Abstract: Transforming growth factor-β1 (TGF-β1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-β type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-β/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-β1 signaling and the role of endoglin for both pathways in endoglin-deficient L6E9 cells. In this myoblastic cell line, TGF-β1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-β1) are two examples. TGF-β1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-β1-induced collagen I expression and (CAGA)12-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-β1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway. Transforming growth factor-β1 (TGF-β1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-β type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-β/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-β1 signaling and the role of endoglin for both pathways in endoglin-deficient L6E9 cells. In this myoblastic cell line, TGF-β1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-β1) are two examples. TGF-β1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-β1-induced collagen I expression and (CAGA)12-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-β1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway. TGF-β 3The abbreviations used are: TGF-β, transforming growth factor-β; MOPS, 3-(N-morpholino)propanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 2-(N-morpholino)ethanesulfonic acid; RT, reverse transcription; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GM, growth medium; DM, differentiation medium; HSC, hepatic stellate cells; PC, parenchymal cells. and BMP belong to the TGF-β superfamily of cytokines regulating a broad spectrum of cellular functions, including proliferation, apoptosis, and differentiation (1Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2112) Google Scholar, 2Blobe G.C. Schiemann W.P. Lodish H.F. N. Engl. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2218) Google Scholar). Mammals possess three different TGF-β isoforms (i.e. TGF-β1, TGF-β2, and TGF-β3) and more than 20 BMP-related proteins. 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We show for the first time that TGF-β1 activates two different signaling pathways in this myogenic cell line. One activates matrix gene expression via Smad3, and the other inhibits myogenic differentiation by up-regulation of Id1 via the Smad1/Smad5 pathway. Additionally, we demonstrate that L6E9 cells are highly responsive to BMP-7. BMP-7 inhibited TGF-β1-mediated (CAGA)12-MLP-Luc reporter gene activation and collagen I expression. Transient expression of endoglin resulted in the inhibition of TGF-β1/Smad3 responses, whereas signaling via the BMP-7/Smad1/5 pathway was enhanced. Cell Culture—L6E9 myoblasts were maintained in the undifferentiated state in growth medium (GM) containing HEPES-buffered DMEM (BioWhittaker, Verviers, Belgium) supplemented with 20% (v/v) fetal calf serum (FCS) (Perbio Science, Cramlington, UK), 4 mmol/liter l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Cambrex, Verviers, Belgium). To induce differentiation, cells were shifted to differentiation medium (DM) composed of HEPES-buffered DMEM supplemented with 0.5% (v/v) FCS. When indicated, the cells were incubated with 5 μm ALK5 inhibitor SB431542 (Tocris Bioscience, Ellisville, MO). Primary hepatic stellate cells (HSC) were isolated as described before and passaged once to obtain myofibroblasts (39Schafer S. Zerbe O. Gressner A.M. Hepatology. 1987; 7: 680-687Crossref PubMed Scopus (252) Google Scholar). Parenchymal liver cells (PC, i.e. hepatocytes) were isolated following the collagenase method of Seglen (40Seglen P.O. Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5280) Google Scholar). All cultures were kept in a humidified atmosphere containing 5% CO2. Plasmids—The following plasmids have been described previously: CA-ALK-1 and CA-ALK-5 encoding constitutive active forms of the human TGF-β receptors ALK-1 and ALK-5 (24Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (926) Google Scholar); the luciferase reporter constructs (CAGA)12-MLP-Luc (41Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1611) Google Scholar) and (BRE)2-Luc (42Korchynskyi O. ten Dijke P. J. Biol. Chem. 2002; 277: 4883-4891Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar); pcDNA-endoglin and pCMV-HA-rTβRIII encoding rat endoglin (43Meurer S.K. Tihaa L. Lahme B. Gressner A.M. Weiskirchen R. J. Biol. Chem. 2005; 280: 3078-3087Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar); and rat betaglycan (33Lopez-Casillas F. Cheifetz S. Doody J. Andres J.L. Lane W.S. Massague J. Cell. 1991; 67: 785-795Abstract Full Text PDF PubMed Scopus (562) Google Scholar). Expression plasmids for rat Smad1 (IRBPp993A072D2) and mouse Id1 (IRAVp968B0979D) were purchased from the RZPD Resource Center (Berlin, Germany). The vector pEGFP-C1 was purchased from Clontech. For cloning pCMV-rSmad2 and pCMV-rSmad3, the coding regions of the respective rat Smads were amplified by RT-PCR using the Expand High Fidelity PCR system (Roche Applied Science, Mannheim, Germany) and the primers listed in supplemental Table I. The cycling conditions were as follows: Smad3, initial denaturation for 2 min at 94 °C and then 35 cycles at 94 °C for 15 s, 54 °C for 30 s, 72 °C for 2.5 min, followed by a final elongation step at 72 °C for 10 min; Smad2, initial denaturation for 2 min at 94 °C and then 35 cycles at 94 °C for 15 s, 52 °C for 30 s, 72 °C for 2 min, followed by a final elongation at 72 °C for 10 min. The resulting PCR fragments were first cloned into the pGEM ®-T Easy vector (Promega, Mannheim, Germany), verified by sequence analysis, and subsequently cloned into pcDNA3 (Invitrogen, Karlsruhe, Germany). Transient Transfections—For transfection, cells were plated in 6-well dishes at a density of 2.5 × 105 cells/well and transfected with 2 μg of DNA per well of indicated expression plasmids using the FuGENE 6™ transfection reagent (Roche Applied Science). After 24 h, the medium was renewed, and cells were extracted after 48 h. Stimulation Experiments—COS-7 and L6E9 cells were cultured in growth medium containing 10 or 20% (v/v) FCS, respectively. The serum was reduced to 0.5% FCS 16 h before stimulation. For short (1 h) and long term stimulation (0–10; 12 and 24 h), the FCS content was lowered to 0.2%, and the medium was supplemented with TGF-β1 (0.05–5 ng/ml) or BMP-7 (5–100 ng/ml). To detect Smad phosphorylation, the cells were extracted 1 h after addition of ligand in lysis buffer (50 mmol/liter Tris-HCl (pH 7.2), 250 mmol/liter NaCl, 2% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, and 2.5 mmol/liter EDTA) containing the Complete™ mixture of proteinase inhibitors (Roche Applied Science) and the phosphatase inhibitor mixture set 2 (Sigma, Taufkirchen, Germany). Luciferase Reporter Gene Assay—L6E9 cells were plated in 6-well plates at a density of 2.5 × 105 cells/well and transfected with 1 μg of (CAGA)12-MLP-Luc and (BRE)2-Luc or 2 μg of CA-ALK-1, CA-ALK-5, and pcDNA-endoglin using the Lipofectamine Plus reagent (Invitrogen). Briefly, transfections were carried out in DMEM without supplements, and the medium was replaced by normal growth medium 6 h post-transfection. After a further 12-h incubation period, the medium was renewed, and 24 h later the cells were starved for 16 h in medium containing 0.5% FCS. Thereafter, stimulation with TGF-β1 and BMP-7 was carried out for indicated times in medium containing 0.2% FCS. Cell extracts were prepared in 350 μl of passive lysis buffer (Promega), and 20 μl were applied to luciferase measurements. All experiments were done in triplicate and normalized to the protein content, and the relative luciferase activities (±S.D.) are given. Western Blot Analysis—For Western blot analysis, cells were washed in ice-cold Hanks' buffered saline solution and extracted in lysis buffer containing proteinase and phosphatase inhibitors. Equal amounts of protein lysates were diluted with nonreducing NuPAGE lithium dodecyl sulfate-electrophoresis sample buffer (Invitrogen), heated at 75 °C for 10 min, and separated on 10 or 4–12% BisTris gels (Invitrogen) using MOPS-SDS running buffer (50 mmol/liter MOPS, 50 mmol/liter Tris-HCl (pH 7.7), 3.47 mmol/liter SDS, and 1.025 mmol/liter EDTA) or MES-SDS running buffer (50 mmol/liter MES, 50 mmol/liter Tris-HCl (pH 7.3), 3.47 mmol/liter SDS, and 1.025 mmol/liter EDTA), respectively. The analysis of collagen I expression was done on 3–8% Tris acetate gels (Invitrogen) in MOPS-SDS running buffer. Proteins were electroblotted using NuPAGE transfer buffer (Invitrogen) onto nitrocellulose membranes (Schleicher & Schuell), and equal protein loading was monitored in Ponceau S stain. Unspecific binding sites were blocked in TBST (10 mm Tris-HCl, 150 mm NaCl, 0.1% (v/v) Tween 20 (pH 7.6)) containing 5% (w/v) nonfat milk powder. Primary antibodies used are listed in supplemental Table 2. The antibody PPabE1 was raised by order against keyhole limpet hemocyanin-coupled peptide LALHPSTLSQEVY corresponding to amino acids 548–560 of rat endoglin (Davids Biotechnologie, Regensburg, Germany). Primary antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) using the SuperSignal West Dura Extended Duration substrate (Perbio Science). In some cases the signals were enhanced with a biotinylated secondary antibody prior to incubation with a streptavidin-horseradish peroxidase conjugate. Signal quantification and normalization were done in a Lumi-Imager™ (Roche Applied Science) using the LumiAnalyst 3.0 software. Affinity Labeling of TGF-β Receptors and Immunoprecipitation—125I-TGF-β1 affinity labeling and cross-linking experiments were performed as described previously (43Meurer S.K. Tihaa L. Lahme B. Gressner A.M. Weiskirchen R. J. Biol. Chem. 2005; 280: 3078-3087Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, confluent monolayers of L6E9 cells were washed and incubated in binding buffer (50 mmol/liter HEPES (pH 7.4), 128 mmol/liter NaCl, 5 mmol/liter KCl, 5 mmol/liter MgSO4, 13 mmol/liter CaCl2, 0.5% (v/v) bovine serum albumin) at 37 °C for 0.5 h. Thereafter, the cells were washed in ice-cold binding buffer, and ligand binding was performed for 3 h at 4 °C using 2.8 ng of 125I-TGF-β1/ml with a activity of 1621 Ci/mmol (Amersham Biosciences Buckinghamshire, UK). After incubation with disuccinimidyl suberate (Perbio Science) for 15 min at 4 °C, proteins were extracted and subjected to immunoprecipitation with receptor-specific antibodies. The precipitates were resolved by SDS-PAGE. The gels were dried and exposed X-Omat AR films (Eastman Kodak Co.). RT-PCR and Data Analysis—For RT-PCR experiments, purified samples of total RNA (1 μg each) were reverse-transcribed at 42 °C for 60 min using the Superscript II reverse transcriptase kit (Invitrogen) and random hexamer primers. Aliquots of first strand cDNAs were subjected to PCR in 1× PCR buffer (10 mmol/liter Tris-HCl (pH 8.3), 50 mmol/liter KCl, 1.5 mmol/liter MgCl2) using 2 μm forward/reverse primers, 0.2 mm each dATP, dCTP, dGTP, and dTTP, and 2.5 units of Taq DNA polymerase (Roche Applied Science). Cycle conditions were as follows: initial denaturation for 5 min at 95 °C; 35 cycles (40 cycles for BMP-RII, ALK-3, ALK-2, and BMP-7; 45 cycles for rS6, ALK-6, ActR-II, and ActR-IIb) at 95 °C for 60 s, 54 °C (55 °C for endoglin) for 60 s, 72 °C for 3 min; final elongation at 72 °C for 10 min. The PCR products were gel-purified and sequenced using the ABI Prism BigDye termination reaction kit as described elsewhere (44Weiskirchen R. Gressner A.M. Biochem. Biophys. Res. Commun. 2000; 274: 655-663Crossref PubMed Scopus (23) Google Scholar). Sequence data analysis was performed at the NCBI using the BLAST algorithm (45Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61086) Google Scholar). Statistics—Results are presented as the mean of three independent experiments (±S.D.). Statistical analyses were performed with an unpaired Student's t test. Differences found were considered as significant (*) or highly significant (**) at p < 0.05 or p < 0.01, respectively. L6E9 Cells Are Deficient for Endoglin and Mediate Smad1/Smad5 Phosphorylation in Response to BMP-7 and TGF-β1—To determine the function of endoglin in BMP-7 and TGF-β1 signaling in L6E9 cells, we first re-evaluated the reported endoglin deficiency at both the RNA and protein level. In rat hepatic stellate cells, we found two specific endoglin transcripts of 3 and 3.6 kb in size, whereas no endoglin signals were obtained in L6E9 cells (not shown). The deficiency for endoglin was further confirmed by RT-PCR (not shown) and Western blot analysis (Fig. 1A). In addition, affinity labeling experiments with iodinated TGF-β1 followed by immunoprecipitation reconfirmed the absence of functional endoglin in L6E9 cells (see below). To focus on the Smad activation pattern in L6E9 cells, we next performed stimulation experiments with TGF-β1 and BMP-7 and analyzed the respective protein extracts with antibodies that exclusively detect carboxyl-terminal Smad phosphorylation (induced by the type I receptor kinase) and not those of the Smad linker regions (induced by mitogen-activated protein kinases). When cells were stimulated with BMP-2 or BMP-7, a strong phosphorylation of Smad1/Smad5 was provoked (Fig. 1B). When L6E9 cells were stimulated with TGF-β1, we observed phosphorylation of Smad2 and Smad1/Smad5 indicating that TGF-β1 activates both Smad signaling pathways. TGF-β1 Dose-dependently Activates the ALK-5 and ALK-1 Pathways—The presence of ALK-1 in L6E9 cells was analyzed by RT-PCR (Fig. 2A) and in cross-linking experiments (Fig. 2B). The cross-linking experiments further confirmed the already mentioned absence of functional endoglin in L6E9 cells. Stimulation of L6E9 cells with increasing amounts of TGF-β1 revealed that Smad1/Smad5 and Smad3 were dose-dependently phosphorylated (Fig. 2C). Both signals were abrogated in the presence of the synthetic ALK-5 inhibitor SB431542. Consistent with this finding, we observed a time-dependent accumulation of PAI-1 in supernatants of TGF-β1-stimulated L6E9 cells and a strong activation of the pSmad3 reporter (CAGA)12-MLP-Luc upon TGF-β1 stimulation (Fig. 2, D and E), which was prevented in the presence of SB431542 (data not shown). Similarly, a strong stimulation was induced by a constitutive active ALK-5 receptor (CA-ALK-5) (Fig. 2F) that induces Smad2 and Smad3 phosphorylation. In conclusion, this set of experiments revealed that TGF-β1 was sufficient to activate Smad3 and Smad1/Smad5. Interestingly, both phosphorylation of Smad3 and Smad1/5 was blocked in the presence of SB431542 (cf. Fig. 2C). Because ALK-1 is insensitive toward this inhibitor (46Laping N.J. Grygielko E. Mathur A. Butter S. Bomberger J. Tweed C. Martin W. Fornwald J. Lehr R. Harling J. Gaster L. Callahan J.F. Olson B.A. Mol. Pharmacol. 2002; 62: 58-64Crossref PubMed Scopus (540) Google Scholar), this finding indicated that ALK-1 signaling itself is ALK-5-dependent in L6E9 cells. BMP-7 Signaling and Receptors in L6E9 Cells—To examine BMP-7 signaling in L6E9 cells, we first analyzed the expression of potential BMP-7 membrane receptors, i.e. ActR-I (ALK-2), BMPR-IA (ALK-3), or BMPR-IB (ALK-6) by RT-PCR. Under the experimental setting, we were able to amplify fragments specific for ALK-3 (677 bp) and to a much lower content ALK-6 (620 bp), but we failed to detect ALK-2 mRNA expression in L6E9 cells (Fig. 3A). In addition, the analysis revealed that the putative type II receptors BMPR-II (682 bp), ActR-IIB (678 bp), and ActR-II (700 bp) were expressed in L6E9 cells. In contrast, BMP-7 transcripts were not found. As positive controls for BMP-7 (507 bp) and ALK-2 (1560 bp), we analyzed cDNAs from rat hepatocytes (PC) or rat HSC that express high contents of corresponding genes. 4O. Scherner, S. K. Meurer, L. Tihaa, A. M. Gressner, and R. Weiskirchen, unpublished observations. Having shown that the necessary receptors for BMP-7 are expressed, we next examined the activation of the downstream effectors Smad1/Smad5 by BMP-7 in Western blot (Fig. 3B). In agreement with Smad1/Smad5 activation, the (BRE)2-Luc reporter harboring a Smad1/Smad5-dependent response element of the Id1 promoter was dose-dependently stimulated by BMP-7 (Fig. 3C), whereas the (CAGA)12-MLP-Luc reporter was left unaffected (data not shown). In contrast to the comparable activation potency of the (CAGA)12-MLP-Luc construct by CA-ALK-5 and TGF-β1 (cf. Fig. 2F), the (BRE)2-Luc reporter was only weakly stimulated by a constitutive active ALK-1 receptor (CA-ALK-1) (Fig. 3D) that potently induced the phosphorylation of Smads involved in BMP signaling. BMP-7 Blocks TGF-β1 Signaling in L6E9 Cells—To analyze the interdependence of BMP-7 and TGF-β1 signaling, we performed advanced reporter assays. TGF-β1 alone strongly activated the (CAGA)12-MLP-Luc-reporter, but when BMP-7 was simultaneously applied, the reporter activity was significantly reduced (Fig. 4A). Consistently, the amount of pSmad3 was lower in the presence of BMP-7 compared with samples that were only treated with TGF-β1 (Fig. 4B). Conversely, BMP-7 induced a strong activation of the (BRE)2-Luc-reporter that was decreased when TGF-β1 was present (Fig. 4C). Likewise, application of TGF-β1 in the presence of BMP-7 resulted in reduced Smad1 and/or Smad5 phosphorylation. In addition, Smad3 phosphorylation was only marginally increased by TGF-β1 in the presence of BMP-7 (Fig. 4D). Differential Effects of BMP-7 and TGF-β1 on Id1 and Collagen I Expression—To further examine BMP-7 and TGF-β1 downs