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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 6, 2001 as doi:10.1096/fj.00-0661fje. |
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,¶2
* Division of Bioengineering and Environmental Health,
Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, and
¶ Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
2Correspondence: MIT, 77 Massachusetts Ave., Building 16–561, Cambridge, MA 02139, USA. E-mail: rams{at}mit.edu
SPECIFIC AIMS
To investigate the effect of heparin/heparan sulfate-like glycosaminoglycans (HLGAGs) derived from smooth muscle cells (SMCs) on fibroblast growth factor 2 (FGF2) signaling, we used HLGAG-degrading enzymes, or heparinases (heps), to depolymerize SMC-derived HLGAGs from the cell surface and assayed their ability to modulate FGF2-mediated proliferation on engineered cell lines expressing defined FGF receptor (FGFR) isoforms.
PRINCIPAL FINDINGS
1. Distinct SMC-derived HLGAG fragments generated by heparinase
digestion
To generate SMC-derived HLGAG fragments, we treated cells
with either hepI, II, or III and harvested 
100 ng of cell surface
HLGAGs for compositional analysis and cell-based assays. As expected
from the known substrate specificities of the heparinases, HLGAG
fragments derived from hepI digestion were less highly sulfated than
those fragments resulting from hepII or III treatment (Table 1
). Accordingly, hepIII fragments were the most highly sulfated, and
hepII treatment resulted in the release of fragments with a composition
intermediate between that of hepI and hepIII. Conversely, hepI
fragments were relatively deficient in 6-O and 2-O sulfation. Notably,
all three heparinase-treated samples generated approximately the same
amount of HLGAG fragments, viz., 50 ng/ml. These same fragments,
isolated from the SMC cell surface, were used in proliferation assays
to determine their ability to promote or inhibit FGF2 signaling. Very
few HLGAG fragments could be isolated from PBS-treated cells,
demonstrating that it is a good negative control for this set of
experiments. With each receptor isoform tested, the response of the
engineered BaF3 cell lines was the same whether the supernatant from
PBS-treated cells was used or whether the BaF3 cells were treated with
DMEM medium, free of exogenous HLGAGs.
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2. Differential response toward heparin by cells expressing defined
FGFR isoforms
Proliferation assays were performed using four separate BaF3 cell
lines, each engineered with a distinct FGFR isoform: FGFR1c, FGFR2b,
FGFR2c, and FGFR3c. To determine the types of receptor isoforms
expressed in each of these cell lines, their FGFR ‘profile’ was
assayed using RT-PCR. Each cell line was found to express exclusively
its designated FGFR isoform and no other splice variants. To
functionally characterize these four cells lines, each was cultured in
the presence or absence of FGF2 with mast cell heparin. It is known
that heparin binds with high affinity to FGF2 and is able to potentiate
FGF2-mediated signaling with certain FGFR isoforms. We find that FGF2
with heparin elicits a significant proliferative response in cells
expressing FGFR1c, 2c, and 3c, but not FGFR2b, consistent with its
known affinity to each receptor isoform.
3. Differential response toward SMC-derived HLGAG fragments by
FGF2-stimulated cells
The proliferative response of cell surface HLGAG fragments
prepared by different heparinase treatments was tested by culturing
transfected BaF3 cells with or without FGF2. For ease of comparison,
all values are expressed as proliferative indexes (PI), where a PI of 1
indicates the maximal response and a PI of 0 indicates a negligible
response. The reference point (PI=1) for this set of experiments was
BaF3 cells stimulated with 50 ng/ml FGF2 and 500 ng/ml heparin. This
point was chosen from our previous study and is based on a rigorous
determination of the dose-response relationship for FGF2/heparin.
Establishing this reference point provides a mechanism by which the
cell data can be standardized so that comparisons between HLGAG
preparations and across receptor isoforms can be accomplished. A
pronounced increase in the PI was observed in all cells upon the
introduction of exogenous FGF2 and/or SMC fragments except for
FGFR2b-transfected cells. In BaF3 cells expressing FGFR1c, HLGAG
fragments derived from either hepII or hepIII treatment elicited a
significantly higher PI than the PBS control (Fig. 1A
), when exogenous FGF2 was added to the cells. The potency
of these fragments rivaled that of heparin itself. Conversely, hepI
fragments were unable to significantly promote FGF2 activity in
FGFR1c-expressing BaF3 cells beyond the response obtained by PBS alone,
where stimulation was observed upon the addition of FGF2.
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BaF3 cells expressing FGFR2c gave a significant response to FGF2
stimulation whether exogenous HLGAG fragments were present or not
(Fig. 1B
). The PI of PBS-treated cells was not significantly
different from those of cells treated with SMC fragments derived from
either hepII or III treatment. Thus, it appears that HLGAG fragments
from SMC cells do not potentiate FGF2 signaling through FGFR2c in this
case, and exogenous HLGAGs at best only slightly promote FGF2 signaling
through FGFR2c. In the presence of hepI-treated HLGAG fragments,
FGF2-mediated proliferation is decreased such that there is no
significant difference in the PI for BaF3/FGFR2c cells in the presence
and absence of FGF2. This decrease may be the result of an inhibitory
effect of hepI-generated fragments in that a significant increase in
the PI was observed in the PBS control upon addition of FGF2.
With BaF3 cells expressing FGFR3c, it appears that HLGAG fragments are
required for significant FGF2 signaling, as was observed in the
FGFR1c-expressing cell line (Fig. 1C
). In direct contrast to
FGFR1c-transfected cells, however, hepI- or II-treated HLGAG fragments
elicited a significantly higher PI than the PBS control in the FGFR3c
engineered cell line whereas hepIII-derived fragments failed to elicit
a significant response in this cell type. In contrast to FGFR1c and 2c
transfected cells, no significant response is seen in FGFR3c cells upon
the addition of FGF2 unless exogenous HLGAGs are present; the PI for
the PBS-treated sample in this case is 0.02 ± 0.17 vs. PIs of
0.78 ± 0.12 and 0.67 ± 0.20 for FGFR1c- and 2c-transfected
cells, respectively (Fig. 1D
).
As a control for the above studies and to investigate whether our results are extendable to other members of the FGF family, we completed experiments with the BaF3 cell lines, but used FGF1 as the ligand instead of FGF2. It was shown earlier that FGF1 signals equally through each FGFR isoform used in this study, including FGFR2b (to which FGF2 does not appreciably bind). With FGFR1c, 2c, and 3c, the results obtained with FGF1 are consistent with the observed effect of the SMC fragments on FGF2-mediated proliferation (data not shown). This is even true when one considers the effect of fragments on FGF-mediated signaling through FGFR2c, where FGF1 is a better ligand than FGF2. Thus, in each case the effect of SMC HLGAG fragments on FGF1-mediated signaling paralleled its effect on FGF2-mediated signaling. The one notable exception was with FGFR2b. Unlike FGF2, FGF1 induced significant proliferation of BaF3 cells expressing the FGFR2b isoform in the presence of either heparin or hepIII-generated SMC fragments (data not shown), consistent with its known receptor specificity and ensuring the authenticity of our findings.
CONCLUSIONS
SMC proliferation plays an important role in the pathophysiology of many disease conditions, including atherosclerosis. Among the important stimulatory molecules for SMC migration and proliferation is FGF2, a potent mitogen that requires interaction with HLGAGs for receptor binding and activation. To explore the structure–function relationship between SMC-derived HLGAG fragments and FGFR-dependent proliferation, we designed a series of experiments using two well-established systems: the BaF3 cell lines, which express distinct FGFR isoforms, and the heparinases, which cleave HLGAGs with known substrate specificity. Our data show that FGF2 signaling is differentially modulated by HLGAG fragments of distinct disaccharide composition through different FGFR isoforms.
In FGFR1c-transfected cells, the observation that hepIII-derived
fragments produced the highest PI and the finding that hepIII-treated
HLGAG fragments contained the most 6-O sulfated disaccharide units
(particularly
U2S-HNS,6S) support the
notion that 6-O sulfated oligosaccharides generated by heparinase
treatment promote FGF2 activity. In contrast, hepI fragments, which are
relatively deficient in 6-O sulfation, produced the lowest PI. This
observation is consistent with biochemical studies indicating that 6-O
sulfation is required for FGF2 activity but not for FGF2 binding.
Unlike cells expressing FGFR1c, 2c-transfected cells in the presence of
FGF2 do not respond to hepII- or III-derived fragments, where the PI
was similar to the PBS-treated control. It is possible that SMCs may
not express the ‘correct’ HLGAG sequences for facilitating FGF2
signaling through FGFR2c. In the presence of hepI-derived HLGAG
fragments, FGF2 signaling is slightly suppressed, so that there is no
significant difference in the PI for FGFR2c-transfected cells in the
presence and the absence of FGF2. Thus, hepI-derived fragments may in
this case actually inhibit FGF2 binding to its cogent receptor.
In contrast to the results obtained with FGFR2c, it appears that with certain receptor isoforms (FGFR1c and 3c), HLGAG fragments of proper length and sulfation pattern are important for signaling to occur. This conclusion is reinforced by the fact that hepI- or II-treated HLGAG fragments significantly increase FGF2 signaling whereas hepIII-derived fragments abrogate it, indicating that sulfation number and position are of utmost importance. These results are in stark contrast to the response of BaF3 cells expressing FGFR1c to FGF2, where hepIII treatment results in the production of SMC-derived HLGAG fragments that strongly potentiate FGF2 activity whereas hepI-derived SMC fragments do not support proliferation. Notably, hepII-derived HLGAG fragments supported a significant FGF2 response in both FGFR1c- and 3c-expressing cells compared with the PBS control, as would be expected from hepII’s broad substrate specificity. Consistent with another study that used porcine mucosal HLGAGs, our results indicate that FGF2 signaling through a given FGFR is differentially modulated by chemically distinct HLGAG fragments.
We extend this prevailing notion to the physiologically relevant form
of HLGAGs found at the cell surface. We find that their ability to
modulate FGF2 signaling is highly dependent on the composition and
length of the fragments as well as the FGFR isoform expressed by the
recipient cells. The modulatory effect of HLGAG fragments is not
restricted to FGF2, but is also true for FGF1 (data not shown). Taken
together, differences in the chemical content of HLGAGs from cell
surface can lead to a potential mechanism for the fine control of FGF2
activity in biological processes. For instance, FGFR1 is predominantly
expressed in proliferating adult rat SMC whereas FGFR3 is the preferred
type in newborn rat SMCs. Thus, cells could take advantage of the
inherent structural diversity of HLGAGs to regulate FGF signaling
through a receptor or set of receptors by dynamically altering the
HLGAG sequences on cell surface. The schematic diagram (Fig. 2
) depicts a model to account for the differential effects on FGF2
signaling mediated separately by HLGAG fragments generated by
hepI and hepIII, respectively. In this model, a proper spatial display
of 2 O-, 6 O-, and N-sulfated groups on HLGAGs would allow optimal
interaction with FGF2 and FGFR isoforms. Further characterization of
‘active’ HLGAG fragments that provide the required specificity for
FGF2 activity would not only enable important insight into identifying
the structural requirement of the ternary complex, but would also aid
the development of therapeutics for targeting proliferative vascular
disorders.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0661fje ; to cite
this article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0661fje 
This article has been cited by other articles:
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 Z. L. Wu, L. Zhang, T. Yabe, B. Kuberan, D. L. Beeler, A. Love, and R. D. Rosenberg The Involvement of Heparan Sulfate (HS) in FGF1/HS/FGFR1 Signaling Complex J. Biol. Chem., May 2, 2003; 278(19): 17121 - 17129. [Abstract] [Full Text] [PDF]
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, P > 0.3 (¥).
D) Summary of the PIs resulting from treatment of each
cell type with FGF2 and the corresponding SMC HLGAG
oligosaccharide preparation.