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Tumor Biology |
Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, Texas 77030-3303 [X. W., M. K., F. W., C. J., C. Y., W. L. M.]; Graduate School of Biomedical Sciences, The University of Texas-Houston Health Science Center, Houston, Texas 77030 [X. W., C. Y.]; and Central Research Laboratories, Zeria Pharmaceutical Company, Limited, Kohnan-machi, Ohsato-Gun, Saitama 360-0111, Japan [M. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both biochemical and structural approaches have shown that the FGFR-signaling complex is a tripartite oligomer comprised of subunits of the FGFR kinase ectodomain, pericellular matrix HS, and FGF (3 , 7, 8, 9, 10, 11, 12) . HS chains exhibit an FGF-independent, divalent cation-dependent high affinity interaction with Ig module II of self-associated oligomers of the FGFR kinase ectodomain to form a complex that will bind FGF (7, 8, 9 , 13) . Most recently (14) , evidence has been presented that heparin and presumably HS also interacts with Ig module III of FGFR. The HS subunit of the FGFR complex has been shown to play multiple and related roles in the regulation of activity of the complex that, in combination, places restrictions on it in respect to length and composition. HS stabilizes and protects the FGFR ectodomain against proteolysis (10) , holds the unliganded complex in an inactive state, which maintains dependence of the complex on FGF for activation by trans-phosphorylation between kinases (10) , and is conditionally required for FGF binding to the complex (8 , 10) . The HS subunit in a binary complex with the FGFR ectodomain has also been demonstrated to be a cell- and FGFR-specific determinant of the selectivity of the complex for different FGFs (9) . Thus, it follows that alterations in the HS subunit may contribute to alterations in stability, activity, and ligand-specificity of the FGFR complex that impacts tumor progression.
Preliminary characterization experiments indicated that less than 50% of the fraction of crude heparin, which has anticoagulant properties (about 30%), can form a binary complex with FGFR1 that binds FGF (13) . This suggests that there is a specific structural requirement in addition to and including the antithrombin (AT) III-binding motif for formation of the binary FGFR complex (13) . In view of these specific characteristics and the fact that the HS subunit may be cell type- and FGFR-specific, we sought to determine whether premalignant prostate epithelial cells exhibit the complementary HS for tumor-promoting FGFR1 that is foreign to them. Moreover, to understand the nature of the structural requirement within the HS chain for participation in the FGFR complex and nature of the potential alterations that occur during progression to malignancy, it was essential to enrich HS chains based on affinity for FGFR and then to identify the core proteins to which they are attached. In this study, we report for the first time the isolation and identification of the core protein of an HSPG based on ability of its HS chains to bind to and form a complex with the ectodomain of the FGFR kinase that is capable of binding FGF. The HSPG was isolated from premalignant DTE epithelial cells from the nonmalignant, androgen-responsive rat prostate tumor (Dunning R3327PAP) in which FGFR1 is not normally expressed. However, when FGFR1 appears in these cells, in which FGFR2IIIb is the resident isotype, a slow progression to malignancy occurs concurrent with loss of FGFR2 (6) .4 Once acquired, FGFR1 also supports the fully hormone-independent malignant phenotype (5) . A combination of ion exchange, AT, gel filtration, and FGFR1 module II affinity chromatography yielded an HS chain attached to a core protein with a single NH2-terminal sequence characteristic of rat syndecan-1. An estimated purification factor of over 600-fold from fractions of cell lysates enriched on ion exchange columns in respect to sugar chain confirmed that the syndecan-1 HS chain capable of formation of the FGFR complex with ectopic FGFR1 in the premalignant epithelial cells was rare. Expression of recombinant GST-tagged syndecan-1 in the DTE cells from cDNA confirmed that the HS chains on DTE cell syndecan-1 formed a complex with FGFR1 competent to bind FGF and also showed that syndecan-1 bears HS chains that complement resident FGFR2.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation and Purification of HSPG.
DTE cells were cultured as monolayers in RD medium (1:1 RPMI and DMEM; Life Technologies, Inc.) supplemented with 4% fetal bovine serum, 3.3 mg/ml of HEPES, 110 µg/ml of Na-pyruvate, 20 mM NaHCO3, and kanamycin (100 µg/ml) in humidified 95% air and 5% CO2 at 37°C. When cells were subconfluent, the RD medium was replaced with medium F-12 (Life Technologies, Inc.) supplemented with 2% dialyzed fetal bovine serum and 20 mM NaHCO3 without kanamycin for 18 h. Then, 2 µCi/ml [35S]sulfate (sulfuric acid; specific activity 1325 Ci/mmol) was added and incubated for 18 h. Cells were extracted with 1% Triton X-100 containing 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM (2-aminoethyl)-benzenesulfonyl fluoride, 10 mM
N-ethylmaleimide, 100 mM 
-aminocapronic acid, 10 µg/ml leupeptin, and 5 mM benzamidine-HCl at room temperature for 10 min to release HSPG. The lysate was clarified by centrifugation at 2500 rpm for 10 min. Separate experiments in which HS was traced with 35S revealed that the Triton X-100 procedure extracted over 90% of the DTE cell-associated radioactivity. Less than 10% was associated with the extracted monolayers.
The clarified lysate was applied to a 0.8 x 2-cm DEAE-Sepharose column (weak ion exchanger; Fast Flow; Pharmacia Biotech). The column was prepared by washing with 1 M NaCl in PBS (buffer 3) and then equilibration with PBS containing 1% Triton X-100 (buffer 4) before use. The loaded column was extensively washed with buffer 4, and the bound HSPG was eluted by buffer 3 and then dialyzed against distilled H2O for fractionation by ATIII affinity.
The sample was applied to an ATIII affinity column comprised of ATIII conjugated to agarose beads (Sigma Chemical Co., St. Louis, MO). Before loading the sample, a 0.5-ml bed column was washed with 50 mM Tris-HCl (pH 8.0) containing 1 M NaCl, 1% Triton X-100 (buffer 5) and then equilibrated with buffer 1, and the sample was loaded in buffer 1. After incubation for 1 h at room temperature, the beads were extensively washed with buffer 1, and then the bound HSPG was eluted with buffer 5. The eluate was diluted 2-fold and then mixed with benzamidine-conjugated Sepharose 6B beads (Pharmacia Biotech) to eliminate any residual proteases that traveled with the HS and may have been enriched by the ATIII-HS combination. The beads were collected by centrifugation, and the supernatant was then dialyzed against distilled H2O. The sample was adjusted to PBS containing 10 mM MgCl2 for subsequent FGFR1 affinity purification.
R1L2-GST was prepared fresh and immobilized on GSH-Sepharose 4B beads just before use. The sample from ATIII chromatography was applied to a 100-µl column of beads. After standing for 1 h at room temperature, the column was then extensively washed with buffer 1, and then the bound HSPG was eluted with 1 M NaCl in PBS containing 1% Triton X-100 and 10 mM MgCl2 (buffer 6). The sample was then dialyzed against distilled water.
The dialyzed sample was then subjected to a second round of ATIII affinity chromatography as described above, except the HSPG was eluted with buffer 3 after extensive washing of the column with PBS. The sample was then dialyzed against water and freeze-dried. The dried sample was reconstituted in PBS containing 0.5% CHAPS and then applied to a high-performance liquid chromatography gel filtration column (7.8 x 300 mm; Biosil SEC-400; Bio-Rad, Richmond, CA), which was previously equilibrated with PBS containing 0.1% CHAPS. The column was developed with PBS containing 0.5% CHAPS and 1 M NaCl at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected.
The GAG content of HSPG fractions was monitored by the carbazol reaction (9) and staining with the cationic dye Safranin O in dot blots (15) . Protein content was estimated by SDS-PAGE and silver stain. The bicinchoninic acid Protein Assay (Pierce) was applied when quantities of protein were sufficient. Sulfated material was monitored by spiking samples with extracts from cells labeled with [35S]sulfate. The activity of HSPG was monitored by the ability to form a binary complex with immobilized FGFR1 competent to bind 125I-labeled FGF1.
Binding of 125I-labeled FGF1 to Heparin- or HSPG-FGFR Complexes.
The preparation, iodination, and quality control of FGF1 and methods for measurement of the activity of HSPG or heparin in the FGFR complex assembly assay have been described in detail elsewhere (7, 8, 9, 10
, 14)
. Briefly, FGFR-GST fusion protein was extracted from Sf9 cells and immobilized on GSH beads. After extensive washing, the beads were preincubated with heparin or HSPG. The heparin- or HSPG-bound beads then were washed with PBS containing 1% Triton X-100 and 10 mM MgCl2 and incubated with iodinated FGF1. After washing with PBS, the FGF1-incorporated beads were incubated with disuccinimidyl suberate and extracted with protein sample buffer for SDS-PAGE. The assay measures two stages in the formation of the liganded FGFR complexes. First, it measures the ability of the HSPG to form an FGF-independent binary complex with immobilized FGFR1 or its structural modules. Second, it measures the subsequent binding of radiolabeled FGF to the binary complex (9
, 10
, 14)
. Specificity of the FGF binding was periodically checked by competition with unlabeled FGF and covalent affinity cross-linking analysis as described (7, 8, 9, 10)
.
Expression of Recombinant Syndecan-1.
Full-length rat syndecan-1 cDNA was a gift of Dr. Magnus Höök (Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX). A cDNA coding for syndecan-1 fused to GST at the COOH terminus was constructed as in Fig. 4
. A KpnI site was introduced to the cDNA by PCR. The PCR fragment was generated with 5' primer pSW1 (5'-AACTACCAATCAGCTTCCTGCAGG-3'), 3' primer pSW2 (5'-TCCGGTACCGGCGTAGAACTCTTCCTGCCTGGT-3'), and the syndecan-1 cDNA template. After digestion with restriction enzymes KpnI and PstI, the PCR fragment was ligated with the upstream coding sequence of syndecan-1 at a PstI site and the GST coding sequence at the KpnI site, respectively. The cDNA coding for syndecan-1-GST was cloned into pBluescript SK vector at HindIII and EcoRI sites, the sequence determined, and then the cDNA was cloned into pcDNAzeo3.1 (Invitrogen, San Diego, CA) at PstI and XhoI sites.
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Expression of Syndecan-1 mRNA.
Total RNA was extracted from one 75-cm2 flask of premalignant prostate tumor epithelial cells (DTE), stromal cells from the same nonmalignant Dunning R3327PAP tumor (DTS), and cells from fully malignant AT3 tumors (AT), respectively, using the ULTRASPEC RNA Isolation Kit (Biotecx Laboratories, Inc., Houston, TX). RNA was separated in a 1% agarose gel containing 8% formaldehyde, blotted onto positively charged nylon membranes (Ambion, Austin, TX), and UV cross-linked. The blot membranes were hybridized in ULTRAhyb hybridization solution (Ambion) at 42°C for overnight with 1 x 106 cpm/ml of a 298-bp rat syndecan-1 ectodomain cDNA probe. Blots were washed twice at 42°C with 2 x SSC, 0.1% SDS, followed by one wash at 42°C for 15 min and one wash at 65°C for 2 h in 0.1 x SSC with 0.1% SDS, and then exposed to film (Kodak Biomax MS) with an intensifying screen at -80°C. The membranes were rehybridized with a 32P-labeled 286-bp fragment of rat ß-actin probe to evaluate the relative amount of RNA/lane.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and described in "Materials and Methods." Activity was monitored by the ability to form a stable complex with immobilized recombinant Ig module II of FGFR1 (R1L2) isolated from baculoviral-infected Sf9 insect cells. After the removal of unbound material, the ability of the HSPG-R1L2 complex to bind radiolabeled FGF1 was monitored by specific binding and covalent affinity cross-linking. It should be emphasized that this assay method is essential to monitor specifically the HS motif that interacts with the FGFR ectodomain to form a composite binary complex that is competent to bind FGF in absence of added HS or its mimic heparin. The formation of binary complexes before introduction of the radiolabeled FGF into assays allows FGFR and, in this case, R1L2 to affinity select HS chains exhibiting the specific FGFR-binding motif and discard interfering motifs that may only bind FGF but not FGFR (14)
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. A specific activity of the crude cell lysate could not be assigned because of either the low specific activity or substances interfering with FGF1 binding. The specific activity of the fraction representing about 25% of the 35S-labeled material from the crude extract that was recovered after anion exchange (DEAE) chromatography was therefore assigned a value of 1. The weak ion exchange column and stringent washing conditions were used to minimize contaminating proteins and poorly sulfated HS that conceivably interfered with the measurement of a specific activity. Preparations were sequentially scaled up to 2.5 x 1011 DTE cells pooled from 500 75-cm2 tissue culture flasks. Every tenth cell culture in a preparation was metabolically labeled with [35S]sulfate. AT affinity chromatography increased the activity for FGFR complex formation by 20-fold, and FGFR1L2 affinity chromatography yielded an additional 10-fold increase for a total of a 200-fold increase in specific activity over the pool recovered from DEAE chromatography. A repeat of the AT affinity step and a final fractionation based on size resulted in an additional increase of 2.5-fold with a final purification of 622-fold (Table 1)
. Eluted fractions from a final gel filtration step were subjected to analysis by ability to bind FGFR1L2 and support FGF1 binding and by SDS-PAGE (Fig. 1)
. The protein content of fractions 8 to 12 across the main peak of activity was below the limits of detection by both spectrophotometric and staining methods. Analysis by SDS-PAGE and silver staining revealed that fractions 13 to 16 exhibited two main bands of apparent mass of 40 and 60 kDa, whereas fractions 17 to 20 exhibited predominantly a 40-kDa band.
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. A homology search using BIC2 in the SWISSPROT database revealed that 11 of the 13 residues matched those in the NH2-terminal sequence of the rat syndecan-1 core protein deduced from cDNA (Fig. 2)
. The abrupt loss of sequence yield at cycle 14 was consistent with glycosylation at that site, which is a glycosylated serine in syndecan-1 (16)
. This was within the predicted signal sequence (22 to 27 amino acids) deduced from rat syndecan-1 cDNA according to the rules of signal sequence removal.5
Consistent with the rules of signal sequence removal, our results suggest that the signal sequence of rat syndecan-1 consists of 23 amino acids. To date, subsequent analysis of cloned syndecan-1 cDNAs is identical to the reported rat syndecan-1 cDNA in GenBank. This suggests that residues 3 and 13 may be a sequence error, although we cannot eliminate the possibility of rare variants of syndecan-1 core protein that appear in tumors.
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. Expression of recombinant mRNA in selected cell lines was confirmed before further analysis. DEAE ion exchange chromatography and GSH-affinity chromatography were used to recover HSPG from the cell lysates. Expression product was monitored by 35SO4 and analyzed on SDS-PAGE by autoradiography and immunoblot with anti-GST antiserum. Lysates of transfected cells yielded about 1.5 times the total amount of 35S-labeled material that was present in untransfected cells. The GSH-affinity step resulted in a recovery of about 6% of 35S-labeled material from the lysates of transfected cells enriched by DEAE chromatography. Radiolabeled material recovered from control samples from the same number of untransfected cells was negligible. Autoradiographic analysis on 5% SDS-PAGE revealed Hase I- and Case ABC-sensitive material spanning about 85 kDa to greater than 210 kDa, which was the most intensely labeled area (Fig. 4A
, arrow). Immunoanalysis confirmed the presence of the GST tag in the core protein (Fig. 4B)
. Treatment with Hase and Case caused an expected reduction in the higher molecular weight material with the concurrent increase in intensity of bands of lower molecular weight (Fig. 4B
, arrows). Hase cleaves highly sulfated polysaccharide chains containing 1–4 linkages to 2-O-sulfated iduronic acid residues but does not completely digest the HS chains. Some of the residual carbohydrate chains may also be incompletely digested chondroitin sulfate chains. Analysis on 7.5% SDS-PAGE revealed distinct bands of about 80 kDa and lower (Fig. 4C
, open arrow). These bands are presumably poorly glycosylated or unglycosylated syndecan-1 core proteins that fail to be retained on the 5% gels in Fig. 4B
. The predicted molecular mass of the syndecan-1-GST protein core from cDNA without carbohydrate is 57 kDa, but syndecan core proteins exhibit an artificially high molecular mass on SDS-PAGE (16)
. Treatment with Hase, Case, or the combination (data not shown) had no effect on the bands of 80 kDa and lower. Treatment with Hase in the absence of Case and subsequent analysis on 7.5% gels resulted in appearance of a broad band with mean molecular mass of 147 kDa (Fig. 4C
, black arrow). This presumably reflects syndecan-1 core proteins that penetrate the 7.5% gel bearing partially digested HS chains and native chondroitin sulfate chains.
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. Activity was compared from lysates from the indicated number of transfected cell lines and untransfected control cells after enrichment by DEAE chromatography. Activity was markedly higher for the lysate from cells transfected with GST-syndecan-1 cDNA (DTE-Syn1) than the untransfected DTE cells (Fig. 5A)
. Material from the enrichment by DEAE ion exchange that was subsequently subjected to purification by GSH-affinity exhibited similar differential activity between transfected and untransfected DTE cells (Fig. 5B)
. Treatment of the purified preparation before the incubation with FGFR1L2 with Hase I, which left significant levels of presumably poorly sulfated HS and chondroitin sulfate chains on the core protein (Fig. 4)
, destroyed all of the activity.
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confirms that DTE cell syndecan-1 also formed binary complexes with FGFR2ßIIIb competent to support binding monitored by covalent affinity cross-linking to radiolabeled FGF1 and FGF7.
Malignant AT3 Tumor Cells Exhibit Decreased Levels of Syndecan-1 mRNA.
To determine whether fully malignant cells exhibit a change in syndecan-1 core protein expression from the premalignant DTE cells, we compared levels of syndecan-1 mRNA by Northern hybridization. Expression was compared in DTE cells derived from slow-growing, hormone-responsive Dunning R3327PAP tumors, stromal cells (DTS) from the same tumors, and cells from fully malignant AT3 tumors (AT), which arise from the parent tumors after castration of male hosts, or prolonged passage of DTE cells through animals in absence of stromal cells (2)
. Fig. 6
confirms that the level of syndecan-1 mRNA is significantly reduced in AT cells to about the levels observed in the DTS cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Distinct changes occur within the FGF family in model nonmalignant, hormone-responsive rat prostate tumors during their slow progression to malignancy. These include changes that underlie both loss of stromal instructions, which maintain a balance between epithelial cell growth and the growth-limiting processes of apoptosis, morphogenesis, and differentiation, and gain of factors which bypass the latter and drive the malignant phenotypes (Fig. 1A)
. In the Dunning R3327PAP model, a major hallmark is the loss of resident epithelial cell FGFR2 through splice switching that abrogates recognition of stromal FGF7 and FGF10 in some cells and, of higher impact, the loss of expression of the FGFR2 gene altogether (1, 2, 3, 4)
. This directionally specific paracrine communication can promote growth under some conditions but otherwise has a net growth-regulating effect that maintains homeostasis (3
, 6)
. A second hallmark is the appearance of FGFR1 in epithelial cells, the expression of which is normally limited to stromal cells. Artificial expression of FGFR1 in the premalignant epithelial cells accelerates progression to malignancy even in the presence of resident FGFR2 (6
, 17)
. Thus, the abnormal expression of FGFR1 in epithelial cells correlates with the malignant phenotype and is a key element in both progression to and support of the malignant state (3
, 6
, 17)
. The acquisition of a mitogenic response of premalignant epithelial cells to ectopic FGFR1 is a progressive phenomenon. Although FGFR1 in freshly transfected cells binds FGF and exhibits autophosphorylation and transcriptional activity, acquisition of the mitogenic response to FGFR1 and coincident accelerated progression to the malignant phenotype is a time-dependent process (3
, 6)
.4
Therefore, an understanding of the factors involved in FGFR1 signaling in both premalignant and fully malignant epithelial cells where its abnormal presence promotes and supports malignancy, respectively, is critical for understanding the gradual evolution of the malignant state.
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. Although a significant body of knowledge concerning the interaction with heparin, an artificial mimic of HS, has emerged, little is known about the nature and origin of the cellular HS chains that act as an integral subunit of the FGFR complex, in general, and much less is known about those that act in prostate tumor progression, in particular. Here, we show that although FGFR1 is normally not expressed in nonmalignant prostate tumor epithelial cells, the epithelial cells express HS chains that form a binary complex with the FGFR1 ectodomain that is competent to bind FGF1 and FGF2. We exploited two specific properties of FGFR-interactive HS to directly purify by affinity chromatography the native HS from the epithelial cells. These included (a) the specific requirement of the FGFR interaction for anticoagulant HS that can be enriched because of affinity for AT III (13)
and (b) dependence of the interaction of anticoagulant HS with physiological concentrations of divalent cations (10)
. AT affinity chromatography used at two different steps increased the specific activity of the HSPG based on HS content by at least 30 times while reducing the protein content of the HSPG preparation by over 100-fold. For FGFR affinity chromatography, we used the isolated Ig module II of FGFR1, which exhibits a major heparin-binding domain (3
, 8
, 14)
and an affinity for FGF1 equal to FGFR1
and FGFR1ß containing two and three Ig modules, respectively (14)
. This eliminated heterogeneity of the HS part of the preparation with chains that only interact with Ig module III (14
; Fig. 7B
). This affinity purification step increased specific activity of the HSPG preparation 12 times while further reducing the protein and GAG content by 5 and 10 times, respectively. These affinity steps, combined with conventional ion exchange and gel filtration chromatography, resulted in a 622-fold purification of the active GAG from the GAG enriched from cell lysates by ion exchange. Although activity could not be accurately measured in whole cell lysates, the over 3-fold enrichment of GAG by ion exchange may indicate that the procedure may represent purification as high as 1000–2000-fold from the total GAG in cell lysates. These results demonstrate directly that the premalignant epithelial cells exhibit an HS motif that complements the primary HS binding site within Ig module II of ectopic FGFR1. They also pointed out that the motif with those specific properties is rare and may constitute less than 0.6% of total cellular GAG (assuming a 1% activity recovery by the described procedures). The low quantity presents significant challenges in scale-up. However, the specific approach described here yields highly purified HSPG in respect to HS chains and core protein (syndecan-1) for structural identification of HS motifs and HS-core protein relationships involved directly in assembly and regulation of activity of FGFR-signaling complexes among different cell types.
Syndecan-1 Bears the Rare Motif that Combines with FGFR1 in Premalignant Epithelial Cells to Form an FGF-binding Complex.
Generation of a clear single NH2-terminal sequence from the affinity-purified and FGFR1-specific HS required a final fractionation based on size in which peak fractions exhibited an additional 1.6-fold increase in specific activity and in which protein content was reduced below the level of detection by routine methods. The fraction with the highest specific activity exhibited a single NH2-terminal sequence beginning with NH2-terminal residue ileu-24 and, except for two differences, exhibited an NH2 terminus identical to rat syndecan-1. To our knowledge, this is the first report of the NH2-terminal sequence of native syndecan-1. We confirmed that the HS chains of syndecan-1 from the premalignant prostate epithelial cells participate in the formation of a binary complex with Ig module II of FGFR1 by demonstration that recombinant syndecan-1 expressed from external cDNA in the DTE cell also forms the complex. We cannot eliminate the possibility that core proteins bearing active HS chains other than syndecan-1 are present in other active fractions from the final gel filtration step or in the 10% of cell-associated HS that is detergent-resistant. However, our combined results are strongly suggestive that syndecan-1 may be the predominant HS provider for ectopically expressed FGFR1 in the premalignant epithelial cells (8
, 18)
.
Our results provide the first direct evidence that syndecan-1, through rare motifs within its HS chains, can act as a subunit of an FGFR complex through affinity of the HS chains for FGFR. It has been estimated that an epithelial cell displays about 106 syndecan-1 molecules/cell (16) . On the basis of a 1% recovery through the five steps of purification beginning with the initial cell lysates, we estimate that the number of specific syndecan-1 molecules that can act as an integral subunit of the FGFR1 complex may be less than 10,000/cell. Although we cannot eliminate the possibility that the complementary HS subunit for FGFR1 complexes is extremely low, specifically in the nonmalignant epithelial cells where FGFR1 is not normally expressed, the low level of HS molecules approximates the number of FGFR-binding sites expressed on most mammalian cells including prostate epithelial cells (19) . Although the level of endogenous HS motifs that form complexes with the resident FGFR2IIIb in the epithelial cells relative to ectopic FGFR1 is unclear, we showed that the recombinant syndecan-1 expressed in the epithelial cells also bears an HS motif that forms a binary complex competent to bind both FGF1 and stromal-derived FGF7 for which the FGFR2IIIb exhibits specificity relative to FGFR1 (1, 2, 3, 4) . Whether ectopic FGFR1 shares the same motif within a single syndecan-1 HS chain normally allocated to resident FGFR2IIIb and binds to an FGFR-specific motif within the same or different chains on the same syndecan-1 or whether the two FGFRs interact with chains on separate syndecan-1 molecules are challenging questions and under investigation.
Potential Role of Syndecan-1 and Its HS Chains in FGFR-related Progression to Malignancy.
The results presented here, our current knowledge of changes in the FGF ligand and FGFR isoforms that occur during progression to malignancy in prostate tumor models, and current models of the oligomeric FGFR-signaling complex of pericellular matrix HS chains suggest that syndecan-1 may play a multifunctional and multistep role in the process (Fig. 7B)
. As a source of the HS chain subunits for FGFR2IIIb in the epithelial cells of normal prostate and nonmalignant hormone-responsive and well-differentiated prostate tumors, through its HS chains, syndecan-1 plays a key role in support of stromal-dependent homeostasis of the epithelial compartment. On the one hand, alteration in HS chain composition, depression in syndecan-1 GAG content, or syndecan-1 gene expression that depresses FGFR2-specific HS motifs may cause FGF-independent activity of FGFR2 complexes and perturb homeostasis through perturbation of differentiation, morphogenesis, and apoptosis with early and indirect contributions to progression toward malignancy. On the other hand, alterations in FGFR2- and FGF7/FGF10-binding motifs within HS chains may abrogate recognition of stromal instructions through FGF7 and FGF10 to indirectly promote progression as proposed previously (2
, 3)
for the splicing switch from exon IIIb to exon IIIc. However, because of the net tumor growth-controlling role of the FGFR2 kinase, these alterations are insufficient to drive progression to malignancy.
The presence of an FGFR1-binding motif among the HS repertoire in the premalignant epithelial cells suggests that activity of initially low levels of ectopic FGFR1 that do not exceed the level of the HS motifs will be limited and dependent on activation by FGF family members other than FGF7 and FGF10. Rejection of FGF7 and FGF10 by FGFR1 is strict and encoded in the FGFR1 protein structure, independent of the HS partner (14)
. Separate experiments repeatedly show that increasing levels of FGFR expression or depression of HS chain sulfation alters specificity for FGF6
(9)
and results in increasing FGF-independent activity.7
The FGF-independent activity of FGFR1 may arise by simply increased expression of FGFR1 that exceeds the level of the restrictive HS partner we report here that is estimated to be as few as 10,000 sites/cell or changes that result in reduction of the already low FGFR1-interactive motif from syndecan-1. In contrast to the impact of FGFR2-specific HS chains, alterations in FGFR1-specific HS chain composition, depression in syndecan-1 GAG content, or depression of syndecan-1 gene expression that results in depression of FGFR1-specific HS motifs serve to remove additional restrictions on ectopic FGFR1 activity (Fig. 7B)
. High levels of expression of FGFR1 and unresponsiveness to external FGFs coupled with the loss of expression of all of the FGFR2 isoforms is characteristic of the fully malignant state to which the nonmalignant tumors and derived cells progress over time (5
, 6)
.4
In summary, because syndecan-1 bears HS chains that serve as an integral subunit of both resident growth-limiting FGFR2 and ectopic progression-promoting FGFR1, its impact on FGFR-related malignant progression of premalignant epithelial cells likely lies predominantly in FGFR-specific alterations in its HS chains. However, a general depression of syndecan-1 in the advanced stages of malignant progression when FGFR2 and other tumor growth restrictions have been lost will serve to further remove restrictions on FGFR1 activity that includes dependence on FGF. We demonstrated in this report that a depression of syndecan-1 at the mRNA level is characteristic of autonomous, hormone- and stromal-independent tumor cells (AT) relative to their nonmalignant parent DTE cell. Reduction in sulfation (20
, 21) and an overall reduction in syndecan-1 expression (22)
have been generally reported to correlate with poorly differentiated tumors in advanced stages of malignancy. Restoration of syndecan-1 and particularly FGFR- and FGF-specific sulfation patterns within HS may be of utility in restoration of FGF-dependence in advanced malignancies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Public Health Service Grant Nos. DK40739 and DK35310 from the National Institute of Diabetes and Digestive and Kidney Diseases, and CA59971 from the National Cancer Institute. 
2 To whom requests for reprints should be addressed, at Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Boulevard, Houston, TX 77030-3303. Phone: (713) 677-7522; Fax (713) 677-7512; E-mail: wmckeeha{at}ibt.tamu.edu
3 The abbreviations used are: FGF, fibroblast growth factor; HS, heparan sulfate; HSPG, HS proteoglycan; FGFR, FGF receptor; Ig, immunoglobulin; GSH, glutathione; GST, GSH S-transferase; Sf9, Spodoptera frugiperda cells; DEAE, diethylaminoethyl; AT, antithrombin; GAG, glycosaminoglycan; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Hase, heparinase; Case, chondroitinase.
4 F. Wang, K. McKeehan, C. Yu, M. Kan, and W. L. McKeehan. Phospholipase C
-interactive phosphotyrosine 766 is required for acquisition of the proliferative response to ectopic FGFR1 in prostate epithelial cells, submitted for publication.
5 SignalP V1.1 World Wide Web Prediction Server, Center for Biological Sequence Analysis; http://www.cbs.dtu.dk/services/, accessed on 06/02/00.
6 M. Kan, X. Wu, F. Uematsu, F. Wang, and W. L. McKeehan. The heparan sulfate subunit of the FGF receptor complex determines the specificity of prostate stromal and epithelial cells for different FGF isoforms, manuscript in preparation.
Received 11/15/00. Accepted 4/26/01.
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 C. Jin, F. Wang, X. Wu, C. Yu, Y. Luo, and W. L. McKeehan Directionally Specific Paracrine Communication Mediated by Epithelial FGF9 to Stromal FGFR3 in Two-Compartment Premalignant Prostate Tumors Cancer Res., July 1, 2004; 64(13): 4555 - 4562. [Abstract] [Full Text] [PDF]
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C. Jin, K. McKeehan, W. Guo, S. Jauma, M. M. Ittmann, B. Foster, N. M. Greenberg, W. L. McKeehan, and F. Wang Cooperation between Ectopic FGFR1 and Depression of FGFR2 in Induction of Prostatic Intraepithelial Neoplasia in the Mouse Prostate Cancer Res., December 15, 2003; 63(24): 8784 - 8790. [Abstract] [Full Text] [PDF]
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X. Wu, C. Jin, F. Wang, C. Yu, and W. L. McKeehan Stromal Cell Heterogeneity in Fibroblast Growth Factor-mediated Stromal-Epithelial Cell Cross-Talk in Premalignant Prostate Tumors Cancer Res., August 15, 2003; 63(16): 4936 - 4944. [Abstract] [Full Text] [PDF]
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 D. Qiao, K. Meyer, C. Mundhenke, S. A. Drew, and A. Friedl Heparan Sulfate Proteoglycans as Regulators of Fibroblast Growth Factor-2 Signaling in Brain Endothelial Cells. SPECIFIC ROLE FOR GLYPICAN-1 IN GLIOMA ANGIOGENESIS J. Biol. Chem., April 25, 2003; 278(18): 16045 - 16053. [Abstract] [Full Text] [PDF]
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S. Knox, C. Merry, S. Stringer, J. Melrose, and J. Whitelock Not All Perlecans Are Created Equal. INTERACTIONS WITH FIBROBLAST GROWTH FACTOR (FGF) 2 AND FGF RECEPTORS J. Biol. Chem., April 19, 2002; 277(17): 14657 - 14665. [Abstract] [Full Text] [PDF]
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F. Wang, K. McKeehan, C. Yu, and W. L. McKeehan Fibroblast Growth Factor Receptor 1 Phosphotyrosine 766: Molecular Target for Prevention of Progression of Prostate Tumors to Malignancy Cancer Res., March 1, 2002; 62(6): 1898 - 1903. [Abstract] [Full Text] [PDF]
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