Growth Factor Binding Proteoglycans

Since the original observation that members of the FGF family bind heparin, many growth factors have been demonstrated to bind to heparin, and it is now accepted that this interaction is indicative of important regulatory activities of HS chains on HSPG. The importance of these interactions for the prototypic heparin-binding growth factor, FGF-2, was originally revealed in cells that were rendered HS deficient through either genetic mutation or treatment with hepa-rinase or sulfation inhibitor chlorate (68,69). In these instances, it was observed that FGF-2 binding to its receptor was significantly reduced in the absence of HS. The data indicate that HS participates in forming a ternary complex so that binding of FGF-2 is stabilized through the simultaneous binding to both its receptor and HS (70,71). Hence, kinetic analysis of these binding events suggested that the principal impact of HS is to reduce the rate of FGF-2 dissociation from its receptor without significantly altering the association rate (70,72,73). While the majority of the studies on the co-receptor activity of HS have focused on FGF-2, high-affinity binding of a number of growth factors for their receptors has similarly been shown to depend on HS. For example, receptor binding of VEGF is reduced in endothelial cells treated with heparinase or chlorate (43,74-77), and the binding and activity of heparin-binding epidermal growth factor-like growth factor to EGF receptors on vascular smooth muscle cells are reduced by chlorate treatment (78). Thus, HS likely plays a general role in enhancing the sensitivity of receptors for heparin-binding growth factors through its ability to increase the observed affinity for binding.

As one considers the classic principles of pharmacology, which tend to consider parameters such as expression of receptors on target cells along with traditional elimination and tissue adsorption constants, it is clear that additional components will need to be included to model growth factor responsiveness. Moreover, since the therapeutic window of growth factors in vivo is likely very narrow with the potential for toxicity or unwanted responses at high doses, approaches that simply aim to maximize growth factor concentration at the target site for as long as possible will not be effective. In vitro studies have revealed a complex concentration-response relationship for FGF-2 that is further modulated by the relative presence of HSPGs on the cell surface (Fig. 2). Toward this end, we have defined the relevant kinetic parameters that define FGF-2

FIGURE 2 FGF-2 activity shows a narrow therapeutic window that is modulated by HSPG in vitro. Balb/c3T3 cells were treated with (*) or without (o) chlorate (50 mM) for 72 hours to inhibit the production of sulfated HSPGs. Nonsulfated HSPGs produced by chlorate-treated cells cannot bind to FGF-2 or enhance FGF-2 binding to its receptors. FGF-2 was added along with [3H] thymidine, and the cells were incubated for 36 hours. Newly synthesized DNA (3H-DNA) was quantitated and presented on the y-axis as "Thymidine Incorportation." Maximal response to FGF-2 was similar in these cells with and without HSPG present. However, the therapeutic window was shifted approximately 10-fold. Abbreviations: HSPG, heparan sulfate proteoglycan; FGF, fibroblast growth factor; FGF-2 or bFGF, basic FGF. Source: From Ref. 79.

FIGURE 2 FGF-2 activity shows a narrow therapeutic window that is modulated by HSPG in vitro. Balb/c3T3 cells were treated with (*) or without (o) chlorate (50 mM) for 72 hours to inhibit the production of sulfated HSPGs. Nonsulfated HSPGs produced by chlorate-treated cells cannot bind to FGF-2 or enhance FGF-2 binding to its receptors. FGF-2 was added along with [3H] thymidine, and the cells were incubated for 36 hours. Newly synthesized DNA (3H-DNA) was quantitated and presented on the y-axis as "Thymidine Incorportation." Maximal response to FGF-2 was similar in these cells with and without HSPG present. However, the therapeutic window was shifted approximately 10-fold. Abbreviations: HSPG, heparan sulfate proteoglycan; FGF, fibroblast growth factor; FGF-2 or bFGF, basic FGF. Source: From Ref. 79.

binding, internalization, and degradation by cells in culture in the presence and absence of HSPGs and have developed comprehensive mathematical models of these processes (80-82). These models are based on a series of ordinary differential equations that can effectively capture and predict the activation of FGF receptors on cells over time. However, even in these very controlled in vitro cell culture systems, a clear and direct link between receptor complex formation and biological response has been elusive. Indeed, studies by our laboratories and others have demonstrated that FGF-2 can elicit an array of cellular responses that are related to period and extent of activation, some of which may be mediated directly by FGF-2 binding to syndecan 4 independent of FGF receptors (83-85). Continued studies in vitro should allow models of growth factor response to become more sophisticated so that they will provide tools to accurately predict response. Extending these types of dynamic models to the more complex in vivo environment will require more complete understanding of the full range of processes that influence growth factor elimination, degradation, and metabolism in cells.

Mechanism of Co-Receptor Function of HSPGs

The recognition of the importance of HSPGs as co-receptors for FGF-2 has led to proposed approaches for FGF-2 applications that consider this HSPG function. For example, approaches involving the codelivery of heparin, HS, or HSPG, or the use of HS mimetics are being considered. Hence, a detailed understanding of the underlying mechanism of the HSPG co-receptor process is required. The chemical and physical mechanisms of this process have received considerable attention over the past 10 to 15 years. The use of oligosaccharides and chemically modified forms of heparin and HS has demonstrated that a pentasaccharide with N-sulfated glucosamine residues and at least one iduronic acid containing a sulfate in the 2-O-position is the minimal heparin unit that can bind FGF-2 (86,87). However, longer oligosaccharides (dodecasaccharides) containing 6-O sulfate groups in addition to 2-O and N sulfation are required to facilitate FGF-2 binding to its receptor (86,88). The additional requirements for ternary complex formation seemingly reflect the 6-O sulfated glucosamine-dependent binding of HS to FGF receptors (71,86,89,90). Thus, the HS-FGF-2-receptor complex likely consists of two FGF-2 molecules bound to two FGF receptors with a sufficiently long HS chain that makes contacts with all the protein components to effectively stabilize the complex. However, the exact stoichiometry and physical orientation of the various components remain an area of open investigation in spite of the availability of several FGF receptor crystal structures (91-96). The distinct HS structural requirements for binding to FGF-2 versus those for enhanced receptor binding provide an explanation for the fact that some HS species facilitate FGF-2 activity while others act in an inhibitory manner. Indeed, short heparin-derived oligosaccharides and 6-O desulfated heparin have been shown to be potent inhibitors of FGF-2 through the ability to sequester FGF-2 from its receptor (86,97). In some instances, even heparin- and HS-containing sequences capable of binding both FGF-2 and its receptor can inhibit receptor binding and activation. This is generally the case under conditions where heparin or HS molecules are not able to associate with the cell surface and are thus unable to come into proximity of the receptors (67,82,98,99). Thus, HS structure and physical localization, as well as the state of the cell surface of the target cell, are all likely to contribute to the ability of particular HS species to modulate growth factor-receptor binding. An additional consequence of this is that the state of the ECM, in particular HSPGs, will be an important determining factor in how it will interact with and modulate growth factors such as FGF-2.

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