A central question in the field of proteoglycan biology has been whether the complex sequence of modifications that occur during glycosaminoglycan synthesis serves a functional role in vivo, particularly in the tissue specific regulation of growth factor signaling. The integral role of heparan sulfate as a component of the FGF signaling complex, where it assembles with the FGF and the FR, suggests that the generation of specific sulfation patterns within the chain by specific cell types may regulate assembly of FGF family members with their receptors. To address the question of whether tissue-specific HS regulates FGF binding, exogenous FGF-2 and FGF-4 were used to probe and identify FGF-specific HS in the developing mouse embryo. Although both FGF-2 and FGF-4 recognize heparin in vitro (and in mast cells in vivo)
, distinct differences exist in the ability of FGF-2 versus FGF-4 to bind HS in vivo. In the E18 mouse, FGF-2 recognizes HS in a ubiquitous manner, suggesting that essentially all HS binds this growth factor. In contrast, whereas FGF-4 binds HS in many of the same sites as FGF-2, there are other sites where FGF-4 fails to bind. A stark contrast is seen in the vascular system where FGF-4 fails to bind to HS in the heart and large blood vessels, as well as to aortic endothelial cells in culture. Importantly, the failure of FGF-4 to bind HS on these cells correlates with a failure of FGF-4 to stimulate a response in these cells. However, a response can be stimulated, when heparin is added, suggesting that the correct FRs are indeed expressed on these cells, but that the endogenous HS is inappropriate for FGF to bind and signal. This result is surprising, as FGF-4, also known as k-FGF (Delli-Bovi et al., 1988
) or hst
(Miyagawa et al., 1988
), was originally identified as an angiogenic factor with activity toward endothelial cells. However, these studies typically added heparin along with the FGF to achieve activity (Delli-Bovi et al., 1988
; Yoshida et al., 1994
; Dell'Era et al., 2001
). Interestingly, FGF-4 binding to endothelial HS seems to vary among blood vessels of different origin, as FGF-4 does bind to HS in capillaries of the brain. A more detailed investigation will be necessary to determine whether this extends to all capillaries, or whether this is a function of the tissue type, as the phenotype of endothelial cells in various tissues is clearly affected by the cadre of neighboring cells (Aird et al., 1997
It is also surprising that FGF-4 fails to bind in the heart because of its role in early heart development. There is nothing known about FGF-specific HS expression during heart development, although it is clear that FGF induction of precardiac differentiation in vitro requires HS (Zhu et al., 1996
). Heart development from precardiac mesenchyme is initiated before gastrulation by paracrine signaling originating in the endoderm and acting on the adjacent mesodermal layer. This has been studied mostly in the chick embryo in which the underlying endoderm at stage 5 expresses FGF-4, along with FGF-1 and FGF-2. Any of these FGFs will induce the proliferation and differentiation of cardiac myocytes from the precardiac mesenchyme (Lough et al., 1996
; Zhu et al., 1996
). These same FGFs reappear later during heart chamber formation, where they are expressed in the myocardium with autocrine roles in cardiomyocyte proliferation and differentiation (Zhu et al., 1996
). Expression of these FGFs in the heart is subsequently lost at later stages of development. Interestingly, FGF expression in the chick is largely paralleled by expression of FR1, which peaks at stage 24, the stage when heart chamber formation is completed, but persists until day 7 (Sugi et al., 1995
). Expression then declines in the ventricle but persists in the atrium. Importantly, these stages of FGF-4 expression in the chick compare with E9–E11 in the mouse, which precede the mouse E18 stage examined here. Because FGF-4 fails to bind the heart HS and FR1c recognizes it only rarely when bound elsewhere in the E18 embryo, it suggests that the HS structure may change with development. This is indeed suggested by our preliminary data (unpublished data).
These results suggest that specific differences exist in HS of both the heart and large blood vessels such that FGF-2, but not FGF-4, is recognized. The ability of FGF-2 to bind to most, if not all HS, indicates either that the FGF-2 binding motif in HS is a common HS sequence that is present in all tissues, or that FGF-2 is able to recognize multiple HS sulfation patterns, such that binding is not dramatically affected by variations in HS structure. Where examined in detail using isolated heparin or HS fragments, it has been shown that FGF-2 binding is dependent on the minimum of a pentasaccharide containing 2-O
-sulfation (Turnbull et al., 1992
; Guimond et al., 1993
; Maccarana et al., 1993
) ( B). Thus, the presence of 2-O
-sulfation may be sufficient for FGF-2 binding, regardless of what other sulfate groups are present.
The failure of FGF-4 to bind in the vascular tissue suggests that the binding motif for FGF-4 is different from that of FGF-2, and that this motif is lacking in the heart and major blood vessels. Although there are fewer data regarding the HS binding requirements of FGF-4 than FGF-2, previous studies have suggested that binding of this growth factor is dependent on HS containing a high content of N
-sulfate groups (Guimond et al., 1993
). This supports the findings here that FGF-2 and FGF-4 recognize different sites; however, these prior experiments, which are aimed primarily at specific types of sulfation (i.e., 2-O
-sulfation, or 6-O
-sulfation) rather than specific motifs within the HS chain, provide insufficient information on what the specific motifs might be.
Overall, these results suggest that the HS in the walls of vascular elements has very different FGF binding capabilities, which may have far-reaching implications during vascular development and tumor-mediated angiogenesis, where FGFs are known to play an important role (Slavin, 1995
; Beckner, 1999
). Additionally, the finding that tissue-specific HS regulates FGF binding is likely to have a major impact not only on the 23 FGFs, but also on other HS-binding growth factors, including BMPs, wnts, and hedgehogs, among others (Bernfield et al., 1999
The ability of FGFs to bind tissue-specific HS fulfills only part of the requirement necessary for activation of FGF signaling, however, as FRs must also recognize specific HS structures in order to bind to and be activated by a particular FGF. Previous studies have shown that FGF-2 activity via FR1c requires a dodecasaccharide (twice the length necessary for binding alone) bearing glucosaminyl-6-O
-sulfates in addition to the iduronysyl-2-O
-sulfates necessary for FGF-2 binding (Guimond et al., 1993
; Pye et al., 1998
; Turnbull et al., 1992
) ( B). This additional length and sulfation requirement represents a second level of HS specificity that is likely to be important for assembly with FRs leading to signaling. Indeed, heparin depleted of 6-O
-sulfates will bind FGF-2 essentially as well as native heparin or HS, but will inhibit the growth factor by failing to assemble with the FR (Guimond et al., 1993
). Importantly, recent evidence suggests that it is not merely the presence of 6-O
-sulfates that is critical for signaling, but also the location of the 6-O
-sulfates on the HS chain that plays a critical role in FR activation (Guimond and Turnbull, 1999
In the current study, FR1c recognizes FGF-2–HS complexes throughout the E18 mouse embryo; this recognition is duplicated by FR2c. Given these data alone, it would be tempting to conclude that HS serves as nothing more than a nonspecific partner for the FGF and receptor. However, When taken in the context of the inability of FR1c to recognize FGF-4–HS in the vast majority of sites within the embryo, a different story emerges. The fact that FR1c recognizes FGF-4–heparin both in vitro and in vivo, but fails to recognize FGF-4–HS at most sites suggests that FRs do indeed require specific HS sulfation sequences in order to recognize a specific FGF. Additionally, the data suggest that the HS sequence necessary for FGF recognition differs between FRs, as FR2c does recognize FGF-4–HS throughout the embryo. Indeed, one wonders if FR2c and FR1c are binding exactly the same sites even when they bind to the FGF-2–HS complexes. It is entirely possible that these are actually distinct HS chains, or at least distinct sites on HS chains.
Previous studies provide at least partial explanations for the two tissues, namely the liver and the kidney, where FR1c does recognize FGF-4. In the liver, the structure of HS has been characterized as being highly sulfated, and in fact, heparin-like (Lyon et al., 1994
). As a result, it is likely that the rare sulfation sequence necessary for FR1c recognition of FGF-4, which exists in heparin, also exists on the heparin-like HS chains present in this site. In the kidney, it has been shown that the HS is heterogeneous, with the detection of at least five different HS species by antibodies generated via phage display (van Kuppevelt et al., 1998
). The presence of an HS sequence in the kidney that promotes FR1c–FGF-4 complex formation, when combined with the knowledge that both FGF-4 and FR1c are expressed simultaneously in the developing kidney (Cancilla et al., 1999
), supports the notion that HS has a regulatory role in FGF-4 signaling during kidney development. The fact that mice that lack the enzyme necessary for 2-O
-sulfation of HS fail to develop functional kidneys may also implicate the HS in binding either FGFs or FRs (Bullock et al., 1998
The lack of binding specificity in the case of FR1c recognition of FGF-2 or FR2c recognition of either FGF-2 or FGF-4 suggests several possibilities. In the case of FGF-2, it is likely that the minimum HS requirements necessary for receptor recognition are common components of HS biosynthesis. This is an intuitive result, as FGF-2 is one of the most widely expressed FGF family members and likely serves to signal in a wide variety of tissues and under a wide variety of physiological and pathological conditions (Baird, 1994
; Szebenyi and Fallon, 1999
). In this case, HS may serve as a facilitator of FGF-2 signaling rather than as a regulator. However, in the case of FGF-4, HS appears to be serving a regulatory role. It is clear that HS allows recognition of FGF-4 by FR1c in only very specific sites in the E18 stage embryo, whereas FR2c recognizes FGF-4–HS on a much broader level. In each of these cases, it will be of interest to examine both FGF and receptor binding at earlier stages of development and in tissues where the FGFs and FRs are expressed and known to play a role, such as limb development in the case of FGF-4 (Martin, 1998
). Further studies using other potential FGF receptors (including splice variants) as well as other FGF family members should identify additional specificity of these HS moieties in the regulation of FGF signaling.
Although it is widely accepted that HS is required for the formation of a high affinity FGF–FR signaling complex, there are few in vivo data regarding the ability of specific HS structures to regulate complex formation. The method used here provides a highly useful approach for mapping differences in HS structure and relating them directly to FGF activity. Although it is difficult to be certain that the binding of exogenous FGFs to the tissue sections is dependent only on the HS and is not influenced also by endogenous FRs, this seems unlikely as the FGFs bind to sites in the matrix where FRs are not expressed. In addition, we have shown that the putative FGF–HS complexes that are formed can be recognized by the exogenous FR probes. Furthermore, the predictions derived from the use of these FGF and FR probes are verified by activity studies using the BaF3 cells expressing FR1c or FR2c. Thus, it seems apparent that tissue-specific HS differentially regulates the binding of FGF-2 and FGF-4 in the developing mouse and also regulates the recognition of these FGFs by FR1c and FR2c in a tissue-specific manner. These results suggest a new paradigm where the formation of specific FGF–FR signaling complexes is regulated not only by the presence of HS, but also by site specific expression of distinct HS sequences necessary for complex formation.