MATBIO-01021; No of Pages 5 Matrix Biology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Matrix Biology journal homepage: www.elsevier.com/locate/matbio
Mini review
The role of glypicans in Hedgehog signaling Jorge Filmus ⁎, Mariana Capurro Platform of Biological Sciences, Sunnybrook Research Institute, ON, Canada Dept. of Medical Biophysics, University of Toronto, ON, Canada
a r t i c l e
i n f o
Article history: Received 19 August 2013 Received in revised form 18 December 2013 Accepted 18 December 2013 Available online xxxx Keywords: Glypicans Proteoglycans Hedgehog
a b s t r a c t Glypicans (GPCs) are a family of proteoglycans that are bound to the cell surface by a glycosylphosphatidylinositol anchor. Six glypicans have been found in the mammalian genome (GPC1 to GPC6). GPCs regulate several signaling pathways, including the pathway triggered by Hedgehogs (Hhs). This regulation, which could be stimulatory or inhibitory, occurs at the signal reception level. In addition, GPCs have been shown to be involved in the formation of Hh gradients in the imaginal wing disks in Drosophila. In this review we will discuss the role of various glypicans in specific developmental events in the embryo that are regulated by Hh signaling. In addition, we will discuss the mechanism by which loss-of-function GPC3 mutations alter Hh signaling in the Simpson–Golabi–Behmel overgrowth syndrome, and the molecular basis of the GPC5-induced stimulation of Hh signaling and tumor progression in rhabdomyosarcomas. © 2014 Elsevier B.V. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . 2. Hedgehog signaling . . . . . . . . . . . . . . . . . . . . 3. Regulation of Hedgehog signaling by Drosophila glypicans . . . 4. Glypican-3 and the Simpson–Golabi–Behmel syndrome . . . . 5. The role of Glypican-5 and Glypican-1 in Hh signaling . . . . . 6. Are vertebrate glypicans involved in Hh secretion and transport? 7. Final remarks . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
1. Introduction Glypicans are a family of proteoglycans that are bound to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Proteoglycans are proteins that carry glycosaminoglycan (GAG) chains. These are polymers of disaccharide repeats that are usually highly sulfated. Various types of GAG chains have been identified based on the structure of the disaccharides (Esko et al., 2009). The most common ones are heparan sulfate (HS) and chondroitin sulfate (CS). The mammalian genome includes six glypicans (GPC1 to GPC6) (Filmus et al., 2008; Filmus and Capurro, 2012), and ortholog genes have been identified across Metazoans, including two in Drosophila (Dally and Dlp). Glypicans do not display domains with obvious
⁎ Corresponding author at: Sunnybrook Health Sciences Centre, Room S220, 2075 Bayview Ave., Toronto, ON, M4N 3M5, Canada. E-mail address: jorge.fi[email protected] (J. Filmus).
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
0 0 0 0 0 0 0 0
homology to characterized domains found in other proteins, suggesting that these proteoglycans have unique functions. Although the sequence identity between mammalian glypicans could be as low as 25%, the three dimensional structure seems to be similar across the family. For example, the localization of 14 cysteine residues is highly conserved (Veugelers et al., 1999; Filmus et al., 2008). Another feature shared by all glypicans is the position of the insertion sites for the GAG chains. These sites are located close to the C-terminus, suggesting that the GAG chains could mediate the interaction of these proteoglycans with other cell membrane proteins (Filmus and Capurro, 2012). The number of GAG insertion sites in each glypican, however, varies across the family (from 2 sites in GPC3, to 5 sites in GPC5). The functional implications of this variation are still not understood. In general, glypicans display HS chains, but GPC5 produced by rhabdomyosarcoma (RMS) cells also exhibits CS chains (Li et al., 2011). Most glypicans can be secreted to the extracellular environment by a lipase called Notum, which cleaves the GPI anchor (Kreuger et al., 2004; Traister et al., 2008). In addition, glypicans can be cleaved by a furin-like
0945-053X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matbio.2013.12.007
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
2
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx
convertase. This cleavage generates two subunits that remain attached to each other by one or more disulfide linkages. Genetic and biochemical studies have shown that glypicans regulate the activity of the signaling pathways triggered by Hedgehogs, Wnts, bone morphogenetic proteins, and fibroblast growth factors (Filmus et al., 2008). In addition, glypicans play an important role in axon guidance (Smart et al., 2011; Wilson and Stoeckli, 2013), and in the formation of excitatory synapses (Allen et al., 2012). 2. Hedgehog signaling The Hedgehog (Hh) signaling pathway plays a critical role in embryonic morphogenesis (Ingham et al., 2011; Briscoe and Therond, 2013). In addition, hyperactivation of this pathway has been shown to promote the progression of various cancer types (Teglund and Toftgard, 2010; Ng and Curran, 2011). Three Hhs have been identified in mammals: Sonic (Shh), Indian (Ihh), and Desert (Dhh) (Nieuwenhuis and Hui, 2005). Whereas Shh is produced by many tissues, the expression of Ihh and Dhh is restricted to a few cell types. Ihh is the main Hh found in developing bone (St-Jacques et al., 1999). Mature Hh is covalently bound at its C terminus to cholesterol, and to palmitic acid at its N terminus (Ryan and Chiang, 2012). It is well established that, in addition to acting in an autocrine and paracrine manner, Hh can travel and act on cells that are up to 300 μm away from the secretion point (Briscoe and Therond, 2013). Different mechanisms of Hh transport have been described, but the attachment of cholesterol and palmitic acid is known to play a key role in the regulation of long-range transport (Ryan and Chiang, 2012; Therond, 2012). Hh signaling is triggered by the binding of the ligands to the cell surface receptor Patched, which is a 12-pass transmembrane protein. High affinity binding of Hh to Patched requires the presence of at least one of three co-receptors: CDO, BOC or GAS1. CDO and BOC are transmembrane proteins, and GAS1, which is specific to vertebrates, binds to the cell membrane through a GPI anchor (Zheng et al., 2010; Allen et al., 2011; Izzi et al., 2011). In the absence of Hh, Patched suppresses the activity of Smoothened (Smo), a member of the G protein-coupled receptor family, through a poorly characterized mechanism. Hh binding to Patched abrogates its Smo-inhibitory function. Activated Smo then triggers a signaling cascade that ultimately results in the accumulation of Gli1 and Gli2, and in the depletion of the inhibitory form of Gli3. The Gli family of transcription factors regulates the expression of cell-type specific genes that control cell proliferation, migration and differentiation (Briscoe and Therond, 2013). In vertebrates, Hh binds to Patched at the primary cilium (Nozawa et al., 2013), and it has been demonstrated that Hh cannot activate signaling activity in cells without this appendage (Toftgard, 2009). 3. Regulation of Hedgehog signaling by Drosophila glypicans The initial report of the regulatory activity of a glypican in Hh signaling was the result of an RNAi screen for proteins that regulate Hh reporter activity in Drosophila cultured cells (Lum et al., 2003). One of the proteins identified by this screen was Dlp. Interestingly, Dally, the other Drosophila glypican, was not able to replace Dlp activity in the same Hh signaling assay. Soon after this report, genetic evidence was provided that Dlp, but not Dally, was required for the reception of Hh signal in Drosophila embryos (Desbordes and Sanson, 2003). Several mechanisms for the secretion of Hh in the imaginal wing disk of Drosophila have been described (Han et al., 2004; Ayers et al., 2010; Callejo et al., 2011). It is now well established that the longdistance activity of lipid-modified Hh requires its oligomerization or its inclusion in lipoprotein particles (Eugster et al., 2007; Briscoe and Therond, 2013). Both Dlp and Dally have been shown to play a role in these two processes through their GAG chains (Han et al., 2004; Eugster et al., 2007), but the precise mechanism of oligomerized Hh transport in the imaginal wing disk remains controversial. Guerrero and collaborators have proposed that Hh is first secreted from the apical
side, but that then the morphogen undergoes a glypican-mediated trancytosis to the basolateral region, where the long-distance gradient is finally formed (Callejo et al., 2011). These authors also propose that filopodia-like structures called cytonemes are required for the basolateral long-distance transport of Hh (Callejo et al., 2011). Alternatively, Therond and colleagues have proposed the existence of two types of Hh gradients in the imaginal disk. An apical one, which acts at long distance and requires Notum-mediated secretion of Dally (Ayers et al., 2010), and a basolateral gradient that acts at close-range and requires Dlp for apicobasal trancytosis of Hh (Gallet et al., 2008). 4. Glypican-3 and the Simpson–Golabi–Behmel syndrome The Simpson–Golabi–Behmel syndrome (SGBS) is a rare X-linked condition characterized by pre- and post-natal overgrowth and a broad and variable spectrum of developmental abnormalities, including distinctive craniofacial features, skeletal anomalies, heart defects, supernumerary nipples, renal dysplasia, and urinary tract malformations (Li et al., 2001; Mariani et al., 2003; Young et al., 2006). Loss-of-function mutations in GPC3 were identified as the cause of SGBS (Pilia et al., 1996). Given the critical role that insulin-like-growth factor II (IGF-II) plays in the regulation of embryonic growth, it was initially proposed that GPC3 was an inhibitor of IGF-II, and that the overgrowth observed in the SGBS patients was due to an increase in IGF-II signaling caused by the loss of functional GPC3 (Pilia et al., 1996; Eggenschwiler et al., 1997). This model was initially questioned by our findings that GPC3 does not interact with IGF-II, and that the GPC3-null embryos display normal levels of IGF-II (Song et al., 1997; Cano-Gauci et al., 1999). Definitive proof that the overgrowth in SGBS patients is independent of IGF was provided by Efstratiadis and collaborators, who crossed the GPC3-null mice with various mouse strains that lacked critical components of the IGF signaling pathway, and did not find any genetic interaction (Chiao et al., 2002). Based on the findings described above indicating that glypicans regulate Hh signaling in Drosophila, and on the knowledge that the Hh signaling pathway promotes embryonic growth (Milenkovic et al., 1999; Desbordes and Sanson, 2003), Capurro et al. hypothesized that GPC3 acts as an inhibitor of Hh signaling in the embryo, and that the overgrowth found in the SGBS patients is due, at least in part, to hyperactivation of Hh signaling caused by the loss of functional GPC3. Strong support to this hypothesis was provided by the finding that Hh signaling activity is elevated in GPC3-null mice (Capurro et al., 2008). Furthermore, it was also demonstrated that GPC3 binds with high affinity to Shh and Ihh, and that it competes with Patched for Hh binding (Capurro et al., 2008, 2009). The binding of Hh to GPC3 triggers the endocytosis and degradation of the GPC3/Hh complex (Capurro et al., 2008). Consistent with this finding, the GPC3-null embryos display higher levels of Shh and Ihh protein than normal littermates (Capurro et al., 2008, 2009). Significantly, the overgrowth of the GPC3-null embryos is partially reverted in embryos that also lack Ihh (Capurro et al., 2009). Additional support for the finding that GPC3 is a negative regulator of Hh signaling was more recently provided by experiments performed in cultured Drosophila cells (Williams et al., 2010). These studies also revealed that the GPC3-induced inhibition of Hh signaling requires the attachment of this glypican to the cell surface through a GPI anchor (Capurro et al., 2008). Recently, a GPC3 gene displaying a point mutation that inhibits the addition of the GPI anchor to the core protein was identified in an SGBS patient (Penisson-Besnier et al., 2008). Consistent with the essential role of the GPI-mediated anchorage in GPC3 function, this GPC3 mutant does not inhibit Hh signaling (Shi and Filmus, 2009). Based on investigations performed in Drosophila with mutants that were deficient in the production of HS chains, it was initially proposed that the glypican-induced regulation of Hh signaling was exclusively mediated by these chains (Nybakken and Perrimon, 2002). However, the HS chains are not essential for the regulatory effect of GPC3,
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx
although they are required for optimal activity (Capurro et al., 2008). Moreover, surface plasmon resonance experiments showed that Shh and Ihh bind with high affinity to the core protein of GPC3 (Capurro et al., 2008, 2009). More recently, an interaction between the core protein of Dlp and Hh was also reported (Yan et al., 2010). In addition, this study demonstrated that the HS chains were not required for the regulatory activity of Dlp in the reception of Hh signaling (Yan et al., 2010). It should be noted, however, that another study concluded that Dlp core protein does not bind to Hh in a pull-down assay (Williams et al., 2010). The reason for this discrepancy is not clear. The mechanism by which the GPC3/Hh complex is endocytosed has been recently investigated (Capurro et al., 2012). Most GPI-anchored proteins are localized in cholesterol-sphingolipid-rich membrane domains called lipid rafts (Mayor and Riezman, 2004). In general, GPIanchored proteins in lipid rafts are internalized by clathrin-independent
A
3
mechanisms (Bhagatji et al., 2009). Surprisingly, however, GPC3 is mostly found outside of the lipid rafts in fibroblasts (Capurro et al., 2012). Consistent with this finding, it was demonstrated that the endocytosis of the GPC3/Hh complex is mediated by the low-density-lipoprotein receptorrelated protein-1 (LRP1) in a clathrin-dependent manner. LRP1 is a well characterized endocytic receptor that mediates the internalization of a large number of proteins (May et al., 2007). This study revealed that GPC3 constitutively interacts with LRP1 through its GAG chains, but that the internalization of the complex only occurs in the presence of Hh. Based on this observation, and on the findings reported for the endocytosis of other protein complexes that include GPI-anchored proteins and are mediated by LRP1 (Czekay et al., 2001; Taylor and Hooper, 2008), it was proposed that only in the presence of Hh is the GPC3/ LRP1 protein complex stable enough to be internalized. Another notable discovery of this study was that the predominant localization of GPC3
Hh
Off state
Patched
On state
Patched
Smo
X
X
Smo
Signaling
Hh
B
Patched
X
Smo
Signaling
GPC3 competes with Patched for Hh binding
GPC5 facilitates/stabilizes Hh-Patched interaction
Hh
Hh
GPC5
GPC3 Patched
X
Decreased signaling
Smo
Patched
X
Smo
Increased signaling
Fig. 1. Opposite effects of GPC3 and GPC5 on Hh signaling. A — In the absence of Hedgehog (Hh) (Off-state), Patched suppresses the activity of Smoothened (Smo), a member of the G protein-coupled receptor family whose activity is required for downstream signaling. Binding of Hh to Patched (On-state) releases the inhibition on Smo and turns on the signaling cascade. B — GPC3 at the cell membrane binds to Hh but does not interact with Ptc, reducing the amount of Hh available for binding to Ptc with the consequent decreased signaling (left). GPC5 at the cell membrane interacts with both Hh and Patched, facilitating or stabilizing Hh–Patched interaction with the consequent increased signaling (right).
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
4
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx
outside of the lipid rafts is due to its GAG-mediated interaction with LRP1, and that the removal of the GAG chains allows GPC3 to migrate to lipid rafts (Capurro et al., 2012). A similar change in subcellular localization induced by the removal of GAG chains had been previously described for GPC1 (Mertens et al., 1996). Based on the findings that LRP1 is required for GPC3 endocytosis and degradation, and that the interaction between these two proteins is mediated by the GAG chains of GPC3, it was expected that a nonglycanated GPC3 (GPC3ΔGAG) would completely lose its Hh-inhibitory activity. However, it had been previously reported that GPC3ΔGAG still displays some Hh-inhibitory activity (Capurro et al., 2008). Because LRP1 can transiently associate with lipid rafts, it is possible that Hh could trigger the formation of a protein complex between LRP1 and GPC3ΔGAG by acting as a bridge. This complex would then move outside of the lipid rafts to undergo clathrin-mediated endocytosis. Another possibility is that the GPC3ΔGAG/Hh complex is internalized at a low rate by an LRP1-independent process.
biliary atresia (BA) has suggested that GPC1 regulates Hh signaling in cholangiocytes. BA is a progressive fibroinflammatory disorder that affects the extrahepatic and intrahepatic biliary tree. It is believed to be the result of environmental factors acting on genetically susceptible infants (Cui et al., 2013). BA patients display high levels of Hh activity in the biliary tree. Recently, Cui et al. observed deletions at 2q37.3 in a significant number of BA patients that resulted in the deletion of one copy of GPC1 (Cui et al., 2013). Furthermore, downregulation of GPC1 in zebrafish led to developmental biliary defects, and injection of this fish with Shh generated biliary defects similar to those of GPC1 morphants. Cholangiocytes from BA patients have reduced levels of apical GPC1 compared to healthy individuals. Based on these results it was proposed that, like GPC3, GPC1 acts as a negative regulator of Hh signaling in the biliary tree, although it is still not known whether Hhs are present in the bile.
5. The role of Glypican-5 and Glypican-1 in Hh signaling
There are no reports to date demonstrating that glypicans play a role in the secretion and transport of Hh in vertebrates. However, there are a few studies showing that certain types of HS could be involved in these events. One study has shown that a mixture of HS chains extracted from E11.5 mouse embryos (but not from older embryos), can induce the oligomerization of ShhN monomers (Dierker et al., 2009). In addition, other studies have shown that the binding of Hh to HS restricts the movement of Hh across the extracellular matrix (Koziel et al., 2004; Danesin et al., 2006; Ohlig et al., 2012). Whether the HS of glypicans contribute to the roles attributed to the mixed HS population implicated in these studies remains to be investigated.
Another member of the glypican family that has been implicated in Hh signaling is GPC5. This glypican is highly expressed in the brain, kidney, testis, and pituitary gland (Saunders et al., 1997). Williamson et al. have shown that GPC5 is significantly upregulated in rhabdomyosarcoma (RMS) (Williamson et al., 2007). This is a softtissue sarcoma that affects mostly children and adolescents. Because Patched-null mice tend to develop RMS, this tumor has been associated with the hyperactivation of Hh signaling (Hahn et al., 1998). Li et al. have recently reported that GPC5 stimulates RMS cell proliferation by activating Hh signaling (Li et al., 2011) (Fig. 1). These authors demonstrated that GPC5 increases the binding of Shh to its signaling receptor Patched. Consistent with this, it was found that GPC5 can interact with Shh. In addition, it was shown that, unlike GPC3, GPC5 can also interact with Patched (Li et al., 2011). It should be noted that in the context of Hh signaling GPC5 behaves like Dlp, which also stimulates Hh activity, and interacts with both Hh and Patched (Yan et al., 2010). By using a nonglycanated mutant it was found that the GAG chains are required for the binding of GPC5 to Hh and Patched. An important observation made in this study was that GPC5 localizes to the primary cilium, something that is consistent with the fact that Hh binds to Patched at this location (Li et al., 2011). Significantly, it was also observed that GPC3 cannot be found in cilia. This is consistent with our previous finding that GPC3 competes with Patched for Hh binding. This study also showed that a non-glycanated GPC5, which does not bind to Patched and does not stimulate Hh signaling, resides outside of the cilia (Li et al., 2011). Because GPC3, like GPC5, is highly glycanated, these results raised the question as to why the GAG chains of GPC5 bind to Patched, and those of GPC3 do not. A possible answer to this question was provided by the finding that the HS chains of GPC5 display a higher degree of sulfation than those of GPC3 (Li et al., 2011). It is well known that the negative charge provided by the sulfate groups is responsible of most of the interactions involving HS chains (Esko and Linhardt, 2009). Another possibility is that the dissimilar type of sulfate modifications, or number of GAG chains in GPC3 and GPC5 are responsible for the differential interaction with Patched. More recently, GPC5 was also shown to act in Shh signal reception in cerebellar granule precursor cells, where Hh signaling promotes cell proliferation (Witt et al., 2013). In addition, this study showed that in the cerebellar cells GPC5 localizes at the base of the cilia. Notably, these authors also demonstrated that the HS chains of GPC5 require 2-O-sulfo-iduronic acid residues at the reducing end to display Shh-stimulatory activity (Witt et al., 2013). GPC1 has also been shown to regulate Hh signaling. One study has demonstrated that this glypican acts as a Shh co-receptor in commissural neurons, where Hh signaling mediates repulsive guidance cues (Wilson and Stoeckli, 2013). Another study on the molecular basis of
6. Are vertebrate glypicans involved in Hh secretion and transport?
7. Final remarks It is well established that the Hh signaling pathway plays a critical role in embryonic development and in cancer. Given the fact that glypicans regulate Hh activity, it is not surprising that some of these proteoglycans have been implicated in genetic syndromes or human malignancies. Recently, loss-of-function mutations in GPC6 have been identified in patients with autosomal recessive omodysplasia (CamposXavier et al., 2009). This syndrome is characterized by short-limbed short stature, and craniofacial dysmorfism. This phenotype is consistent with reduced Hh activity in the bones. It is therefore reasonable to speculate that a reduction in Hh signaling could, at least in part, play an important role in this syndrome. References Allen, B.L., et al., 2011. Overlapping roles and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway function. Dev. Cell 20, 775–787. Allen, N.J., Bennett, M.L., Foo, L.C., Wang, G.X., Chakraborty, C., Smith, S.J., Barres, B.A., 2012. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414. Ayers, K.L., et al., 2010. The long-range activity of hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum. Dev. Cell 18, 605–620. Bhagatji, P., et al., 2009. Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins. J. Cell Biol. 186, 615–628. Briscoe, J., Therond, P.P., 2013. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat. Rev. Mol. Cell Biol. 14, 418–431. Callejo, A., et al., 2011. Dispatched mediates Hedgehog basolateral release to form the long-range morphogentic gradient in the Drosophila wing disk epithelium. Proc. Natl. Acad. Sci. U. S. A. 108, 12591–12598. Campos-Xavier, A.B., et al., 2009. Mutations in the heparan-sulfate proteoglycan glypican-6 (GPC6) impair endochodral ossification and cause recessive omodysplasia. Am. J. Hum. Genet. 84, 760–770. Cano-Gauci, D.F., et al., 1999. Glypican-3-deficient mice exhibit the overgrowth and renal abnormalities typical of the Simpson–Golabi–Behmel syndrome. J. Cell Biol. 146, 255–264. Capurro, M.I., et al., 2008. Glypican-3 inhibits hedgehog signaling during development by competing with Patched for Hedgehog binding. Dev. Cell 14, 700–711. Capurro, M.I., et al., 2009. Overgrowth of a mouse model of Simpson–Golabi–Behmel syndrome is partly mediated by Indian Hedgehog. EMBO Rep. 10, 901–907.
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx Capurro, M.I., et al., 2012. LRP1 mediates Hedgehog-induced endocytosis of the GPC3-Hedgehog complex. J. Cell Sci. 125, 3380–3389. Chiao, E., et al., 2002. Overgrowth of a mouse model of the Simpson–Golabi–Behmel syndrome is independent of IGF signaling. Dev. Biol. 243, 185–206. Cui, S., et al., 2013. Evidence from human zebrafish that GPC1 is a biliary atresia susceptibility gene. Gastroenterology 144, 1107–1115. Czekay, R.P., et al., 2001. Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity. Mol. Biol. Cell 12, 1467–1479. Danesin, C., et al., 2006. Ventral neural progenitors switch toward an oligodendroglial fate in response to increased sonic hedgehog (Shh) activity: involvement of sulfatase 1 in modulating Shh signaling in the ventral spinal cord. J. Neurosci. 26, 5037–5048. Desbordes, S.C., Sanson, B., 2003. The glypican Dally-like is required for hedgehog signalling in the embryonic epidermis of Drosophila. Development 130, 6245–6255. Dierker, T., et al., 2009. Heparan sulfate and transglutaminase activity are required for the formation of covalently cross-linked hedgehog oligomers. J. Biol. Chem. 47, 32562–32571. Eggenschwiler, J., et al., 1997. Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes. Genes Dev. 11, 3128–3142. Esko, J.D., Linhardt, R.J., 2009. Proteins that bind sulfated glycosaminoglycans. In: Varki, A., et al. (Eds.), Essentials of Glycobiology. Cold Spring Harbor Press. Esko, J.D., et al., 2009. Proteoglycans and sulfated glycosaminoglycans. In: Varki, A., et al. (Eds.), Essentials of Glycobiology. Cold Spring Harbor Press. Eugster, C., et al., 2007. Lipoprotein–heparan sulfate interactions in the Hh pathway. Dev. Cell 13, 57–71. Filmus, J., Capurro, M., 2012. The glypican family. In: Karamanos, Nikos K. (Ed.), Extracellular Matrix: Pathobiology and Signaling. De Gruyter, Berlin/Boston, pp. 209–220. Filmus, J., et al., 2008. Glypicans. Genome Biol. 9, 224. Gallet, A., et al., 2008. Cellular trafficking of the glypican Dally-like is required for fullstrength hedgehog signaling and wingless transcytosis. Dev. Cell 14, 712–725. Hahn, H., et al., 1998. Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin syndrome. Nat. Med. 4, 619–622. Han, C., et al., 2004. Drosophila glypicans control the cell-to-cell movement of hedgehog by a dynamin-independent process. Development 131, 601–611. Ingham, P.W., et al., 2011. Mechanisms and functions of hedgehog signalling across the metazoa. Nat. Rev. Genet. 12, 393–406. Izzi, L., et al., 2011. Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Dev. Cell 20, 788–801. Koziel, L., et al., 2004. Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification. Dev. Cell 6, 801–813. Kreuger, J., et al., 2004. Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity. Dev. Cell 7, 503–512. Li, M., et al., 2001. GPC3 mutation analysis in a spectrum of patients with overgrowth expands the phenotype of Simpson–Golabi–Behmel syndrome. Am. J. Med. Genet. 102, 161–168. Li, F., et al., 2011. Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling. J. Cell Biol. 192, 691–704. Lum, L., et al., 2003. Identification of hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045. Mariani, S., et al., 2003. Genotype/phenotype correlations of males affected by Simpson–Golabi–Behmel syndrome with GPC3 gene mutations: patient report and review of the literature. J. Pediatr. Endocrinol. Metab. 16, 225–232. May, P., et al., 2007. The LDL receptor-related protein (LRP) family: an old family of proteins with new physiological functions. Ann. Med. 39, 219–228. Mayor, S., Riezman, H., 2004. Sorting GPI-anchored proteins. Nat. Rev. Mol. Cell Biol. 5, 110–120. Mertens, G., et al., 1996. Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content. J. Cell Biol. 132, 487–497.
5
Milenkovic, L., et al., 1999. Mouse patched controls body size determination and limb patterning. Development 126, 4431–4440. Ng, J.M.Y., Curran, T., 2011. The Hedgehog's tale: developing strategies for targeting cancer. Nat. Rev. Cancer 11, 493–501. Nieuwenhuis, E., Hui, C.C., 2005. Hedgehog signaling and congenital malformations. Clin. Genet. 67, 193–208. Nozawa, Y.I., Lin, C., Chuang, P.T., 2013. Hedgehog signaling from the primary cilium to the nucleus: an emerging picture of ciliary localization, trafficking and transduction. Curr. Opin. Genet. Dev. 23, 429–437. Nybakken, K., Perrimon, N., 2002. Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila. Biochim. Biophys. Acta 1573, 280–291. Ohlig, S., et al., 2012. An emerging role of sonic hedgehog shedding as a modulator of heparan sulfate interactions. J. Biol. Chem. 287, 43708–43719. Penisson-Besnier, I., et al., 2008. Carotid artery dissection in an adult with the Simpson–Golabi–Behmel syndrome. Am. J. Med. Genet. A 146A, 464–467. Pilia, G., et al., 1996. Mutations in GPC3, a glypican gene, cause the Simpson–Golabi– Behmel overgrowth syndrome. Nat. Genet. 12, 241–247. Ryan, K.E., Chiang, C., 2012. Hedgehog secretion and signal transduction in vertebrates. J. Biol. Chem. 287, 17905–17913. Saunders, S., Paine-Saunders, S., Lander, A.D., 1997. Expression of the cell surface proteoglycan glypican-5 is developmentally regulated in kidney, limb, and brain. Dev. Biol. 190, 78–93. Shi, W., Filmus, J., 2009. A patient with the Simson–Golabi–Behmel syndrome displays a loss-of-function point mutation in GPC3 that inhibits the attachment of this proteoglycan to the cell surface. Am. J. Med. Genet. 149A, 552–554. Smart, A.D., et al., 2011. Heparan sulfate proteoglycan specificity during axon pathway formation in Drosophila embryo. Dev. Neurobiol. 71, 608–618. Song, H.H., et al., 1997. OCI-5/rat glypican-3 binds to fibroblast growth factor-2 but not to insulin-like growth factor-2. J. Biol. Chem. 272, 7574–7577. St-Jacques, B., et al., 1999. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13, 2072–2086. Taylor, D.R., Hooper, N.M., 2008. The low-density lipoprotein receptor-related protein 1 (LRP1) mediates the endocytosis of the cellular prion protein. Biochem. J. 402, 17–23. Teglund, S., Toftgard, R., 2010. Hedgehog beyond medulloblastoma and basal cell carcinoma. Biochim. Biophys. Acta 1805, 181–208. Therond, P.P., 2012. Release and transportation of hedgehog molecules. Curr. Opin. Cell Biol. 24, 173–180. Toftgard, R., 2009. Two sides to cilia in cancer. Nat. Med. 15, 994–996. Traister, A., et al., 2008. Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. Biochem. J. 410, 503–511. Veugelers, M., et al., 1999. Glypican-6, a new member of the glypican family of cell surface proteoglycans. J. Biol. Chem. 274, 26968–26977. Williams, E.H., et al., 2010. Dally-like core protein and its mammalian homologues mediate stimulatory and inhibitory effects on Hedgehog signal response. Proc. Natl. Acad. Sci. U. S. A. 107, 5869–5874. Williamson, D., et al., 2007. Role for amplification and expression of glypican-5 in rhabdomyosarcoma. Cancer Res. 67, 57–65. Wilson, N.H., Stoeckli, E.T., 2013. Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner. Neuron 79, 478–491. Witt, R.M., Hecht, M.L., Pazyra-Murphy, M.F., Cohen, S.M., Noti, C., van Kuppevelt, T.H., Fuller, M., Chan, J.A., Hopwood, J., Seeberger, P.H., Segal, R.A., 2013. Heparan sulfate proteoglycans containing a Glypican 5 core and 2-O-sulfo-iduronic acid function as sonic hedgehog co-receptors to promote proliferation. J. Biol. Chem 288, 26275–26288. Yan, D., et al., 2010. The cell-surface proteins Dally-like and Ihog differentially regulate Hedgehog signaling strength and range during development. Development 137, 2033–2044. Young, E.L., et al., 2006. Expanding the clinical picture of Simpson–Golabi–Behmel syndrome. Pediatr. Neurol. 34, 139–142. Zheng, X., et al., 2010. Genetic and biochemical definition of the Hedgehog receptor. Genes Dev. 24, 57–71.
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
Contents lists available at ScienceDirect
Matrix Biology journal homepage: www.elsevier.com/locate/matbio
Mini review
The role of glypicans in Hedgehog signaling Jorge Filmus ⁎, Mariana Capurro Platform of Biological Sciences, Sunnybrook Research Institute, ON, Canada Dept. of Medical Biophysics, University of Toronto, ON, Canada
a r t i c l e
i n f o
Article history: Received 19 August 2013 Received in revised form 18 December 2013 Accepted 18 December 2013 Available online xxxx Keywords: Glypicans Proteoglycans Hedgehog
a b s t r a c t Glypicans (GPCs) are a family of proteoglycans that are bound to the cell surface by a glycosylphosphatidylinositol anchor. Six glypicans have been found in the mammalian genome (GPC1 to GPC6). GPCs regulate several signaling pathways, including the pathway triggered by Hedgehogs (Hhs). This regulation, which could be stimulatory or inhibitory, occurs at the signal reception level. In addition, GPCs have been shown to be involved in the formation of Hh gradients in the imaginal wing disks in Drosophila. In this review we will discuss the role of various glypicans in specific developmental events in the embryo that are regulated by Hh signaling. In addition, we will discuss the mechanism by which loss-of-function GPC3 mutations alter Hh signaling in the Simpson–Golabi–Behmel overgrowth syndrome, and the molecular basis of the GPC5-induced stimulation of Hh signaling and tumor progression in rhabdomyosarcomas. © 2014 Elsevier B.V. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . 2. Hedgehog signaling . . . . . . . . . . . . . . . . . . . . 3. Regulation of Hedgehog signaling by Drosophila glypicans . . . 4. Glypican-3 and the Simpson–Golabi–Behmel syndrome . . . . 5. The role of Glypican-5 and Glypican-1 in Hh signaling . . . . . 6. Are vertebrate glypicans involved in Hh secretion and transport? 7. Final remarks . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
1. Introduction Glypicans are a family of proteoglycans that are bound to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Proteoglycans are proteins that carry glycosaminoglycan (GAG) chains. These are polymers of disaccharide repeats that are usually highly sulfated. Various types of GAG chains have been identified based on the structure of the disaccharides (Esko et al., 2009). The most common ones are heparan sulfate (HS) and chondroitin sulfate (CS). The mammalian genome includes six glypicans (GPC1 to GPC6) (Filmus et al., 2008; Filmus and Capurro, 2012), and ortholog genes have been identified across Metazoans, including two in Drosophila (Dally and Dlp). Glypicans do not display domains with obvious
⁎ Corresponding author at: Sunnybrook Health Sciences Centre, Room S220, 2075 Bayview Ave., Toronto, ON, M4N 3M5, Canada. E-mail address: jorge.fi[email protected] (J. Filmus).
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
0 0 0 0 0 0 0 0
homology to characterized domains found in other proteins, suggesting that these proteoglycans have unique functions. Although the sequence identity between mammalian glypicans could be as low as 25%, the three dimensional structure seems to be similar across the family. For example, the localization of 14 cysteine residues is highly conserved (Veugelers et al., 1999; Filmus et al., 2008). Another feature shared by all glypicans is the position of the insertion sites for the GAG chains. These sites are located close to the C-terminus, suggesting that the GAG chains could mediate the interaction of these proteoglycans with other cell membrane proteins (Filmus and Capurro, 2012). The number of GAG insertion sites in each glypican, however, varies across the family (from 2 sites in GPC3, to 5 sites in GPC5). The functional implications of this variation are still not understood. In general, glypicans display HS chains, but GPC5 produced by rhabdomyosarcoma (RMS) cells also exhibits CS chains (Li et al., 2011). Most glypicans can be secreted to the extracellular environment by a lipase called Notum, which cleaves the GPI anchor (Kreuger et al., 2004; Traister et al., 2008). In addition, glypicans can be cleaved by a furin-like
0945-053X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matbio.2013.12.007
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
2
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx
convertase. This cleavage generates two subunits that remain attached to each other by one or more disulfide linkages. Genetic and biochemical studies have shown that glypicans regulate the activity of the signaling pathways triggered by Hedgehogs, Wnts, bone morphogenetic proteins, and fibroblast growth factors (Filmus et al., 2008). In addition, glypicans play an important role in axon guidance (Smart et al., 2011; Wilson and Stoeckli, 2013), and in the formation of excitatory synapses (Allen et al., 2012). 2. Hedgehog signaling The Hedgehog (Hh) signaling pathway plays a critical role in embryonic morphogenesis (Ingham et al., 2011; Briscoe and Therond, 2013). In addition, hyperactivation of this pathway has been shown to promote the progression of various cancer types (Teglund and Toftgard, 2010; Ng and Curran, 2011). Three Hhs have been identified in mammals: Sonic (Shh), Indian (Ihh), and Desert (Dhh) (Nieuwenhuis and Hui, 2005). Whereas Shh is produced by many tissues, the expression of Ihh and Dhh is restricted to a few cell types. Ihh is the main Hh found in developing bone (St-Jacques et al., 1999). Mature Hh is covalently bound at its C terminus to cholesterol, and to palmitic acid at its N terminus (Ryan and Chiang, 2012). It is well established that, in addition to acting in an autocrine and paracrine manner, Hh can travel and act on cells that are up to 300 μm away from the secretion point (Briscoe and Therond, 2013). Different mechanisms of Hh transport have been described, but the attachment of cholesterol and palmitic acid is known to play a key role in the regulation of long-range transport (Ryan and Chiang, 2012; Therond, 2012). Hh signaling is triggered by the binding of the ligands to the cell surface receptor Patched, which is a 12-pass transmembrane protein. High affinity binding of Hh to Patched requires the presence of at least one of three co-receptors: CDO, BOC or GAS1. CDO and BOC are transmembrane proteins, and GAS1, which is specific to vertebrates, binds to the cell membrane through a GPI anchor (Zheng et al., 2010; Allen et al., 2011; Izzi et al., 2011). In the absence of Hh, Patched suppresses the activity of Smoothened (Smo), a member of the G protein-coupled receptor family, through a poorly characterized mechanism. Hh binding to Patched abrogates its Smo-inhibitory function. Activated Smo then triggers a signaling cascade that ultimately results in the accumulation of Gli1 and Gli2, and in the depletion of the inhibitory form of Gli3. The Gli family of transcription factors regulates the expression of cell-type specific genes that control cell proliferation, migration and differentiation (Briscoe and Therond, 2013). In vertebrates, Hh binds to Patched at the primary cilium (Nozawa et al., 2013), and it has been demonstrated that Hh cannot activate signaling activity in cells without this appendage (Toftgard, 2009). 3. Regulation of Hedgehog signaling by Drosophila glypicans The initial report of the regulatory activity of a glypican in Hh signaling was the result of an RNAi screen for proteins that regulate Hh reporter activity in Drosophila cultured cells (Lum et al., 2003). One of the proteins identified by this screen was Dlp. Interestingly, Dally, the other Drosophila glypican, was not able to replace Dlp activity in the same Hh signaling assay. Soon after this report, genetic evidence was provided that Dlp, but not Dally, was required for the reception of Hh signal in Drosophila embryos (Desbordes and Sanson, 2003). Several mechanisms for the secretion of Hh in the imaginal wing disk of Drosophila have been described (Han et al., 2004; Ayers et al., 2010; Callejo et al., 2011). It is now well established that the longdistance activity of lipid-modified Hh requires its oligomerization or its inclusion in lipoprotein particles (Eugster et al., 2007; Briscoe and Therond, 2013). Both Dlp and Dally have been shown to play a role in these two processes through their GAG chains (Han et al., 2004; Eugster et al., 2007), but the precise mechanism of oligomerized Hh transport in the imaginal wing disk remains controversial. Guerrero and collaborators have proposed that Hh is first secreted from the apical
side, but that then the morphogen undergoes a glypican-mediated trancytosis to the basolateral region, where the long-distance gradient is finally formed (Callejo et al., 2011). These authors also propose that filopodia-like structures called cytonemes are required for the basolateral long-distance transport of Hh (Callejo et al., 2011). Alternatively, Therond and colleagues have proposed the existence of two types of Hh gradients in the imaginal disk. An apical one, which acts at long distance and requires Notum-mediated secretion of Dally (Ayers et al., 2010), and a basolateral gradient that acts at close-range and requires Dlp for apicobasal trancytosis of Hh (Gallet et al., 2008). 4. Glypican-3 and the Simpson–Golabi–Behmel syndrome The Simpson–Golabi–Behmel syndrome (SGBS) is a rare X-linked condition characterized by pre- and post-natal overgrowth and a broad and variable spectrum of developmental abnormalities, including distinctive craniofacial features, skeletal anomalies, heart defects, supernumerary nipples, renal dysplasia, and urinary tract malformations (Li et al., 2001; Mariani et al., 2003; Young et al., 2006). Loss-of-function mutations in GPC3 were identified as the cause of SGBS (Pilia et al., 1996). Given the critical role that insulin-like-growth factor II (IGF-II) plays in the regulation of embryonic growth, it was initially proposed that GPC3 was an inhibitor of IGF-II, and that the overgrowth observed in the SGBS patients was due to an increase in IGF-II signaling caused by the loss of functional GPC3 (Pilia et al., 1996; Eggenschwiler et al., 1997). This model was initially questioned by our findings that GPC3 does not interact with IGF-II, and that the GPC3-null embryos display normal levels of IGF-II (Song et al., 1997; Cano-Gauci et al., 1999). Definitive proof that the overgrowth in SGBS patients is independent of IGF was provided by Efstratiadis and collaborators, who crossed the GPC3-null mice with various mouse strains that lacked critical components of the IGF signaling pathway, and did not find any genetic interaction (Chiao et al., 2002). Based on the findings described above indicating that glypicans regulate Hh signaling in Drosophila, and on the knowledge that the Hh signaling pathway promotes embryonic growth (Milenkovic et al., 1999; Desbordes and Sanson, 2003), Capurro et al. hypothesized that GPC3 acts as an inhibitor of Hh signaling in the embryo, and that the overgrowth found in the SGBS patients is due, at least in part, to hyperactivation of Hh signaling caused by the loss of functional GPC3. Strong support to this hypothesis was provided by the finding that Hh signaling activity is elevated in GPC3-null mice (Capurro et al., 2008). Furthermore, it was also demonstrated that GPC3 binds with high affinity to Shh and Ihh, and that it competes with Patched for Hh binding (Capurro et al., 2008, 2009). The binding of Hh to GPC3 triggers the endocytosis and degradation of the GPC3/Hh complex (Capurro et al., 2008). Consistent with this finding, the GPC3-null embryos display higher levels of Shh and Ihh protein than normal littermates (Capurro et al., 2008, 2009). Significantly, the overgrowth of the GPC3-null embryos is partially reverted in embryos that also lack Ihh (Capurro et al., 2009). Additional support for the finding that GPC3 is a negative regulator of Hh signaling was more recently provided by experiments performed in cultured Drosophila cells (Williams et al., 2010). These studies also revealed that the GPC3-induced inhibition of Hh signaling requires the attachment of this glypican to the cell surface through a GPI anchor (Capurro et al., 2008). Recently, a GPC3 gene displaying a point mutation that inhibits the addition of the GPI anchor to the core protein was identified in an SGBS patient (Penisson-Besnier et al., 2008). Consistent with the essential role of the GPI-mediated anchorage in GPC3 function, this GPC3 mutant does not inhibit Hh signaling (Shi and Filmus, 2009). Based on investigations performed in Drosophila with mutants that were deficient in the production of HS chains, it was initially proposed that the glypican-induced regulation of Hh signaling was exclusively mediated by these chains (Nybakken and Perrimon, 2002). However, the HS chains are not essential for the regulatory effect of GPC3,
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx
although they are required for optimal activity (Capurro et al., 2008). Moreover, surface plasmon resonance experiments showed that Shh and Ihh bind with high affinity to the core protein of GPC3 (Capurro et al., 2008, 2009). More recently, an interaction between the core protein of Dlp and Hh was also reported (Yan et al., 2010). In addition, this study demonstrated that the HS chains were not required for the regulatory activity of Dlp in the reception of Hh signaling (Yan et al., 2010). It should be noted, however, that another study concluded that Dlp core protein does not bind to Hh in a pull-down assay (Williams et al., 2010). The reason for this discrepancy is not clear. The mechanism by which the GPC3/Hh complex is endocytosed has been recently investigated (Capurro et al., 2012). Most GPI-anchored proteins are localized in cholesterol-sphingolipid-rich membrane domains called lipid rafts (Mayor and Riezman, 2004). In general, GPIanchored proteins in lipid rafts are internalized by clathrin-independent
A
3
mechanisms (Bhagatji et al., 2009). Surprisingly, however, GPC3 is mostly found outside of the lipid rafts in fibroblasts (Capurro et al., 2012). Consistent with this finding, it was demonstrated that the endocytosis of the GPC3/Hh complex is mediated by the low-density-lipoprotein receptorrelated protein-1 (LRP1) in a clathrin-dependent manner. LRP1 is a well characterized endocytic receptor that mediates the internalization of a large number of proteins (May et al., 2007). This study revealed that GPC3 constitutively interacts with LRP1 through its GAG chains, but that the internalization of the complex only occurs in the presence of Hh. Based on this observation, and on the findings reported for the endocytosis of other protein complexes that include GPI-anchored proteins and are mediated by LRP1 (Czekay et al., 2001; Taylor and Hooper, 2008), it was proposed that only in the presence of Hh is the GPC3/ LRP1 protein complex stable enough to be internalized. Another notable discovery of this study was that the predominant localization of GPC3
Hh
Off state
Patched
On state
Patched
Smo
X
X
Smo
Signaling
Hh
B
Patched
X
Smo
Signaling
GPC3 competes with Patched for Hh binding
GPC5 facilitates/stabilizes Hh-Patched interaction
Hh
Hh
GPC5
GPC3 Patched
X
Decreased signaling
Smo
Patched
X
Smo
Increased signaling
Fig. 1. Opposite effects of GPC3 and GPC5 on Hh signaling. A — In the absence of Hedgehog (Hh) (Off-state), Patched suppresses the activity of Smoothened (Smo), a member of the G protein-coupled receptor family whose activity is required for downstream signaling. Binding of Hh to Patched (On-state) releases the inhibition on Smo and turns on the signaling cascade. B — GPC3 at the cell membrane binds to Hh but does not interact with Ptc, reducing the amount of Hh available for binding to Ptc with the consequent decreased signaling (left). GPC5 at the cell membrane interacts with both Hh and Patched, facilitating or stabilizing Hh–Patched interaction with the consequent increased signaling (right).
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
4
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx
outside of the lipid rafts is due to its GAG-mediated interaction with LRP1, and that the removal of the GAG chains allows GPC3 to migrate to lipid rafts (Capurro et al., 2012). A similar change in subcellular localization induced by the removal of GAG chains had been previously described for GPC1 (Mertens et al., 1996). Based on the findings that LRP1 is required for GPC3 endocytosis and degradation, and that the interaction between these two proteins is mediated by the GAG chains of GPC3, it was expected that a nonglycanated GPC3 (GPC3ΔGAG) would completely lose its Hh-inhibitory activity. However, it had been previously reported that GPC3ΔGAG still displays some Hh-inhibitory activity (Capurro et al., 2008). Because LRP1 can transiently associate with lipid rafts, it is possible that Hh could trigger the formation of a protein complex between LRP1 and GPC3ΔGAG by acting as a bridge. This complex would then move outside of the lipid rafts to undergo clathrin-mediated endocytosis. Another possibility is that the GPC3ΔGAG/Hh complex is internalized at a low rate by an LRP1-independent process.
biliary atresia (BA) has suggested that GPC1 regulates Hh signaling in cholangiocytes. BA is a progressive fibroinflammatory disorder that affects the extrahepatic and intrahepatic biliary tree. It is believed to be the result of environmental factors acting on genetically susceptible infants (Cui et al., 2013). BA patients display high levels of Hh activity in the biliary tree. Recently, Cui et al. observed deletions at 2q37.3 in a significant number of BA patients that resulted in the deletion of one copy of GPC1 (Cui et al., 2013). Furthermore, downregulation of GPC1 in zebrafish led to developmental biliary defects, and injection of this fish with Shh generated biliary defects similar to those of GPC1 morphants. Cholangiocytes from BA patients have reduced levels of apical GPC1 compared to healthy individuals. Based on these results it was proposed that, like GPC3, GPC1 acts as a negative regulator of Hh signaling in the biliary tree, although it is still not known whether Hhs are present in the bile.
5. The role of Glypican-5 and Glypican-1 in Hh signaling
There are no reports to date demonstrating that glypicans play a role in the secretion and transport of Hh in vertebrates. However, there are a few studies showing that certain types of HS could be involved in these events. One study has shown that a mixture of HS chains extracted from E11.5 mouse embryos (but not from older embryos), can induce the oligomerization of ShhN monomers (Dierker et al., 2009). In addition, other studies have shown that the binding of Hh to HS restricts the movement of Hh across the extracellular matrix (Koziel et al., 2004; Danesin et al., 2006; Ohlig et al., 2012). Whether the HS of glypicans contribute to the roles attributed to the mixed HS population implicated in these studies remains to be investigated.
Another member of the glypican family that has been implicated in Hh signaling is GPC5. This glypican is highly expressed in the brain, kidney, testis, and pituitary gland (Saunders et al., 1997). Williamson et al. have shown that GPC5 is significantly upregulated in rhabdomyosarcoma (RMS) (Williamson et al., 2007). This is a softtissue sarcoma that affects mostly children and adolescents. Because Patched-null mice tend to develop RMS, this tumor has been associated with the hyperactivation of Hh signaling (Hahn et al., 1998). Li et al. have recently reported that GPC5 stimulates RMS cell proliferation by activating Hh signaling (Li et al., 2011) (Fig. 1). These authors demonstrated that GPC5 increases the binding of Shh to its signaling receptor Patched. Consistent with this, it was found that GPC5 can interact with Shh. In addition, it was shown that, unlike GPC3, GPC5 can also interact with Patched (Li et al., 2011). It should be noted that in the context of Hh signaling GPC5 behaves like Dlp, which also stimulates Hh activity, and interacts with both Hh and Patched (Yan et al., 2010). By using a nonglycanated mutant it was found that the GAG chains are required for the binding of GPC5 to Hh and Patched. An important observation made in this study was that GPC5 localizes to the primary cilium, something that is consistent with the fact that Hh binds to Patched at this location (Li et al., 2011). Significantly, it was also observed that GPC3 cannot be found in cilia. This is consistent with our previous finding that GPC3 competes with Patched for Hh binding. This study also showed that a non-glycanated GPC5, which does not bind to Patched and does not stimulate Hh signaling, resides outside of the cilia (Li et al., 2011). Because GPC3, like GPC5, is highly glycanated, these results raised the question as to why the GAG chains of GPC5 bind to Patched, and those of GPC3 do not. A possible answer to this question was provided by the finding that the HS chains of GPC5 display a higher degree of sulfation than those of GPC3 (Li et al., 2011). It is well known that the negative charge provided by the sulfate groups is responsible of most of the interactions involving HS chains (Esko and Linhardt, 2009). Another possibility is that the dissimilar type of sulfate modifications, or number of GAG chains in GPC3 and GPC5 are responsible for the differential interaction with Patched. More recently, GPC5 was also shown to act in Shh signal reception in cerebellar granule precursor cells, where Hh signaling promotes cell proliferation (Witt et al., 2013). In addition, this study showed that in the cerebellar cells GPC5 localizes at the base of the cilia. Notably, these authors also demonstrated that the HS chains of GPC5 require 2-O-sulfo-iduronic acid residues at the reducing end to display Shh-stimulatory activity (Witt et al., 2013). GPC1 has also been shown to regulate Hh signaling. One study has demonstrated that this glypican acts as a Shh co-receptor in commissural neurons, where Hh signaling mediates repulsive guidance cues (Wilson and Stoeckli, 2013). Another study on the molecular basis of
6. Are vertebrate glypicans involved in Hh secretion and transport?
7. Final remarks It is well established that the Hh signaling pathway plays a critical role in embryonic development and in cancer. Given the fact that glypicans regulate Hh activity, it is not surprising that some of these proteoglycans have been implicated in genetic syndromes or human malignancies. Recently, loss-of-function mutations in GPC6 have been identified in patients with autosomal recessive omodysplasia (CamposXavier et al., 2009). This syndrome is characterized by short-limbed short stature, and craniofacial dysmorfism. This phenotype is consistent with reduced Hh activity in the bones. It is therefore reasonable to speculate that a reduction in Hh signaling could, at least in part, play an important role in this syndrome. References Allen, B.L., et al., 2011. Overlapping roles and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway function. Dev. Cell 20, 775–787. Allen, N.J., Bennett, M.L., Foo, L.C., Wang, G.X., Chakraborty, C., Smith, S.J., Barres, B.A., 2012. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414. Ayers, K.L., et al., 2010. The long-range activity of hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum. Dev. Cell 18, 605–620. Bhagatji, P., et al., 2009. Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins. J. Cell Biol. 186, 615–628. Briscoe, J., Therond, P.P., 2013. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat. Rev. Mol. Cell Biol. 14, 418–431. Callejo, A., et al., 2011. Dispatched mediates Hedgehog basolateral release to form the long-range morphogentic gradient in the Drosophila wing disk epithelium. Proc. Natl. Acad. Sci. U. S. A. 108, 12591–12598. Campos-Xavier, A.B., et al., 2009. Mutations in the heparan-sulfate proteoglycan glypican-6 (GPC6) impair endochodral ossification and cause recessive omodysplasia. Am. J. Hum. Genet. 84, 760–770. Cano-Gauci, D.F., et al., 1999. Glypican-3-deficient mice exhibit the overgrowth and renal abnormalities typical of the Simpson–Golabi–Behmel syndrome. J. Cell Biol. 146, 255–264. Capurro, M.I., et al., 2008. Glypican-3 inhibits hedgehog signaling during development by competing with Patched for Hedgehog binding. Dev. Cell 14, 700–711. Capurro, M.I., et al., 2009. Overgrowth of a mouse model of Simpson–Golabi–Behmel syndrome is partly mediated by Indian Hedgehog. EMBO Rep. 10, 901–907.
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
J. Filmus, M. Capurro / Matrix Biology xxx (2014) xxx–xxx Capurro, M.I., et al., 2012. LRP1 mediates Hedgehog-induced endocytosis of the GPC3-Hedgehog complex. J. Cell Sci. 125, 3380–3389. Chiao, E., et al., 2002. Overgrowth of a mouse model of the Simpson–Golabi–Behmel syndrome is independent of IGF signaling. Dev. Biol. 243, 185–206. Cui, S., et al., 2013. Evidence from human zebrafish that GPC1 is a biliary atresia susceptibility gene. Gastroenterology 144, 1107–1115. Czekay, R.P., et al., 2001. Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity. Mol. Biol. Cell 12, 1467–1479. Danesin, C., et al., 2006. Ventral neural progenitors switch toward an oligodendroglial fate in response to increased sonic hedgehog (Shh) activity: involvement of sulfatase 1 in modulating Shh signaling in the ventral spinal cord. J. Neurosci. 26, 5037–5048. Desbordes, S.C., Sanson, B., 2003. The glypican Dally-like is required for hedgehog signalling in the embryonic epidermis of Drosophila. Development 130, 6245–6255. Dierker, T., et al., 2009. Heparan sulfate and transglutaminase activity are required for the formation of covalently cross-linked hedgehog oligomers. J. Biol. Chem. 47, 32562–32571. Eggenschwiler, J., et al., 1997. Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes. Genes Dev. 11, 3128–3142. Esko, J.D., Linhardt, R.J., 2009. Proteins that bind sulfated glycosaminoglycans. In: Varki, A., et al. (Eds.), Essentials of Glycobiology. Cold Spring Harbor Press. Esko, J.D., et al., 2009. Proteoglycans and sulfated glycosaminoglycans. In: Varki, A., et al. (Eds.), Essentials of Glycobiology. Cold Spring Harbor Press. Eugster, C., et al., 2007. Lipoprotein–heparan sulfate interactions in the Hh pathway. Dev. Cell 13, 57–71. Filmus, J., Capurro, M., 2012. The glypican family. In: Karamanos, Nikos K. (Ed.), Extracellular Matrix: Pathobiology and Signaling. De Gruyter, Berlin/Boston, pp. 209–220. Filmus, J., et al., 2008. Glypicans. Genome Biol. 9, 224. Gallet, A., et al., 2008. Cellular trafficking of the glypican Dally-like is required for fullstrength hedgehog signaling and wingless transcytosis. Dev. Cell 14, 712–725. Hahn, H., et al., 1998. Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin syndrome. Nat. Med. 4, 619–622. Han, C., et al., 2004. Drosophila glypicans control the cell-to-cell movement of hedgehog by a dynamin-independent process. Development 131, 601–611. Ingham, P.W., et al., 2011. Mechanisms and functions of hedgehog signalling across the metazoa. Nat. Rev. Genet. 12, 393–406. Izzi, L., et al., 2011. Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Dev. Cell 20, 788–801. Koziel, L., et al., 2004. Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification. Dev. Cell 6, 801–813. Kreuger, J., et al., 2004. Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity. Dev. Cell 7, 503–512. Li, M., et al., 2001. GPC3 mutation analysis in a spectrum of patients with overgrowth expands the phenotype of Simpson–Golabi–Behmel syndrome. Am. J. Med. Genet. 102, 161–168. Li, F., et al., 2011. Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling. J. Cell Biol. 192, 691–704. Lum, L., et al., 2003. Identification of hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045. Mariani, S., et al., 2003. Genotype/phenotype correlations of males affected by Simpson–Golabi–Behmel syndrome with GPC3 gene mutations: patient report and review of the literature. J. Pediatr. Endocrinol. Metab. 16, 225–232. May, P., et al., 2007. The LDL receptor-related protein (LRP) family: an old family of proteins with new physiological functions. Ann. Med. 39, 219–228. Mayor, S., Riezman, H., 2004. Sorting GPI-anchored proteins. Nat. Rev. Mol. Cell Biol. 5, 110–120. Mertens, G., et al., 1996. Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content. J. Cell Biol. 132, 487–497.
5
Milenkovic, L., et al., 1999. Mouse patched controls body size determination and limb patterning. Development 126, 4431–4440. Ng, J.M.Y., Curran, T., 2011. The Hedgehog's tale: developing strategies for targeting cancer. Nat. Rev. Cancer 11, 493–501. Nieuwenhuis, E., Hui, C.C., 2005. Hedgehog signaling and congenital malformations. Clin. Genet. 67, 193–208. Nozawa, Y.I., Lin, C., Chuang, P.T., 2013. Hedgehog signaling from the primary cilium to the nucleus: an emerging picture of ciliary localization, trafficking and transduction. Curr. Opin. Genet. Dev. 23, 429–437. Nybakken, K., Perrimon, N., 2002. Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila. Biochim. Biophys. Acta 1573, 280–291. Ohlig, S., et al., 2012. An emerging role of sonic hedgehog shedding as a modulator of heparan sulfate interactions. J. Biol. Chem. 287, 43708–43719. Penisson-Besnier, I., et al., 2008. Carotid artery dissection in an adult with the Simpson–Golabi–Behmel syndrome. Am. J. Med. Genet. A 146A, 464–467. Pilia, G., et al., 1996. Mutations in GPC3, a glypican gene, cause the Simpson–Golabi– Behmel overgrowth syndrome. Nat. Genet. 12, 241–247. Ryan, K.E., Chiang, C., 2012. Hedgehog secretion and signal transduction in vertebrates. J. Biol. Chem. 287, 17905–17913. Saunders, S., Paine-Saunders, S., Lander, A.D., 1997. Expression of the cell surface proteoglycan glypican-5 is developmentally regulated in kidney, limb, and brain. Dev. Biol. 190, 78–93. Shi, W., Filmus, J., 2009. A patient with the Simson–Golabi–Behmel syndrome displays a loss-of-function point mutation in GPC3 that inhibits the attachment of this proteoglycan to the cell surface. Am. J. Med. Genet. 149A, 552–554. Smart, A.D., et al., 2011. Heparan sulfate proteoglycan specificity during axon pathway formation in Drosophila embryo. Dev. Neurobiol. 71, 608–618. Song, H.H., et al., 1997. OCI-5/rat glypican-3 binds to fibroblast growth factor-2 but not to insulin-like growth factor-2. J. Biol. Chem. 272, 7574–7577. St-Jacques, B., et al., 1999. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13, 2072–2086. Taylor, D.R., Hooper, N.M., 2008. The low-density lipoprotein receptor-related protein 1 (LRP1) mediates the endocytosis of the cellular prion protein. Biochem. J. 402, 17–23. Teglund, S., Toftgard, R., 2010. Hedgehog beyond medulloblastoma and basal cell carcinoma. Biochim. Biophys. Acta 1805, 181–208. Therond, P.P., 2012. Release and transportation of hedgehog molecules. Curr. Opin. Cell Biol. 24, 173–180. Toftgard, R., 2009. Two sides to cilia in cancer. Nat. Med. 15, 994–996. Traister, A., et al., 2008. Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. Biochem. J. 410, 503–511. Veugelers, M., et al., 1999. Glypican-6, a new member of the glypican family of cell surface proteoglycans. J. Biol. Chem. 274, 26968–26977. Williams, E.H., et al., 2010. Dally-like core protein and its mammalian homologues mediate stimulatory and inhibitory effects on Hedgehog signal response. Proc. Natl. Acad. Sci. U. S. A. 107, 5869–5874. Williamson, D., et al., 2007. Role for amplification and expression of glypican-5 in rhabdomyosarcoma. Cancer Res. 67, 57–65. Wilson, N.H., Stoeckli, E.T., 2013. Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner. Neuron 79, 478–491. Witt, R.M., Hecht, M.L., Pazyra-Murphy, M.F., Cohen, S.M., Noti, C., van Kuppevelt, T.H., Fuller, M., Chan, J.A., Hopwood, J., Seeberger, P.H., Segal, R.A., 2013. Heparan sulfate proteoglycans containing a Glypican 5 core and 2-O-sulfo-iduronic acid function as sonic hedgehog co-receptors to promote proliferation. J. Biol. Chem 288, 26275–26288. Yan, D., et al., 2010. The cell-surface proteins Dally-like and Ihog differentially regulate Hedgehog signaling strength and range during development. Development 137, 2033–2044. Young, E.L., et al., 2006. Expanding the clinical picture of Simpson–Golabi–Behmel syndrome. Pediatr. Neurol. 34, 139–142. Zheng, X., et al., 2010. Genetic and biochemical definition of the Hedgehog receptor. Genes Dev. 24, 57–71.
Please cite this article as: Filmus, J., Capurro, M., The role of glypicans in Hedgehog signaling, Matrix Biol. (2014), http://dx.doi.org/10.1016/ j.matbio.2013.12.007
