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Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro
Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro Jos´e M. Mu˜noz-F´elix, Cristina Cuesta, Nuria Perretta-Tejedor, Mariela Subileau, Francisco J. L´opez-Hern´andez, Jos´e M. L´opez-Novoa, Carlos Mart´ınez-Salgado PII: DOI: Reference:
S0898-6568(16)30116-4 doi: 10.1016/j.cellsig.2016.05.015 CLS 8693
To appear in:
Cellular Signalling
Received date: Revised date: Accepted date:
24 November 2015 12 May 2016 17 May 2016
Please cite this article as: Jos´e M. Mu˜ noz-F´elix, Cristina Cuesta, Nuria Perretta-Tejedor, Mariela Subileau, Francisco J. L´opez-Hern´ andez, Jos´e M. L´opez-Novoa, Carlos Mart´ınezSalgado, Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro, Cellular Signalling (2016), doi: 10.1016/j.cellsig.2016.05.015
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Identification of bone morphogenetic protein 9
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(BMP9) as a novel profibrotic factor in vitro
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José M. Muñoz-Félix1,2, Cristina Cuesta1,2*, Nuria Perretta-Tejedor1,2*, Mariela Subileau3, Francisco J. López-Hernández1,2,4, José M. López-Novoa1,2, Carlos
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Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain;
Unidad de Fisiopatología Renal y Cardiovascular, Instituto Reina Sofía de
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Martínez-Salgado1,2,4
Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain; 3Inserm, U1036, CEA, DSV, Irtsv, Laboratoire Biologie du Cancer et de l’Infection, Université Joseph Fourier, Grenoble, F-38054 France; 4Instituto de Estudios de Ciencias de la Salud de Castilla y León (IECSCYL),
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Hospital Universitario de Salamanca, Salamanca, Spain.
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Short title: BMP9 induces fibrosis in vitro
Correspondence to: Carlos Martínez-Salgado, Unidad de Investigación, Hospital
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Universitario de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain. Phone: +34 923 294500 ext. 1945, email: [email protected]
*These authors have contributed equally to this work
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ABSTRACT Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in
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fibrosis development has been extensively studied, the involvement of other members
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of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptorlike kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is
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unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways.
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In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM
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synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of
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ALK1.
Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic
receptors.
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factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5
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Keywords: BMP9, ALK1, ALK5, fibroblasts, fibrosis, extracellular matrix proteins
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1. INTRODUCTION
Fibrotic diseases include a wide spectrum of entities characterized by the progressive
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and uncontrolled accumulation of fibrotic tissue in one or several organs. The resulting
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fibrosis disrupts the normal architecture of the affected organs, leading to a progressive loss of function [1]. Myofibroblasts are the normal source of ECM protein synthesis [2, 3]. Several cytokines regulate ECM protein synthesis and other processes involved in
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tissue fibrosis –such as inflammation, proliferation and apoptosis [4]. Transforming growth factor beta 1 (TGF-β1) is a pleiotropic cytokine that has a pivotal involvement
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in the development of fibrosis [5, 6]. Other members of the TGF-β superfamily are the bone morphogenetic proteins (BMPs), multifunctional cytokines with important roles in development and cellular physiology. Aberrant BMP effects result in numerous human diseases [7]. While TGF-β1 is possibly the best studied pro-fibrotic member [5], BMPs -such as BMP7 or BMP6- have shown antifibrotic properties, although the
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pathophysiological role of other BMPs in fibrosis has not been fully elucidated [8-11]. Bidart et al. demonstrated that BMP9 is synthesized in liver–mainly in hepatocytes and
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bile duct cells- and actively circulates in the bloodstream [12]. Although the involvement of BMP9 in pathologies such as hepatocellular carcinoma [13], pancreatic
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islet carcinoma or epithelial ovarian carcinoma [14] has been described, its role in fibrosis is practically unknown [15]. One of the major differences between TGF-β1 and BMP9 is the signaling pathway they
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use. TGF-β1 transduces signals normally through the type I receptor ALK5 –and occasionally through ALK1 in several cell types [16], whereas BMPs transduce signals mainly through the ALK2, ALK3 or ALK6 receptors [17]. BMP9, also known as growth differentiation factor 2 (GDF-2), was identified as a potent ligand for the TGF-β1 type I receptor receptor ALK1 in endothelial cells [18, 19]. BMP9 promotes Smad1/5/8 phosphorylation in several cell types [14], such as endothelial cells [18] or hepatocarcinoma cells [13, 20], regulating different cell functions. Activation of the Smad1/5/8 pathway has been shown to exert pro- or antifibrotic effects, depending on the biological context and cell type. This pathway plays an anti-fibrotic role in different experimental models of renal fibrosis [8, 10, 21, 22] or in in vitro experiments [23, 24]. However, in other biological contexts, Smad1 activation promotes ECM protein synthesis in liver cells [25, 26], glomerular mesangial
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cells [27, 28] or dermal fibroblasts [29-31]. BMP9 also inhibits angiogenesis in different conditions [18, 19, 32]. In contrast to the Smad1/5/8 signaling pathway, the TGF-β1-induced Smad2/3 pathway
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plays an important role in fibrosis development and in ECM protein expression [33-35].
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In this scenario, BMP receptors (ALK3, ALK6) expression is decreased in experimental fibrosis [10, 36] whereas the expression of TGF-β receptors or co-receptors such as ALK5 or endoglin is increased [35-37]. We have recently reported that the ALK1
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receptor participates in renal fibrosis development [35], and negatively regulates ECM protein expression in mouse embryo fibroblasts (MEFs) [38].
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Because the ALK1 receptor can be activated by both TGF-β1 and BMP9, and because the effects of BMP9 on organ fibrosis have never been investigated, the aim of the present study was to elucidate the role of BMP9 in the regulation of ECM production. For this purpose, we have analyzed in MEFs the effects of BMP9 on ECM protein expression, as well as the receptors and the signaling pathways involved. Our data
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indicate that BMP9 stimulates collagen I, fibronectin and CTGF/CCN2 expression in MEFs through both ALK1 and ALK5 and the activation of Smad1/5/8, Smad2/3 and
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MAPK/Erk1/2 pathways. These data identifies BMP9 as a novel profibrotic factor in
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vitro that breaks new ground in the study of fibrotic diseases.
2. MATERIAL AND METHODS
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2.1. Cell culture and experimental conditions Wild type (WT) and ALK1+/- MEFs, and NIH3T3 fibroblasts were subcultured and immortalized as previously reported [38]. When cultures achieved 80-90% confluence, cells were serum-starved for 24 h and treated with either human recombinant BMP9 at different concentrations, human recombinant TGF-β1 (1 ng/ml) or control vehicle during 30 min or 24 h. The ALK5 inhibitor SB431542 (5 µM), the Smad3 inhibitor SIS3 (4 µM), the ALK1/2/3/6 inhibitor dorsomorphin-1 (1µM) or the MAPK/Erk1/2 inhibitor U0126 (20 µM) were added 30 minutes before BMP9 or TGF-β1 stimulation. These concentrations were chosen according to previous studies [13, 38, 39].
2.2. Quantitative real-time RT-PCR Total RNAs were extracted from cells using the Nucleospin RNA XS kit (MachereyNagel, Düren, Germany) and first-strand cDNA were synthesized from one microgram
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total RNA by reverse transcription using the iScript system (BioRad, Hercules CA, USA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction was performed using a BioRad CFX96 apparatus and qPCR Master Mix
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from Promega. Values obtained for specific genes were normalized to hypoxanthine
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phosphoribosyl transferase (HPRT) expression level. Sequences of the PCR primers used are listed in Table 1.
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2.3. Western blot
Cell extracts were homogenized in magnesium lysis buffer (MLB, from Millipore,
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Billerica, MA, USA) supplemented with 80% glycerol, 1 mg/mL leupeptin, 1 mg/mL aprotinin, 10 mM PMSF, 1 mmol/L Na3VO4 and 25 mmol/L NaF, and centrifuged at 14000 g during 20 min. Supernatants were recovered and the protein amount was quantified. Lysates (20 µg per lane) were loaded onto polyacrylamide gels and the proteins were transferred to nitrocellulose membranes (Millipore) by electroblotting.
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Next, membranes were blocked in bovine serum albumin and were incubated overnight at 4ºC with the following antibodies: rabbit anti-collagen type I (dilution 1:1000), rabbit
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anti-fibronectin (1:1000) from Chemicon International (Temecula, CA, USA); goat antiCTGF (1:1000), rabbit anti-human endoglin (H-300), mouse anti-phospho-Erk1/2 and
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mouse anti-ALK5 from Santa Cruz Biotechnology (Madrid, Spain); rabbit antiphospho-Smad2 (1:1000) from Upstate Biotechnology (Barcelona, Spain); rabbit antiphospho-Smad3 (1:1000) and rabbit anti-phospho-Smad1/5/8 from Cell Signaling
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(Boston, MA, USA), and rabbit anti-ALK1 (1:1000) from Abgent (San Diego, CA, USA). PS1 antiserum was used also for phospho-Smad3 detection, as it was previously described [40-43]. Membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10000) and images were developed using ECL chemiluminescence reagent (Amersham Biosciences, Barcelona, Spain) on X-ray films (Fujifilm Spain, Barcelona, Spain) for densitometric analysis (Scion Image software, Frederick, MD, USA). Erk1/2 (probed with rabbit anti-Erk1/2, Santa Cruz Biotechnology) was used as loading control in cell extracts as it has been previously described and recommended for expression studies of extracellular matrix proteins, since the expression of conventional loading controls such as alpha-actin or tubulin varies upon stimulation with cytokines of the TGF- family [39, 44].
2.4. DNA transfection and dual luciferase assay
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We performed the luciferase assay in order to evaluate promoter activation of target genes induced by BMP9 or TGF-β1 through Smad1/5/8 and Smad2/3 pathways. The promoter contains Smad1/5/8 (BRE element) or Smad2/3 response elements (CAGA
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promoter). NIH-3T3 cells (70000 in 24 microwell plates) were transfected in Opti-
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MEM culture medium (Invitrogen, Madrid, Spain) using lipofectamine (Invitrogen) with 0.18 µg pGL3(BRE)-luc (BMP activation reporter that contains the crucial Smad1specific response elements of the Id1 promoter) [45], or 0.35 µg pGL3(CAGA)12-luc
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(Smad2/3 activation reporter with TGF-β-inducible Smad3 and Smad4 binding sites of PAI-1 promoter) [46], 0.02 µg pRL-TK-luc (containing the Renilla luciferase gene,
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used to normalize the results), and 0.005 µg of either pcDNA3 empty vector, pALK1 or pALK5 vectors (in order to overexpress ALK1 or ALK5 receptors). Four hours after transfection, cells were treated with the appropriate ligand (BMP9 or TGF-β1) at different concentrations Firefly and renilla luciferase activities were measured sequentially with the Dual-Luciferase reporter assay (Promega, Madison, WI, USA).
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Results are expressed as ratios of firefly luciferase activity over renilla luciferase
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activity.
2.5. RNA interference
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MEFs (200000 cells in 60 mm plates) were transfected in DMEM with 10% fetal calf serum without antibiotics, using Dharmafect 4 (Dharmacon, Chicago, IL) with 2 µM of either siRNA directed against mouse ALK1 (Dharmacon), ALK5 (Ambion-Life
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Technologies, Carlsbad, CA) or negative siRNA for 24 hours. Later, MEFs were treated with 20 ng/ml BMP9 30 minutes before protein or RNA extraction.
2.6. Statistical methods Data are expressed as mean ± standard error of the mean (SEM). Comparison of means was performed by two way analysis of variance (ANOVA) and Bonferroni post-test. Statistical significant differences between two groups were assessed by the Student “t” test. Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California, USA). A “p” value lower than 0.05 was considered statistically significant.
3. RESULTS
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3.1. ALK receptors expression in MEFs We analyzed by qPCR the mRNA expression of ALK1 (BMP9, BMP10 and TGF-β1
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receptor), ALK2 (BMP9 receptor when ALK1 is absent), ALK5 (TGF-β1 receptor),
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ALK3 and ALK6 (BMP7 receptor). All these receptors are expressed in MEFs. ALK1
3.2. BMP9-induced effects in MEFs
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expression is higher than ALK2 and ALK5 expressions in MEFs (Suppl. Fig. 1a).
20 ng/ml BMP9 (30 minutes) stimulated Smad1/5/8 phosphorylation as well as Smad2,
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Smad3 and Erk1/2 phosphorylation in MEFs (Fig. 1a, b and Suppl. Fig.2). The same stimulus increased collagen I, fibronectin and CTGF/CCN2 expression in MEFs (Fig. 1c). BMP9 induced a slight activation of the CAGA promoter in NIH3T3 cells, while TGF-β1 clearly activated this promoter (Suppl. Fig. 1b). These results are supported by the fact that the effect of BMP9-inducing Smad2/3 phosphorylation was lower than the
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effect induced by TGF-β1 on this pathway (Fig. 1b). On the other hand, BMP9-induced increase in Smad1/5/8 phosphorylation was similar to the TGF-β1-induced effect on these proteins (Fig. 1b). However, we did not detect BRE activation in NIH3T3
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fibroblasts after TGF-β1 stimulation, but we detected BRE activation after BMP9
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treatment (Suppl. Fig. 1c). These results indicate that TGF-β1-induced activation of Smad1/5/8 is transient, as it has been previously described [18, 47]; moreover, timelapse curve experiments show that after 24 h-stimulation with TGF-β1, Smad1/5/8 is
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not detectable (Suppl. Fig. 2b). BMP9 and TGF-β1 effects on ECM protein expression are not synergistic. While both BMP9 and TGF-β1 treatments induced a higher CAGA activation than TGF-β1 alone and a lower BRE activation that BMP9 alone, we have not found differences in Smad2/3 and Smad1/5/8 activation. Interestingly, we have observed that BMP9 prestimulated cells do not show changes in collagen I and fibronectin expressions after TGF-β1 treatment, suggesting a possible competition between both cytokines (Fig. 1c). All these data indicate that BMP9 stimulation leads to activation of Smad1/5/8 and Smad2/3 -to a lower extent than that induced by TGF-β1-, and an increase in ECM protein synthesis.
3.3. BMP9-induced ECM protein synthesis is dependent of ALK5 and MAPK/Erk1/2
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In order to analyze the possible contribution of the ALK5/Smad2/3 and MAPK/Erk1/2 pathways in the BMP9-induced profibrotic effects, we stimulated MEFs with 20 ng/ml BMP9 after a pre-incubation with either 5 µM SB431542 (ALK5 inhibitor [33, 48],
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with inhibitory effects on ALK4 and ALK7), 4µM SIS3 (Smad3 inhibitor [49]), or 20
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µM U0126 (phospho-Erk inhibitor [50]). BMP9 stimulation after pretreatment with the inhibitors SB431542, SIS3 and U0126 did not induce any effect on collagen I, fibronectin and CTGF expressions (Fig. 2a). ALK5 inhibition with SB431542
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suppressed BMP9-induced Smad2 phosphorylation, but did not modify BMP9-induced Smad1/5/8 phosphorylation (Fig. 3a). ALK5 knockdown lead to an inhibition of BMP9-
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induced Smad2 phosphorylation without modifying BMP9-induced Smad1/5/8 phosphorylation (Fig. 3b).
3.4. BMP9-induced ECM protein synthesis is also due to the activation of a BMP receptor type I (BMPRI)
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We studied the role of the BMP receptors ALK1, ALK2, ALK3 and ALK6 in BMP9induced ECM protein synthesis and in Smads and MAPK phosphorylation after pre-
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incubation of the cells with the ALK1/2/3/6 inhibitor dorsomorphin-1(1µM) [13, 51]. Dorsomorphin-1 inhibited BMP9-induced increase in collagen I, fibronectin and CTGF
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expressions (Fig. 4a). Moreover, dorsomorphin-1 also inhibited BMP9-induced increase in phospho-Smad1/5/8 and phospho-Erk1/2 expressions, and reduced phospho-Smad2 and phospho-Smad3 expressions after 30 min (Fig. 4b). Phospho-Smad1/5/8 activation
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is detectable even after 24 hours of BMP9 stimulation, being this activation much lower in cells pretreated with dorsomorphin-1 (Fig. 4a). All these data suggest that ALK1/2/3/6 receptors are also necessary for BMP9 to phosphorylate Smad1/5/8, Smad2/3 and Erk1/2 proteins and to induce ECM protein expression.
3.5. BMP9-induced Smad1/5/8, Smad2/3 and MAPK phosphorylation is ALK1 dependent We assessed whether ALK1 was the main receptor involved in BMP9 induction of Smads phosphorylation in MEFs, using two different approaches. First, we analyzed Smad1/5/8, Smad2, Smad3 and Erk1/2 phosphorylation in ALK1 heterozygous MEFs (ALK1+/-). BMP9-induced phosphorylation of Smad1/5/8, Smad2, Smad3 and Erk1/2
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was much lower in ALK1+/- MEFs than in WT MEFs, suggesting a relevant role of the ALK1 receptor in the observed BMP9 effects in fibroblasts (Fig. 5a).
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Next, we analyzed Smad1/5/8, Smad2, Smad3 and Erk1/2 phosphorylation in MEFs
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after ALK1 knockdown with siRNA. ALK1 knockdown led to a reduced phosphorylation of Smad1/5/8, Smad2, Smad3 and Erk1/2 after BMP9 stimulation. Although ALK1 expression, analysed by RT-PCR, was practically inhibited (Suppl.
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Fig. 3), Smad phosphorylation in response to BMP9 was not completely abolished (Fig.
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5b).
On the other hand, NIH3T3 cells overexpressing ALK1 showed a higher activation of the CAGA promoter without changes in the BRE promoter after BMP9 treatment. We only observed a higher activation of BRE promoter in NIH3T3 cells overexpressing ALK1 in basal conditions. Moreover, NIH3T3 fibroblasts overexpressing ALK5
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showed a slightly higher induction of the CAGA promoter than control cells after BMP9 treatment (Suppl. Fig. 4), suggesting the role of both ALK1 and ALK5 receptors
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4. DISCUSSION
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in BMP9-induced effects in fibroblasts.
Our results identify BMP9 as a new and potentially relevant mediator of degenerative diseases involving fibrosis. Our study demonstrates that BMP9 induces ECM proteins
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synthesis in fibroblasts through the activation of Smad1/5/8, Smad2/3 and MAPK/Erk1/2 pathways. These three pathways seem to be essential for BMP9induction of ECM proteins synthesis. ALK1 and ALK5 receptors are also necessary for Smad1/5/8, Smad2/3 and MAPK/Erk1/2 phosphorylation. Several cytokines have been traditionally described as profibrotic, such as TGF-β1 [5] and its canonical Smad signalling pathway [34, 52], PDGF [53] and CTGF [54]. Bone morphogenetic proteins such as BMP7 [8, 9, 55-57], BMP6 or BMP2 have been described as antifibrotic [58, 59]. On the other hand, other BMPs such as BMP4 have been characterized as profibrotic [60, 61]. The activation of Smad2/3 pathway is closely related to organ fibrosis and ECM protein expression [35], but Smad1 pathway activation also induces ECM protein expression in several contexts [29-31, 60, 62]. Our results show that both Smad1/5/8 and Smad2/3 pathways are necessary for BMP9-induction of ECM protein expression. In all cellular
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types studied, BMP9 induces stimulation of the Smad1/5/8 pathway [14], whereas the Smad2/3 pathway has been mainly associated to TGF-β1signaling [63]. On the other hand, it has been reported that BMP9 also activates the Smad2/3 pathway in several cell
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types [64, 65].
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Regarding the relationship between BMP9 and Erk1/2, some authors have demonstrated that Erk1/2 inhibition was related to a decrease in BMP9-induced osteogenic differentiation [66, 67], and an activation of MAPK/Erk1/2 has been demonstrated in
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endothelial cells after BMP9 stimulation [65]. In our in vitro studies with fibroblasts, BMP9 treatment leads to an increase of Erk1/2 phosphorylation, a signalling pathway
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further related to ECM protein expression [29, 39]. Moreover, our data after inhibition of ALK1,2,3 and 6 with dorsomorphin-1 indicates that BMP9-induced Erk1/2 activation is Smad1/5/8 dependent.
BMP9 and TGF-β effects on the induction of ECM protein expression are not synergistic. While treatment with both BMP9 and TGF-β1 induced a higher CAGA
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activation than TGF-β alone, and a lower BRE activation that BMP9 alone, we have not found any difference in Smad2/3 and Smad1/5/8 activation after stimulation with both BMP9 and TGF-β1 in comparison with TGF-β1 alone. Interestingly, we have observed
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that TGF-β1 has no effect on collagen I and fibronectin expression in BMP9 pre-
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stimulated cells, suggesting a possible competition between these ligands. After pharmacological inhibition of ALK4, 5 and 7 with SB431542 and ALK1, 2, 3 and 6 with dorsomorphin-1, we have observed that both type I receptors transducing TGF-
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β1 signals (ALK4,5,7) and receptors transducing BMP signals (ALK1,2,3,6) are necessary for BMP9-induced Smad2/3 (but not Smad1/5/8) phosphorylation and subsequent ECM protein expression. These results suggest two different mechanisms: First, BMP9 might activate Smad1/5/8 trough ALK1 receptors (possibly forming a complex with BMPRII receptors). Second, BMP9 might activate Smad2/3 through ALK5 receptors, but ALK1 is essential to promote this BMP9-induced activation, thus suggesting the formation of an ALK1/ALK5 receptor complex (Fig. 6). ALK5-ALK2/3 complexes were demonstrated to be necessary to induce TGFβ1/Smad1/5/8 signaling in epithelial cells [68]. A new combination of complexes ALK2/3-ALK5/7 that are essential for BMP2 induced Smad2/3 signaling in numerous non-transformed and transformed cell lines has been recently described [7]. We detected mRNA expression of ALK1, 2, 3, 5 and 6 in MEFs, being the last receptor barely detectable. After inhibition with SB431542 and overexpression studies it seems that the
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ALK5 receptor –directly involved in ECM protein expression [33]- may be one of the TGF-β receptors participating in the described BMP9 effects. On the other hand, either knockdown, overexpression or ALK1 heterozygosity demonstrated that ALK1 is
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necessary for BMP9 to induce the activation of both Smad1/5/8, Smad2/3 and Erk1/2
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pathways. Thus, we suggest the existence of an ALK1/ALK5 complex in fibroblasts that is necessary for BMP9 to induce Smad signaling and ECM protein expression. This ALK1/ALK5 complex, which has been previously described in endothelial cells,
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participates in the TGF-β1-regulated balance between the activated and quiescent endothelium [69-71]. A similar complex was described in scleroderma fibroblasts that
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was responsible for TGF-β1-inducedSmad1 phosphorylation [29]. Summarizing, this is the first study that identifies BMP9 as a profibrogenic factor and describes its relationship with ALK1 in a non-endothelial cell type. Moreover, we show that BMP9 –traditionally related with liver diseases- might also play a role in other organs such as the kidney, due to its profibrotic function. Moreover, due to the ability of
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BMP9 to activate Smad proteins and to bind to receptors involved in fibrosis, BMP9 excels as a potential target to consider in the search of new antifibrotic therapies. The
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data shown in this paper support the idea that BMP9 behaves as a profibrotic factor in vitro. In order to confirm the relevant role of this cytokine in these fibrotic pathologies,
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additional in vivo studies should be performed.
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ACKNOWLEDGEMENTS This work was supported by grants from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, PS09/01067, PI12/00959 and Retic RD012/0021 RedinRen, cofunded by FEDER) and Junta de Castilla y León (Excellence Group GR100 and IES095U14). JMMF, CC and NPT are supported by Junta de Castilla y León and European Social Fund.
CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest
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FIGURE LEGENDS
Fig. 1. Effects of BMP9 stimulation on ECM protein expression and Smads
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phosphorylation.
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a) Expression of phospho-Smad1/5/8 and phospho-Smad2 after stimulation with BMP9 stimulation (1, 10 and 20 ng/ml) for 30 minutes, and expression of phospho-Smad3 and phospho-Erk1/2 after BMP9 stimulation (1 and 20 ng/ml). b) Expression of phospho-
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Smad1/5/8 and phospho-Smad2 after BMP9 stimulation with 20 ng/ml BMP9, and 1 ng/ml TGF-β1 during 30 minutes. c) Expression of collagen I, fibronectin and
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CTGF/CCN2 after stimulation with 20 ng/ml BMP9 and 1 ng/ml TGF-β1 for 24 hours. Erk1/2 was used as loading control as reported in methods section. Histograms represent the mean ± SEM of the optical density of the bands of five experiments expressed as percentage over basal values. *P < 0.01 vs. MEFs in basal conditions.
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Fig. 2. Effects of ALK4/5/7 inhibition with SB431542 on ECM protein expression and Smads phosphorylation
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Expression of collagen I, fibronectin and CTGF/CCN2 after BMP9 stimulation with 20 ng/mL in cells pre-inhibited with the ALK4/5/7 inhibitor SB431542 (5µM), the Smad3
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inhibitor SIS3 (4µM) and the MEK/Erk1/2 inhibitor U0126 (20µM). Erk1/2 was used as loading control. Histograms represent the mean ± SEM of the optical density of the bands of five experiments expressed as percentage over basal values. *P < 0.01 vs.
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MEFs in basal conditions.
Fig. 3. Effects of ALK4/5/7 inhibition with SB431542 and ALK5 knockdown on Smads phosphorylation a) Expression of phospho-Smad1/5/8, phospho-Smad2 after BMP9 stimulation with 20 ng/ml in cells pre-inhibited with the ALK4/5/7 inhibitor SB431542 (5µM). b) Expression of phospho-Smad1/5/8 and phospho-Smad2 after BMP9 stimulation with 20 ng/ml in cells transfected with ALK5 siRNA (siALK5). Erk1/2 was used as loading control. *P < 0.01 vs. MEFs in basal conditions. #P < 0.01 vs. MEFs with ALK5 inhibition.
Fig. 4. Effects of ALK1/2/3/6 inhibition with dorsomorphin-1 on ECM protein expression and Smads phosphorylation.
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a) Expression of collagen I, fibronectin and CTGF/CCN2 after BMP9 stimulation with 20 ng/ml for 24 hours in cells pre-inhibited with the ALK1/2/3/6 inhibitor dorsomorphin-1 (1µM). b) Level of phospho-Smad1/5/8, phospho-Smad2, phospho-
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Smad3 and phospho-Erk1/2 after BMP9 stimulation with 20 ng/ml for 30 minutes in
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cells pre-inhibited with the ALK1/2/3/6 inhibitor dorsomorphin-1 (1µM). Erk1/2 was used as loading control. Histograms represent the mean ± SEM of the optical density of the bands of five experiments expressed as percentage over basal values. *P < 0.01 vs.
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MEFs in basal conditions.
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Fig. 5. Effects of ALK1 heterozygosity and ALK1 knockdown on BMP9-induced Smad signalling.
a) Expression of phospho-Smad1/5/8, phospho-Smad2, phospho-Smad3 and phosphoErk1/2 after BMP9 stimulation with 20 ng/ml for 30 minutes in ALK1 heterozygous cells (ALK1+/-) and their respective controls (ALK1+/+). b) Levels of phospho-
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Smad1/5/8, phospho-Smad2, phospho-Smad3 and phospho-Erk1/2 after stimulation BMP9 stimulation with 20 ng/ml in cells transfected with ALK1 siRNA (siALK1) and
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their respective controls (siCtrl). Erk1/2 was used as loading control. Histograms in (a) represent the mean ± SEM of the optical density of the bands of six experiments expressed as percentage over basal values. Blots in (b) are representative from 6
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different experiments. *P < 0.01 vs. ALK1+/+ MEFs in basal conditions. #P < 0.01 vs.
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ALK1+/- MEFs in basal conditions. +P < 0.01 vs. ALK1+/- MEFs in basal conditions. Fig. 6. Representative scheme of the proposed “BMP9/ALK1/ALK5 complex” and its downstream mechanism. BMP9 binds ALK1 and activates the Smad1/5/8 and Smad2/3 pathways. ALK5 is also necessary for BMP9 to promote Smads activation. Moreover, BMP9 also activates the MAPK/Erk1/2 pathway, perhaps through ALK1 activation. All these pathways contribute to the regulation of ECM protein expression in fibroblasts.
SUPPORTING INFORMATION
Supplementary figure 1. (a) ALK1, ALK2, ALK3, ALK5 and ALK6 mRNA expression in MEFs. Data (ALK vs. HPRT mRNA relative expression) shows 1 representative histogram of 3 similar performed PCR analysis. BMP9-induced
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activation of the CAGA (b) and BRE (c) promoters. NIH3T3 cells were transiently transfected with pGL3 (BRE)-luc and pRL-TK-luc and were treated with BMP9 (1, 10, 20 and 40 ng/mL) or TGF-β1 (1 ng/mL) for 15 hours. The firefly luciferase activity was
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normalized to renilla luciferase activity. Data (expressed as mean ± SEM) show one
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representative experiment of two performed.
Supplementary figure 2. Effects of TGF-β1 stimulation on Smads phosphorylation.
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a) Expression of phospho-Smad1/5/8, phospho-Smad2 and phospho-Erk1/2 after stimulation with 20 ng/ml BMP9 at different time points (30 min, 2, 6 and 24 hours). b)
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Expression of phospho-Smad1/5/8, phospho-Smad2 and phospho-Erk1/2 after stimulation with 1 ng/ml TGF-β1 at different time points (30 min, 2, 6 and 24 hours).
Supplementary figure 3. ALK1 knockdown with siRNA. Analysis of ALK1 mRNA expression in cells transfected with negative siRNA (siCtrol)
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or siALK1 with four different Dharmafect reagents (1-4). After this analysis we used
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Dharmafect4 due to its higher ability to silencing ALK1 expression.
Supplementary figure 4. BMP9 activation of the BRE and the CAGA promoter is
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dependent of ALK1.
NIH-3T3 cells were transiently transfected with pGL3 (BRE)-luc and pRL-TK-luc and either pCDNA3 empty vector, pALK1 or pALK5. After 4 hours, cells were treated with
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BMP9 (20 ng/mL) for 15 hours. Data (expressed as mean ± SEM) show one representative experiment of two performed.
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Highlights:
BMP9 increases collagen I, fibronectin and CTGF/CCN2 expression in
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fibroblasts. BMP9 promotes both Smad1/5/8 and Smad2/3 phosphorylation in fibroblasts.
ALK1 is necessary for BMP9 to induce Smad1/5/8 and Smad2/3
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phosphorylation.
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Overexpression of endoglin increases BMP9-induced Smad1/5/8 and Smad2/3
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phosphorylation and its profibrotic properties.
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S0898-6568(16)30116-4 doi: 10.1016/j.cellsig.2016.05.015 CLS 8693
To appear in:
Cellular Signalling
Received date: Revised date: Accepted date:
24 November 2015 12 May 2016 17 May 2016
Please cite this article as: Jos´e M. Mu˜ noz-F´elix, Cristina Cuesta, Nuria Perretta-Tejedor, Mariela Subileau, Francisco J. L´opez-Hern´ andez, Jos´e M. L´opez-Novoa, Carlos Mart´ınezSalgado, Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro, Cellular Signalling (2016), doi: 10.1016/j.cellsig.2016.05.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Identification of bone morphogenetic protein 9
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(BMP9) as a novel profibrotic factor in vitro
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José M. Muñoz-Félix1,2, Cristina Cuesta1,2*, Nuria Perretta-Tejedor1,2*, Mariela Subileau3, Francisco J. López-Hernández1,2,4, José M. López-Novoa1,2, Carlos
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Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain;
Unidad de Fisiopatología Renal y Cardiovascular, Instituto Reina Sofía de
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Martínez-Salgado1,2,4
Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain; 3Inserm, U1036, CEA, DSV, Irtsv, Laboratoire Biologie du Cancer et de l’Infection, Université Joseph Fourier, Grenoble, F-38054 France; 4Instituto de Estudios de Ciencias de la Salud de Castilla y León (IECSCYL),
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Hospital Universitario de Salamanca, Salamanca, Spain.
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Short title: BMP9 induces fibrosis in vitro
Correspondence to: Carlos Martínez-Salgado, Unidad de Investigación, Hospital
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Universitario de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain. Phone: +34 923 294500 ext. 1945, email: [email protected]
*These authors have contributed equally to this work
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ABSTRACT Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in
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fibrosis development has been extensively studied, the involvement of other members
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of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptorlike kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is
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unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways.
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In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM
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synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of
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ALK1.
Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic
receptors.
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factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5
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Keywords: BMP9, ALK1, ALK5, fibroblasts, fibrosis, extracellular matrix proteins
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1. INTRODUCTION
Fibrotic diseases include a wide spectrum of entities characterized by the progressive
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and uncontrolled accumulation of fibrotic tissue in one or several organs. The resulting
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fibrosis disrupts the normal architecture of the affected organs, leading to a progressive loss of function [1]. Myofibroblasts are the normal source of ECM protein synthesis [2, 3]. Several cytokines regulate ECM protein synthesis and other processes involved in
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tissue fibrosis –such as inflammation, proliferation and apoptosis [4]. Transforming growth factor beta 1 (TGF-β1) is a pleiotropic cytokine that has a pivotal involvement
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in the development of fibrosis [5, 6]. Other members of the TGF-β superfamily are the bone morphogenetic proteins (BMPs), multifunctional cytokines with important roles in development and cellular physiology. Aberrant BMP effects result in numerous human diseases [7]. While TGF-β1 is possibly the best studied pro-fibrotic member [5], BMPs -such as BMP7 or BMP6- have shown antifibrotic properties, although the
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pathophysiological role of other BMPs in fibrosis has not been fully elucidated [8-11]. Bidart et al. demonstrated that BMP9 is synthesized in liver–mainly in hepatocytes and
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bile duct cells- and actively circulates in the bloodstream [12]. Although the involvement of BMP9 in pathologies such as hepatocellular carcinoma [13], pancreatic
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islet carcinoma or epithelial ovarian carcinoma [14] has been described, its role in fibrosis is practically unknown [15]. One of the major differences between TGF-β1 and BMP9 is the signaling pathway they
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use. TGF-β1 transduces signals normally through the type I receptor ALK5 –and occasionally through ALK1 in several cell types [16], whereas BMPs transduce signals mainly through the ALK2, ALK3 or ALK6 receptors [17]. BMP9, also known as growth differentiation factor 2 (GDF-2), was identified as a potent ligand for the TGF-β1 type I receptor receptor ALK1 in endothelial cells [18, 19]. BMP9 promotes Smad1/5/8 phosphorylation in several cell types [14], such as endothelial cells [18] or hepatocarcinoma cells [13, 20], regulating different cell functions. Activation of the Smad1/5/8 pathway has been shown to exert pro- or antifibrotic effects, depending on the biological context and cell type. This pathway plays an anti-fibrotic role in different experimental models of renal fibrosis [8, 10, 21, 22] or in in vitro experiments [23, 24]. However, in other biological contexts, Smad1 activation promotes ECM protein synthesis in liver cells [25, 26], glomerular mesangial
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cells [27, 28] or dermal fibroblasts [29-31]. BMP9 also inhibits angiogenesis in different conditions [18, 19, 32]. In contrast to the Smad1/5/8 signaling pathway, the TGF-β1-induced Smad2/3 pathway
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plays an important role in fibrosis development and in ECM protein expression [33-35].
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In this scenario, BMP receptors (ALK3, ALK6) expression is decreased in experimental fibrosis [10, 36] whereas the expression of TGF-β receptors or co-receptors such as ALK5 or endoglin is increased [35-37]. We have recently reported that the ALK1
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receptor participates in renal fibrosis development [35], and negatively regulates ECM protein expression in mouse embryo fibroblasts (MEFs) [38].
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Because the ALK1 receptor can be activated by both TGF-β1 and BMP9, and because the effects of BMP9 on organ fibrosis have never been investigated, the aim of the present study was to elucidate the role of BMP9 in the regulation of ECM production. For this purpose, we have analyzed in MEFs the effects of BMP9 on ECM protein expression, as well as the receptors and the signaling pathways involved. Our data
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indicate that BMP9 stimulates collagen I, fibronectin and CTGF/CCN2 expression in MEFs through both ALK1 and ALK5 and the activation of Smad1/5/8, Smad2/3 and
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MAPK/Erk1/2 pathways. These data identifies BMP9 as a novel profibrotic factor in
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vitro that breaks new ground in the study of fibrotic diseases.
2. MATERIAL AND METHODS
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2.1. Cell culture and experimental conditions Wild type (WT) and ALK1+/- MEFs, and NIH3T3 fibroblasts were subcultured and immortalized as previously reported [38]. When cultures achieved 80-90% confluence, cells were serum-starved for 24 h and treated with either human recombinant BMP9 at different concentrations, human recombinant TGF-β1 (1 ng/ml) or control vehicle during 30 min or 24 h. The ALK5 inhibitor SB431542 (5 µM), the Smad3 inhibitor SIS3 (4 µM), the ALK1/2/3/6 inhibitor dorsomorphin-1 (1µM) or the MAPK/Erk1/2 inhibitor U0126 (20 µM) were added 30 minutes before BMP9 or TGF-β1 stimulation. These concentrations were chosen according to previous studies [13, 38, 39].
2.2. Quantitative real-time RT-PCR Total RNAs were extracted from cells using the Nucleospin RNA XS kit (MachereyNagel, Düren, Germany) and first-strand cDNA were synthesized from one microgram
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total RNA by reverse transcription using the iScript system (BioRad, Hercules CA, USA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction was performed using a BioRad CFX96 apparatus and qPCR Master Mix
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from Promega. Values obtained for specific genes were normalized to hypoxanthine
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phosphoribosyl transferase (HPRT) expression level. Sequences of the PCR primers used are listed in Table 1.
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2.3. Western blot
Cell extracts were homogenized in magnesium lysis buffer (MLB, from Millipore,
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Billerica, MA, USA) supplemented with 80% glycerol, 1 mg/mL leupeptin, 1 mg/mL aprotinin, 10 mM PMSF, 1 mmol/L Na3VO4 and 25 mmol/L NaF, and centrifuged at 14000 g during 20 min. Supernatants were recovered and the protein amount was quantified. Lysates (20 µg per lane) were loaded onto polyacrylamide gels and the proteins were transferred to nitrocellulose membranes (Millipore) by electroblotting.
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Next, membranes were blocked in bovine serum albumin and were incubated overnight at 4ºC with the following antibodies: rabbit anti-collagen type I (dilution 1:1000), rabbit
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anti-fibronectin (1:1000) from Chemicon International (Temecula, CA, USA); goat antiCTGF (1:1000), rabbit anti-human endoglin (H-300), mouse anti-phospho-Erk1/2 and
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mouse anti-ALK5 from Santa Cruz Biotechnology (Madrid, Spain); rabbit antiphospho-Smad2 (1:1000) from Upstate Biotechnology (Barcelona, Spain); rabbit antiphospho-Smad3 (1:1000) and rabbit anti-phospho-Smad1/5/8 from Cell Signaling
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(Boston, MA, USA), and rabbit anti-ALK1 (1:1000) from Abgent (San Diego, CA, USA). PS1 antiserum was used also for phospho-Smad3 detection, as it was previously described [40-43]. Membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10000) and images were developed using ECL chemiluminescence reagent (Amersham Biosciences, Barcelona, Spain) on X-ray films (Fujifilm Spain, Barcelona, Spain) for densitometric analysis (Scion Image software, Frederick, MD, USA). Erk1/2 (probed with rabbit anti-Erk1/2, Santa Cruz Biotechnology) was used as loading control in cell extracts as it has been previously described and recommended for expression studies of extracellular matrix proteins, since the expression of conventional loading controls such as alpha-actin or tubulin varies upon stimulation with cytokines of the TGF- family [39, 44].
2.4. DNA transfection and dual luciferase assay
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We performed the luciferase assay in order to evaluate promoter activation of target genes induced by BMP9 or TGF-β1 through Smad1/5/8 and Smad2/3 pathways. The promoter contains Smad1/5/8 (BRE element) or Smad2/3 response elements (CAGA
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promoter). NIH-3T3 cells (70000 in 24 microwell plates) were transfected in Opti-
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MEM culture medium (Invitrogen, Madrid, Spain) using lipofectamine (Invitrogen) with 0.18 µg pGL3(BRE)-luc (BMP activation reporter that contains the crucial Smad1specific response elements of the Id1 promoter) [45], or 0.35 µg pGL3(CAGA)12-luc
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(Smad2/3 activation reporter with TGF-β-inducible Smad3 and Smad4 binding sites of PAI-1 promoter) [46], 0.02 µg pRL-TK-luc (containing the Renilla luciferase gene,
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used to normalize the results), and 0.005 µg of either pcDNA3 empty vector, pALK1 or pALK5 vectors (in order to overexpress ALK1 or ALK5 receptors). Four hours after transfection, cells were treated with the appropriate ligand (BMP9 or TGF-β1) at different concentrations Firefly and renilla luciferase activities were measured sequentially with the Dual-Luciferase reporter assay (Promega, Madison, WI, USA).
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Results are expressed as ratios of firefly luciferase activity over renilla luciferase
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activity.
2.5. RNA interference
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MEFs (200000 cells in 60 mm plates) were transfected in DMEM with 10% fetal calf serum without antibiotics, using Dharmafect 4 (Dharmacon, Chicago, IL) with 2 µM of either siRNA directed against mouse ALK1 (Dharmacon), ALK5 (Ambion-Life
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Technologies, Carlsbad, CA) or negative siRNA for 24 hours. Later, MEFs were treated with 20 ng/ml BMP9 30 minutes before protein or RNA extraction.
2.6. Statistical methods Data are expressed as mean ± standard error of the mean (SEM). Comparison of means was performed by two way analysis of variance (ANOVA) and Bonferroni post-test. Statistical significant differences between two groups were assessed by the Student “t” test. Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, California, USA). A “p” value lower than 0.05 was considered statistically significant.
3. RESULTS
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3.1. ALK receptors expression in MEFs We analyzed by qPCR the mRNA expression of ALK1 (BMP9, BMP10 and TGF-β1
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receptor), ALK2 (BMP9 receptor when ALK1 is absent), ALK5 (TGF-β1 receptor),
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ALK3 and ALK6 (BMP7 receptor). All these receptors are expressed in MEFs. ALK1
3.2. BMP9-induced effects in MEFs
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expression is higher than ALK2 and ALK5 expressions in MEFs (Suppl. Fig. 1a).
20 ng/ml BMP9 (30 minutes) stimulated Smad1/5/8 phosphorylation as well as Smad2,
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Smad3 and Erk1/2 phosphorylation in MEFs (Fig. 1a, b and Suppl. Fig.2). The same stimulus increased collagen I, fibronectin and CTGF/CCN2 expression in MEFs (Fig. 1c). BMP9 induced a slight activation of the CAGA promoter in NIH3T3 cells, while TGF-β1 clearly activated this promoter (Suppl. Fig. 1b). These results are supported by the fact that the effect of BMP9-inducing Smad2/3 phosphorylation was lower than the
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effect induced by TGF-β1 on this pathway (Fig. 1b). On the other hand, BMP9-induced increase in Smad1/5/8 phosphorylation was similar to the TGF-β1-induced effect on these proteins (Fig. 1b). However, we did not detect BRE activation in NIH3T3
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fibroblasts after TGF-β1 stimulation, but we detected BRE activation after BMP9
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treatment (Suppl. Fig. 1c). These results indicate that TGF-β1-induced activation of Smad1/5/8 is transient, as it has been previously described [18, 47]; moreover, timelapse curve experiments show that after 24 h-stimulation with TGF-β1, Smad1/5/8 is
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not detectable (Suppl. Fig. 2b). BMP9 and TGF-β1 effects on ECM protein expression are not synergistic. While both BMP9 and TGF-β1 treatments induced a higher CAGA activation than TGF-β1 alone and a lower BRE activation that BMP9 alone, we have not found differences in Smad2/3 and Smad1/5/8 activation. Interestingly, we have observed that BMP9 prestimulated cells do not show changes in collagen I and fibronectin expressions after TGF-β1 treatment, suggesting a possible competition between both cytokines (Fig. 1c). All these data indicate that BMP9 stimulation leads to activation of Smad1/5/8 and Smad2/3 -to a lower extent than that induced by TGF-β1-, and an increase in ECM protein synthesis.
3.3. BMP9-induced ECM protein synthesis is dependent of ALK5 and MAPK/Erk1/2
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In order to analyze the possible contribution of the ALK5/Smad2/3 and MAPK/Erk1/2 pathways in the BMP9-induced profibrotic effects, we stimulated MEFs with 20 ng/ml BMP9 after a pre-incubation with either 5 µM SB431542 (ALK5 inhibitor [33, 48],
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with inhibitory effects on ALK4 and ALK7), 4µM SIS3 (Smad3 inhibitor [49]), or 20
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µM U0126 (phospho-Erk inhibitor [50]). BMP9 stimulation after pretreatment with the inhibitors SB431542, SIS3 and U0126 did not induce any effect on collagen I, fibronectin and CTGF expressions (Fig. 2a). ALK5 inhibition with SB431542
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suppressed BMP9-induced Smad2 phosphorylation, but did not modify BMP9-induced Smad1/5/8 phosphorylation (Fig. 3a). ALK5 knockdown lead to an inhibition of BMP9-
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induced Smad2 phosphorylation without modifying BMP9-induced Smad1/5/8 phosphorylation (Fig. 3b).
3.4. BMP9-induced ECM protein synthesis is also due to the activation of a BMP receptor type I (BMPRI)
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We studied the role of the BMP receptors ALK1, ALK2, ALK3 and ALK6 in BMP9induced ECM protein synthesis and in Smads and MAPK phosphorylation after pre-
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incubation of the cells with the ALK1/2/3/6 inhibitor dorsomorphin-1(1µM) [13, 51]. Dorsomorphin-1 inhibited BMP9-induced increase in collagen I, fibronectin and CTGF
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expressions (Fig. 4a). Moreover, dorsomorphin-1 also inhibited BMP9-induced increase in phospho-Smad1/5/8 and phospho-Erk1/2 expressions, and reduced phospho-Smad2 and phospho-Smad3 expressions after 30 min (Fig. 4b). Phospho-Smad1/5/8 activation
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is detectable even after 24 hours of BMP9 stimulation, being this activation much lower in cells pretreated with dorsomorphin-1 (Fig. 4a). All these data suggest that ALK1/2/3/6 receptors are also necessary for BMP9 to phosphorylate Smad1/5/8, Smad2/3 and Erk1/2 proteins and to induce ECM protein expression.
3.5. BMP9-induced Smad1/5/8, Smad2/3 and MAPK phosphorylation is ALK1 dependent We assessed whether ALK1 was the main receptor involved in BMP9 induction of Smads phosphorylation in MEFs, using two different approaches. First, we analyzed Smad1/5/8, Smad2, Smad3 and Erk1/2 phosphorylation in ALK1 heterozygous MEFs (ALK1+/-). BMP9-induced phosphorylation of Smad1/5/8, Smad2, Smad3 and Erk1/2
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was much lower in ALK1+/- MEFs than in WT MEFs, suggesting a relevant role of the ALK1 receptor in the observed BMP9 effects in fibroblasts (Fig. 5a).
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Next, we analyzed Smad1/5/8, Smad2, Smad3 and Erk1/2 phosphorylation in MEFs
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after ALK1 knockdown with siRNA. ALK1 knockdown led to a reduced phosphorylation of Smad1/5/8, Smad2, Smad3 and Erk1/2 after BMP9 stimulation. Although ALK1 expression, analysed by RT-PCR, was practically inhibited (Suppl.
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Fig. 3), Smad phosphorylation in response to BMP9 was not completely abolished (Fig.
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5b).
On the other hand, NIH3T3 cells overexpressing ALK1 showed a higher activation of the CAGA promoter without changes in the BRE promoter after BMP9 treatment. We only observed a higher activation of BRE promoter in NIH3T3 cells overexpressing ALK1 in basal conditions. Moreover, NIH3T3 fibroblasts overexpressing ALK5
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showed a slightly higher induction of the CAGA promoter than control cells after BMP9 treatment (Suppl. Fig. 4), suggesting the role of both ALK1 and ALK5 receptors
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4. DISCUSSION
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in BMP9-induced effects in fibroblasts.
Our results identify BMP9 as a new and potentially relevant mediator of degenerative diseases involving fibrosis. Our study demonstrates that BMP9 induces ECM proteins
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synthesis in fibroblasts through the activation of Smad1/5/8, Smad2/3 and MAPK/Erk1/2 pathways. These three pathways seem to be essential for BMP9induction of ECM proteins synthesis. ALK1 and ALK5 receptors are also necessary for Smad1/5/8, Smad2/3 and MAPK/Erk1/2 phosphorylation. Several cytokines have been traditionally described as profibrotic, such as TGF-β1 [5] and its canonical Smad signalling pathway [34, 52], PDGF [53] and CTGF [54]. Bone morphogenetic proteins such as BMP7 [8, 9, 55-57], BMP6 or BMP2 have been described as antifibrotic [58, 59]. On the other hand, other BMPs such as BMP4 have been characterized as profibrotic [60, 61]. The activation of Smad2/3 pathway is closely related to organ fibrosis and ECM protein expression [35], but Smad1 pathway activation also induces ECM protein expression in several contexts [29-31, 60, 62]. Our results show that both Smad1/5/8 and Smad2/3 pathways are necessary for BMP9-induction of ECM protein expression. In all cellular
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types studied, BMP9 induces stimulation of the Smad1/5/8 pathway [14], whereas the Smad2/3 pathway has been mainly associated to TGF-β1signaling [63]. On the other hand, it has been reported that BMP9 also activates the Smad2/3 pathway in several cell
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types [64, 65].
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Regarding the relationship between BMP9 and Erk1/2, some authors have demonstrated that Erk1/2 inhibition was related to a decrease in BMP9-induced osteogenic differentiation [66, 67], and an activation of MAPK/Erk1/2 has been demonstrated in
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endothelial cells after BMP9 stimulation [65]. In our in vitro studies with fibroblasts, BMP9 treatment leads to an increase of Erk1/2 phosphorylation, a signalling pathway
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further related to ECM protein expression [29, 39]. Moreover, our data after inhibition of ALK1,2,3 and 6 with dorsomorphin-1 indicates that BMP9-induced Erk1/2 activation is Smad1/5/8 dependent.
BMP9 and TGF-β effects on the induction of ECM protein expression are not synergistic. While treatment with both BMP9 and TGF-β1 induced a higher CAGA
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activation than TGF-β alone, and a lower BRE activation that BMP9 alone, we have not found any difference in Smad2/3 and Smad1/5/8 activation after stimulation with both BMP9 and TGF-β1 in comparison with TGF-β1 alone. Interestingly, we have observed
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that TGF-β1 has no effect on collagen I and fibronectin expression in BMP9 pre-
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stimulated cells, suggesting a possible competition between these ligands. After pharmacological inhibition of ALK4, 5 and 7 with SB431542 and ALK1, 2, 3 and 6 with dorsomorphin-1, we have observed that both type I receptors transducing TGF-
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β1 signals (ALK4,5,7) and receptors transducing BMP signals (ALK1,2,3,6) are necessary for BMP9-induced Smad2/3 (but not Smad1/5/8) phosphorylation and subsequent ECM protein expression. These results suggest two different mechanisms: First, BMP9 might activate Smad1/5/8 trough ALK1 receptors (possibly forming a complex with BMPRII receptors). Second, BMP9 might activate Smad2/3 through ALK5 receptors, but ALK1 is essential to promote this BMP9-induced activation, thus suggesting the formation of an ALK1/ALK5 receptor complex (Fig. 6). ALK5-ALK2/3 complexes were demonstrated to be necessary to induce TGFβ1/Smad1/5/8 signaling in epithelial cells [68]. A new combination of complexes ALK2/3-ALK5/7 that are essential for BMP2 induced Smad2/3 signaling in numerous non-transformed and transformed cell lines has been recently described [7]. We detected mRNA expression of ALK1, 2, 3, 5 and 6 in MEFs, being the last receptor barely detectable. After inhibition with SB431542 and overexpression studies it seems that the
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ALK5 receptor –directly involved in ECM protein expression [33]- may be one of the TGF-β receptors participating in the described BMP9 effects. On the other hand, either knockdown, overexpression or ALK1 heterozygosity demonstrated that ALK1 is
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necessary for BMP9 to induce the activation of both Smad1/5/8, Smad2/3 and Erk1/2
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pathways. Thus, we suggest the existence of an ALK1/ALK5 complex in fibroblasts that is necessary for BMP9 to induce Smad signaling and ECM protein expression. This ALK1/ALK5 complex, which has been previously described in endothelial cells,
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participates in the TGF-β1-regulated balance between the activated and quiescent endothelium [69-71]. A similar complex was described in scleroderma fibroblasts that
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was responsible for TGF-β1-inducedSmad1 phosphorylation [29]. Summarizing, this is the first study that identifies BMP9 as a profibrogenic factor and describes its relationship with ALK1 in a non-endothelial cell type. Moreover, we show that BMP9 –traditionally related with liver diseases- might also play a role in other organs such as the kidney, due to its profibrotic function. Moreover, due to the ability of
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BMP9 to activate Smad proteins and to bind to receptors involved in fibrosis, BMP9 excels as a potential target to consider in the search of new antifibrotic therapies. The
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data shown in this paper support the idea that BMP9 behaves as a profibrotic factor in vitro. In order to confirm the relevant role of this cytokine in these fibrotic pathologies,
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additional in vivo studies should be performed.
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ACKNOWLEDGEMENTS This work was supported by grants from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, PS09/01067, PI12/00959 and Retic RD012/0021 RedinRen, cofunded by FEDER) and Junta de Castilla y León (Excellence Group GR100 and IES095U14). JMMF, CC and NPT are supported by Junta de Castilla y León and European Social Fund.
CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest
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FIGURE LEGENDS
Fig. 1. Effects of BMP9 stimulation on ECM protein expression and Smads
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phosphorylation.
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a) Expression of phospho-Smad1/5/8 and phospho-Smad2 after stimulation with BMP9 stimulation (1, 10 and 20 ng/ml) for 30 minutes, and expression of phospho-Smad3 and phospho-Erk1/2 after BMP9 stimulation (1 and 20 ng/ml). b) Expression of phospho-
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Smad1/5/8 and phospho-Smad2 after BMP9 stimulation with 20 ng/ml BMP9, and 1 ng/ml TGF-β1 during 30 minutes. c) Expression of collagen I, fibronectin and
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CTGF/CCN2 after stimulation with 20 ng/ml BMP9 and 1 ng/ml TGF-β1 for 24 hours. Erk1/2 was used as loading control as reported in methods section. Histograms represent the mean ± SEM of the optical density of the bands of five experiments expressed as percentage over basal values. *P < 0.01 vs. MEFs in basal conditions.
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Fig. 2. Effects of ALK4/5/7 inhibition with SB431542 on ECM protein expression and Smads phosphorylation
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Expression of collagen I, fibronectin and CTGF/CCN2 after BMP9 stimulation with 20 ng/mL in cells pre-inhibited with the ALK4/5/7 inhibitor SB431542 (5µM), the Smad3
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inhibitor SIS3 (4µM) and the MEK/Erk1/2 inhibitor U0126 (20µM). Erk1/2 was used as loading control. Histograms represent the mean ± SEM of the optical density of the bands of five experiments expressed as percentage over basal values. *P < 0.01 vs.
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MEFs in basal conditions.
Fig. 3. Effects of ALK4/5/7 inhibition with SB431542 and ALK5 knockdown on Smads phosphorylation a) Expression of phospho-Smad1/5/8, phospho-Smad2 after BMP9 stimulation with 20 ng/ml in cells pre-inhibited with the ALK4/5/7 inhibitor SB431542 (5µM). b) Expression of phospho-Smad1/5/8 and phospho-Smad2 after BMP9 stimulation with 20 ng/ml in cells transfected with ALK5 siRNA (siALK5). Erk1/2 was used as loading control. *P < 0.01 vs. MEFs in basal conditions. #P < 0.01 vs. MEFs with ALK5 inhibition.
Fig. 4. Effects of ALK1/2/3/6 inhibition with dorsomorphin-1 on ECM protein expression and Smads phosphorylation.
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a) Expression of collagen I, fibronectin and CTGF/CCN2 after BMP9 stimulation with 20 ng/ml for 24 hours in cells pre-inhibited with the ALK1/2/3/6 inhibitor dorsomorphin-1 (1µM). b) Level of phospho-Smad1/5/8, phospho-Smad2, phospho-
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Smad3 and phospho-Erk1/2 after BMP9 stimulation with 20 ng/ml for 30 minutes in
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cells pre-inhibited with the ALK1/2/3/6 inhibitor dorsomorphin-1 (1µM). Erk1/2 was used as loading control. Histograms represent the mean ± SEM of the optical density of the bands of five experiments expressed as percentage over basal values. *P < 0.01 vs.
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MEFs in basal conditions.
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Fig. 5. Effects of ALK1 heterozygosity and ALK1 knockdown on BMP9-induced Smad signalling.
a) Expression of phospho-Smad1/5/8, phospho-Smad2, phospho-Smad3 and phosphoErk1/2 after BMP9 stimulation with 20 ng/ml for 30 minutes in ALK1 heterozygous cells (ALK1+/-) and their respective controls (ALK1+/+). b) Levels of phospho-
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Smad1/5/8, phospho-Smad2, phospho-Smad3 and phospho-Erk1/2 after stimulation BMP9 stimulation with 20 ng/ml in cells transfected with ALK1 siRNA (siALK1) and
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their respective controls (siCtrl). Erk1/2 was used as loading control. Histograms in (a) represent the mean ± SEM of the optical density of the bands of six experiments expressed as percentage over basal values. Blots in (b) are representative from 6
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different experiments. *P < 0.01 vs. ALK1+/+ MEFs in basal conditions. #P < 0.01 vs.
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ALK1+/- MEFs in basal conditions. +P < 0.01 vs. ALK1+/- MEFs in basal conditions. Fig. 6. Representative scheme of the proposed “BMP9/ALK1/ALK5 complex” and its downstream mechanism. BMP9 binds ALK1 and activates the Smad1/5/8 and Smad2/3 pathways. ALK5 is also necessary for BMP9 to promote Smads activation. Moreover, BMP9 also activates the MAPK/Erk1/2 pathway, perhaps through ALK1 activation. All these pathways contribute to the regulation of ECM protein expression in fibroblasts.
SUPPORTING INFORMATION
Supplementary figure 1. (a) ALK1, ALK2, ALK3, ALK5 and ALK6 mRNA expression in MEFs. Data (ALK vs. HPRT mRNA relative expression) shows 1 representative histogram of 3 similar performed PCR analysis. BMP9-induced
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activation of the CAGA (b) and BRE (c) promoters. NIH3T3 cells were transiently transfected with pGL3 (BRE)-luc and pRL-TK-luc and were treated with BMP9 (1, 10, 20 and 40 ng/mL) or TGF-β1 (1 ng/mL) for 15 hours. The firefly luciferase activity was
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normalized to renilla luciferase activity. Data (expressed as mean ± SEM) show one
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representative experiment of two performed.
Supplementary figure 2. Effects of TGF-β1 stimulation on Smads phosphorylation.
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a) Expression of phospho-Smad1/5/8, phospho-Smad2 and phospho-Erk1/2 after stimulation with 20 ng/ml BMP9 at different time points (30 min, 2, 6 and 24 hours). b)
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Expression of phospho-Smad1/5/8, phospho-Smad2 and phospho-Erk1/2 after stimulation with 1 ng/ml TGF-β1 at different time points (30 min, 2, 6 and 24 hours).
Supplementary figure 3. ALK1 knockdown with siRNA. Analysis of ALK1 mRNA expression in cells transfected with negative siRNA (siCtrol)
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or siALK1 with four different Dharmafect reagents (1-4). After this analysis we used
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Dharmafect4 due to its higher ability to silencing ALK1 expression.
Supplementary figure 4. BMP9 activation of the BRE and the CAGA promoter is
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dependent of ALK1.
NIH-3T3 cells were transiently transfected with pGL3 (BRE)-luc and pRL-TK-luc and either pCDNA3 empty vector, pALK1 or pALK5. After 4 hours, cells were treated with
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BMP9 (20 ng/mL) for 15 hours. Data (expressed as mean ± SEM) show one representative experiment of two performed.
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Highlights:
BMP9 increases collagen I, fibronectin and CTGF/CCN2 expression in
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fibroblasts. BMP9 promotes both Smad1/5/8 and Smad2/3 phosphorylation in fibroblasts.
ALK1 is necessary for BMP9 to induce Smad1/5/8 and Smad2/3
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phosphorylation.
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Overexpression of endoglin increases BMP9-induced Smad1/5/8 and Smad2/3
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phosphorylation and its profibrotic properties.
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