cells were plated at a density of 4 x 104 cells/cm2 on standard culture dishes and 24-well plates (Nunc, Napierville, IL) which had been previously coated with 0.06 µg/mm2 type I collagen (Sigma, St. Louis, MO). Cells were allowed to attach and spread overnight at 5%CO2, 37°C. Attached cells were washed once with DMEM and treated with serum-free, hormonally-defined (SF) medium as described previously (25). Briefly, the medium consisted of a mixture of Ham's F12 and DMEM (1:1, vol/vol) supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 1 µM hydrocortisone, 0.5 mM cAMP, 25 nM selenious acid, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, and 10 µg/ml Gentamicin. The only component missing from the original description (25) was epidermal growth factor. Some mixtures were further supplemented with 0.005–500 µg/ml of high (13,500–15,000 MW) molecular weight heparin (Calbiochem Corp., La Jolla, CA), 100 ng/ml FGF-1, or 100 ng/ml FGF-2. For all experiments, the initial 24-h attachment period (no experimental treatments) will be designated "Day 0," and subsequent days (experimental treatments) as 1, 2, and so on.

Cell Counts
Cells were cultured in 24-well plates and terminated on Days 0 or 3 (with or without FGF-1/-2, with or without heparin as above) by a single wash in phosphate-buffered saline (PBS) followed by 15 min fixation with 2% formaldehyde. Cell contrast was enhanced with Mayer's hematoxylin. The cell numbers were determined under a phase-contrast microscope (Olympus, Tokyo, Japan) by averaging five random fields per well in triplicate wells. Statistical differences between groups were determined using Student's t test. These results were confirmed by comparing them with those from separate experiments which defined the number of detached cells and total number of attached cells. Cells were isolated as above and cell counts performed on the media removed on Days 1 and 3. These numbers were compared with the number of cells still attached at Day 3 (attached cells were detached with 0.01 mg/ml trypsin at 37°C for 20 min. with gentle agitation and counted in a hemacytometer). In addition, phenotypes were determined on cells fixed with cold acetone and immunostained with selected antibodies. Type II cell phenotype was confirmed with anti–surfactant protein-D (Santa Cruz Biotech, Santa Cruz, CA) and anti–JBR-1 antigen (generous gift of Dr. Ray Peterson, University of S. Alabama, Mobile, AL). Fibroblast phenotype was defined with anti-vimentin (Santa Cruz Biotech) and macrophage with ED1 (Chemicon, Temecula, CA). All antibodies were visualized with Vectastain Peroxidase ABC detection kit (Vector Laboratories, Burlingame, CA).

[3H]Thymidine Incorporation Assay
To assess DNA synthesis, freshly isolated type II cells were seeded to 24-well plates as above. After a 24-h adhesion period, the cells were switched to serum-free medium with/without heparin treatment (in triplicate for each group) containing 2 µCi/ml of [3H]thymidine (ICN, Costa Mesa, CA) and continuously cultured with or without FGF-1/-2, with or without heparin as above at 37°C in 5% CO2. Experiments were terminated with washing (3x in PBS) and lysing cells as described previously (20). The incorporated [3H]thymidine radioactivity in cell lysates was measured with an LKB 1,219 Scintillation Counter (Wallac, Turku, Finland) on a per-well basis. Results were analyzed as previously described (20).

Total Protein and FGF-2 Assay
Treated cells in culture dishes were harvested at Day 1 and Day 3 by scraping, followed by low-speed centrifugation. The cells were washed with 1x PBS once, centrifuged, and the supernatant discarded. The resulting pellets were resuspended with 100 µl of a 50 mM Tris-HCl (pH7.4), 1 mM MgCl, 0.85% NaCl, and 1.5% NP-40 lysing buffer at 4°C for 15 min. The insoluble cellular debris was removed by high-speed centrifugation (12,000 x g) at 4°C for 10 min. For protein assay, the cell lysate samples were diluted 40x with diH2O and measured with the Micro BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions. FGF-2 protein was measured in cell lysates diluted 10x with a diluent (HD1–3) from a Quantikine FGF-2 ELISA kit (R&D Systems, Minneapolis, MN) and assayed by ELISA according to the manufacturer's instructions. All above assays were performed in triplicate for each time point.

RNA Isolation
After 3 d treatment, cells in culture dishes were washed once with cold 1x PBS, lysed, and extracted with TRI Reagent (Sigma) according to the instructions of the manufacturer. The resulting RNA pellets were suspended with 25 µl of RNase-free diH2O and treated with 5 U RQ1 DNase (Promega, Madison, WI) at 37°C for 30 min to remove residual DNA. The RNA was measured using GeneQuant (Pharmacia, Piscataway, NJ), and its integrity confirmed by formaldehyde denaturing gel electrophoresis (26).

Reverse Transcriptase-Polymerase Chain Reaction
A quantity of 2 µg total RNA from each sample was used for cDNA synthesis. To avoid the interference of heparin in the reverse transcriptase–polymerase chain reaction (RT-PCR), RNA was pretreated with 5 U heparinase I to remove heparin residues (27). The RT reaction was performed with random hexamer primers and Avian Myeloblastosis Virus reverse transcriptase following product instruction (Promega). RT products were stored at -20°C for later PCR use.

Oligonucleotide primers (Table 1) for ß-actin, FGF-1 and -2, and FGF receptors (R) -1 and -2 were selected with oligo design software based on appropriate gene sequences and synthesized by Retrogen Inc. (San Diego, CA). The PCR primers for the FGFRs were designed to detect all splice variants for these two isoforms. The PCR assay was performed with Qiagen Taq-PCR core kit (Qiagen, Chatsworth, CA) as previously described (9). To optimize the assay conditions and avoid overrun, a series of PCRs was conducted to determine the adequate cycle numbers needed for different genes. Accordingly, formal PCR assay was done for ß-actin, FGF-1 and -2, and FGF-R1 and -R2 with corresponding cycle number 28, 33, 38, 35, and 35, respectively. PCR products were analyzed with 1.5% agarose gel electrophoresis.


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  		TABLE 1 Primers used for PCR amplification

 
Data Analysis
All experiments were repeated a minimum of three times. Statistical mean value ± SE were developed for the various treatment groups. To demonstrate the overall significance of the heparin treatments, data was also expressed as percent of control (No-treatment), so that % Control = ([mean value of treatment - mean value of untreated control] ÷ mean value of untreated control) x 100, so that positive values would represent increases and negative values, decreases. Each experimental group was compared with its control group using a paired t test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Type II Cell Cultures
Except for the study of the relationship between heparin and cell density, a seeding density of 4 x 104 cells/cm2 was used in all experiments. After an initial 24-h attachment period (Day 0), cells typically became 30–40% confluent. Approximately 90% of them were confirmed microscopically to be type II alveolar cells with typically numerous lamellar bodies. As experiments progressed, cells cultured without heparin gradually spread and became completely confluent at Day 3 in SF medium without growth factor addition—primarily through spreading. FGF-1 or -2 addition stimulated increases in real cell numbers and thus more rapidly achieved confluence. Cells treated with heparin in SF media without growth factors spread but did not become confluent, nor did they appear to increase in number by Day 3 (Figures 1A–1B) . Those with FGF-1 or -2 added achieved confluence and appeared to increase in number over the same time period (Figures 1C–1D). Only some cells treated with heparin alone spread (Figures 1E–1F); when combined with FGF-1 or -2, however, they all spread, but showed only minimal increase in numbers with FGF-1 (Figures 1G–1H) and even less with FGF-2 (data not shown). Less than 1% of the total cells detached between Days 0 and 3 regardless of treatment. The phenotype of cells in SF media remained constant from Days 0–3. Type II cells were > 95% of the total population as detected by SP-D and JBR-1 antigen, whereas macrophages constituted 0.3–0.4% and fibroblasts 1.5–3.3%.



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  		Figure 1. Type II cells were seeded at low density in serum-containing medium for 24 h (A, C, E, G). The exact fields were photographed again after 48 h in either serum-free (SF) medium (B), 100 ng/ml FGF-1 (D), SF and 500 µg/ml heparin (F), or 100 ng/ml FGF-1 and 500 µg/ml heparin (H). Cells spread but did not increase in number in SF medium (A and B), spread and increased in number in FGF-1 (C and D), spread very little in heparin alone (E and F), and spread and modestly increased in number with FGF-1 and heparin (G and H). Bars = 15 µm.

 
[3H]Thymidine Incorporation into DNA
High molecular weight heparin was added to cultured cells in different concentrations at Day 0 and the cultures were terminated and analyzed at Day 2. During the experimental period the medium was not changed nor heparin replenished. FGF-1 or -2 treatment stimulated incorporation of [3H]thymidine into DNA without heparin, with FGF-1 providing the greatest increase above serum-free controls (> 200%)(Figure 2A) . Heparin addition to the medium resulted in reduced [3H]thymidine incorporation in a concentration-dependent fashion under all culture conditions with and without FGFs (Figures 2A and 2B), with low heparin concentration having minimal effect and high concentration the greatest. The FGF-1–treated cells were less sensitive to heparin inhibition than those treated with FGF-2. Cells treated with heparin alone (no growth factors) were inhibited the most. In cells treated with 500 µg/ml heparin, the level of inhibition was 29%, 75%, and 82% in FGF-1, FGF-2, and SF, respectively, in cultures compared with their heparin-free controls (P < 0.01) (Figure 2B).



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  		Figure 2. Without heparin, FGF-1 or FGF-2 treatments increased [3H]thymidine incorporation by isolated rat type II cells significantly compared with serum/growth factor–free controls (*P < 0.01). Incorporation declined steadily with increasing heparin concentration (A). The significant inhibitory quality of heparin is enhanced when incorporation is displayed as a percent of the heparin-free controls (B) (*P < 0.01).

 
To determine if cell density altered heparin's effects, isolated type II cells were plated at three different concentrations (400,000, 80,000, and 20,000 cells/cm2). At the start of Day 1, cells were exposed to [3H]thymidine containing SF medium ± 200 µg/ml of heparin without addition of growth factors. After two days, incorporation of [3H]thymidine was inhibited to a greater degree (> 80% of untreated control) in the lower seeding density (20,000 cells/cm2) than in the higher (> 30% of untreated control) seeding density (400,000 cells/cm2) (Figure 3) .



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  		Figure 3. At a constant concentration of 200 µg heparin/ml, the [3H]thymidine incorporation rate was decreased more (83%) at a seeding density of 20,000 cells/cm2 than at 80,000 cells/cm2 (46%) or at 400,000 cells/cm2 (33.4%) compared with heparin-free controls (P < 0.01).

 
Changes in Protein Content and Cell Number
To better understand type II cell responses to FGF-1 and -2 and high-dose heparin, changes in the total protein of type II cell lysates and absolute cell numbers were examined. Results indicated that heparin treatment caused a sharp decline of total extractable protein. In cultures not treated with FGFs, total protein content in 500 µg/ml heparin treatments was 52% less than SF alone at 3 d (Figure 4) . In both FGF-1– and FGF-2–treated cells, total protein yield was higher than that of SF controls, but when coupled with high-dose heparin treatment there were significant decreases of total protein when expressed as a percent of heparin-free controls (25% and 30%, respectively)(Figure 4). These changes corresponded to decreases in cell number, as heparin in SF was over 50% less than SF alone from Days 1–3 (Figure 5) . Heparin treatment combined with FGF-1 or -2 resulted in cell counts that were ~ 25% less than those resulting from treatments with FGF-1 and FGF-2 alone (Figure 5). It should be noted that the data is expressed as a percent of heparin-free controls, in which FGF-1 consistently was a more potent stimulus of DNA synthesis, total protein expression, and cell number than FGF-2 or SF/growth factor–free treatments. Separate experiments demonstrated that less than 1% of the cells detached between Days 0 and 3, and that when the total number of cells per well were counted following trypsinization, the percent decrease in cell number of heparin-free control remained the same ± 10% (data not shown).



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  		Figure 4. At a constant 500 µg heparin/ml, total protein in type II cell cultures was reduced 52% in serum/growth factor–free treatments. Treatment with 100 ng/ml FGF-1 or -2 resulted in 25% and 30% decreases, respectively, in protein compared with heparin-free treatments (*P < 0.01).

 


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  		Figure 5. FGF-1 and -2 resulted in significant increases in total type II cell numbers between Days 1 and 3 compared with SF controls (A) (*P < 0.05). 500 µg heparin/ml decreased cell numbers significantly in SF controls and in FGF-1 and -2 treatments such that they were not different from SF/heparin-free control (*P < 0.01). The significance of heparin's inhibitory effects is demonstrated when the data is displayed as a percent of the heparin-free control (B) (*P < 0.01).

 
FGF-2 Protein Production
FGF-1 has previously been shown to stimulate FGF-2 production (21). Results showed that 500 µg/ml heparin in SF cultures reduced FGF-2 production in type II cells by 90% at Day 1 and by 62% at Day 3 compared with SF alone. The addition of heparin to FGF-1 treatments resulted in 40% and 34% decreases in FGF-2 production at Days 1 and 3, respectively, compared with FGF-1 alone in SF media (Figure 6) . Thus, heparin reduces the otherwise stimulatory effects of FGF-1 and -2 on FGF-2 protein production. Detection of FGF-2 in FGF-2–treated cells was not possible due to the obvious interference of the assay by exogenously applied FGF-2.



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  		Figure 6. FGF-2 protein production by type II cells was significantly decreased by 500 µg heparin/ml between Days 1 (> 90%) and 3 (62%) in SF treatments when expressed as a percent of heparin-free control (*P < 0.001). Similarly, heparin caused significant decreases in FGF-2 production with FGF-1 treatment between Days 1 (40%) and 3 (34%) compared with heparin-free controls (*P < 0.001).

 
FGF-1 and -2 and FGF-R1 and -R2 Gene Expression
RT-PCR analysis indicated that heparin influenced both FGF-1 and -2 gene expression in a concentration-dependent fashion, with higher levels being more inhibitory and lower levels being less inhibitory to slightly stimulatory (Figures 7A–7F) . In all culture conditions, the decrease caused by heparin in FGF-2 mRNA was greater than that in FGF-1 mRNA (Figures 7A–7F). In general, the comparative degree of inhibition of FGF-1 and -2 mRNA between treatment groups was greatest in SF > FGF-2 > FGF-1. For the most part, FGF-receptor mRNA is less sensitive to heparin treatments compared with FGF-1 and -2, again in a generally concentration-dependent manner (Figures 7A–7F). The exception was FGF-R1 expression in SF media, which actually was stimulated by heparin—with higher concentrations being more stimulatory than lower ones (Figure 7A). It should be noted that increased heparin treatment significantly decreased the total amount of RNA, so that equal loading of RNA in most cases likely represents a greater percentage of decrease than was actually demonstrated in respective samples.



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  		Figure 7. RT-PCR products resolved in agarose gel demonstrated that heparin treatments altered mRNAs expressed by type II cells in all culture conditions in a concentration-dependent fashion (A, C, E). Plots of densitometric tracings of gels (expressed as percentage of heparin-free controls) indicated that FGF-2 mRNA was reduced to the greatest degree (88% decrease), whereas FGF-R1 mRNA was increased by heparin (38% increase) in serum-free/growth factor–free media (B). mRNA expression was not dramatically affected by heparin in FGF-1 treatments (C), with FGF-2 being altered the most at high concentration (40% decrease) (D). mRNA expression was altered significantly by heparin in FGF-2 treatments (E), with FGF-1 (44% decrease), FGF-2 (65% decrease), and FGF-R1 (39% decrease), with the greatest effect at high heparin concentration (F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Microdomains within the basement membrane (BM) region associated with type II cells of mammalian lungs have been shown to be less sulfated than that of type I cells (21–23). It has been suggested that such compositional differences within the ECM microenvironment could act to potentiate/promote type II cell responses to appropriate growth factors while at the same time retarding/inhibiting similar responses by type I cells (19, 22). Such an arrangement could reflect mechanisms which help maintain stable cell populations in the pulmonary alveolus (19). The results reported here confirm that the model sulfated ECM heparin exerts antiproliferative activity on type II alveolar cells in vitro. Previous evidence indicated that high or low molecular weight heparin significantly reduced DNA synthesis induced by FGF-1, -2, or -7 in a fashion that was sulfate-dependent, as chemically desulfated forms of heparin had little or no effect (19), or in some cases were actually stimulatory (19, 23). Heparin's effects are shown here to be concentration-dependent for FGF-1 and -2, with the latter showing greater sensitivity. Not surprisingly, the lowest concentration had little effect, whereas the highest concentration tested (500 µg/ml) resulted in significant decreases in [3H]thymidine incorporation into DNA, with growth factor/SF-treated (-82%) and FGF-2–treated (-75%) affected more than FGF-1–treated (-29%) compared with their heparin-free controls. The observation that heparin has significant inhibitory effects on DNA synthesis in isolated type II cells which increase with concentration further supports the notion that sulfated ECMs in alveolar BMs influence epithelial responses to growth factors. It could be interpreted from the data that a lower/reduced level content of sulfated ECMs in alveolar BMs would facilitate type II cell responses to FGF-1 or -2, whereas higher/increased levels would retard/inhibit such a response by either type I or type II cells. As both growth factors are present either in type II cells (FGF-1) or bound extracellularly within the BM (FGF-2), they are ideally located within the alveolar microenvironment to influence the process of normal cell turnover in concert with sulfated ECMs (8, 19). The state of sulfate composition of the BM could therefore modify alveolar cell turnover by predisposing type II cells (residing on undersulfated BM) to undergo DNA synthesis/proliferation but not nearby type I or newly-dividing type II cells (residing on or in contact with more heavily sulfated BMs).

The precise mechanism(s) involved in the complex interactions between alveolar epithelium and their ECM environment are less obvious. Heparin, a useful model for sulfated ECMs, has been demonstrated to inhibit FGF-2 binding to its receptor in Balb/c3T3 cells at high concentrations similar to that used in the present study (33). At lower concentrations, heparin had little effect on binding, whereas at intermediate levels it was shown to facilitate binding (33). These data reflect the importance of the low-affinity receptor (HSPG) portion of the dual receptor system operative in cells responsive to members of the fibroblast growth factor family. In such studies, heparin was shown to be a useful "substitute/surrogate" for HS-depleted cells treated with sodium chlorate, which prevents sulfation of newly synthesized proteoglycans. Type II cells depleted in this fashion do not all spread and have depressed DNA synthesis in media without specific growth factors added (34). This approach differs from that used in the present study, where heparin was used as an additive in excess of existing sulfated proteoglycans in the cultures—effectively competing for binding sites on soluble FGFs and cell surface receptors and resulting in a concentration-dependent reduction in DNA synthesis. This suggests that sulfated proteoglycans positively influence FGF activities in a relatively narrow concentration range—having a negative effect or no effect at low concentrations (chlorate treatment) and clearly negative effects at high concentrations.

It has been demonstrated that specific saccharide sequences within heparin/heparan sulfate containing 2-0- and 6-0-sulfate groups are particularly important for the FGF-2 binding/signaling in 3T3 fibroblasts (16). Specific desulfation of exogenously applied heparin prevented its promitogenic effects (16). In addition to the saccharide sequences, the number and positions of individual sulfate groups determine the affinity of HS for the various FGFs (35). The synthesis of sulfated components of the pericellular and subendothelial extracellular matrix are controlled by intracellular sulfotransferases, and are key in defining sequestration, dimerization, and stimulation of cell proliferation by FGF-2 (18). Type II cells express N-deacetylase/N-sulfotransferase (NDST) and 3-0-sulfotransferase (3–0ST), and the former is necessary for their spreading and transdifferentiation (34). Targeted disruption of the NDST-1 gene leads to defective heparan sulfate biosynthesis (36), pulmonary hypoplasia, neonatal respiratory distress, and death (36, 37). Neonates with this deletion had underdeveloped type II cells lacking SP production, leading to the conclusion that certain HSPGs were necessary for their maturation (37). This might in fact be an oversimplification, as such deletions would negatively impact the activity of a variety of HS-binding growth factors, including the FGFs. Although these observations were made from the perspective of the absence of HS/heparin-like molecules, they nonetheless are relevant in the current context. The antimitogenic effects described here and previously for heparin were on mature type II cells not treated with sodium chlorate, so that heparin acted to compete for binding sites on FGFs and reduce their access to the cell-bound/associated HSs that act as the low-affinity receptor binding sites (19). The final outcome in each case is essentially the same: FGFs cannot effectively perform their biologic functions in the absence or excess of HS/heparin-like molecules; a critical, moderate/intermediate concentration range appears to be important. These collective observations support the notion that the sulfated quality and quantity of ECM components would have a predictably important influence over the activity of FGFs on overlying epithelial cells in both developing and adult lung.

The second portion of the dual receptor construct is one of four types of FGF receptors with intrinsic tyrosine kinase activity, all derived from separate genes with varying affinity for each member of the FGF family (38). The mechanism(s) for differential expression of these genes has not been clearly defined. In human liver fibroblasts, retinal pigmented epithelial cells and rat alveolar type II cells, FGF-R1 has been shown to be significantly upregulated in response to FGF-1 or TGF-ß1 stimulation (9, 39, 40). FGF-R1 gene expression was reduced with combined heparin and FGF-1 treatment in a concentration-dependent fashion. Perhaps not surprisingly, gene and protein expression of its relevant ligand, FGF-2, were also downregulated by heparin treatment in combination with FGF-1. These observations serve to emphasize the importance of previous work that demonstrated autocrine upregulation of FGF-2 and its FGF-R1 receptor by FGF-1 (9). Furthermore, the present results suggest an additional potential autocrine pathway, wherein sulfated ECMs biosynthesized by type II cells and released into the pericellular environment could act to modulate the activity of FGFs and their relevant receptors. This would expand current views of autocrine regulatory mechanisms, and lend even more support for the role(s) ECMs play in influencing alveolar epithelial cell biology.

In conclusion, the data presented here demonstrate that the model sulfated ECM, heparin, can significantly influence DNA synthesis, gene expression, and protein expression in FGF-1– or FGF-2–treated cells. These results support the proposed paradigm that low levels of sulfated ECMs known to exist within microdomains of the alveolar BM beneath type II cells promote their response to growth factors, especially members of the FGF family. Conversely, higher levels of sulfated ECMs found with the BM beneath type I cells would retard their potential response(s) to the same conditions. This arrangement would be expected to constitute a significant modulatory mechanism in normal cell turnover, maintenance of stable cell numbers, and responses to injury in the mammalian alveolus.


   Acknowledgments
 
This study was supported by Public Health Service Grant HL-44497 and grants from the State of North Carolina.

Received in original form January 15, 2002

Received in final form April 10, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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