Glycosylphosphatidylinositol-anchored Proteins Regulate Transforming Growth Factor-β Signaling in Human Keratinocytes (2003)

Glycosylphosphatidylinositol (GPI)-anchored proteins have been demonstrated to bind transforming growth factor-β (TGF-β) in certain cell lines. However, the identity of these GPI-anchored proteins and the role they may play in TGF-β signaling remain unknown. We have previously reported the presence of GPI-anchored TGF-β-binding proteins on human skin fibroblasts and keratinocytes (Tam, B. Y. Y., and Philip, A. (1998) J. Cell. Physiol. 176, 553–564; Tam, B. Y. Y., Germain, L., and Philip, A. (1998) J. Cell. Biochem. 70, 573–586). On human keratinocytes, we identified a 150-kDa GPI-anchored TGF-β1-binding protein (r150) and demonstrated that it can form a heteromeric complex with the type I and II TGF-β signaling receptors. To explore whether GPI-anchored proteins modulate TGF-β signaling in keratinocytes, we created keratinocytes defective in GPI anchor biosynthesis (GPI mutant cells) by chemical mutagenesis of HaCaT cells. Mutant clones were selected by fluorescence-activated cell sorting analysis based on the loss of a CD59 marker. In comparison with parental HaCaT cells, GPI mutant cells demonstrated a significant loss of r150 expression. In contrast, the levels of the type I and II TGF-β receptors and their ligand affinities, cell morphology, and doubling time remained unchanged. Importantly, GPI mutant cells displayed enhanced gene transcriptional activity and Smad2 and Smad3 activation in response to TGF-β1 treatment in a dose-dependent manner. Taken together, our results indicate that GPI-anchored protein(s) inhibit TGF-β signaling and implicate r150 as the GPI-anchored protein responsible for this inhibition in human keratinocytes. Glycosylphosphatidylinositol (GPI)-anchored proteins have been demonstrated to bind transforming growth factor-β (TGF-β) in certain cell lines. However, the identity of these GPI-anchored proteins and the role they may play in TGF-β signaling remain unknown. We have previously reported the presence of GPI-anchored TGF-β-binding proteins on human skin fibroblasts and keratinocytes (Tam, B. Y. Y., and Philip, A. (1998) J. Cell. Physiol. 176, 553–564; Tam, B. Y. Y., Germain, L., and Philip, A. (1998) J. Cell. Biochem. 70, 573–586). On human keratinocytes, we identified a 150-kDa GPI-anchored TGF-β1-binding protein (r150) and demonstrated that it can form a heteromeric complex with the type I and II TGF-β signaling receptors. To explore whether GPI-anchored proteins modulate TGF-β signaling in keratinocytes, we created keratinocytes defective in GPI anchor biosynthesis (GPI mutant cells) by chemical mutagenesis of HaCaT cells. Mutant clones were selected by fluorescence-activated cell sorting analysis based on the loss of a CD59 marker. In comparison with parental HaCaT cells, GPI mutant cells demonstrated a significant loss of r150 expression. In contrast, the levels of the type I and II TGF-β receptors and their ligand affinities, cell morphology, and doubling time remained unchanged. Importantly, GPI mutant cells displayed enhanced gene transcriptional activity and Smad2 and Smad3 activation in response to TGF-β1 treatment in a dose-dependent manner. Taken together, our results indicate that GPI-anchored protein(s) inhibit TGF-β signaling and implicate r150 as the GPI-anchored protein responsible for this inhibition in human keratinocytes. TGF-β 1The abbreviations used are: TGF-βtransforming growth factor-βGPIglycosylphosphatidylinositolPAI-1plasminogen activator inhibitor-1MLPmajor late promoterPBSphosphate-buffered salineEMSethyl methanesulfonateFACSfluorescence-activated cell sortingGPI MGPI mutantGPI NMGPI non-mutantSTATsignal transducer and activator of transcription. is a 25-kDa multifunctional growth factor that plays an important role in cell growth and differentiation, extracellular matrix deposition, cell adhesion, and immunomodulation (1.Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors I. Springer-Verlag New York, Inc., New York1990Google Scholar). It is a member of the TGF-β superfamily, which includes activin, inhibin, bone morphogenic protein, growth and differentiation factor-1, and glial derived neurotropic growth factor (2.Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1731) Google Scholar). Three distinct isoforms of TGF-β (TGF-β1, -β2, and -β3) have been described in mammals. There is ∼70% homology between the isoforms, but they appear to have distinct activities in vivo as suggested by their differences in receptor binding; potencies; developmental expression; and the dissimilar phenotypes exhibited by mice null for TGF-β1, -β2, or -β3 genes (3.Roberts A.B. Sporn M.B. Mol. Reprod. Dev. 1992; 32: 91-98Crossref PubMed Scopus (156) Google Scholar, 4.Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3988) Google Scholar). transforming growth factor-β glycosylphosphatidylinositol plasminogen activator inhibitor-1 major late promoter phosphate-buffered saline ethyl methanesulfonate fluorescence-activated cell sorting GPI mutant GPI non-mutant signal transducer and activator of transcription. TGF-β signaling is transduced by a pair of transmembrane serine/threonine kinases known as type I and type II receptors, which are present in almost all cell types analyzed. The type I receptor does not bind TGF-β in the absence of the type II receptor. According to the current model, the binding of TGF-β to the type II receptor, a constitutively active kinase, results in the recruitment and phosphorylation of the type I receptor in its "GS sequence" upstream of the kinase domain, resulting in its activation. The activated type I receptor then phosphorylates its intracellular substrates, Smad2 and Smad3, which are released from the receptor and form a complex with Smad4. This heteromeric Smad complex then translocates to the nucleus and interacts with DNA to regulate target gene expression by recruiting transcriptional coactivators such as CBP (cAMP-responsive element-binding protein-binding protein)/p300 and corepressors such as TGF-β-induced factor, Ski, and SnoN (5.Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3349) Google Scholar, 6.Miyazono K. Cytokine Growth Factor Rev. 2000; 11: 15-22Crossref PubMed Scopus (236) Google Scholar, 7.Zimmerman C.M. Padgett R.W. Gene (Amst.). 2000; 249: 17-30Crossref PubMed Scopus (151) Google Scholar). Other cell-surface TGF-β-binding proteins that have been identified include betaglycan (type III TGF-β receptor) and endoglin (CD105), which have a more limited tissue distribution and are considered to be accessory receptors. Betaglycan, a membrane proteoglycan, binds all three TGF-β isoforms with high affinity and is thought to facilitate TGF-β binding to the type II TGF-β receptor (8.Lopez-Casillas F. Wrana J.L. Massagué J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (776) Google Scholar, 9.Lopez-Casillas F. Payne H.M. Andres J.L. Massagué J. J. Cell Biol. 1994; 124: 557-568Crossref PubMed Scopus (349) Google Scholar). Endoglin shows 70% homology to betaglycan and binds TGF-β1 and TGF-β3 with high affinity through its association with the type II receptor (10.Cheifetz S. Hernandez H. Laiho M. ten Dijke P. Iwata K.K. Massagué J. J. Biol. Chem. 1990; 265: 20533-20538Abstract Full Text PDF PubMed Google Scholar, 11.Yamashita H. Ichijo H. Grimsby S. Moren A. ten Dijke P. Miyazono K. J. Biol. Chem. 1994; 269: 1995-2001Abstract Full Text PDF PubMed Google Scholar). Furthermore, endoglin appears to interact with activin and bone morphogenic protein in the presence of their cognate receptors (12.Letamendía A. Lastres P. Botella L.M. Raab U. Langa C. Velasco B. Attisano L. Bernabeu C. J. Biol. Chem. 1998; 273: 33011-33019Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Interestingly, overexpression of endoglin attenuates TGF-β responses in monocytes and myoblasts (13.Li C. Hampson L. Bernabeu C. Kumar S. FASEB J. 2000; 14: 55-64Crossref PubMed Scopus (226) Google Scholar), whereas overexpression of betaglycan enhances TGF-β responses in these cells (9.Lopez-Casillas F. Payne H.M. Andres J.L. Massagué J. J. Cell Biol. 1994; 124: 557-568Crossref PubMed Scopus (349) Google Scholar). Glycosylphosphatidylinositol (GPI)-anchored proteins, which lack transmembrane and cytoplasmic domains, have also been shown to bind TGF-β. These proteins have been reported in several cell lines (14.Cheifetz S. Massagué J. J. Biol. Chem. 1991; 266: 20767-20772Abstract Full Text PDF PubMed Google Scholar, 15.Dumont N. O'Connor-McCourt M.D. Philip A. Mol. Cell. Endocrinol. 1995; 111: 57-66Crossref PubMed Scopus (31) Google Scholar, 16.Tam B.Y.Y. Philip A. J. Cell. Physiol. 1998; 176: 553-564Crossref PubMed Scopus (18) Google Scholar, 17.Tam B.Y.Y. Germain L. Philip A. J. Cell. Biochem. 1998; 70: 573-586Crossref PubMed Scopus (14) Google Scholar), but the identity of these GPI-anchored proteins and the role they may play in TGF-β signaling remain unknown. We have shown recently that GPI-anchored proteins bind TGF-β in early passage human endometrial stromal cells (15.Dumont N. O'Connor-McCourt M.D. Philip A. Mol. Cell. Endocrinol. 1995; 111: 57-66Crossref PubMed Scopus (31) Google Scholar), skin fibroblasts (16.Tam B.Y.Y. Philip A. J. Cell. Physiol. 1998; 176: 553-564Crossref PubMed Scopus (18) Google Scholar), and keratinocytes (17.Tam B.Y.Y. Germain L. Philip A. J. Cell. Biochem. 1998; 70: 573-586Crossref PubMed Scopus (14) Google Scholar). In addition, we have characterized a 150-kDa GPI-anchored TGF-β1-binding protein on human keratinocytes (designated as r150) that can form a heteromeric complex with the type I and II TGF-β receptors (17.Tam B.Y.Y. Germain L. Philip A. J. Cell. Biochem. 1998; 70: 573-586Crossref PubMed Scopus (14) Google Scholar). In the present study, we have generated a GPI anchor mutant form of a human keratinocyte cell line (HaCaT) and have demonstrated that these cells display reduced levels of r150 on their cell surface relative to parental HaCaT cells. Furthermore, these cells show an enhanced responsiveness to TGF-β1. Our results suggest that GPI-anchored proteins regulate TGF-β signaling and implicate r150 as an inhibitor of TGF-β1 signal transduction in human keratinocytes. Plasmids—The 3TP-lux and pCMV-βgal constructs were gifts from Dr. M. D. O'Connor-McCourt (Biotechnology Research Institute, Montreal, Quebec, Canada). The 3TP-lux construct contains three 12-O-tetradecanoylphorbol-13-acetate-responsive elements and TGF-β-responsive elements from the PAI-1 promoter fused in tandem to a luciferase reporter (18.Wrana J.L. Attisano L. Wieser R. Ventrus F. Massagué J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2114) Google Scholar). (CAGA)12-lux and the empty vector MLP-lux were gifts from Dr. S. Huet (Laboratoire Glaxo Wellcome, France). (CAGA)12-lux is a luciferase construct specific for Smad3/4-driven signaling and contains 12 repeats of the sequence CAGA upstream of the MLP of adenovirus and a luciferase gene (19.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1583) Google Scholar, 20.Dennler S. Huet S. Gauthier J.-M. Oncogene. 1999; 18: 1643-1648Crossref PubMed Scopus (154) Google Scholar). Dominant-negative Smad3 and its empty vector pcDNA3 were gifts from Dr. J. J. Lebrun (McGill University). Dominant-negative Smad3 has its C-terminal region deleted (21.Valderrama-Carvaji H. Cockolakis E. Lacerte A. Lee E.-H. Krystal G. Ali S. Lebrun J.J. Nat. Cell Biol. 2002; 4: 963-969Crossref PubMed Scopus (137) Google Scholar). Cell Culture—HaCaT cells were obtained from Dr. P. Boukamp (German Cancer Research Center, Heidelberg, Germany). HaCaT cells are a spontaneously immortalized cell line and show no major differences in differentiation compared with normal keratinocytes. They exhibit a transformed phenotype, but are not tumorigenic (22.Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3476) Google Scholar). HaCaT cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 mm sodium pyruvate, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin (Invitrogen). Cell Growth Assay—Cells (8 × 105) were seeded in 60-mm dishes (Falcon, Mississauga, Ontario, Canada). At the indicated times, cells were trypsinized and counted on a hemocytometer. Affinity Labeling of Cells with125I-TGF-β—Affinity cross-link labeling was performed as described by Dumont et al. (15.Dumont N. O'Connor-McCourt M.D. Philip A. Mol. Cell. Endocrinol. 1995; 111: 57-66Crossref PubMed Scopus (31) Google Scholar). Briefly, cell monolayers were washed three times for 10 min each with ice-cold binding buffer (PBS with Ca2+ and Mg2+, pH 7.4 (Dulbecco's PBS) containing 0.1% bovine serum albumin). Cells were then incubated at 4 °C for 3 h with 100 pm125I-TGF-β1. Incubations were also done in the presence of excess nonradioactive TGF-β1 (Genzyme Corp., Cambridge, MA) to confirm specific binding (data not shown). The receptor-ligand complexes were then cross-linked with 1 mm bis(sulfosuccinimidyl)suberate (BS3, Pierce). After 10 min, the reaction was stopped by the addition of 500 mm glycine (final concentration of 10 mm) for 5 min. The supernatant was aspirated, and cells were washed twice with Dulbecco's PBS. Cells were lysed with solubilization buffer (20 mm Tris-HCl, pH 7.4, containing 1% Triton X-100, 10% glycerol, 1 mm EDTA, 10 μm phenylmethylsulfonyl fluoride, 200 μg/ml bovine serum albumin, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml benzamide, and 2 μg/ml pepstatin). The solubilized material was mixed with 0.2 volume of 5× electrophoresis sample buffer (0.25 m Tris-HCl, pH 6.8, 5% SDS, 50% glycerol, and trace bromphenol blue). Samples were resolved by 3–11% SDS-PAGE under nonreducing or reducing (in the presence of 5% β-mercaptoethanol) conditions. The gels were analyzed by autoradiography, and radioactive bands were quantitated by densitometry. Isolation and Cloning of GPI Anchor Mutant Keratinocytes—The preparation and selection of cells mutated in GPI biosynthesis was based on the method described by Stevens (23.Stevens V.L. Methods Mol. Biol. 1999; 116: 13-22PubMed Google Scholar). HaCaT cells grown to 50–60% confluence in a 75-cm2 flask (Falcon) were treated with 300 μg/ml EMS for 24 h at 37 °C in an atmosphere of 5% CO2 and air. Cells were allowed to recover for 48 h in normal growth medium. FACS was performed to select cells that lost expression of GPI-anchored proteins on their cell surface. GPI anchor mutant cells were negatively sorted for a GPI-anchored protein (CD59) known to be expressed on human keratinocytes. Briefly, cells were trypsinized, and ∼2 × 106 cells were resuspended in 1 ml of FACS buffer (PBS containing 1% fetal bovine serum). Cells were incubated on ice for 45 min with 4 μg of fluorescein isothiocyanate-conjugated mouse monoclonal anti-human IgG2a (negative control) or anti-CD59 antibody (Pharmingen, Mississauga). Cells were pelleted by centrifugation, washed, and resuspended in 1 ml of FACS buffer. Propidium iodide (3 μg/ml; Sigma) was added to the cells prior to sorting, which was performed using the FACS Vantage system (BD Biosciences, Mississauga). Cell sorting was repeated twice. At the third and final FACS step, a single CD59-negative cell was seeded into each well of a 96-well plate using the FACS Vantage automated cell deposition unit. The surviving clones were analyzed by flow cytometry to determine which cells expressed deficient levels of CD59. Affinity cross-link labeling with125I-TGF-β1 was performed to confirm reduced expression of r150. Transient Transfection Assays—HaCaT cells were seeded at 2.5 × 105 cells/well in 12-well plates and allowed to attach for 24 h. Cells were transiently transfected with 1.0 μg each of 3TP-lux and pCMV-βgal using Superfect reagent (QIAGEN, Mississauga) according to the manufacturer's instructions. To determine whether the effects of TGF-β1on 3TP-lux reporter activity involve activation of Smad3, cells were transiently transfected with 0.5 μg each of 3TP-lux, pCMV-βgal, and either dominant-negative Smad3 or pcDNA3 (empty vector). Alternatively, cells were transfected with 0.5 μg each of (CAGA)12-lux or MLP-lux (control reporter) and pCMV-βgal. Cells were allowed to recover overnight and then serum-starved for 3 h prior to treatment with various doses of TGF-β1 or TGF-β2 (Austral Biochemicals Inc.) for the indicated times. Cells were solubilized in 150 μl of lysis buffer (Pharmingen) for 30 min at 4 °C. Samples were centrifuged for 10 min at 10,000 × g, and supernatants were assayed for luciferase and β-galactosidase activities using commercially available kits (BD Biosciences). Western Blotting of Phosphorylated Smad2—HaCaT and GPI M cells were grown in 60-mm dishes to 70–80% confluency and serum-starved overnight. Cells were washed with PBS and treated with various doses of TGF-β1 or TGF-β2 for the indicated times. Cells were solubilized with 500 μl of lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 50 mm NaF, 50 mm β-glycerophosphate, 1 mm sodium orthovanadate, 1 mm dithiothreitol, 5 mm EDTA, pH 8.0, 1% Nonidet P-40, 10% glycerol, 10 μm phenylmethylsulfonyl fluoride, 200 μg/ml bovine serum albumin, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml benzamide, and 2 μg/ml pepstatin) for 30 min at 4 °C with mild agitation. Cell lysates were collected and centrifuged for 10 min at 10,000 × g. Protein concentrations were determined using the Bio-Rad protein assay kit. Lysates containing equivalent protein concentrations were mixed with 0.2 volume of 5× electrophoresis sample buffer containing 5% β-mercaptoethanol and boiled for 5 min. Samples were resolved on 7.5% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked for 3 h in blocking buffer (30 mm Tris, pH 7.5, 150 mm NaCl, and 0.05% (w/v) Tween 20 (Tris-buffered saline/Tween) containing 5% (w/v) skim milk powder). The blot was incubated for 60 min at room temperature with a 1:2000 dilution of anti-phospho-Smad2 antibody (a gift from Dr. S. Souchelnytskyi, Ludwig Institute for Cancer Research, Uppsala, Sweden). Membranes were also probed with goat polyclonal anti-Smad2 or rabbit polyclonal anti-STAT3 antibody (Santa Cruz Biotechnology) to confirm equal protein loading. Blots were washed with Tris-buffered saline/Tween and incubated for 45 min at room temperature with a 1:5000 dilution of the appropriate secondary antibody (rabbit or goat) conjugated to horseradish peroxidase (Pierce). Chemiluminescence detection of the immune complexes was performed using the ECL system (Amersham Biosciences) according to the manufacturer's instructions. In Vitro Kinase Assay—HaCaT and GPI M cells grown in 75-cm2 flasks were serum-starved overnight and treated with or without 100 TGF-β1 for 20 min at 37 °C. Cells were in 200 μl of lysis buffer and incubated for an 30 min on The cells were and the supernatants were with μl of protein (Amersham Biosciences) and 3 μg/ml rabbit polyclonal antibody (Santa Cruz Biotechnology) for 2 h at 4 °C. of protein was incubated with 3 μg/ml II TGF-β receptor antibody (Santa Cruz Biotechnology) overnight at 4 °C. complexes were incubated with 50 μl of protein for 2 h at 4 °C. The were pelleted and washed three times with 0.5 ml of lysis buffer and time with 0.5 ml of kinase buffer (50 mm Tris, pH 7.4, 10 mm and 1 mm After the were resuspended in 20 μl of kinase buffer and incubated with 10 of for 30 min at 30 °C. The reaction was stopped by the addition of 5 μl of 5× electrophoresis sample buffer containing 5% β-mercaptoethanol and boiled for 5 min. The were on a 7.5% SDS-polyacrylamide and analyzed by Isolation and Cloning of HaCaT Cells in GPI Anchor cells were treated with 300 μg/ml EMS for 24 h as described under EMS is an that has been used to mutagenesis of in the biosynthesis of the GPI anchor (23.Stevens V.L. Methods Mol. Biol. 1999; 116: 13-22PubMed Google Scholar, A. PubMed Scopus (201) Google Scholar). a GPI-anchored protein expressed on keratinocytes, was used as a for the of GPI cells. CD59 is an inhibitor of the membrane complex J. J. 1994; PubMed Scopus Google Scholar, N. J. 1998; 111: Full Text Full Text PDF PubMed Scopus Google Scholar). After EMS cells were with fluorescein isothiocyanate-conjugated anti-CD59 antibody and negatively sorted by FACS Cells selected from CD59-negative were using the FACS Vantage automated cell deposition unit. clones and were for CD59 expression by flow clones displayed reduced expression of CD59 on their cell surface and were to be mutated in GPI anchor These clones were as GPI M and GPI the flow analysis of parental HaCaT and GPI M cells. the expression of CD59 was not in GPI M cells, the of CD59 was of that obtained for HaCaT cells results were obtained with GPI cells (data not shown). To confirm that the GPI mutant cells display reduced levels of r150 on their cell we HaCaT and GPI M cells and r150 levels were compared by shows a significant in r150 levels in GPI M cells relative to parental cells, whereas the relative of type and III receptors in HaCaT GPI M cells remained Cell Growth and in GPI Anchor Mutant or loss of GPI-anchored proteins as a of EMS mutagenesis has no reported effects on cell or cell in cell types (23.Stevens V.L. Methods Mol. Biol. 1999; 116: 13-22PubMed Google Scholar). To confirm that these are not in GPI M cells, the doubling times and cell of HaCaT and GPI M cells were that HaCaT and GPI M cells have growth with equivalent doubling times In addition, the of HaCaT and GPI M cells are GPI-anchored 3TP-lux transcriptional reporter contains TGF-β-responsive elements from the PAI-1 promoter and is used to TGF-β signaling L. J. R. J. Cell Biol. 2000; PubMed Scopus Google Scholar). To the of GPI-anchored proteins on TGF-β signaling in keratinocytes, we transfected HaCaT and GPI M cells with the 3TP-lux reporter construct and compared luciferase activities 4 and h of TGF-β1 HaCaT cell to the EMS but not mutated in GPI anchor biosynthesis (GPI was as a GPI M cells exhibited a in luciferase activity relative to HaCaT cells a treatment with 10 TGF-β1 At 100 TGF-β1, GPI M cells an in luciferase activity relative to HaCaT cells Interestingly, the of luciferase activity in HaCaT cells treated with 10 and 100 TGF-β1 remained and whereas GPI M cells displayed a response with and h of TGF-β1 GPI M cells displayed a enhanced response to 100 TGF-β1 compared with HaCaT cells GPI M cells exhibited a which is twice that of HaCaT and GPI cells for GPI M in response to 100 TGF-β1. In contrast, GPI and GPI M cells no significant in response to 100 TGF-β2 In addition, HaCaT and GPI M cells displayed a dose-dependent response to 10 and 100 TGF-β1 at this time (data not shown). After treatment with 10 TGF-β1 for GPI M cells displayed a of luciferase with HaCaT and GPI cells and in luciferase for GPI M GPI Anchor Mutant Cells Smad2 at of determine whether the loss of GPI-anchored proteins has on the phosphorylation of HaCaT and GPI M cells were treated with TGF-β1 for 20 min, and was performed with anti-phospho-Smad2 demonstrated in GPI M cells displayed enhanced phosphorylation of Smad2 compared with HaCaT cells. In cell Smad2 phosphorylation at 2 TGF-β1 and in a dose-dependent manner. However, GPI M cells exhibited enhanced Smad2 phosphorylation in response to and 5 and (50 doses of TGF-β1. with an anti-Smad2 antibody that and of Smad2 demonstrated equivalent protein and that the enhanced Smad2 phosphorylation was not a of expression of protein of HaCaT and GPI M cell lysates was also with anti-STAT3 antibody the is not by TGF-β treatment the time used J.J. E. 2002; PubMed Scopus Google Scholar). Importantly, TGF-β2 treatment not in enhanced phosphorylation of Smad2 in GPI M cells compared with HaCaT cells, as GPI M and HaCaT cells demonstrated the of Smad2 phosphorylation Smad2 in GPI Anchor Mutant whether the GPI cells exhibited enhanced Smad2 phosphorylation to TGF-β1 In response to 100 TGF-β1, of Smad2 phosphorylation in HaCaT cells at 10 min and was for min. In comparison with HaCaT cells, GPI M cells displayed Smad2 which was at 45 min and was for to min. The GPI anchor mutant (GPI also exhibited enhanced Smad2 phosphorylation in response to TGF-β1 In this GPI cells displayed an of Smad2 phosphorylation compared with HaCaT cells 20 min, and this was for to min. On the Smad2 phosphorylation in GPI cells in response to TGF-β1 was to that in HaCaT cells at all time with anti-STAT3 antibody demonstrated equal protein of the cell These results show that the GPI cells (GPI M and GPI but not GPI cells, exhibit enhanced phosphorylation of Smad2 in comparison with parental HaCaT cells. GPI Anchor Mutant Cells Smad3 at of Smad2 and Smad3 as of responses by they have been shown to have L. J. R. J. Cell Biol. 2000; PubMed Scopus Google Scholar, E. J. D. M. C. R. Roberts A.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Smad3 binds DNA whereas Smad2 does not (19.Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1583) Google Scholar). the described that GPI mutant cells displayed enhanced Smad2 it was of to determine whether these cells Smad3 activation. To Smad3 we transfected GPI M and HaCaT cells with the (CAGA)12-lux reporter construct specific for signaling and compared luciferase activity in response to TGF-β1 shows that GPI M cells a luciferase activity HaCaT cells at and 50 TGF-β1 and of in GPI Anchor Mutant Cells then determined whether Smad3 is in the of PAI-1 transcriptional activity by TGF-β in GPI M cells. The PAI-1 promoter present in the 3TP-lux reporter construct contains three binding for Smad3, but is also to such as J. Y. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). To determine whether the enhanced 3TP-lux reporter activity in GPI