Processing by Convertases Is Not Required for Glypican-3-induced Stimulation of Hepatocellular Carcinoma Growth

Abstract

Glypicans are a family of heparan sulfate proteoglycans that are bound to the cell surface by a lipid anchor. Six members of this family have been identified in mammals (GPC1-GPC6). Glypicans act as regulators of the activity of various cytokines, including Wnts, Hedgehogs, and bone morphogenetic proteins. It has been reported that processing by a convertase is required for GPC3 activity during convergent extension in zebrafish embryos, for GPC3-induced regulation of Wnt signaling, and for the binding of GPC3 to Wnt5a. In our laboratory, we have recently demonstrated that GPC3 promotes the growth of hepatocellular carcinomas (HCCs) by stimulating canonical Wnt signaling. Because there is increasing evidence indicating that the structural requirements for GPC3 activity are cell type specific, we decided to investigate whether GPC3 needs to be processed by convertases to stimulate cell proliferation and Wnt signaling in HCC cells. We report here that a mutant GPC3 that cannot be processed by convertases is still able to play its stimulatory role in Wnt activity and HCC growth. Glypicans are a family of heparan sulfate proteoglycans that are bound to the cell surface by a lipid anchor. Six members of this family have been identified in mammals (GPC1-GPC6). Glypicans act as regulators of the activity of various cytokines, including Wnts, Hedgehogs, and bone morphogenetic proteins. It has been reported that processing by a convertase is required for GPC3 activity during convergent extension in zebrafish embryos, for GPC3-induced regulation of Wnt signaling, and for the binding of GPC3 to Wnt5a. In our laboratory, we have recently demonstrated that GPC3 promotes the growth of hepatocellular carcinomas (HCCs) by stimulating canonical Wnt signaling. Because there is increasing evidence indicating that the structural requirements for GPC3 activity are cell type specific, we decided to investigate whether GPC3 needs to be processed by convertases to stimulate cell proliferation and Wnt signaling in HCC cells. We report here that a mutant GPC3 that cannot be processed by convertases is still able to play its stimulatory role in Wnt activity and HCC growth. Glypicans are a family of heparan sulfate proteoglycans that are linked to the exocytoplasmic surface of the plasma membrane by a glycosyl-phosphatidylinositol anchor (1Lander A.D. Stipp C.S. Ivins J.K. Perspect. Dev. Neurobiol. 1998; 1: 1-7Google Scholar, 2Veugelers M. David G. Trends Glycosc. Glycothec. 1998; 10: 145-152Crossref Scopus (11) Google Scholar, 3Filmus J. Selleck S.B. J. Clin. Investig. 2001; 108: 497-501Crossref PubMed Scopus (330) Google Scholar, 4Filmus J. Song H.H. Proteoglycans. 2000; : 161-176Google Scholar). To date six glypicans have been identified in mammals (GPC1-GPC6) (4Filmus J. Song H.H. Proteoglycans. 2000; : 161-176Google Scholar, 5Veugelers M. De Cat B. Ceulemans H. Bruystens A.M. Coomans C. Durr J. Vermeesch J. Marynen P. David G. J. Biol. Chem. 1999; 274: 26968-26977Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In general, glypicans are expressed predominantly during development. Their expression levels change in a stage- and tissue-specific manner, suggesting that they are involved in morphogenesis (4Filmus J. Song H.H. Proteoglycans. 2000; : 161-176Google Scholar, 6Song H.H. Filmus J. Biochim. Biophys. Acta. 2002; 1573: 241-246Crossref PubMed Scopus (120) Google Scholar). Genetic and functional studies performed in Drosophila, Xenopus, zebrafish, and mammals have demonstrated that glypicans can stimulate the signaling activity of Wnts, Hedgehogs, and bone morphogenetic proteins (7Jackson S.M. Nakato H. Sugiura M. Jannuzi A. Oakes R. Kaluza V. Golden C. Selleck S.B. Development (Camb.). 1997; 124: 4113-4120Crossref PubMed Google Scholar, 8Baeg G.H. Perrimon N. Curr. Opin. Cell Biol. 2000; 12: 575-580Crossref PubMed Scopus (94) Google Scholar, 9Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (656) Google Scholar, 10Paine-Saunders S. Viviano B.L. Zupicich J. Skarnes W.C. Saunders S. Dev. Biol. 2000; 225: 179-187Crossref PubMed Scopus (154) Google Scholar, 11Lum L. Yao S. Mozer B. Rovescalli A. Von Kessler D. Nirenberg M. Beachy P.A. Science. 2003; 299: 2039-2045Crossref PubMed Scopus (457) Google Scholar, 12Desbordes S.C. Sanson B. Development (Camb.). 2003; 130: 6245-6255Crossref PubMed Scopus (121) Google Scholar, 13Topczewsky J. Sepich D.S. Myers D.C. Walker C. Amores A. Lele Z. Hammerschmidt M. Postlethwait J. Solnica-Krezel L. Dev. Cell. 2001; 1: 251-264Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 14Ohkarawa B. Yamamoto T.S. Tada M. Ueno N. Development (Camb.). 2003; 130: 2129-2138Crossref PubMed Scopus (151) Google Scholar, 15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar, 16Kramer K.L. Yost H.J. Annu. Rev. Genet. 2003; 37: 461-484Crossref PubMed Scopus (111) Google Scholar, 17Baeg G.H. Lin X. Khare N. Baumgartner S. Perrimon N. Development (Camb.). 2001; 128: 87-94Crossref PubMed Google Scholar). In the specific case of Wnts, glypicans have been reported to stimulate both the canonical and non-canonical pathways (8Baeg G.H. Perrimon N. Curr. Opin. Cell Biol. 2000; 12: 575-580Crossref PubMed Scopus (94) Google Scholar, 13Topczewsky J. Sepich D.S. Myers D.C. Walker C. Amores A. Lele Z. Hammerschmidt M. Postlethwait J. Solnica-Krezel L. Dev. Cell. 2001; 1: 251-264Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 14Ohkarawa B. Yamamoto T.S. Tada M. Ueno N. Development (Camb.). 2003; 130: 2129-2138Crossref PubMed Scopus (151) Google Scholar, 18Song H.H. Shi W. Xiang Y. Filmus J. J. Biol. Chem. 2005; 280: 2116-2125Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Because Wnts are known to bind to heparan sulfate (19Cumberledge S. Reichsman F. Trends Genet. 1997; 13: 421-423Abstract Full Text PDF PubMed Scopus (47) Google Scholar), it was originally proposed that the stimulatory activity of glypicans is based on their ability to act as facilitators of the interaction between Wnts and their receptors (8Baeg G.H. Perrimon N. Curr. Opin. Cell Biol. 2000; 12: 575-580Crossref PubMed Scopus (94) Google Scholar). This hypothesis has been supported by the finding that several Wnts can co-immunoprecipitate with glypicans (14Ohkarawa B. Yamamoto T.S. Tada M. Ueno N. Development (Camb.). 2003; 130: 2129-2138Crossref PubMed Scopus (151) Google Scholar, 15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar, 18Song H.H. Shi W. Xiang Y. Filmus J. J. Biol. Chem. 2005; 280: 2116-2125Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 20Ai X. Do A.T. Lozynska O. Kusche-Gullberg M. Lindhal U. Emerson C.P. J. Cell Biol. 2003; 162: 341-351Crossref PubMed Scopus (359) Google Scholar, 21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar), although it is now known that at least in certain cell systems the heparan sulfate chains are not required for the glypican-Wnt interaction (18Song H.H. Shi W. Xiang Y. Filmus J. J. Biol. Chem. 2005; 280: 2116-2125Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). This lack of requirement of the heparan sulfate chains for the interaction with a growth factor has also been reported for perlecan, another heparan sulfate proteoglycan that can regulate cell proliferation (22Gonzalez E.M. Mongiat M. Slater S.J. Baffa R. Iozzo R.V. J. Biol. Chem. 2003; 278: 38113-38116Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In addition to acting as facilitators of ligand-receptor interactions, glypicans have been shown to play a role during development in the transport of Wnts, Hedgehogs, and bone morphogenetic proteins for the purpose of morphogen gradient formation (23Fujise M. Takeo S. Kamimura K. Matsuo T. Aigaki T. Izumi S. Nakato H. Development (Camb.). 2003; 130: 1515-1522Crossref PubMed Scopus (189) Google Scholar, 24Han C. Belenkaya T.Y. Wang B. Li X. Development (Camb.). 2004; 131: 601-611Crossref PubMed Scopus (189) Google Scholar, 25Blair S.S. Curr. Biol. 2005; 15: R92-R93Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 26Baeg G.H. Selva E.M. Goodman R.M. Dasgupta R. Perrimon N. Dev. Biol. 2004; 276: 89-100Crossref PubMed Scopus (90) Google Scholar, 27Han C. Yan D. Belenkaya T.Y. Lin X. Development (Camb.). 2005; 132: 667-679Crossref PubMed Scopus (164) Google Scholar, 28Kirkpatrick C.A. Dimitroff B.D. Rawson J.M. Selleck S.B. Dev. Cell. 2004; 7: 513-523Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). One of the highly conserved structural features of the glypicans is the location of the insertion sites for the heparan sulfate chains, which seem to be restricted to the last 55 amino acids, placing such chains in proximity to the cell surface (6Song H.H. Filmus J. Biochim. Biophys. Acta. 2002; 1573: 241-246Crossref PubMed Scopus (120) Google Scholar). Another conserved feature of this protein family is the location of 14 cysteine residues, suggesting that glypicans share a highly similar three-dimensional structure (6Song H.H. Filmus J. Biochim. Biophys. Acta. 2002; 1573: 241-246Crossref PubMed Scopus (120) Google Scholar). Western blot analysis of glypicans has unveiled the existence of an internal cleavage site in at least three family members (GPC1, GPC3, and GPC4) (29David G. Lories V. Decock B. Marynen P. Cassiman J. Van Den Berghe H. J. Cell Biol. 1990; 111: 3165-3176Crossref PubMed Scopus (218) Google Scholar, 30Filmus J. Shi W. Wong Z.M. Wong M.J. Biochem. J. 1995; 311: 561-565Crossref PubMed Scopus (72) Google Scholar, 31Watanabe K. Yamada H. Yamaguchi Y. J. Cell Biol. 1995; 130: 1207-1218Crossref PubMed Scopus (136) Google Scholar). Cleavage at this site generates an ∼40-kDa N-terminal subunit and an ∼30-kDa C-terminal subunit containing the heparan sulfate chains (Fig. 1A). After cleavage both subunits remain linked to each other by one or more disulfide bonds and the N-terminal subunit is not released into the conditioned medium, but the subunits can be separated in reducing conditions (31Watanabe K. Yamada H. Yamaguchi Y. J. Cell Biol. 1995; 130: 1207-1218Crossref PubMed Scopus (136) Google Scholar). A recent study by De Cat et al. (15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar) on GPC3 shows that this endoproteolytic site can be cleaved by members of the convertase protein family. Moreover, this study shows that cleavage by a convertase is required for GPC3 activity during convergent extension in zebrafish embryos, for the binding of GPC3 to Wnt5a, and for GPC3-induced regulation of Wnt signaling (15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar). Convertases are ubiquitously expressed subtilisin-like serine endoproteases that activate proproteins by cleavage at specific recognition sites (32Taylor N.A. Van de Ven W.J.M. Creemers J.W.M. FASEB J. 2003; 17: 1215-1227Crossref PubMed Scopus (190) Google Scholar). The first mammalian convertase that was characterized was furin, but at least six other members of this family have been identified after furin. Many classes of proteins have been shown to be processed by convertases, including neuropeptides, hormones, growth factors, metalloproteases, and adhesion molecules (32Taylor N.A. Van de Ven W.J.M. Creemers J.W.M. FASEB J. 2003; 17: 1215-1227Crossref PubMed Scopus (190) Google Scholar). These proteins are usually synthesized as inactive precursors, and their processing by convertases is required for activation. It is now well established that convertases play a critical role in many physiological processes and that they are involved in numerous pathologies, including cancer (32Taylor N.A. Van de Ven W.J.M. Creemers J.W.M. FASEB J. 2003; 17: 1215-1227Crossref PubMed Scopus (190) Google Scholar, 33Khatib A.M. Siegfried G. Chretien M. Metrakos P. Seidah N.G. Am. J. Pathol. 2002; 160: 1921-1935Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Our laboratory has recently reported that GPC3 is up-regulated in most human hepatocellular carcinomas (HCC) 2The abbreviations used are: HCChepatocellular carcinomaGPC3glypican-3HAhemagglutinin AGPIglycosil-phosphatidylinositolEFelongation factorFACSfluorescence-activated cell sortingwtwild-type.2The abbreviations used are: HCChepatocellular carcinomaGPC3glypican-3HAhemagglutinin AGPIglycosil-phosphatidylinositolEFelongation factorFACSfluorescence-activated cell sortingwtwild-type. (34Capurro M. Wanless I.R. Sherman M. Deboer G. Shi W. Miyoshi E. Filmus J. Gastroenterology. 2003; 125: 81-90Abstract Full Text Full Text PDF Scopus (711) Google Scholar). In addition, we showed that GPC3 promotes the growth of HCC by stimulating canonical Wnt signaling (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). The growth-stimulating activity of GPC3 in HCC cells has also been reported by another laboratory (35Huang J.S. Chao C.C. Su T.L. Yeh S.H. Chen D.S. Chen C.T. Chen P.J. Jou Y.S. Biochem. Biophys. Res. Commun. 2004; 315: 950-958Crossref PubMed Scopus (123) Google Scholar). One interesting finding of our study was that the heparan sulfate chains were not essential for the GPC3-induced activation of Wnt signaling and for the GPC3-induced stimulation of HCC growth in vivo. Similarly, the heparan sulfate chains were shown to be dispensable for the regulatory activity of GPC4 in convergent extension during Xenopus gastrulation, a process that is also known to be driven by Wnt signaling (14Ohkarawa B. Yamamoto T.S. Tada M. Ueno N. Development (Camb.). 2003; 130: 2129-2138Crossref PubMed Scopus (151) Google Scholar). Contrarily, in the study by De Cat et al. (15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar) it was shown the heparan sulfate chains are essential for the regulatory activity of GPC3 on Wnt signaling in Chinese hamster ovary cells. Collectively, these results suggest that the structural requirements for GPC3-induced regulation on Wnt signaling are cell context specific. We decided to investigate whether the processing of GPC3 by convertases is for its regulatory activity of Wnt signaling in the context of HCC cells. We report here that a mutant GPC3 that cannot be cleaved by convertases is still able to stimulate the growth of HCC cells. Moreover, we also that cleavage by convertases is not required for the interaction of GPC3 with and and for the of canonical Wnt signaling. hepatocellular A factor cell hepatocellular A factor cell Cell and and cell were in essential with with essential amino and The cells were in and The and cell were the and the cell was by Miyoshi of cells with or as were and in containing To conditioned medium, the cells were at for cells with or were J. containing N-terminal A GPC3, the mutant GPC3 that cannot be and the mutant GPC3 the glycosyl-phosphatidylinositol were A. M. Shi W. Song H. Filmus J. J. Cell Biol. 1998; PubMed Scopus Google Scholar). of the GPC3 the endoproteolytic cleavage site in the N-terminal GPC3 and were to by and were by To that this mutant is convertase cells were with the GPC3 the GPC3 or with were in and the by Western blot or as (34Capurro M. Wanless I.R. Sherman M. Deboer G. Shi W. Miyoshi E. Filmus J. Gastroenterology. 2003; 125: 81-90Abstract Full Text Full Text PDF Scopus (711) Google Scholar). of HCC Cell and cells were with expression containing the GPC3 the GPC3 the or with as a cells were and as (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). The expression levels of GPC3 in the cell was also by as (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). Cell were into in the cells were and with a was at least three by cells were into the of a the the of the were by the and a was to the of between the of was by the and the of was as and were performed in with were in the of for with and with a cell were as (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar), and the of was by Western blot with the 14 was used as for cells were in at a of and with a driven by the V. N. P.J. D. de R. B. H. Science. 1997; PubMed Scopus Google Scholar), a expression and GPC3 GPC3 or One after the cells were for with conditioned cells or cells with as were and activity and activity were was for was performed three by To the with cells we a similar to that for the that we used for and for stimulating Wnt signaling the cells were with cells or cells with as for cells were with GPC3 or GPC3 mutant and or expression after cell were in in containing of protein were with protein during at After GPC3 was the at After the were and the of Wnt in the by Western blot an To for of GPC3 and Wnt levels were by Western blot analysis of of the GPC3 mutant GPC3 that cannot be processed by convertases was by and in the processing site with The of this mutant GPC3 to processing was by Western blot analysis of cells. shown in the an in the is used for Western blot analysis in reducing GPC3 generates a that to which to the of the of the (Fig. 1A). It also be that a of the to the of the GPC3, analysis with the shows that not the GPC3 that is on the cell surface is processed by convertases in cells. In addition, the Western blot analysis the of a This most the GPC3 protein that has not been Western blot analysis of the GPC3 mutant with the shows the of the of the with a of that is the GPC3 that GPC3 cleavage and the of the and of its not the site by the convertases is the cell containing GPC3 was with an that the at the the of the is This is the cannot the subunit and its the other the a that to the as a of cleavage of In addition, a of to GPC3 is of the GPC3 mutant with the that this mutant cannot be cleaved by GPC3 Cleavage by Convertases for the of HCC Cell have shown that GPC3 the in and in growth of and HCC cell that and GPC3, (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). cells are able to process GPC3, as by the that GPC3 generates in these cells with a (Fig. In addition, the ability of the HCC cells to GPC3 can be by the of a in Western with the (Fig. To a of the of GPC3 that is cleaved by convertases, we the HCC cell with a mutant GPC3 that cannot be (22Gonzalez E.M. Mongiat M. Slater S.J. Baffa R. Iozzo R.V. J. Biol. Chem. 2003; 278: 38113-38116Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). on the of the ∼30-kDa that to the we that of the GPC3 expressed by the and cell is cleaved by convertases (Fig. To whether the processing of GPC3 by convertases is for its we also and cells with the GPC3 of cells levels of mutant GPC3 were by and the expression of similar GPC3 levels in the various cell was by Western blot and analysis (Fig. It be that the cells seem to be able to heparan sulfate chains, the of of the in these cells is that for cells. the of GPC3 on the proliferation of the HCC cells was shown in we that both the GPC3 and the mutant GPC3 were able to stimulate the proliferation of the HCC cell to the Moreover, or mutant GPC3 the of cells in (Fig. We that the processing of GPC3 by convertases is not for its in and in activity in HCC cells. These are with the hypothesis that the structural requirements for activity are cell context specific. In it be that processing of GPC3 in the HCC cells is not required the levels of GPC3 are It be that we have shown that the levels of GPC3 expressed by and cells are the physiological they are that in a HCC cell (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). The GPC3-induced of Wnt in HCC of Cleavage by recently reported that GPC3 promotes HCC growth by stimulating the canonical Wnt (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). Because De Cat et al. (15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar) showed that convertase activity is required for GPC3-induced regulation of canonical Wnt activity and it is well established that glypicans can also the activity of other binding growth factors, we decided to investigate whether GPC3 also HCC cell proliferation by Wnt To this we the of or mutant GPC3 on the levels of in the and cells. shown in the mutant GPC3 is able to the of levels of similar to We that the processing of GPC3 by convertases is not required for the stimulation of canonical Wnt signaling in HCC cells. De Cat al. (15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar) showed that processing by convertases is required for the GPC3-induced regulation of canonical in Chinese hamster ovary and cells. Because and cells not (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar), it be proposed that the lack of requirement of convertase processing for GPC3-induced activation of Wnt signaling in HCC cells that we is to the that this requirement is restricted for the regulation of To this we the of GPC3 and the mutant in the of cells to Because is we decided to the cells with cells with a expression To the of the cells to we these cells with the canonical We that the mutant GPC3 was as as the GPC3 in stimulating activity (Fig. GPC3 by Convertases to Wnt in whether the lack of requirement of convertase processing for GPC3-induced stimulation of Wnt activity is a restricted to HCC we decided to study the of the mutant GPC3 on the to canonical Wnt in cell To this we activity in cells with the and various GPC3 expression cells was used as the of shows that GPC3 the of cells to and that the mutant GPC3 a similar a we also the of a mutant GPC3 the This mutant cannot be to the cell membrane and not stimulate Wnt signaling in HCC cells (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). was to stimulate activity in cells (Fig. Western blot analysis of GPC3 in the cells showed that the GPC3 and the mutant similar expression levels (Fig. for the of GPC3 and has been proposed that glypicans stimulate Wnt signaling by the interaction of Wnts and their signaling receptors (8Baeg G.H. Perrimon N. Curr. Opin. Cell Biol. 2000; 12: 575-580Crossref PubMed Scopus (94) Google Scholar). with we have demonstrated that in cells GPC3 with and (21Capurro M. Xiang Y.Y. Lobe C. Filmus J. Cancer Res. 2005; 65: 6245-6254Crossref PubMed Scopus (375) Google Scholar). De Cat et al. (15De Cat B. Muyldermans S.Y. Coomans C. Degeest G. Vanderschueren B. Creemers J. Biemar F. Peers B. David G. J. Cell Biol. 2003; 163: 625-635Crossref PubMed Scopus (138) Google Scholar) have shown that in cancer cells the GPC3 not co-immunoprecipitate with Wnt5a. We decided to whether the mutant GPC3 with and in cells. To this we or mutant GPC3 and and into cells. GPC3 was the cell the and the of Wnt in the was by Western blot with an shows that both and with or mutant We have shown here that in the context of HCC GPC3 not to be processed by convertases to be able to canonical Wnt signaling and to its in and in Moreover, we have shown that GPC3 can co-immunoprecipitate with Wnts and stimulate their signaling activity in indicating that the activity of GPC3 is not specific for HCC cells. we not convertase processing is required for GPC3 activity in but not in The interaction of Wnts with their signaling receptors is highly and other proteins that regulate have been identified R. Annu. Rev. Cell Dev. Biol. 2004; PubMed Scopus Google Scholar). We that the requirement of convertase processing for GPC3 activity be by the of the ligand-receptor in a cell context by the expression levels of the of such