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Am J Physiol Lung Cell Mol Physiol 293: L1314-L1320, 2007. First published August 31, 2007; doi:10.1152/ajplung.00211.2007
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Fibroblast growth factor-binding protein and N-deacetylase/N-sulfotransferase-1 expression in type II cells is modulated by heparin and extracellular matrix

Donna R. Newman, Eric Walsh, K. B. C. Apparao, and Philip L. Sannes

Department of Molecular Biomedical Sciences, Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

Submitted 29 May 2007 ; accepted in final form 30 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fibroblast growth factors (FGFs) play critical roles in development, maintenance, and repair following injury or disease in the lung. Their activity is modulated by a variety of factors, including FGF-binding protein (FGF-BP; HBp-17) and N-deacetylase/N-sulfotransferase-1 (NDST-1). Functionally, FGF-BP shuttles FGFs from binding sites in ECMs to cell surfaces and enhances FGF binding and signaling, whereas NDST-1 adds sulfate groups to FGF coreceptor proteoglycans and modulates alveolar type II (ATII) cell maturation and differentiation. Since the sulfated nature of ECMs is a critical determinant of their relationship with FGFs, we predicted that ECMs and their sulfation would modulate the expression of FGF-BP and NDST-1. To examine this question, selected culture conditions of rat ATII cells were manipulated [with and without coculture with rat lung fibroblasts (RLFs)] by treatment with heparin or sodium chlorate (inhibitor of sulfation) for 24–96 h. In addition, ECMs biosynthesized by RLFs for up to 10 days before coculture were used as model intervening barriers to communication between alveolar cells and fibroblasts. FGF-BP expression was enhanced in ATII cells by coculture with RLF cells and least suppressed by desulfated heparin. NDST-1 expression in ATII cells was most sensitive to the amount of sulfation in medium and ECM and enhanced by fully sulfated heparin. Preformed ECM appears to supply factors that modify subsequent treatment effects. These results demonstrate a potentially important modulatory influence of sulfated ECMs and fibroblasts on FGF-BP and NDST-1 at the gene expression level.

coculture; alveolus; sulfation

THE ALVEOLAR SURFACE OF THE lung is covered by an unusual simple epithelium populated by large, squamous type I pneumocytes (ATI cells) and cuboidal type II (ATII) cells. The latter engage in a variety of functions including acting as epithelial progenitors (12), producing surfactant (27), and supporting regional host defense (51). Key to understanding the factors that influence and control ATII function is defining the complex microenvironment of the alveolus, which involves important cell-to-ECM and cell-to-cell relationships (11, 20). The alveolar basal lamina is a critical barrier as well as a conduit that modulates the passage of molecular information between alveolar and vascular epithelial surfaces and the underlying interstitial fibroblasts. These cells are not only in close proximity to one another, but also form numerous contacts (43, 45). Each of these cells can produce a variety of growth factors, chemokines, and other biological response modifiers and is capable of altering its own environment and that of its neighbors. This has been dramatically demonstrated in lung development, wherein mesenchyme has been shown to control branching morphogenesis and epithelial cell differentiation (41). These effects are, in part, due to members of the fibroblast growth factor (FGF) family of growth factors, which are known to play key roles in lung development and repair following injury (33, 48–50). Their expression and activity is modulated by a variety of factors including sulfated ECM, FGF-binding protein (FGF-BP; HBp-17), and N-deacetylase/N-sulfotransferase-1 (NDST-1). A considerable body of evidence has demonstrated that the sulfated character of ECMs is of particular importance in imparting their effects on FGFs, including receptor binding (13), signaling mechanisms (29), induced DNA synthesis (25, 29, 40), and stimulated gene expression (23, 25) and protein synthesis (25).

Like sulfated ECMs, FGF-BP and NDST-1 are capable of altering the activities of FGFs although in very different ways. FGF-BP is released into the pericellular environment where it can bind, sequester, and transport FGFs (16, 22). It is known to be important in embryogenesis (22), epithelial repair (7), and tumorigenesis (1, 9, 22) and is expressed in the lung (3). In the embryo, FGF-BP is localized primarily in epithelium although some mesenchymal localization occurs in early development (4).

NDST-1 influences FGF activity in a less direct fashion by defining the patterns of sulfation of nascent heparan sulfate chains during biosynthesis, which determines their ability to interact effectively with target molecules (21) such as FGFs (30). This has been demonstrated by NDST-1 knockout mice, which die at birth due to lung hypoplasia caused by disruption of normal sulfation of key ECM components such as basal laminae (21, 36). In the eye, interference with NDST-1 expression disrupts FGF signaling during early lens development, leading to lens hypoplasia and anophthalmia (31). Furthermore, NDST-1, FGFs, and FGF receptors colocalize at sites of morphogenetic change during embryogenesis (18). The potential for interplay between sulfated ECMs and FGF-BP and NDST-1 has not been examined.

It was the central aim of this study to determine whether sulfated ECMs, as modeled by soluble heparin or biosynthesized ECMs, influenced the gene and protein expression of FGF-BP and/or NDST-1. Since epithelial-fibroblast interactions as well as the sulfated ECMs they produce are suspected of playing roles in the control of the alveolar microenvironment, isolated rat ATII cells were cocultured with rat lung fibroblasts (RLFs) for 24–96 h. Conditions were also manipulated to model increases in the sulfated ECMs, as might occur during inflammatory events in the form of increased cell surface shedding (8, 19) or enhanced ECM deposition during fibrogenesis (17), by addition of heparin. In some cases, RLF monolayers were treated with TGF-beta to enhance ECM biosynthesis and its sulfate content or with sodium chlorate to inhibit 6-O-sulfation of ECM components (e.g., heparan sulfate) during biosynthesis (37). Results indicated that treatment with heparin and/or coculture with RLFs and/or their preformed ECMs altered gene expression of two potentially key biological modifiers of FGF activity in the lung: FGF-BP and NDST-1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Coculture of ATII cells and RLF cells. All animal procedures were approved by the North Carolina State University Institutional Animal Care and Use Committee and performed in accordance with National Institutes of Health guidelines. The physicochemical interactions between epithelial cells, interstitial fibroblasts, and the intervening ECM are critical determinants of homeostasis and repair following injury in the pulmonary alveolus. To examine how these complex relationships impact specific biological response modifiers, such as FGF-BP and NDST-1, an established coculture system was employed to exploit the capacity of the fibroblast to biosynthesize components of ECM. Accordingly, polyester (PET) Transwell-Clear Inserts (Costar-Corning, Acton, MA) with 0.4-µm pores were precoated on the upper surface with type I collagen from calf skin (C-8919; Sigma, St. Louis, MO) or rat tail collagen (BD Biosciences, Franklin Lakes, NJ) at a density of 0.06 µg/mm2 and allowed to dry, irradiated to sterilize, and used immediately or stored at 4°C. RLFs were isolated from mixed lung cells by differential adherence and light trypsinization and passaged twice to near purity before cryopreservation. These isolated cells express the markers characteristic of RLF, i.e., vimentin, desmin, leptin, and after prolonged cultivation, {alpha}-smooth muscle actin. Cryopreserved RLF were thawed and cultured for 3 days in complete DMEM (DMEM + 10% FBS + antibiotic/antimycotic) before seeding at a density of 70,000–80,000 per cm2 to the bottoms of inverted Transwell inserts. Careful replacement of the well plate allowed the drops of cell suspension to spread to cover the inserts. After attachment and spreading at 37°C (4 h), inserts were righted, and cells were cultured for 10 days in complete DMEM with TGF-beta (10 ng/ml) to stimulate additional matrix production or in complete DMEM without TGF-beta. As established in previous studies from this lab, high molecular weight heparin (Calbiochem, La Jolla, CA) at 500 µg/ml (3 µM), de-N-sulfated heparin (Sigma) at 500 µg/ml, fully desulfated heparin (28) at 500 µg/ml, or sodium chlorate (Fisher Scientific, Suwanee, GA) at 20 mM were included in growth medium during ECM formation, each treatment in duplicate or triplicate.

ATII cells were isolated from Fischer rats (Harlan, Indianapolis, IN or Charles River, Wilmington, MA) by elastase digestion according to Dobbs (10). After panning on rat IgG plates, nonattached cells were resuspended in complete DMEM and seeded onto inserts for coculture with RLFs and onto additional control inserts ("ATII only") at a density of 150,000 ATII per cm2. After overnight culture in complete DMEM, cells were gently rinsed in DMEM and treated in DMEM + 5% FBS or DMEM/F-12 (50:50) + 5% FBS with heparins and/or chlorate (no TGF-beta) and incubated for 48 h with no further medium changes.

DNA synthesis. A fundamental response of ATII cells to FGFs and their attendant modifiers, such as FGF-BP and NDST-1, is DNA synthesis and proliferation. In the present context, we assessed DNA synthesis in ATII cells alone and in coculture on various preformed matrices by treatment of cells in medium containing [3H]thymidine at 2 µCi/ml during the incubation period. After 48 h, PBS-rinsed cells were fixed in ice-cold 5% TCA. RLF cells were wiped from the undersides of inserts, and ATII cells were lysed directly on the inserts in 1 N NaOH and neutralized in 1 N acetic acid. Complete RLF removal and ATII lysis were confirmed microscopically. The incorporated radioactivity in equal volumes of recovered ATII lysates was measured with an LKB 1219 liquid scintillation counter (Wallac, Turku, Finland).

Quantitative real-time PCR. To assess expression of FGF-BP and NDST-1 in ATII cells cultured under parallel or similar conditions described above, RLF cells were scraped from the undersides of inserts, and ATII cells were lysed in Buffer RLT (RNeasy; Qiagen, Valencia, CA) directly in inserts. Total RNA was prepared from pooled triplicates (12-well plates) or from a single insert (6-well plates) with on-column DNase digestion. cDNAs were prepared from equal amounts of RNA using the cDNA Archive Kit (Applied Biosystems, Foster City, CA). Gene expression was assessed using TaqMan assays (Applied Biosystems). An amount of cDNA equivalent to 100 ng of input total RNA was used in each 25-µl quantitative real-time PCR reaction. TaqMan assays for rat fgf-bp, ndst-1, and beta-actin (for normalization of RNA expression) were performed in triplicate on the iCycler (Bio-Rad) using the TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems) according to the instructions of the manufacturer. PCR efficiencies were assumed to be 100%.

Statistical methods. Relative gene expression analysis was performed using the Relative Expression Software Tool-Multiple Condition Solver (REST-MCS) (32) with beta-actin as the reference gene and the untreated, 0-h, control sample used to normalize expression. Relative gene expression values were graphed in Microsoft Excel. Confidence intervals of 95% were considered to be 4 x SE of the means for n ≤ 3, and P < 0.05 was considered statistically significant. For GoGoFig. 3, absolute gene regulation values were graphed, as no relative expression between the two genes was implied.


Figure 1
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  		Fig. 1. Coculture and sulfated glycoproteins/ECMs strongly affect DNA synthesis. Alveolar type II (ATII) cells, alone or with rat lung fibroblast (RLF) cells in same-day coculture or on matrices preformed for 10 days (10D ECM) in the presence of heparin (500 µg/ml), de-N-sulfated heparin (de-N-sulf; 500 µg/ml), or sodium chlorate (20 mM) and with (E+TGFbeta) or without TGF-beta (10 ng/ml) to stimulate ECM production, were cultured on Transwell inserts for 48 h after attachment in media containing [3H]thymidine and the same treatments (no TGF-beta). DNA synthesis of a representative experiment (n = 3) is shown. Error bars denote standard deviations. NA, no added treatments.

 

Figure 2
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  		Fig. 2. A and B: gene expression in ATII cells at 48 h relative to untreated ATII cells cultured alone (ATII only, NA). A: gene expression of fgf-bp, examined by quantitative real-time PCR (qRT-PCR) in ATII cells alone or in coculture with RLF cells and preformed matrices, is strongly increased by coculture. Desulf, fully desulfated heparin; FGF-BP, FGF-binding protein. Error bars denote 95% confidence intervals (CI), which equal 4 x SE for n = 3. B: gene expression of ndst-1 in the same experiments is strongly increased in proportion to the addition of sulfated glycoproteins. Error bars denote 95% CI, n = 3. NDST-1, N-deacetylase/N-sulfotransferase-1.

 

Figure 3
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  		Fig. 3. The influence of aging on gene expression of fgf-bp and ndst-1 in ATII cells is shown. Freshly isolated rat lung ATII cells were cultured on Transwell inserts and harvested after overnight attachment ("hr 0") and 24, 48, and 96 h later. Gene expression was assessed by qRT-PCR and graphed relative to the 0-h sample (reference condition). Error bars represent 95% CI, n = 2.

 
Protein analysis. The effects of heparin on fgf-bp and ndst-1 gene expression over time were confirmed by Western blot analysis of total proteins isolated from a representative experiment. Cell lysates in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA) were sonicated (4 x 5 s) and centrifuged (14,000 rpm for 20 min at 4°C). The total protein in each supernatant was quantified by Micro BCA assay (Pierce Biotechnology, Rockford, IL). Equal amounts of protein (10 µg) from each sample were separated by SDS-PAGE on 4–12% Bis-Tris gels in MES running buffer (Invitrogen, Grand Island, NY) followed by transfer to nitrocellulose membranes. After blocking for 1 h in 4% BSA or 5% milk in TBS-T [20 mM Tris·HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20], the blots were probed overnight at 4°C with rabbit anti-rat FGF-BP (a generous gift of Dr. Anton Wellstein) at 1:1,000 dilution in TBS-T-0.4% BSA or with rabbit anti-NDST (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1,000 dilution in TBS-T-5% milk. Anti-rabbit secondary antibody conjugated to horseradish peroxidase (Cell Signaling Technology) was diluted 1:3,000 in TBS-T with 0.4% BSA or 5% nonfat dry milk. Specific bands were detected by chemiluminescence using SuperSignal West Pico (Pierce Biotechnology) and visualized by autoradiography. For normalization and quantification, the same blots were hybridized with anti-GAPDH monoclonal antibody (Santa Cruz Biotechnology) and anti-mouse secondary antibody (Cell Signaling Technology). Bitmapped images of specific bands on autoradiographs were quantified in LabWorks imaging and analysis software (UVP, Upland, CA), and integrated optical densities, normalized for GAPDH expression, were graphed in Microsoft Excel as relative to "0-h, NA" levels.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cell coculture. Both ATII cells and RLFs maintained morphological characteristics typical of their cell lineage throughout the time course of the various treatments. Within the relatively brief time periods assessed, no differences in ATII or RLF cell morphology among treatment groups were observed (data not shown).

DNA synthesis. Isolated ATII cells cocultured for 48 h with lung fibroblasts on the opposite side of Transwell insert membranes were found to incorporate [3H]thymidine at rates greatly exceeding those of ATII cells cultured alone, regardless of the heparin or chlorate treatment (Fig. 1). DNA synthesis was dramatically enhanced by coculture for 48 h with or without preformed ECMs compared with ATII cells alone (Fig. 1). Preformed ECMs stimulated DNA synthesis in untreated ATII cells as did coculture with freshly seeded RLFs. Compared with untreated ATII cells, addition of heparin at 500 µg/ml (~3 µM) significantly reduced [3H]thymidine incorporation. However, ECM formed in the presence of TGF-beta was less stimulatory of untreated ATII cells, and, in combination with this highly developed ECM, heparin was more inhibitory than in the other two coculture conditions. De-N-sulfated heparin (500 µg/ml) proved less suppressive than fully sulfated heparin or chlorate, especially on the preformed matrices, implying a role for sulfate in the suppressive effects of heparin on ATII proliferation (Fig. 1).

Relative gene expression due to matrix composition and treatments. Fgf-bp expression was increased by coculture itself. Heparin treatment nonsignificantly increased fgf-bp in ATII cells grown alone but was significantly (P < 0.01) suppressive in coculture with RLFs (Fig. 2A). All treatments counteracted the stimulatory effects of coculture on fgf-bp expression. Desulfated heparin was significantly less suppressive (P < 0.05) of fgf-bp than fully sulfated heparin in same-day coculture, and this significance increased (P < 0.01) on the ECM with TGF-beta. In summary, fgf-bp was enhanced in ATII cells primarily by coculture with RLF cells and least suppressed by factors produced by RLFs/myofibroblasts (MFs) in response to less sulfated environments.

Unlike fgf-bp, ndst-1 expression was not stimulated by coculture but appeared to be regulated more by heparins and by the amount and type of sulfate available to the cells. In each culture condition, compared with untreated cells, ndst-1 expression was significantly increased (P < 0.01) by fully sulfated heparin (Fig. 2B). De-N-sulfated heparin and chlorate also significantly increased ndst-1, especially on preformed ECMs, but to a lesser extent than did heparin. Fully desulfated heparin was significantly different from no treatment only on preformed ECMs (P < 0.05). These results indicate that ndst-1 expression increases in parallel with sulfation. Coculture with untreated RLFs or untreated MFs moderately decreased ndst-1 expression in ATII cells (Fig. 2B), but a similar pattern of ndst-1 response to treatments was seen in each of the culture conditions, with minor modifications, implying that coculture itself has a lesser effect than sulfate on ndst-1 expression. In summary, ndst-1 expression in ATII cells was most strongly enhanced by fully sulfated heparin and least by desulfated heparin and was downregulated by the presence of freshly cocultured fibroblasts. Interestingly, desulfated heparin significantly decreased ndst-1 expression (P < 0.05) in ATII cells cocultured with MFs (Fig. 2B).

Relative gene expression due to aging, coculture, and heparin. To isolate the effects of aging on gene expression, untreated ATII cells were grown on inserts and harvested at 24, 48, and 96 h. Analysis of the relative gene expression of ATII cells as a function of time revealed that the expression of fgf-bp quickly dropped and remained at low levels (Fig. 3A), whereas ndst-1, after a modest drop at 24 h, increased over 96 h to significantly exceed the 0-h time point level (Fig. 3B).

Coculture had no significant effect on gene expression of either fgf-bp or ndst-1 at 24 h (Fig. 4). However, coculture significantly enhanced expression of fgf-bp at 48 and 96 h over the levels expressed by ATII cells alone (Fig. 4A). Ndst-1 was increased by aging and enhanced further by coculture at 48 h but was not significantly altered by coculture at any time point (Fig. 4B).


Figure 4
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  		Fig. 4. A and B: the influence of coculture on selected gene expression in aging ATII cells. A: the influence of coculture on fgf-bp gene expression in ATII cells was assessed by qRT-PCR. Error bars represent 95% CI for n = 3. **P < 0.01. Coculture significantly increases fgf-bp mRNA expression at 48 and 96 h. B: the influence of coculture on ndst-1 gene expression in ATII cells was assessed by qRT-PCR. Error bars represent 95% CI for n = 3. There are no significant differences in ndst-1 mRNA expression at any time point due to coculture.

 
To examine the effects of heparin on ATII cells in the absence of coculture, freshly isolated ATII cells were plated in collagen-coated dishes, allowed to attach overnight, and treated with heparin (500 µg/ml) every 48 h. Cells were harvested along with untreated controls at 24, 48, and 96 h. Heparin treatment was found to significantly counteract the effects of ATII aging on fgf-bp expression at 48 h (Fig. 5A) and to strongly increase ndst-1 expression at 24 and 48 h (Fig. 5B). Western blots of proteins isolated from one of these experiments (Fig. 6A) similarly showed a reversal in the decrease of FGF-BP at 48 h with heparin treatments (Fig. 6B) and a strong increase in NDST-1 protein expression due to heparin at 48 and 96 h (Fig. 6C).


Figure 5
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  		Fig. 5. A and B: the influence of heparin on selected gene expression in aging ATII cells. A: the influence of heparin on fgf-bp gene expression in ATII cells cultured on collagen-coated dishes was assessed by qRT-PCR. Error bars represent 95% CI for n = 3. *P < 0.05. Heparin significantly increases fgf-bp mRNA expression only at 48 h. B: the influence of heparin on ndst-1 gene expression. Error bars represent 95% CI for n = 3. *P < 0.05. Heparin significantly increases ndst-1 mRNA expression at 24 and 48 h.

 

Figure 6
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  		Fig. 6. A–C: protein expression time course in heparin-treated ATII cells. A: Western blots of FGF-BP and NDST-1 protein expression in ATII cells cultured on collagen-coated dishes, untreated or treated with heparin (+; 500 µg/ml). GAPDH, loading control for normalization of integrated optical densities (IODs) for quantitation. B: densitometry of total FGF-BP-specific protein bands, IODs normalized to GAPDH and graphed as fold change from 0-h reference value. Pattern is similar to that of fgf-bp gene expression (Fig. 5A). C: densitometry of NDST-1-specific band, IODs normalized to GAPDH and graphed as fold change from 0-h reference value. Pattern is similar to that of ndst-1 gene expression (Fig. 5B) while demonstrating a lag in protein synthesis behind gene upregulation.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As demonstrated by others (2, 41, 42), the presence of a preexisting layer of fibroblasts and/or preformed ECM significantly increased DNA synthesis in isolated ATII cells. The addition of heparin significantly reduced this increase, not unlike results demonstrated in previous studies (25). The inclusion of chlorate in the medium, which interferes with 6-O-sulfation of newly biosynthesized proteoglycans (37), moderately reduced DNA synthesis as did a lower level of heparin (data not shown), suggesting that the amount of sulfation is a key determinant of the inhibitory qualities of heparin. In addition, the sulfate linkage may be important, as removal of N-linked sulfates partially ameliorated the inhibitory effects of heparin, as shown previously (23, 29), although it is unclear whether this may instead reflect the less suppressive effect of a reduced number of sulfate molecules.

Interestingly, preformed 10-day ECMs enhanced DNA synthesis in ATII cells more than did same-day cocultures, whereas TGF-beta-treated RLF/MFs formed ECMs that were less stimulatory than 10-day ECMs without TGF-beta. This could be due to the increased presence of sulfated components, such as heparan and chondroitin sulfates, in ECMs produced by TGF-beta-stimulated fibroblasts (44, 47). In vivo, ECM supports cells with a dynamic supply of sulfate (heparan sulfate proteoglycans) and embedded growth factors, such as FGF-2, TGF-beta, and IGF-1. These, as well as numerous binding proteins and cofactors, would be expected to influence DNA synthesis directly or indirectly.

Although the growth factors produced by one cell type are presumably capable of stimulating the other cell type in coculture in the absence of intervening matrix, ECMs pose more of a barrier to FGF-2 than to keratinocyte growth factor (5, 14). This is primarily due to the different affinities of the FGFs for heparan sulfate, a major component of basal laminae and interstitial ECM in the lung (38, 39, 46, 47). Because heparin by itself is capable of freeing FGF-2 bound to heparan sulfate in ECMs, as can oligosaccharides derived from depolymerized heparin or heparin fragments as small as tetrasaccharides (6), shed cell surface ECMs may play important roles as biological modifiers (8). It is therefore important to consider potential roles of other modulatory or adapter molecules that can influence FGF activity. One modulator of FGF activity is FGF-BP, which can compete for binding of ECM-bound FGFs (16, 22). When secreted, FGF-BP can compete for FGFs from matrix stores, mobilize, and guide them to cell surfaces where they can bind to their cognate receptors (1). This ability to enhance the activity of low concentrations of FGFs has led to the suggestion that fgf-bp upregulation following injury may be important in epithelial repair (7). Expressed in normal lung, FGF-BP is known to be important in tumor growth, but its role in homeostasis and repair following injury remains to be fully understood (1). Fgf-bp mRNA expression in ATII cells was found to be responsive to coculture with fibroblasts on inserts and to heparin on collagen-coated culture dishes, both of which delayed its downregulation with time in culture. This responsiveness may be particularly important during inflammatory events when there is increased shedding of cell surface sulfated ECMs (8, 19) or sulfated ECM deposition, as in fibrosis (17). Increased fgf-bp expression may help insure adequate FGF-stimulated re-epithelialization during repair processes (24, 52). The mechanism behind the effects of sulfated ECMs on fgf-bp expression is unclear, but because sulfated ECMs are important for signaling of certain Wnts (35), one possible connection is via the Wnt/beta-catenin pathway, which has been shown to upregulate FGF-BP (34). More work is needed in this area to clarify this important issue.

A second potentially important modulator, NDST-1 is crucial in development of the lung (36). It plays a role in the addition of N-linked sulfates to nascent proteoglycans and is important in the synthesis of low affinity heparan sulfate receptors for FGFs (21). Recent evidence indicated that targeted disruption of ndst-1 reduced the sulfation of heparan sulfates, which resulted in diminished binding of some FGFs to their receptors and disruption of growth factor signaling (31). Furthermore, targeted disruption of NDST-1 in endothelial cells altered pathological angiogenesis in experimental tumors by reducing their ability to bind FGF-2 and VEGF (15). It was interesting that the amount of sulfate available to ATII cells influenced ndst-1 expression at 48 h, such that lower than normal levels (desulfated heparin or sodium chlorate-treated) had little effect on ndst-1 expression, whereas increased levels (heparin addition) strongly enhanced expression over no treatment controls (Fig. 2B). This is supported by earlier studies that demonstrated that inhibition of sulfation with sodium chlorate reduced ATII cell spreading and transdifferentiation to ATI cells, presumably by altering ndst-1 expression (26).

Taken together, changes observed here in fgf-bp and ndst-1 expression in response to increased sulfated ECMs may provide important clues about how ATII cells adapt to changes in their microenvironment during inflammation. Whereas highly sulfated environments may act to reduce some gene expression (23, 25), protein expression (25), and signaling events (29) in the ATII cell, they also act, perhaps selectively, to increase others. FGF-BP and NDST-1 may be of particular importance in helping to define pathways that support both specific proliferative events induced by FGFs as well as differentiation induced by structural, sulfated ECMs. The present study lays the groundwork for future studies of these relationships.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-44497 and the State of North Carolina.


   ACKNOWLEDGMENTS
 
We thank Dr. Anton Wellstein of the Lombardi Comprehensive Cancer Center, Georgetown University, for the generous gift of the FGF-BP antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. L. Sannes, Dept. of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606 (e-mail: philip_sannes{at}ncsu.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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