Abstract

The name glypican has been assigned to a family of heparan sulfate (HS) proteoglycans that are linked to the cell membrane by a glycosyl-phosphatidylinositol anchor. To date, six family members of this family have been identified in mammals (GPC1 to GPC6) and two in Drosophila. Glypicans are expressed predominantly during development, and they are thought to play a role in morphogenesis. As HS-carrying molecules, glypicans were initially considered potential regulators of heparin-binding growth factors. This has been recently confirmed by genetic interaction experiments showing that glypicans regulate wingless signaling in Drosophila. The involvement of glypicans in the in vivo regulation of other heparin-binding growth factors, such as fibroblast growth factors, remains to be determined. Interestingly and unexpectedly, a role for GPC3 in the regulation of insulin-like growth factors has been proposed. This hypothesis is based on the phenotype of patients with Simpson-Golabi-Behmel syndrome (SGBS), an overgrowth and dysmorphic syndrome in which the GPC3 gene is mutated. Thus, it is possible that glypicans regulate different kinds of growth factors in a tissue-specific manner. In addition to its involvement in SGBS, down-regulation of GPC3 has been recently associated with the progression of several types of malignant tumors, including mesotheliomas and ovarian cancer. A role for GPC1 in pancreatic cancer progression has also been proposed.

Accepted on December 15, 2000;

The glypican family

Glypicans are a family of heparan sulfate proteoglycans (HSPGs) that are linked to the exocytoplasmic surface of the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. The size of the core proteins of glypicans is similar (60 to 70 kDa), and, as expected, they all contain an N-terminal secretory signal peptide and a hydrophobic domain in the C-terminal region required for the insertion of the GPI anchor. Although the degree of amino acid homology between most glypicans is moderate (Veugelers et al., 1999), the location of 14 cysteine residues is conserved, suggesting the existence of a highly similar three-dimensional structure. Another shared feature of glypicans is the location of the heparan sulfate (HS) insertion sites, which seems to be restricted to the last 50 amino acids in the C-terminus, placing the HS chains close to the cell membrane (Saunders et al., 1997).

To date, six members of the glypican family (GPC1 to GPC6) have been identified in mammals (Table I), and two in Drosophila (Nakato et al., 1995; Baeg et al., 2001). The greater degree of homology between the primary amino acid sequences of GPC3 and GPC5 suggests that these glypicans share unique structural features that distinguish them from the other family members (Paine-Saunders et al., 1999). In addition, it is interesting to note that GPC3 and GPC4 are clustered on chromosome Xq26 (Veugelers et al., 1998), and GPC5 and GPC6 on chromosome 13q32 (Veugelers et al., 1999). This suggests that the glypican family arose as a result of gene duplication.

In general, glypicans are expressed predominantly during development. Their expression levels change in a stage- and tissue-specific manner, suggesting that glypicans are involved in the regulation of morphogenesis (Li et al., 1997; Saunders et al., 1997; Litwack et al., 1998).

The function of glypicans

During the last few years it has been clearly established that cell-surface HSPGs are required for the optimal activity of heparin-binding growth factors, such as fibroblast growth factors (FGFs) and Wnts (Yayon et al., 1991; Schlessinger et al., 1995; Reichsman et al., 1996; Binari et al., 1997). In the case of FGF2, a model has been proposed in which the HS chains interact with both the ligand and the high-affinity FGF receptor. This interaction increases FGF2–FGF receptor binding and also promotes FGF receptor dimerization (Venkataraman et al., 1999; Schlessinger, 2000; Stauber et al., 2000).

In addition to glypicans, there are other types of HSPGs present on the cell surface. Unfortunately, the experiments that established the requirement of HSPGs for optimal FGF signaling in vivo did not identify the specific HSPGs involved(Lin et al., 1999b). Thus, although it is very clear that in tissue cultured cells glypicans can bind FGF2 and increase its mitogenic activity(Steinfeld et al., 1996; Song et al., 1997; Bonneh-Barkay et al., 1997), there is still no proof that glypicans are required for FGF activity in vivo. With regard to Wnt signaling, on the other hand, an in vivo role for glypicans has been clearly demonstrated. The experimental evidence was generated by studying the role of dally, a Drosophila glypican (Lin and Perrimon, 1999; Tsuda et al., 1999). Reduction of dally expression as a result of mutation or the use of RNA interference is associated with segment polarity defects similar to the ones caused by the loss of wingless activity (Lin and Perrimon, 1999; Tsuda et al., 1999). Further genetic interaction experiments suggest that dally regulates wingless extracellular interactions (Baeg and Perrimon, 2000), which is consistent with a previous report indicating that cell-surface HS is required for optimal Wnt signaling (Reichsman et al., 1996). The existence of a dally-like (dly)gene in Drosophila has been recently reported (Baeg et al., 2001). Although dly mutants are not available yet, results of RNA interference experiments suggest that dly can also regulate wingless activity.

Genetic interaction experiments have also implicated glypicans in the regulation of the activity of bone morphogenetic proteins (BMPs). Work in Drosophila with dally mutants has shown that this glypican regulates the signalingof dpp (a Drosophila BMP) in specific tissues during development (Jackson et al., 1997; Tsuda et al., 1999). Furthermore, when GPC3-deficient mice are mated with BMP4 heterozygote mice, the offspring displays polydactyly and rib malformations with high penetrance (Paine-Saunders et al., 2000). These abnormalities are not observed in either parental strain.

Taken together, the studies described above provide strong evidence in favor of genetic interactions between Wnt and dpp/BMP pathways, and glypicans. However, the biochemical basis for these interactions remains to be defined.

The peculiar case of GPC3

In 1996 Pilia et al. reported that GPC3 is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS) (Pilia et al., 1996). This is an X-linked disorder characterized by pre- and postnatal overgrowth and a broad spectrum of clinical manifestations that vary from a very mild phenotype in carrier females to infantile lethal forms in some males (reviewed in Neri et al., 1998). The list of clinical manifestations of SGBS can include a distinct facial appearance, macroglossia (enlarged tongue), cleft palate, syndactyly (incomplete separation of digits), polydactyly (more digits than normal), supernumerary nipples, cystic and dysplastic kidneys, congenital heart defects, rib and vertebral abnormalities, and umbilical/inguinal hernias.

Most of the GPC3 mutations identified to date are point mutations or microdeletions encompassing a varying number of exons (Hughes-Benzie et al., 1996; Lindsay et al., 1997; Veugelers et al., 2000). Given the lack of correlation between phenotype and the location of the mutations, it has been proposed that SGBS is caused by a nonfunctional GPC3 protein, with additional genetic factors responsible for the intra- and interfamilial phenotypic variation (Hughes-Benzie et al., 1996). Strong support for this hypothesis has been provided by the generation of GPC3-deficient mice (Cano-Gauci et al., 1999; Paine-Saunders et al., 2000). These mice display several of the abnormalities found in SGBS patients, including developmental overgrowth (Figure 1) and cystic and dysplastic kidneys. Starting from early stages of development there is a persistent increase in the proliferation rate of epithelial cells in the ureteric bud/collecting system (Cano-Gauci et al., 1999). This observation supports the idea that GPC3 is a negative regulator of cell proliferation, which is obviously consistent with the general overgrowth observed in the SGBS patients and the GPC3-deficient mice.

Some of the clinical features of the SGBS patients, such as syndactyly and the presence of multiple nipples, suggest that GPC3 may act to regulate cell survival in certain tissues during development. Our laboratory recently provided experimental support to this hypothesis by demonstrating that GPC3 can induce apoptosis in a cell type–specific manner (Duenas Gonzales et al., 1998).

SGBS shares some clinical features with the Beckwith-Wiedemann syndrome (BWS), another overgrowth syndrome (Weng et al., 1995). Because overexpression of insulin-like growth factor II (IGF-II) is thought to be one of the contributing factors to BWS (Weksberg and Squire, 1997), it has been proposed that GPC3 negatively regulates IGF-II activity by competing for IGF-II binding with the signaling receptor and that the loss-of-function mutations of GPC3 are equivalent to overexpression of IGF-II (Weksberg et al., 1996). Further support for this idea was provided by the generation of mice overexpressing IGF-II (Eggenschwiler et al., 1997). In addition to the phenotypic features of BWS, these mice display skeletal defects that are typical of SGBS. It has to be noted, however, that the SGBS patients and the GPC3-null mice display severe kidney abnormalities that are not found in the IGF-II transgenic mice.

Another piece of evidence suggesting an involvement of GPC3 in IGF-II signaling comes from the finding that IGF-II receptor (IGF2A)–deficient mice display the same degree of developmental overgrowth than the GPC3-null mice (Wang et al., 1994; Lau et al., 1994). The IGF2R is a well-characterized negative regulator of IGF-II. It binds this growth factor and down-regulates its activity by endocytosis and degradation (Ludwig et al., 1996). Thus, the IGF2R-deficient mice display increased levels of IGF-II in blood and tissues. In the case of the GPC3-null embryos, however, no significant alteration in IGF-II levels has been found (Cano-Gauci et al., 1999). Furthermore, no direct interaction between IGF-II and GPC3 has been detected (Song et al., 1997). It can be concluded, therefore, that if GPC3 inhibits IGF-II signaling, it does so by a mechanism that is fundamentally different than that used by the IGF2R.

Given the complex clinical features of SGBS and the differences in the kidney phenotype between the IGF-II transgenic and the GPC3-null mice, it is also possible that GPC3 regulates other growth factors in addition to IGF-II. As discussed above, there is already one report demonstrating a genetic interaction between BMP4 and GPC3 (Paine-Saunders et al., 2000). In addition, it is important to note that there is some experimental evidence suggesting that the HS chains are not required for all the activities of GPC3 (Duenas Gonzales et al., 1998). Thus, it can be proposed that the protein core of GPC3 interacts with other molecule(s) independent of the HS chains. In addition, because GPC3, like other glypicans, can be secreted (Filmus et al., 1995), it is possible that the secreted form of GPC3 may act via a mechanism distinct from that of the cell surface form.

Glypicans and cancer

Because GPC3 is an inhibitor of cell proliferation and can induce apoptosis in certain types of tumor cells (Duenas Gonzales et al., 1998; Cano-Gauci et al., 1999), recent reports indicating that GPC3 expression is down-regulated in tumors from different origin were not surprising. Lin et al. showed that, although GPC3 is expressed in normal ovary, its expression is undetectable in a significant proportion of ovarian cancer cell lines (Lin et al., 1999a). In all cases where GPC3 expression was lost, the GPC3 promoter was hypermethylated, and no mutations were found in the coding region. GPC3 expression was restored by treatment with a demethylating agent. In addition, the authors demonstrated that ectopic expression of GPC3 inhibits colony-forming activity in several ovarian cancer cell lines.

Another report associating GPC3 with cancer was originated from a differential mRNA display study on normal rat mesothelial cells and mesothelioma cell lines (Murthy et al., 2000). In this study it was found that GPC3 was consistently down-regulated in the tumor cell lines. Moreover, a similar down-regulation was found in primary rat mesotheliomas and in cell lines derived from human mesotheliomas. Similarly to ovarian cancer, no mutations in the GPC3 coding sequence were found, but most of the cell lines displayed aberrant methylation in the GPC3 promoter region. As reported previously (Duenas Gonzales et al., 1998), this study showed that ectopic expression of GPC3 in mesothelioma cell lines inhibits their colony-forming activity.

Interestingly, another differential mRNA study comparing normal liver and human hepatocellular carcinomas found that, although GPC3 is not expressed in the liver, the expression of this glypican is up-regulated in the majority of tumors (Hsu et al., 1997). In a similar manner, in our laboratory we found that, though normal colon does not express GPC3, a significant proportion of colorectal tumors do (unpublished observations). Because GPC3 is highly expressed in embryonic liver and intestine and is silenced in the corresponding normal adult tissues, these results suggest that in these organs GPC3 is behaving as an oncofetal protein. In general, oncofetal proteins do not seem to play a critical role in tumor progression, but they have been used as tumor markers or as targets for immunotherapy (Coggin, 1992; Matsuura and Hakomori, 1985). In this regard, Hsu et al. (1997) reported that GPC3 is more frequently upregulated in hepatocarcinomas than α-fetoprotein, another oncofetal protein that has been extensively used in the clinic as a tumor marker (Beasley et al., 1981) and that is thought to be a potential target for immunotherapy (Vollmer et al., 1999). It remains to be seen whether the oncofetal behavior of GPC3 can be exploited for clinical use.

In summary, depending on the tissue, GPC3 displays a very different pattern of expression during tumor progression. In cancers originated from tissues that are GPC3-positive in adults, the expression of GPC3 is reduced during tumor progression; this reduction seems to play a role in the generation of the malignant phenotype. On the other hand, in tumors originated from tissues that only express GPC3 in the embryo, GPC3 expression tends to reappear on malignant transformation. Whether GPC3 reexpression plays a role in the progression of these tumors is still not known. We speculate that this tissue-specific differences are due to the fact that GPC3 is regulating different growth and survival factors in each tissue.

Another connection between glypicans and tumor progression has been established by a recent study that showed that GPC1 expression is significantly increased in a large proportion of pancreatic tumors (Kleef et al., 1998). Furthermore, this study also reported that transfection of antisense GPC-1 inhibited the mitogenic response of cultured pancreatic cancer cells to FGF2 and heparin-binding EGF-like growth factor (HB-EGF) (Kleef et al., 1998), and decreased the tumorigenicity of the transfected cells in nude mice (Kleeff et al., 1999). The molecular basis of the reduced tumorigenicity remains to be determined, although it is certainly possible that a reduced response to FGF2 or HB-HGF is the cause of this reduction.

Perspective

A significant amount of genetic evidence has been generated in the last few years demonstrating that glypicans play a critical role in the regulation of cell proliferation and survival, particularly during development and malignant transformation. This role of glypicans seems to be based on their capacity to modulate the activity of various growth and survival factors. The challenge in the future will be to define more precisely which growth and survival factors are regulated by each glypican in vivo and to uncover the precise biochemical basis of such regulation.

Acknowledgments

I thank Norman Rosemblum and Howard Song for critically reviewing this manuscript and Sophie Ku for assistance in its preparation. The work in my laboratory has been supported by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the March of Dimes.

Abbreviations

BMP, bone morphogenetic protein; BWS, Beckwith-Wiedemann syndrome; dly, dally-like; FGF, fibroblast growth factor; GPI, glycosyl-phosphatidylinositol; HB-GBF, heparin-binding; EGF, epidermal growth factor; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; IGF-II, insulin-like growth factor II; IGF2R, IGF-II receptor; SGBS, Simpson-Golabi-Behmel syndrome.

Fig. 1. Developmental overgrowth in GPC3-deficient mice. Representative picture of day 14 embryos. Left: GPC3-deficient; Right: Wild type.

Fig. 1. Developmental overgrowth in GPC3-deficient mice. Representative picture of day 14 embryos. Left: GPC3-deficient; Right: Wild type.

Table 1.

Glypican family

Name Original Designation Expression in Embryo Expression in Adult Reference 
Glypican 1 Glypican Bone, bone marrow, muscle epidermis, kidney Most tissues David et al., 1990; Litwack et al., 1994 
Glypican 2 Cerebroglycan Nervous system Not detected Stipp et al., 1994; Ivins et al., 1997 
Glypican 3 OCI-5 Most tissues Ovary, mammary gland, mesothelium, lung, kidney Filmus et al., 1998; Pellegrini et al., 1998; Li et al., 1997; Filmus (unpublished observations) 
Glypican 4 K-glypican Brain, kidney, lung Most tissues Watanabe et al., 1995; Veugelers et al., 1998; Siebertz et al., 1999 
Glypican 5  Brain, lung, liver, kidney, limb Brain Veugelers et al., 1997; Saunders et al., 1997 
Glypican 6  Many tissues, including liver and kidney Many tissues including ovary, kidney, liver, and intestine Paine-Saunders et al., 1999; Veugelers et al., 1999 
Name Original Designation Expression in Embryo Expression in Adult Reference 
Glypican 1 Glypican Bone, bone marrow, muscle epidermis, kidney Most tissues David et al., 1990; Litwack et al., 1994 
Glypican 2 Cerebroglycan Nervous system Not detected Stipp et al., 1994; Ivins et al., 1997 
Glypican 3 OCI-5 Most tissues Ovary, mammary gland, mesothelium, lung, kidney Filmus et al., 1998; Pellegrini et al., 1998; Li et al., 1997; Filmus (unpublished observations) 
Glypican 4 K-glypican Brain, kidney, lung Most tissues Watanabe et al., 1995; Veugelers et al., 1998; Siebertz et al., 1999 
Glypican 5  Brain, lung, liver, kidney, limb Brain Veugelers et al., 1997; Saunders et al., 1997 
Glypican 6  Many tissues, including liver and kidney Many tissues including ovary, kidney, liver, and intestine Paine-Saunders et al., 1999; Veugelers et al., 1999 

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