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Glycobiology Pages 1149-1155  

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts

Heparan sulfate upregulates platelet-derived growth factor receptors on human lung fibroblasts

Johan Malmström and Gunilla Westergren-Thorsson1

Department of Cell and Molecular Biology, Lund University, Sweden

Received on November 21, 1997; revised on April 27, 1998; accepted on April 28, 1998

Heparan sulfate is a molecule that possesses a large structural variability and which has been shown to inhibit the proliferation of fibroblasts in vitro. The aim of this study was to determine whether the anti-proliferative effects of heparan sulfate were exerted by regulation of the activity of the platelet-derived growth factor and/or of the platelet-derived growth factor receptors. Both l-iduronate-rich, anti-proliferative and the l-iduronate-poor, non-anti-proliferative heparan sulfate species, were incubated with confluent human embryonic lung fibroblasts for 24 h. The mRNA levels for PDGF-AA, PDGF-BB, and their receptors were measured. Binding studies were performed with [125I]-PDGF-BB and [125I]-EGF for 2 h at 4°C in cultures preincubated with both types of heparan sulfate for 24 h. In separate experiments, cultures were incubated together with heparan sulfate and [125I]-PDGF-BB for 2 h at 4°C. Increases of two- to threefold in the mRNA levels for both the [alpha]- and the [beta]-receptors of PDGF was obtained after treatment with both types of heparan sulfate, whereas the mRNA levels of both the PDGF-AA and the PDGF-BB were essentially unaffected. A sixfold increase in binding was only noted for [125I]-PDGF-BB in cultures pre-treated with the anti-proliferative heparan sulfate for 24 h, whereas no effect was noted with use of the non-anti-proliferative heparan sulfate. Incubating the [125I]-PDGF-BB and the anti-proliferative heparan sulfate together for 2 h resulted in a smaller, threefold increase in binding. This indicates that the anti-proliferative heparan sulfate both stabilizes and increases expression of the PDGF receptors. To investigate whether the increased number of PDGF receptors could affect cell activity, cells were preincubated with anti-proliferative heparan sulfate and then treated with PDGF-BB. This resulted in an increase in mitogenicity compared to cells treated only with PDGF-BB. Neither an increase in binding for [125I-EGF] nor an increase in the mitogenic response of EGF could be observed in cultures pre-treated with the anti-proliferative heparan sulfate. The results indicate that the extracellular matrix itself may regulate important biological phenomena such as cell proliferation and matrix production through affecting the expression of receptors of PDGF, which initiate both stimulatory and inhibitory signals.

Key words: cell growth/fibroblast/heparan sulfate/ platelet-derived growth factor/platelet-derived growth factor receptors

Introduction

It is well known that heparin inhibits the proliferation of many cell types (Wright et al., 1989a; Floege et al., 1993). The degree of inhibition depends on both the length and the charge of the polysaccharides (Wright et al., 1989b). Westergren-Thorsson et al. (1991) have reported that glycosaminoglycans (GAGs) rich in iduronic acid (IdoUA), in particular certain species of heparan sulfate (HS), inhibit the proliferation of human fibroblasts in vitro to an even greater extent than heparin. The anti-proliferative effect also occurs in defined media in which various growth factors serve as mitogens (Westergren-Thorsson et al., 1993a).

HS binds to both high affinity and to low affinity sites on the cell surface and is internalized through endocytosis (Castellot et al., 1985; Vanuuchi et al., 1988; Redini et al., 1989; Arroyo-Yanguas et al., 1997). The internalization of HS results in degradation to smaller fragments (Fedarko and Conrad, 1986; Hiscock et al., 1994). These degraded fragments may subsequently be transported to the nucleus (Fedarko and Conrad 1986; Busch et al., 1992). The binding and/or internalization of anti-proliferative HS has a number of different effects on various signal transduction pathways in the cell. One of the most prominent effects is the inhibition of MAPkinase activity (Ottlinger et al., 1993; Miralem et al., 1996; Ueda et al., 1996), possibly due to a decrease in the signaling of protein kinase C (Wright et al., 1989c; Pukac et al., 1990, 1997; Herbert et al., 1996).

HS also affects the activity of various mitogens. This is best documented for the basic fibroblast growth factor (bFGF) which, in order to exert its growth-promoting effects, is dependent on binding to a saccharide sequence containing the sulfated IdoUA residues found in HS/heparin (Guimond et al., 1993; Maccarana et al., 1993; Aviezer et al., 1994; Walker et al., 1994,). Platelet-derived growth factor (PDGF) has also been shown to bind to both HS and heparin. The specific binding to a sequence translated from the sixth exon of the long PDGF A-chain isoform and PDGF B chain has been shown (Raines and Ross, 1992; Kelly et al., 1993) The carbohydrate sequence required for interaction with the long PDGF-A isoform contains IdoUA (2-OSO3)-GlcNSO3(6-SO3) (Feyzi et al., 1997).

The properties of HS just described, those of specific internalization and processing, effects on different signal transduction pathways and binding directly to different mitogens, are all biological phenomena that appear to be related to the growth factor PDGF. PDGF belongs to a family of signaling molecules of particular importance for the growth and motility of connective tissue cells. The inhibitory actions of HS on fibroblasts that have been observed may thus directly or indirectly involve PDGF. In the present study we aimed to test the hypothesis that HS could modify PDGF receptors. We found that when human fibroblasts were treated with anti-proliferative heparan sulfate, active PDGF receptors were both stabilized and upregulated.

Table I. Chemical data on the sulfated glycosaminoglycans employed
Sample O-SO3 N-SO3 IdoUA IdoUA-O-SO3 Mr (kDa)
HexN (mol/mol) HexN (mol/mol) HexUA (%) HexUA (%)
HS 2 0.30 0.26 30 10 20
HS 6 0.91 0.72 65 60 20
Hex, Hexosamine; HexUA, hexuronic acid; O-SO3, ester sulfate; N-SO3, N-sulfamate. Relative molecular mass (Mr) was determined by gel chromatography on Superose 6 calibrated using HS-species of known molecular mass (determined by light scattering).

Results

Characterization of GAGs

HS consists of a common core structure (-4GlcUA[beta]1-4GlcNAc[alpha]1-)n which, to varying degrees, can be modified by exchange of the N-acetyl group for N-sulfate by the 5-epimerization of d-glucuronic acid (GlcUA) to l-IdoUA and by the additional O-sulfation of both sugars (Table I). The HS2 preparation employed contained approximately one-third IdoUA, most of which was non-sulfated. In contrast, the HS6 preparation contained approximately two-thirds IdoUA, most of which was 2-O-sulfated (Table I).

IdoUA-rich heparan sulfate (HS6) but not IdoUA-poor (HS2) affects the growth rate of human embryonic lung fibroblasts

Serum-starved cells incubated at a density of 5000 cells/well were placed in medium that contained 5% serum with or without HS and were incubated for periods of up to 96 h. HS6 was found to inhibit the growth of cells in a dose-dependent manner from 1 to 100 µg/ml (Westergren-Thorsson et al., 1991). Maximal inhibition induced by HS6 was 35%, and it was obtained using 100 µg of polysaccharide/ml of medium (Figure 1). In contrast, no effect on the growth rate of the cells was observed for HS2 (Figure 1). A similar inhibition of the growth of normal lung fibroblasts with HS6 has been noted in a defined medium without serum (Westergren-Thorsson et al., 1993a).


Figure 1. Effect of exogenously added heparan sulfates on growth rate. Serum-starved embryonic lung fibroblasts at a density of 5000 cells/well were incubated either with or without 100 µg/ml HSs in medium containing 5% serum for periods of up to 96 h. Cell numbers were estimated using crystal violet; for further information see Materials and methods. The substances tested were the IdoUA- and sulfate-rich HS (HS6) and the IdoUA- and sulfate- poor HS (HS2). The values represent the mean ± SEM (n = 6).

Gene expression of the PDGF-receptor subtypes in human embryonic lung fibroblasts is upregulated by both types of heparan sulfate

Northern blot analysis showed that 24 h treatment with HS2 and HS6 increased the mRNA expression of the 6.5 kb PDGF-[alpha] receptor transcript 1.9 ± 0.4-fold and 3.0 ± 0.4-fold respectively, relative to control cells (p < 0.035 and p < 0.002, Figure 2A). A significant difference between the effect of HS2 and HS6 (p < 0.04) was also noticed. Furthermore, the mRNA expression of the 5.5 kb PDGF-[beta] receptor transcript was also increased after treatment with HS2 and HS6 by 2.1 ± 0.4-fold and 2.2 ± 0.5-fold, respectively, compared to the corresponding controls (both p < 0.03, Figure 2B). Intensities obtained after Northern blot analysis using probes for the [alpha]- and the [beta]-receptor were all related to ribosomal RNA (Figure 2C).


Figure 2. Effect of exogenously added heparan sulfates on the mRNA level of the PDGF-[alpha] (A) and the PDGF-[beta] receptor (B) in human embryonic lung fibroblasts. Confluent cultures were incubated for 24 h without or with HS2 or HS6 at a dose of 100 µg/ml. The total RNA was isolated and was separated by gel electrophoresis, transferred to nylon filters, and finally hybridized with a [32P]-labeled cDNA probe for PDGF-[alpha] and PDGF-[beta] receptors. The blots were scanned and quantified and related to the sum of 18S and 28S rRNA quantified from gels stained with EtBr. The data are shown as a relative change in mRNA presented as mean ± SEM (n = 6). The inset shows representative Northern blots hybridized with cDNA for the PDGF-[alpha] receptor (A), the PDGF-[beta] receptor (B) and an EtBr gel showing 18S and 28S rRNA (C) in cultures treated with or without HSs.

Only a slight increase in the mRNA level in relation to ribosomal RNA for both PDGF-AA and the PDGF-BB was noted after treatment with HS6, an increase of 1.6 ± 0.6-fold and 1.4 ± 0.3-fold, respectively, relative to the control culture (Table II). However, the difference between control and HS6 treated cultures was not significant. No effect on the mRNA level in either PDGF-AA or PDGF-BB was noted after HS2 treatment as compared with control cultures (Table II).

Table II. Effect of exogenously added HSs on the relative change in mRNA for PDGF-AA and PDGF-BB in human embryonic lung fibroblasts
Sample PDGF-AA PDGF-BB
Control 1 1
HS2 1.0 ± 0.2 1.0 ± 0.2
HS6 1.6 ± 0.6 1.4 ± 0.3
The values represent mean ± SEM (n = 6).

IdoUA-rich (HS6) but not IdoUA-poor (HS2) heparan sulfate increases the binding of [125I]-PDGF-BB to human embryonic lung fibroblasts

Quiescent, confluent cultures of human lung fibroblasts were incubated for 24 h with either HS6 or HS2 (100 µg/ml). The HS was removed by changing the culture medium and the cells were further incubated with 0.4-30 nM [125I]-PDGF-BB or with 0.08-1.6 nM [125I]-EGF for 2 h at 4°C. The [125I]-PDGF-BB bound in a dose-dependent manner and saturation was reached when approximately 10 nM [125I]-PDGF-BB had been added to both control and the HS2- and HS6-treated cultures (Figure 3). At saturation, the maximal binding of [125I]-PDGF-BB was approximately 500 fmol/106 cells in both the control and HS2-treated cultures, whereas it was 6 times greater (3000 fmol/106 cells) in the HS6-treated cultures (Figure 3). After 12 h incubation, an approximately 3-fold increase in the binding of [125I]-PDGF-BB in HS6-treated cultures, as compared with control cultures, could be observed (data not shown).


Figure 3. Effect of exogenously added heparan sulfates on the binding of [125I]-PDGF-BB or [125I]-EGF (inset) to human embryonic lung fibroblasts. Confluent cultures were incubated for 24 h with or without HS2 or HS6 at a dose of 100 µg/ml. The medium was then removed and the cultures were incubated further for 2 h in 4°C with 0.4-30 nM [125I]-PDGF-BB or 0.08-1.6 nM [125I]-EGF (inset). The radioactivity found in the cell layer was determined and the activity attained, considered as bound material. For further details, see Materials and methods. The results for the binding of [125I]-PDGF-BB that are shown are from one representative experiment out of nine.

To study whether the upregulation of the PDGF receptors following HS6 treatment was due to specific upregulation of the growth factor receptors, the binding of [125I]-EGF was tested. [125I]-EGF bound in a dose-dependent manner and saturation was reached at approximately 0.8 nM [125I]-EGF in both the control and HS6- and HS2-treated cultures. The maximal binding of [125I]-EGF at saturation was approximately 80 fmol/106 cells in all three cases (Figure 3, inset). Treatment with HS6 did not increase the binding of [125I]-EGF to the fibroblasts compared to the control and HS2-treated cultures, this result was in contrast to the binding of [125I]-PDGF-BB (Figure 3, insert).

Scatchard analyses were performed to determine the binding parameters (Kd and sites/cell) of [125I]-PDGF-BB in cultures preincubated for 24 h with or without HS. No significant differences between the Kd-values in control, HS2- and HS6-treated cultures were found, the Kd values obtained being 0.55 ± 0.09 nM, 0.64 ± 0.14 nM, and 0.94 ± 0.22 nM, respectively (Figure 4). Cells treated with HS6 showed approximately a 6-fold increase in the number of sites/cell from 43,000 ± 5000 in control cultures to 265,000 ± 38,000 sites/cell (p < 0.001, Figure 4) in the HS6-treated culture. No effects on the number of sites/cell were observed after treatment with HS2 as compared with the control culture (Figure 4).


Figure 4. Scatchard analysis of [125I]-PDGF-BB binding to human embryonic lung fibroblasts after treatment with heparan sulfates. Confluent cultures were incubated for 24 h with or without HS2 or HS6 at a dose of 100 µg/ml. The medium was then removed and the cultures were incubated further for 2 h in 4°C with 0.04-1.6 nM of [125I]-PDGF-BB. The radioactivity found in the cell layer was considered as bound material B, the radioactivity found in the medium being considered as free material F. For further details see Materials and methods. The figure shows one representative experiment out of nine.

To investigate whether the observed increase could be attributed to an increased binding activity of receptors already present at the cell surface, cells were incubated together with [125I]-PDGF-BB and HS2 or HS6 for 2 h at 4°C without preincubation of either HS. Under these conditions, a 3-fold increase in the number of sites/cell was noted in cultures treated with HS6 as compared to control cultures, the values ranging from 55,000 ± 5000 to 170,000 ± 20,000 sites/cell (p < 0.001). No increase in the number of sites/cell was obtained in cultures treated with HS2 as compared with control cultures (data not shown). The Kd values obtained when the cultures were incubated with both HS2 or HS6 and PDGF together were the same as those obtained with preincubation (see above).

IdoUA-rich (HS6) heparan sulfate produces an increase in mitogenic response to PDGF-BB but not to EGF

To study whether the increase in the number of receptors for PDGF-BB obtained after treatment with HS6 resulted in an increase in cell proliferation, cultures were incubated with or without HS2 or HS6 for 24 h. The medium was then removed, and the cultures were further incubated with or without medium containing PDGF-BB (0.04-1.6 nM) for an additional 48 h. In a control experiment, cultures were incubated with EGF (0.16-6.2 nM). The percentage change in cell growth was obtained for controls with or without PDGF or EGF, and for heparan sulfate-treated cultures in the presence or absence of PDGF or EGF.

Growth in both the control and the HS6-treated cultures responded to PDGF-BB in a dose-dependent manner, maximal growth being obtained at 0.4 nM of PDGF-BB in both the control and the HS6 pretreated cultures (Figure 5A). At 0.04 nM a 4-fold increase in cell growth compared with controls (p < 0.01) was obtained in cultures preincubated with HS6. At higher doses of PDGF-BB the increase was only approximately 1.5-fold (Figure 5A) giving an average increase of 2-fold. No effect on cell proliferation was noted in cultures pretreated with HS2 followed by incubation with PDGF-BB (data not shown).


Figure 5. Mitogenic response of fibroblasts to PDGF-BB (A) and EGF (B) following treatment with IdoUA-rich heparan sulfate. Serum-starved embryonic lung fibroblasts at a density of 5000 cells/well were incubated with or without HS6 at a dose of 100 µg/ml in F12-IT medium for 24 h. At this point an inhibition of growth of 31% was obtained in the HS6-treated cultures. The control medium or the medium containing heparan sulfate was then removed and either 0.04-1.6 nM PDGF-BB (A), 0.16-6.2 nM EGF (B) or new medium alone was added. After 48 h, cell numbers were estimated using the crystal violet method. As there was an inhibition of growth in the HS6-treated cultures data obtained are given as the change in cell growth (%) by comparing controls with or without PDGF or EGF, with the heparan sulfate-treated culture with or without PDGF or EGF. The values represent the mean ± SEM (n = 5).

The growth of control cultures responded to EGF in a dose-dependent manner, maximal growth being obtained at doses of EGF of 0.8 nM or more (Figure 5B). In cultures in which treatment with HS6 was followed by incubation with various doses of EGF, in contrast to subsequent incubation with PDGF-BB, growth was not enhanced as compared with control cultures. Instead, a slight inhibitory effect was observed on cultures that were treated with HS6 for 24 h followed by EGF treatment (Figure 5B).

Discussion

The present experiments analyze the capacity of HS to modify the expression and activity of various proliferative mediators such as PDGF and its receptors. HS exerts many important biological effects, such as processing anti-proliferative activity toward many cell types (Wright et al., 1989a). Both the structure and the size of heparan sulfate are of importance for its anti-proliferative actions (Wright et al., 1989b; Westergren-Thorsson et al., 1991). Our experiments demonstrated that HS2 and HS6 had no effect on the mRNA level of either PDGF-AA or PDGF-BB. However, the mRNA for the [alpha]- and the [beta]-receptors for PDGF were upregulated by the exposure of cells to both anti-proliferative HS6 and the non-anti-proliferative HS2. The binding to the receptor protein was only found to be increased by treatment with anti-proliferative HS6. An important question to be investigated was whether the increase in the number of receptors contributed to the increase in the proliferation of fibroblasts pretreated with the anti-proliferative HS6. Experiments showed that after removal of HS6 a larger response to PDGF could be observed than in control cultures. This indicates that the upregulated receptors also promote cell division. This upregulation of the receptors of PDGF was not a general effect on growth factor receptors, since those of EGF were unaffected. This is further illustrated by the fact that EGF did not enhance, but rather inhibited, cell proliferation of cells preincubated with HS6.

The paradoxical observation that the anti-proliferative HS6 decreased cell proliferation and increased the number of active PDGF receptors may be explained by the fact that PDGF exerts a number of biological functions, i.e., differentiation, cell proliferation, and cell migration (Heldin, 1997). Thus PDGF has distinct signal transduction pathways that are associated with migration as opposed to proliferation (Bornfeldt et al., 1995; Heldin, 1997). Proliferation is associated with the MAPkinase pathway whereas migration is associated with phosphatidylinositol 3-kinase (PI3 ) and with calcium release. Since the anti-proliferative HS is known to inhibit the MAPkinase pathway, it could be expected that other pathways are stimulated, such as the PI3 pathway, with the subsequent release of calcium resulting in cell migration instead of cell proliferation. Furthermore a significant crosstalk between the different signaling transduction pathways occurs, and stimulatory signals are often initiated in parallel to inhibitory ones (Heldin, 1997). What determines how the cell reacts is unclear, but differential regulation of the expression of molecules involved in the different pathways represents one possible explanation. This can be mediated by differences in the composition of the extracellular matrix where PDGF has been shown to induce increases in some types of integrin receptors on cells growing on a matrix of fibronectin, whereas other types of receptors are expressed in an environment of collagen (Xu and Clark, 1996).

This can be illustrated by the role of HS in the wound repair process. HS chains are released from syndecans during cell migration into the wound site (Elenius, 1991). This enables the cells to migrate since their tight connection to the extracellular matrix is reduced. The released HSs may subsequently bind to cell receptors inducing an increase in PDGF receptors enhancing the effect of PDGF on cell migration. Only later does HS decrease cell proliferation after suitable intracellular processing (Arroyo-Yanguas et al., 1997). This implies that the HS side chain residues play an important and varying role at different stages of tissue regeneration, an area of ongoing research.

The fact that only HS6 that was highly sulfated and was rich in IdoUA upregulated the amount of receptors, measured as [125I]-PDGF binding, indicates the effect to be structure-specific. This is also true for the anti-proliferative effects, the effects when using the sulfate and IdoUA-rich HS6 being reached within 3-4 days (Westergren-Thorsson et al., 1991). This effect requires binding, internalization, and processing (Arroyo-Yanguas et al., 1997). Whether the internalization and processing of HS are needed to obtain upregulation of the PDGF receptors has not been investigated. The increase in binding is clearly noticeable, however, after 2 h at 4°C, where activity of synthesis and transport across the cell membrane is prevented. It points, therefore, to the possibility that HS6 has a direct effect on receptor activity. This early increase in the amount of receptors is only threefold, whereas the total increase in binding after 24 h of preincubation with anti-proliferative HS6 is sixfold. This additional increase appears attributable to the upregulation of mRNA. Since HS2 low in sulfate and the IdoUA did not increase the number of receptors, there must be translational or posttranslational processes that require certain specific HS-structures. Of interest is the fact that HS6 upregulates mRNA for the PDGF-[alpha] receptor to a greater extent than that of [beta]-receptor. Furthermore, the effect induced by HS2 on the [alpha]-receptors is smaller than that induced by HS6. This indicates that the effect noted on binding of PDGF may be attributed to an increase of the [alpha]-receptor, which was upregulated mainly by HS6.

The [alpha]-receptor has also been shown to be up-regulated by interleukin-1 (IL-1) (Lindroos et al., 1995). Due to the rapid onset of the HS effect on the receptors of PDGF, it is however not likely that IL-1 is involved in this process. Rather, this suggests a direct pathway for HS by means of gene activation.

The modulation of cytokine activity by HS, like that of PDGF, is most likely an important biological principle, of which the mechanisms of action and the required specific structures of HS must be further investigated.

Materials and methods

Materials

The HSs (HS2, HS6) employed were prepared from beef lung using methods described by Fransson and Sjöberg (1980). The final step in preparation involved separation on a Sepharose 6 column under dissociative conditions to remove the cytoactive components bound to the HSs. Chemical data for the sulfated GAGs employed are shown in Table I. The procedure for the determination of total hexosamine, uronic acid, and sulfate content has been described earlier (see Fransson and Sjöberg, 1980; Westergren-Thorsson et al., 1991, 1993a; and Arroyo-Yanguas et al., 1997).

cDNA probes from PDGF-AA, PDGF-BB, PDGF-[alpha]-, and PDGF-[beta] receptors were used. The cDNA probe from PDGF-AA was an EcoRI fragment of 1300 bp (Betzholtz et al., 1986) and that from PDGF-BB a BamHI fragment of 2000 bp (Collins et al., 1985). The cDNA from the PDGF [alpha]-receptor was an EcoRI and an AccI fragment of 750 bp (Claesson-Welsch et al., 1989) and that from the PDGF [beta]-receptor a BamHI restriction fragment of 751 bp. The cDNA was labeled [32P]-CTP (AA 0005, Amersham, UK) with a specific activity of about 8 × 108 c.p.m./µg DNA, using a random priming DNA labeling kit (Boehringer Mannheim Scandinavia, Bromma, Sweden).

The recombinant human PDGF-BB and EGF were purchased from Novakemi AB, Enskede, Sweden and the [125I]-PDGF BB (IM 213) and [125I]-EGF (IM 196) from Amersham, UK.

Cell culture

Fibroblasts were obtained from human lung embryonic tissue and were grown in 25 cm2 dishes in Eagle's minimal essential medium (EMEM) supplemented with 10% newborn calf serum and 1% glutamine, at 37°C in a humidified incubator in atmosphere of 5% CO2 and 95% air. The experiments were performed on cells between passages 5 and 20. Cells were checked regularly for mycoplasma, using a GEN-PROBE Rapid Detection System (Gen Probe, San Diego, CA). Cell counts were made in a Bûrker-chamber.

Cell proliferation assays

Fibroblasts were seeded into 96 well microplates containing EMEM supplemented with 10% newborn calf serum and 1% glutamine at a density of 5000 cells/well. After 5 h the cells were washed twice with PBS and placed in serum-free medium for an additional 24 h. The medium was then changed and the cells were incubated for periods up to 96 h with or without HSs (100 µg/ml) in EMEM containing 5% newborn calf serum.

To study the mitogenic effect of PDGF-BB after HS treatment, serum-starved embryonic lung fibroblasts were incubated with or without HSs (100 µg/ml) in Ham's F12-IT medium (F12 supplemented with 10 µg/ml insulin and 25 µg/ml transferrin) for 24 h. At this point the medium was changed and the cells were incubated for an additional 48 h with or without 0.04-1.6 nM PDGF-BB or, as in the control experiment, with or without 0.16-6.2 nM EGF. Cell numbers were determined using a method based on the adsorption of crystal violet to the nuclei. The amount of adsorbed dye was measured using a microplate photometer (Labsystems Multiskan RC) following solubilization of the cells by detergent (Gilles et al., 1986; Westergren-Thorsson et al., 1991, 1993a).

Isolation and analysis of RNA

Confluent lung fibroblasts were incubated with or without HSs (100 µg/ml) in EMEM supplemented by 5% newborn-calf serum for 24 h. RNA was isolated from the cell cultures by guanidine isothiocyanate/phenol/chloroform/isoamylalcohol extraction (Chomzynski and Sacchi, 1987). The RNA (15 µg) was separated by 1% agarose gel electrophoresis. Following electrophoresis, a part of the gel was stained with ethidium bromide (EtBr) to verify that approximately equal amounts of RNA had been added to each well and that the RNA was not degraded. The RNA was then transferred to nylon filters and hybridized overnight with [32P]-labeled cDNA probes (Westergren-Thorsson et al., 1993b). The filters were washed sequentially with 2× SSC (0.15 sodium chloride, 15 mM sodium citrate, pH 7.0) and 0.05% sodium dodecyl sulfate at room temperature and then with 0.2× SSC containing 0.1% sodium dodecyl at 50°C. The filters were exposed to a Fuji Imaging Plate overnight or for longer periods. The intensity of the radioactivity in each band was measured using a Fuji BAS 2000 Bioimaging plate analyzer. These values were related to the sum of the intensities of the 18S and 28S rRNAs estimated with the software Gel-Pro Analyzer after scanning.

Binding assays

Confluent human lung fibroblasts were incubated for 24 h in EMEM supplemented with 5% newborn-calf serum with or without 100 µg/ml of HSs. The medium was then removed, and 0.4-30 nM [125I]-PDGF-BB or 0.08-1.6 nM [125I]-EGF in serum-free EMEM was added to the cultures, which were then incubated for an additional 2 h at 4°C. The possible effect of the HSs in stabilizing the PDGF receptors was studied in a number of experiments in which both HS (100 µg/ml) and 0.4-30 nM [125I]-PDGF-BB were incubated together for 2 h at 4°C. The medium was then removed and the cell monolayer was rinsed twice with fresh EMEM. The cell monolayer was extracted by a buffer containing 4 M guanidinium hydrochloride, 2% (v/v) Triton X-100 and 50 mM NaOAc, pH 5.8 (Arroyo-Yanguas et al.,1997). The [125I]-PDGF-BB or [125I]-EGF remaining in the medium after incubation and rinsing was regarded as free, whereas that which was extracted from the cell monolayer was considered to be bound to the cell surface. The radioactivity of the samples was measured in a 1272 CliniGamma counter. Sites/cell and Kd values were determined by use of Scatchard plots. Specific binding was defined as the difference between total and nonspecific binding. Nonspecific binding was determined as described by Huang et al., 1982.

The induction times for HS6 (100 µg/ml) were determined by incubating the cells with or without HS6 for periods of 12, 24, 48, and 96 h. The culture medium was then removed, and the cells were incubated further with 0.2 nM [125I]-PDGF for 2 h at 4°C. Bound material was determined as described above.

Statistical methods

The results were expressed as the mean ± SEM. Student's t-test was used to evaluate the differences in means between groups.

Acknowledgments

This work was supported by grants from the Swedish Medical Research Council (11550), the Swedish Cancer Fund, the Swedish Society for Medical Research, the J. A. Persson, G. Nilsson, Greta and John Kock, A. Österlund, and Anna-Greta Crafoord Foundations, Riksföreningen mot Rheumatism, Gustaf V.s 80 Årsfond, Heart-Lung Foundation, and the Medical Faculty, Lund University. The expert technical assistance of Urszula Endresén and Susanne Persson is gratefully acknowledged. cDNA probes for PDGF-AA, PDGF-BB, PDGF-[alpha]-, and PDGF-[beta] receptors were kindly provided by Prof. C.-H. Heldin, of the Ludwig Institute for Cancer Research, Biomedical Center in Uppsala, Sweden.

Abbreviations

bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; EMEM, Eagle's minimal essential medium; EtBr, ethidium bromide; F12-IT, Ham's F12 medium supplemented with insulin and transferrin; GAG, glycosaminoglycan; GlcNSO3, N-sulfated glucosamine; HS, heparan sulfate; IdoUA, l-iduronic acid; Il-1, interleukin-1; MAPkinase, mitogen activated protein kinase; PI3, phosphatidylinositol 3-kinase; TGF-[beta], transforming growth factor-[beta]; PDGF, platelet-derived growth factor.

References

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1To whom correspondence should be addressed at: Department of Cell and Molecular Biology, Lund University, P.O. Box 94, S-221 00 Lund, Sweden

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