Regulation of Liver Growth by Glypican 3, CD81, Hedgehog, and Hhex

Previous studies from our laboratory have found glypican 3 (GPC3) as a negative regulator of growth. CD81 was found to be a binding partner for GPC3, and its expression and co-localization with GPC3 increased at the end of hepatocyte proliferation. However, the mechanisms through which these two molecules might regulate liver regeneration are not known. We tested the hypothesis that GPC3 down-regulates the hedgehog (HH) signaling pathway by competing with patched-1 for HH binding. We found decreased GPC3-Indian HH binding at peak proliferation in mice followed by increase in glioblastoma 1 protein (effector of HH signaling). We performed a yeast two-hybrid assay and identified hematopoietically expressed homeobox (Hhex, a known transcriptional repressor) as a binding partner for CD81. We tested the hypothesis that Hhex binding to CD81 keeps it outside the nucleus. However, when GPC3 binds to CD81, CD81-Hhex binding decreases, resulting in nuclear translocation of Hhex and transcriptional repression. In support of this, we found decreased GPC3-CD81 binding at hepatocyte proliferation peak, increased CD81-Hhex binding, and decreased nuclear Hhex. GPC3 transgenic mice were used as an additional tool to test our hypothesis. Overall, our data suggest that GPC3 down-regulates cell proliferation by binding to HH and down-regulating the HH signaling pathway and binding with CD81, thus making it unavailable to bind to Hhex and causing its nuclear translocation. Previous studies from our laboratory have found glypican 3 (GPC3) as a negative regulator of growth. CD81 was found to be a binding partner for GPC3, and its expression and co-localization with GPC3 increased at the end of hepatocyte proliferation. However, the mechanisms through which these two molecules might regulate liver regeneration are not known. We tested the hypothesis that GPC3 down-regulates the hedgehog (HH) signaling pathway by competing with patched-1 for HH binding. We found decreased GPC3-Indian HH binding at peak proliferation in mice followed by increase in glioblastoma 1 protein (effector of HH signaling). We performed a yeast two-hybrid assay and identified hematopoietically expressed homeobox (Hhex, a known transcriptional repressor) as a binding partner for CD81. We tested the hypothesis that Hhex binding to CD81 keeps it outside the nucleus. However, when GPC3 binds to CD81, CD81-Hhex binding decreases, resulting in nuclear translocation of Hhex and transcriptional repression. In support of this, we found decreased GPC3-CD81 binding at hepatocyte proliferation peak, increased CD81-Hhex binding, and decreased nuclear Hhex. GPC3 transgenic mice were used as an additional tool to test our hypothesis. Overall, our data suggest that GPC3 down-regulates cell proliferation by binding to HH and down-regulating the HH signaling pathway and binding with CD81, thus making it unavailable to bind to Hhex and causing its nuclear translocation. Glypican 3 (GPC3) is a heparan sulfate proteoglycan that is bound to cell surface by glycosylphosphatidylinositol.1Fransson L.A. Glypicans.Int J Biochem Cell Biol. 2003; 35: 125-129Crossref PubMed Scopus (75) Google Scholar Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by prenatal and postnatal organ overgrowth, including liver.2Hughes-Benzie R.M. Pilia G. Xuan J.Y. Hunter A.G. Chen E. Golabi M. Hurst J.A. Kobori J. Marymee K. Pagon R.A. Punnett H.H. Schelley S. Tolmie J.L. Wohlferd M.M. Grossman T. Schlessinger D. MacKenzie A.E. Simpson-Golabi-Behmel syndrome: genotype/phenotype analysis of 18 affected males from 7 unrelated families.Am J Med Genet. 1996; 66: 227-234Crossref PubMed Scopus (115) Google Scholar, 3Pellegrini M. Pilia G. Pantano S. Lucchini F. Uda M. Fumi M. Cao A. Schlessinger D. Forabosco A. Gpc3 expression correlates with the phenotype of the Simpson-Golabi-Behmel syndrome.Dev Dyn. 1998; 213: 431-439Crossref PubMed Scopus (95) Google Scholar CD81, also known as target of antiproliferative antibody, is a cell surface protein that belongs to tetraspanin 4 superfamily.4Oren R. Takahashi S. Doss C. Levy R. Levy S. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins.Mol Cell Biol. 1990; 10: 4007-4015Crossref PubMed Scopus (336) Google Scholar It is involved in mediating signal transduction events that play a role in regulation of cell development, activation, growth, and motility.5Levy S. Todd S.C. Maecker H.T. CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system.Ann Rev Immunol. 1998; 16: 89-109Crossref PubMed Scopus (435) Google Scholar, 6Yang X. Kovalenko O.V. Tang W. Claas C. Stipp C.S. Hemler M.E. Palmitoylation supports assembly and function of integrin-tetraspanin complexes.J Cell Biol. 2004; 167: 1231-1240Crossref PubMed Scopus (164) Google Scholar Our laboratory has previously demonstrated that GPC3 acts as a negative regulator of liver regeneration.7Liu B. Paranjpe S. Bowen W.C. Bell A.W. Luo J.H. Yu Y.P. Mars W.M. Michalopoulos G.K. Investigation of the role of glypican 3 in liver regeneration and hepatocyte proliferation.Am J Pathol. 2009; 175: 717-724Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Liu B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar This observation was accompanied by an increase in expression and co-localization of CD81 (a binding partner for GPC3) in tandem with GPC3 at the end of hepatocyte proliferation.7Liu B. Paranjpe S. Bowen W.C. Bell A.W. Luo J.H. Yu Y.P. Mars W.M. Michalopoulos G.K. Investigation of the role of glypican 3 in liver regeneration and hepatocyte proliferation.Am J Pathol. 2009; 175: 717-724Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 8Liu B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar However, the mechanisms and pathways through which these two molecules might regulate liver regeneration are not known. In the present study, we sought to identify the mechanisms and potential associated partners through which these two proteins might mediate regulation of liver growth. Previous literature suggests that GPC3 can bind to hedgehog (HH) ligands and inhibit HH signaling during development.9Capurro M.I. Xu P. Shi W. Li F. Jia A. Filmus J. Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding.Dev Cell. 2008; 14: 700-711Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar The HH signaling pathway has been previously reported to play an important role in liver regeneration after partial hepatectomy (PHx) in mice.10Ochoa B. Syn W.K. Delgado I. Karaca G.F. Jung Y. Wang J. Zubiaga A.M. Fresnedo O. Omenetti A. Zdanowicz M. Choi S.S. Diehl A.M. Hedgehog signaling is critical for normal liver regeneration after partial hepatectomy in mice.Hepatology. 2010; 51: 1712-1723Crossref PubMed Scopus (137) Google Scholar We tested the hypothesis that GPC3 down-regulates the HH signaling pathway by competing with patched-1 for HH binding to regulate liver regeneration (Figure 1). On the other hand, GPC3-HH binding decreases to free up HH signaling at the time of proliferation after PHx (Figure 1). To investigate the mechanisms for CD81’s role in regulating liver growth, we performed a yeast two-hybrid assay to identify potential binding partners for CD81. Hematopoietically expressed homeobox protein (Hhex) was a CD81 binding partner under high-stringency binding conditions. Most of the studies describing Hhex’s role are in liver development.11Hunter M.P. Wilson C.M. Jiang X. Cong R. Vasavada H. Kaestner K.H. Bogue C.W. The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis.Dev Biol. 2007; 308: 355-367Crossref PubMed Scopus (111) Google Scholar, 12Paz H. Lynch M.R. Bogue C.W. Gasson J.C. The homeobox gene Hhex regulates the earliest stages of definitive hematopoiesis.Blood. 2010; 116: 1254-1262Crossref PubMed Scopus (29) Google Scholar Hhex is expressed in some adult tissues, including liver, lung, spleen, thymus, and pancreas.13Bedford F.K. Ashworth A. Enver T. Wiedemann L.M. HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human.Nucleic Acids Res. 1993; 21: 1245-1249Crossref PubMed Scopus (131) Google Scholar, 14Hromas R. Radich J. Collins S. PCR cloning of an orphan homeobox gene (PRH) preferentially expressed in myeloid and liver cells.Biochem Biophys Res Commun. 1993; 195: 976-983Crossref PubMed Scopus (69) Google Scholar, 15Crompton M.R. Bartlett T.J. MacGregor A.D. Manfioletti G. Buratti E. Giancotti V. Goodwin G.H. Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells.Nucleic Acids Res. 1992; 20: 5661-5667Crossref PubMed Scopus (155) Google Scholar Hhex mRNA has been previously detected in adult mouse liver and has been shown to be a transcriptional repressor.16Tanaka T. Inazu T. Yamada K. Myint Z. Keng V.W. Inoue Y. Taniguchi N. Noguchi T. cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein.Biochem J. 1999; 339: 111-117Crossref PubMed Scopus (72) Google Scholar, 17Brickman J.M. Jones C.M. Clements M. Smith J.C. Beddington R.S. Hex is a transcriptional repressor that contributes to anterior identity and suppresses Spemann organiser function.Development. 2000; 127: 2303-2315PubMed Google Scholar Its role in adult liver remains unexplored for the most part. Previous studies suggest that the role of Hhex is dependent on its shuttling between cytoplasm and nucleus.18Puppin C. Puglisi F. Pellizzari L. Manfioletti G. Pestrin M. Pandolfi M. Piga A. Di Loreto C. Damante G. HEX expression and localization in normal mammary gland and breast carcinoma.BMC Cancer. 2006; 6: 192Crossref PubMed Scopus (29) Google Scholar However, the mechanisms governing its translocation into the nucleus are not known. We hypothesized that Hhex binding to CD81 during hepatocyte proliferation keeps it outside the nucleus (Figure 1). When GPC3 binds to CD81, less CD81 is available for binding to Hhex, resulting in nuclear translocation of Hhex, in turn causing transcriptional repression (Figure 1). Thus, the present study tests two mechanisms for the role of GPC3 as a growth regulator: GPC3 binds with HH ligand to decrease HH signaling and GPC3 binds to CD81, leading to nuclear translocation of transcriptional repressor Hhex. We used wild-type (WT) and GPC3 transgenic (TG) mice (previously found to exhibit decreased liver regeneration response)8Liu B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar as an additional tool to test the hypotheses. Generation of male GPC3 TG mice is described previously.8Liu B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar These mice show significant over expression of GPC3 protein and mRNA compared with their WT littermates (friend leukemia virus B strain mice).8Liu B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar The Matchmaker GAL4 yeast two-hybrid system (Clontech, Mountain View, CA) was used for identification of CD81-binding partners according to standard protocol.19Van Criekinge W. Beyaert R. Yeast two-hybrid: state of the art.Bio Proc Online. 1999; 2: 1-38Crossref PubMed Scopus (86) Google Scholar As bait for screening, the vector pGBKT7 expressing a fusion protein composed of full-length rat CD81 cDNA linked to the DNA-binding domain was constructed. Positive clones were selected on SD/-Ade/-His/-Leu/-Trp high-stringency YPDA (yeast, peptone, dextrose, adenine) plates with X-Gal. Plasmids from positive clones were subsequently isolated from the yeast, transferred to Escherichia coli Top10 competent cells (Invitrogen, Grand Island, NY), and sequenced. The GenBank/National Center for Biotechnology Information databases were screened for similar sequences using BLAST search. Nuclear and cytoplasmic extracts pooled from at least four mice per time point were prepared using NE-PER nuclear and cytoplasmic extraction kit according to the manufacturer’s protocol (catalog number 78833; Pierce Biotechnology, Rockford, IL). Solubilized protein was subjected to Western blot analysis as described previously.20Bhave V.S. Paranjpe S. Bowen W.C. Donthamsetty S. Bell A.W. Khillan J.S. Michalopoulos G.K. Genes inducing iPS phenotype play a role in hepatocyte survival and proliferation in vitro and liver regeneration in vivo.Hepatology. 2011; 54: 1360-1370Crossref PubMed Scopus (30) Google Scholar Membranes were stained with Ponceau stain and β-actin (for nuclear and cytoplasmic extracts, respectively) to verify equal loading of total protein and transfer efficiency. Horseradish peroxidase–conjugated secondary antibodies were used at a 1:50,000 dilution (Jackson Immunoresearch Laboratories, West Grove, PA). Primary antibodies used with their concentrations were as follows: GPC3 (ARP37665 Aviva Systems Biology Corp., San Diego, CA, 1:1000; SC10456, Santa Cruz Biotechnology, Dallas, TX, 1:200), CD81 (GTX75432 GeneTex, Irvine, CA, 1:1000; SC70803, Santa Cruz, 1:500), Indian HH (IHH) patched-1 and Hhex Cell San Diego, was used to PHx was performed on at least four mice per per time point as described C. H. and for partial hepatectomy in 2008; PubMed Scopus Google Scholar from GPC3 TG and WT were on and end of hepatocyte after PHx for liver were used for was by the in target CA) for The were in protein for followed by with for 1 at The was linked to secondary followed by was used as the which in a liver was in assay with of protein pooled from at least four mice per per time point were to a of and by with from the of and of for 1 at were from with antibodies at followed by at with were and with and in and from of liver protein were by and subjected to Western that the Western of proteins that are using are not of their expression equal Our in on the of bound protein to which in protein To Hhex as a binding partner for CD81 from the yeast two-hybrid we performed a fusion protein protein from (catalog number was used for the The fusion protein was using a to of that with the cell pooled from four WT mice was in of with and was to and using fusion protein protein was to the followed by pooled cell from WT mice The was and at by for an The were and was using with The was used for Western blot analysis to Hhex and to The was and for CD81. are expressed as were by analysis of followed by significant test to which were from other from using the The for was using GPC3 and that is a binding partner for GPC3 (Figure The of GPC3 and patched-1 that bound to at time after PHx to the total of is shown in WT mice a significant decrease in binding on after PHx of compared with thus making available for binding to patched-1 for HH This the increased binding in WT mice on (Figure and In to this, GPC3 TG mice hepatocyte B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google to show a decrease in binding on after PHx compared with increase in binding on was not significant (Figure was GPC3 and less patched-1 bound to in TG mice compared with WT mice on These data a in of HH signaling pathway in the TG on Nuclear an of HH signaling was on in WT mice compared with (Figure and In the TG mice a significant decrease in compared with for TG mice and for WT mice (Figure and was also in TG mice on after PHx by (Figure of HH was in WT mice after GPC3 TG mice a decrease that was not significant (Figure and Overall, the HH signaling pathway to be in WT mice after it was in the GPC3 TG of in WT and GPC3 TG mouse of at and after from our laboratory in the B. Paranjpe S. Bowen W.C. Bell A.W. Luo J.H. Yu Y.P. Mars W.M. Michalopoulos G.K. Investigation of the role of glypican 3 in liver regeneration and hepatocyte proliferation.Am J Pathol. 2009; 175: 717-724Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar we found that GPC3 binds to CD81 by in mouse as (Figure The of CD81 bound to GPC3 at time after PHx to the total of GPC3 is shown in was CD81 bound to GPC3 in TG mice compared with WT mice at time (Figure and the and TG the of CD81 bound to GPC3 decreased on and increased by end of hepatocyte These data support our in B. Paranjpe S. Bowen W.C. Bell A.W. Luo J.H. Yu Y.P. Mars W.M. Michalopoulos G.K. Investigation of the role of glypican 3 in liver regeneration and hepatocyte proliferation.Am J Pathol. 2009; 175: 717-724Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar that GPC3 and CD81 binding the end of hepatocyte proliferation. GPC3 in WT and TG mice on after This is most to loading of GPC3 during of the and not of less GPC3 expression at time However, we are CD81 bound to GPC3 GPC3 on not our Hhex as of the binding partners of CD81 under plates with proteins identified were and We to Hhex for its role as a transcriptional To Hhex as a binding partner for CD81, we performed two additional assay and with CD81. To Hhex as a binding partner for CD81 in mouse liver, we performed a the yeast two-hybrid CD81 cDNA was our bait In the we used fusion protein protein as our bait and it with pooled liver CD81 in the it as a binding partner (Figure and with protein were to CD81 (Figure and that CD81 is a binding partner for Hhex and not Thus, using two and using proteins as we were to show that CD81 and Hhex are binding partners for To study the of CD81-Hhex binding during liver we performed for the two proteins (Figure The of Hhex bound to CD81 at time was to the total CD81 (Figure In WT less Hhex was bound to CD81 on (Figure and was a significant increase in bound Hhex on after PHx followed by its decrease to normal by In to in the TG was Hhex bound to CD81 to with on which decreased on through To test the of Hhex bound to CD81 the with the of nuclear Hhex we at nuclear Hhex by Western blot (Figure to Ponceau stain for equal loading (Figure As nuclear Hhex was in WT mice on compared with In it increased in TG mice on compared with was also nuclear Hhex in TG WT mice at the peak of proliferation These data support our hypothesis that the binding of CD81 to Hhex might it outside the nucleus and its transcriptional repressor of patched-1 by is Li A. E. Zdanowicz M. J. M. J.H. L.M. Diehl A.M. Hedgehog signaling J 2006; PubMed Scopus Google Scholar To test we WT mouse liver and at patched-1 protein by Western blot analysis in hepatocyte and cell using two We were to a with the antibodies in liver hepatocyte and (Figure GPC3 acts as a growth regulator in the of a known signaling domain in the cell GPC3 TG mice have liver regeneration on after PHx compared with WT mice as by B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar Our data at least two pathways through which GPC3 regulates growth. it binds to HH ligand (IHH) and down-regulates HH signaling in it binds to CD81, leading to decreased CD81-Hhex binding, which in turn nuclear translocation of transcriptional repressor Hhex. of TG mice with can phenotype at least be an of Hhex might not result in of growth of GPC3 of and of Hhex other transcriptional repression. To our is the study describing CD81 and Hhex to be binding partners for We using in the present our suggest of HH signaling in the liver as a GPC3 can regulate HH signaling in is not Previous studies have found that are not to HH signaling not the HH Li A. E. Zdanowicz M. J. M. J.H. L.M. Diehl A.M. Hedgehog signaling J 2006; PubMed Scopus Google Scholar Our data suggest that patched-1 (Figure and and B. Syn W.K. Delgado I. Karaca G.F. Jung Y. Wang J. Zubiaga A.M. Fresnedo O. Omenetti A. Zdanowicz M. Choi S.S. Diehl A.M. Hedgehog signaling is critical for normal liver regeneration after partial hepatectomy in mice.Hepatology. 2010; 51: 1712-1723Crossref PubMed Scopus (137) Google Scholar the transcriptional of HH We also expression in WT and GPC3 TG mice by Western blot and not in after PHx not the that the family of are as by HH a other studies have reported that also be of pathway through and in O. J. M. M.E. D. is through mechanisms in and cell survival and 2009; PubMed Scopus Google Scholar an has not been demonstrated in GPC3 TG mice have less on after PHx compared with WT mice and This be to from our laboratory of gene in TG mice compared with WT B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar We to GPC3 TG mice in study to and of GPC3 the mechanisms for its growth Our data suggest that were in WT and GPC3 TG mice at that after a with of GPC3 in TG these mice a of for proliferation as by increased by Western blot and and that the pathway is in TG in WT mice in The HH signaling pathway has been reported to be involved in and cell Li A. E. Zdanowicz M. J. M. J.H. L.M. Diehl A.M. Hedgehog signaling J 2006; PubMed Scopus Google Scholar We were not to of cell in these by which to WT However, we have not at of cells in the of GPC3 TG mice at to the nuclear Hhex repressor) was less in TG mice liver compared with WT mice (Figure and for cell in these TG However, for proliferation not result in a of proliferation at as by in TG and WT mice at shown previously.8Liu B. Bell A.W. Paranjpe S. Bowen W.C. Khillan J.S. Luo J.H. Mars W.M. Michalopoulos G.K. Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.Hepatology. 2010; 52: 1060-1067Crossref PubMed Scopus (60) Google Scholar These in protein expression in of TG mice are of to gene However, at after PHx TG mice were to the HH pathway to a regeneration to in and CD81-Hhex binding of GPC3 in these The that might the in GPC3 binding to its partners are not known and of This study that in protein by of bound protein to of their might regulation of growth. our study that the HH system is in our studies with liver the of cells and In to the potential on binding of GPC3 to HH ligands in the can decrease the of HH ligands for patched-1 binding in HH cells other also leading to an of HH signaling pathway in the hepatocyte proliferation after PHx is dependent on hepatocyte growth by which are known to be HH GPC3-HH binding also hepatocyte proliferation in an