Activins As Dual Specificity TGF- Family Molecules: SMAD-Activation

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Article Activins as Dual Speciﬁcity TGF-β Family Molecules: SMAD-Activation via Activin- and BMP-Type 1 Receptors

Oddrun Elise Olsen 1,2, Hanne Hella 1, Samah Elsaadi 1, Carsten Jacobi 3, Erik Martinez-Hackert 4 and Toril Holien 1,2,* 1 Department of Clinical and Molecular Medicine, NTNU – Norwegian University of Science and Technology, 7491 Trondheim, Norway 2 Department of Hematology, St. Olav’s University Hospital, 7030 Trondheim, Norway 3 Novartis Institutes for BioMedical Research Basel, Musculoskeletal Disease Area, Novartis Pharma AG, CH-4056 Basel, Switzerland 4 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA * Correspondence: [email protected]; Tel.: +47-924-21-162

Received: 19 February 2020; Accepted: 27 March 2020; Published: 29 March 2020

Abstract: Activins belong to the transforming growth factor (TGF)-β family of multifunctional cytokines and signal via the activin receptors ALK4 or ALK7 to activate the SMAD2/3 pathway. In some cases, activins also signal via the bone morphogenetic protein (BMP) receptor ALK2, causing activation of the SMAD1/5/8 pathway. In this study, we aimed to dissect how activin A and activin B homodimers, and activin AB and AC heterodimers activate the two main SMAD branches. We compared the activin-induced signaling dynamics of ALK4/7-SMAD2/3 and ALK2-SMAD1/5 in a multiple myeloma cell line. Signaling via the ALK2-SMAD1/5 pathway exhibited greater differences between ligands than signaling via ALK4/ALK7-SMAD2/3. Interestingly, activin B and activin AB very potently activated SMAD1/5, resembling the activation commonly seen with BMPs. As SMAD1/5 was also activated by activins in other cell types, we propose that dual specificity is a general mechanism for activin ligands. In addition, we found that the antagonist follistatin inhibited signaling by all the tested activins, whereas the antagonist cerberus specifically inhibited activin B. Taken together, we propose that activins may be considered dual specificity TGF-β family members, critically affecting how activins may be considered and targeted clinically.

Keywords: activin; SMAD; BMP; ALK2; ACVR1; signal transduction

1. Introduction The transforming growth factor (TGF)-β family consists of more than 30 members in humans, including TGF-βs, bone morphogenetic proteins (BMPs), activins and growth diﬀerentiation factors (GDFs) [1]. The ligands mainly signal through two canonical pathways of SMAD transcription factors, but there is also compelling evidence that non-canonical activation can occur when a ligand–receptor signaling complex is formed [2]. The determining factor for which speciﬁc SMAD pathway will relay the signal is which type 1 receptor is activated in the signaling complex [3]. The SMAD2/3 pathway is activated by the type 1 receptors ALK4, ALK5 and ALK7, whereas the SMAD1/5/8 pathway is activated by the type 1 receptors ALK1, ALK2, ALK3 and ALK6 [3]. The approved HUGO (Human Genome Organisation) gene names for TGF-β family receptors are provided in Supplementary Table S1 for reference. The TGF-βs signal through ALK5 and activate the SMAD2/3 pathway but have also been shown to form complexes with ALK1 and/or ALK2 and activate the SMAD1/5/8 pathway. BMPs mostly form complexes with ALK1, ALK2, ALK3 or ALK6 and activate the SMAD1/5/8 pathway.

Biomolecules 2020, 10, 519; doi:10.3390/biom10040519 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 519 2 of 15

Activins activate the SMAD2/3 pathway through the type 1 activin receptors ALK4 and ALK7 [4,5]. Activin A was originally found to interact with ALK2, although no activation of the SMAD1/5/8 pathway was detected in these studies [4,6,7]. It was also reported that activin A could act as an antagonist of BMP signaling by competing for receptor binding [8–11]. Recently, it has become clear that activin A and activin B also activate SMAD1/5 through mutated and wild type ALK2 [11–15]. Knockdown of BMPR2 caused potentiation of activin-induced SMAD1/5, but not SMAD2/3, activity in multiple myeloma and hepatocytic carcinoma cells [14,16]. We have shown that BMPs induce growth arrest and/or apoptosis in myeloma cells via activation of the SMAD1/5 pathway which in turn leads to c-MYC downregulation [17,18]. Maintaining c-MYC levels is important for the survival of myeloma cells [19]. We also reported that some myeloma cells are sensitive to activin A- and activin B-induced apoptosis via ALK2-SMAD1/5 [16]. Together, these studies suggest that activins are dual specificity TGF-β family ligands that can activate both SMAD branches, depending on the context. Here, we aimed to characterize and compare activation of both SMAD branches by activins. Using an activin-responsive myeloma cell line, IH-1, we compared the effects of activin A, B, and C homo- and heterodimers on signaling dynamics and amplitude. We then used small molecule inhibitors with different specificities to further establish the involvement of different receptors in both IH-1 myeloma cells and HepG2 liver carcinoma cells. We further transiently knocked down type I receptors to establish receptor dependencies and compared the effects of the well-known activin antagonist follistatin and the less characterized activin B antagonist cerberus on downstream activation of SMAD2/3 and SMAD1/5 signaling. Although our results show that activins can act as BMP antagonists, we also find that in certain contexts activins may be considered dual specificity members of the TGF-β family. In these cases, ALK2 relays the signal from activins to SMAD1/5.

2. Materials and Methods

2.1. Cells and Reagents In this study, we used the human multiple myeloma cell lines IH-1 and INA-6. IH-1 was established in our laboratory [20]. INA-6 cells were a kind gift from Dr. M. Gramatzki (University of Erlangen-Nurnberg, Erlangen, Germany) [21]. IH-1 cells were cultured in 10% human serum (HS) (Department of Immunology and Transfusion Medicine, St. Olav’s University Hospital, Trondheim, Norway) in RPMI-1640 (Sigma-Aldrich Norway, Oslo, Norway) supplemented with 2 mM L-glutamine (Sigma-Aldrich), hereafter termed RPMI, whereas INA-6 cells were cultured in 10% fetal calf serum (FCS) in RPMI. For both myeloma cell lines, interleukin (IL)-6 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) (1 ng/mL) was added to the growth medium. The hepatocyte carcinoma cell line HepG2 was from European Collection of Authenticated Cell Cultures (ECACC; Salisbury, UK). HepG2 cells were grown in 10% FCS in Eagle’s minimum essential medium (EMEM) supplemented with 2 mM glutamine and non-essential amino acids (Sigma-Aldrich), hereafter termed EMEM. The cells were maintained in 37 ◦C in a humidified atmosphere containing 5% CO2 and tested regularly for mycoplasma. For experiments with myeloma cell lines, 2% HS in RPMI was used as the medium with IL6 (1 ng/mL) added for INA-6, and for HepG2, 0.1% bovine serum albumin (BSA) in EMEM was used. The recombinant human proteins activin B, activin C, and activin AC were from R&D Systems (Bio-Techne, Abingdon, UK), whereas E. coli-produced and refolded activin A (human) [22] and activin AB (human/Xenopus) were kindly provided by Marko Hyvönen’s group at the University of Cambridge, UK. Recombinant human cerberus was expressed and purified as previously described [23]. Recombinant human TGF-β1 and TGFβRII-Fc were from R&D Systems. The inhibitors K02288 and SB431542 were from Sigma-Aldrich, ML347, LDN-193189 and RepSox (Compound 19) were from Selleck Chemicals, Houston, TX, USA, and ZC-47-C95 (resynthesized compound 18a published by Jin et al., 2011 [24]) was from Novartis. Biomolecules 2020, 10, 519 3 of 15

2.2. Western Blotting Treated cells were pelleted, washed once in ice-cold phosphate-buffered saline (PBS), and lysed in a buffer consisting of 1% IGEPAL® CA-630 (Sigma-Aldrich), 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 50 mM NaF and Complete Mini protease inhibitor cocktail (Roche, Basel, Switzerland). The samples were denatured and separated on NuPAGE™ Bis-Tris polyacrylamide gels (Invitrogen, Thermo Fisher Scientific). The gels were then blotted onto 0.45-µm nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and incubated with primary antibodies as indicated at 4 ◦C. Detection was performed with horseradish peroxidase-conjugated secondary antibodies (DAKO Cytomation, Copenhagen, Denmark) and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Images were acquired and analyzed with Odyssey FC and Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA). The primary antibodies that were used were: phospho-SMAD1/5 (RRID: AB_491015, Cat# 9516), phospho-SMAD2 (RRID: AB_490941, Cat# 3108), phospho-SMAD2/3 (RRID: AB_2631089, Cat# 8828), and cleaved caspase-3 (RRID: AB_2070042, Cat# 9664) (Cell Signaling Technology, BioNordika AS, Oslo, Norway), c-MYC (RRID: AB_2148606, Cat# 551102, BD Biosciences, Trondheim, Norway), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (RRID: AB_2107448, Cat# Ab8245, Abcam, Cambridge, UK). Antibodies were diluted 1:1000, except for GAPDH which was diluted 1:30000.

2.3. Cell Viability Relative viable cell numbers were determined by the CellTiter-Glo assay (Promega, Madison, WI, USA), which measures ATP levels, as described [25].

2.4. Transfection of INA-6 Cells INA-6 cells were transfected using the Nucleofector device (Amaxa Biosystems, Cologne, Germany) and Amaxa Cell Line Nucleofector Kit R (Lonza, Basel, Switzerland), as previously described [26]. The siRNAs used were ON-TARGETplus SMARTpool ACVR1/ALK2, siGENOME SMARTpool ACVR1B/ALK4, ACVR1C/ALK7 and TGFBR1/ALK5, and Non-Targeting Pool (Dharmacon, Thermo Fisher Scientiﬁc)

2.5. Transfection of HepG2 Cells HepG2 cells were seeded in 24-well plates and left over night to adhere. Cells at a conﬂuence of about 50% were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the protocol. The siRNAs used were ON-TARGETplus SMARTpool ACVR1/ALK2 and Non-Targeting Pool (Dharmacon). The day after transfection, the cells were treated with the indicated ligands for 1 h and harvested for western blotting or PCR.

2.6. Comparative RT-PCR RNA was isolated using the RNeasy Mini Kit (Qiagen, Crawley, UK) and complementary DNA was synthesized from total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Thermo Fisher Scentiﬁc). PCR was performed using StepOne Real-Time PCR System and Taqman Gene Expression Assays (Applied Biosystems) as described previously [9]. The Taqman assays used were: ACVR1 (Hs00153836_m1), ACVR1B (Hs00244715_m1), ACVR1C (Hs0000899854_m1), TGFB1 (Hs00998133_m1), and GAPDH (Hs99999905_m1). The comparative Ct method was used to calculate relative changes in gene expression with GAPDH as the housekeeping gene.

2.7. Statistical Analysis GraphPad Prism 8 (Graphpad Software, Inc., San Diego, LA) was used to analyze statistical signiﬁcance. The tests used for each experiment are described in the ﬁgure legends. Biomolecules 2020, 10, x 4 of 15 Biomolecules 2020, 10, 519 4 of 15 3. Results

3.3.1. Results Activin Dimers Have Dose-Dependent Effects on IH-1 Cell Viability 3.1. ActivinIH-1 myeloma Dimers Have cells Dose-Dependent were treated with Effects activin on IH-1 hete Cellro- Viabilityand homodimers for three days before cell viability was determined by measuring ATP content in wells. As we have shown before, activin A IH-1 myeloma cells were treated with activin hetero- and homodimers for three days before cell and activin B dose-dependently reduced cell viability, with activin B being the most potent cell viability was determined by measuring ATP content in wells. As we have shown before, activin A and viability inhibitor (Figure 1a,b) [16]. As expected, no difference in cell viability was seen after activin B dose-dependently reduced cell viability, with activin B being the most potent cell viability treatment with activin C at the given doses (Figure 1c). Activin AB reduced cell viability to a similar inhibitor (Figure1a,b) [ 16]. As expected, no difference in cell viability was seen after treatment with extent as activin B (Figure 1d), whereas activin AC was less potent than the other activins (Figure 1e). activin C at the given doses (Figure1c). Activin AB reduced cell viability to a similar extent as activin We further confirmed that the effect of activins on cell number, at least partially, depended on B (Figure1d), whereas activin AC was less potent than the other activins (Figure1e). We further apoptosis due to correlation with SMAD-induced c-MYC downregulation and caspase-3 cleavage confirmed that the effect of activins on cell number, at least partially, depended on apoptosis due to (Figure 1f) [17]. correlation with SMAD-induced c-MYC downregulation and caspase-3 cleavage (Figure1f) [17].

Cell viability (% of control) (% viability Cell Cell viability (% Cellof control) viability (%

(a) (b) (c)

Cell viability (% of control) of (% viability Cell

(d) (e) (f)

FigureFigure 1. 1.Impact Impact ofof activin activin homo-homo- andand heterodimers heterodimers onon IH-1 IH-1 cell cell viability. viability. IH-1 IH-1 myeloma myeloma cellscells were were treated for three days with increasing doses of activins as indicated in the figure. Cell viability was treated for three days with increasing doses of activins as indicated in the figure. Cell viability was measured using CellTiter Glo and the results are plotted relative to control (a–e). The graphs represent measured using CellTiter Glo and the results are plotted relative to control (a–e). The graphs represent mean standard error of the mean (s.e.m.) of n = 3 independent experiments. One-way ANOVA and mean± ± standard error of the mean (s.e.m.) of n = 3 independent experiments. One-way ANOVA and Dunnett’s multiple comparisons test were performed (* p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001, Dunnett’s multiple comparisons test were performed (*≤P ≤ 0.05, **P≤ ≤ 0.01, ***P≤ ≤ 0.001, ****P≤ ≤ 0.0001, ns (not significant) p > 0.05). (f) We also treated IH-1 cells for 24 h with activin A (20 ng/mL), activin B ns (not significant) P > 0.05). (f) We also treated IH-1 cells for 24 h with activin A (20 ng/mL), activin (4 ng/mL), activin C (20 ng/mL), activin AB (5 ng/mL) or activin AC (20 ng/mL), and did western blot to B (4 ng/mL), activin C (20 ng/mL), activin AB (5 ng/mL) or activin AC (20 ng/mL), and did western look at differences in expression of c-MYC, phospho-SMAD1/5 (pSMAD1/5) and cleaved caspase 3, with blot to look at differences in expression of c-MYC, phospho-SMAD1/5 (pSMAD1/5) and cleaved GAPDH as the loading control. The blots shown are representative of n = 3 independent experiments. caspase 3, with GAPDH as the loading control. The blots shown are representative of n = 3 3.2. Activinsindependent Activated experiments. SMAD1 /5 and SMAD2 with Different Dynamics

3.2. ActivinsWe have Activate previouslyd SMAD1/5 shown that and activinSMAD2 A with and activinDifferent B activatedDynamics SMAD1/5 via ALK2 and induced cell death in IH-1 and INA-6 myeloma cell lines [16]. Activation of the SMAD2/3 pathway did not lead to apoptosisWe have in thesepreviously cells, likelyshown due that to mechanismsactivin A and that activin prevent B translocationactivated SMAD1/5 of activated via ALK2 SMAD2 and/3 toinduced the nucleus cell death in myeloma in IH-1 cells and [ 9INA-6,16,27 ].myeloma Extending cell on lines this [16]. finding, Activation activation of ofthe SMAD1 SMAD2/3/5 by pathway activin ABdid and not activin lead to AC apoptosis also correlated in these with cells, reduced likely cell due viability to mechanisms (Figure1c,d,f). that Nevertheless, prevent translocation the activins of activated SMAD2/3 to the nucleus in myeloma cells [9,16,27]. Extending on this finding, activation of Biomolecules 2020, 10, x 5 of 15 Biomolecules 2020, 10, 519 5 of 15 SMAD1/5 by activin AB and activin AC also correlated with reduced cell viability (Figure 1c, d and activatedFigure 1f). both Nevertheless, SMAD branches the activins and weactivated wanted both to compareSMAD branches the signaling and we dynamics wanted to between compare these the two.signaling IH-1 dynamics cells were treatedbetween with these activins two. andIH-1 harvested cells were for treated western with blotting activins at di andfferent harvested time points. for Activinwestern doses blotting were at chosendifferent based time on points. the viability Activin assay doses and were activin chosen C was based omitted on the in viability these experiments assay and sinceactivin we C werewas omitted not able in to these detect experiments any activation since of SMADs we were with not thisable ligand to detect (Figure any 1activationc). Activation of SMADs of the SMAD1with this/5 ligand pathway (Figure peaked 1c). after Activation 2 h for activinof the SMAD1/5 A and activin pathway B, whereas peaked it peakedafter 2 h after for 1activin h for activinA and AB,activin and B, as whereas early as it 0.5 peaked h (or even after earlier) 1 h for foractivin activin AB, AC and (Figure as early2a,b). as Activin0.5 h (or AC even was earlier) altogether for activin not a strongAC (Figure inducer 2a,b). of SMAD1 Activin/5 AC activity was andaltogether the activation not a strong was terminated inducer of quickly SMAD1/5 compared activity to theand other the activins.activation Interestingly, was terminated activation quickly of compared SMAD2 was to the much other more activins. similar Interestingly, amongst the activation different activins of SMAD2 and peakedwas much at 1 more h for similar all the testedamongst ligands the different (Figure2c,d). activins and peaked at 1 h for all the tested ligands (Figure 2c,d).

(a) (b)

Figure 2. Time-series comparing activation of both SMAD branches by activins. IH-1 myeloma cells were treated with activin A (20 ng/mL), activin B (4 ng/mL), activin AB (5 ng/mL) or activin ACFigure (20 2. ng Time-series/mL) for the comparing indicated time-points.activation of Then,both SMAD the phosphorylation branches by activins. statusof IH-1 SMAD1 myeloma/5 (a, bcells) or SMAD2were treated/3 (c,d with) was activin measured A (20 by ng/mL), western activin blot. Representative B (4 ng/mL), activin experiments AB (5 ng/mL) are shown or activin (upper panels)AC (20 andng/mL) relative for the activation indicated was time-points. calculated basedThen, on the signal phosphorylation intensities of status the SMADs of SMAD1/5 and GAPDH (a–b) foror normalizationSMAD2/3 (c–d) (lower was measured panels). Theby western graphs blot. represent Representa mean tives.e.m. experiments of n = 3 independentare shown (upper experiments. panels) ± Two-wayand relative ANOVA activation and Bonferroni’swas calculated multiple based comparisons on signal intensities test were of performed the SMADs (* p and0.05, GAPDH ** p 0.01, for ≤ ≤ ***normalizationp 0.001, **** (lowerp 0.0001, panels). ns The (not gr significant)aphs representp > 0.05). mean ± s.e.m. of n = 3 independent experiments. Two-way≤ ANOVA≤ and Bonferroni’s multiple comparisons test were performed (*P ≤ 0.05, **P ≤ 0.01, 3.3. E***ffectP ≤of 0.001, Small **** MoleculeP ≤ 0.0001, Inhibitors ns (not significant) on Activin-Induced P > 0.05). SMAD Phosphorylation We next sought to characterize the type 1 receptor involved in activation of the SMAD-branches and3.3. Effect used of small Small molecule Molecule inhibitors. Inhibitors on Such Activin-Induced inhibitors are SMAD valuable Phosphorylation tools, but their degree of specificity and sensitivityWe next sought varies, to characterize so we chose the to usetype six 1 receptor different involved inhibitors. in activation The inhibitors of the SMAD-branches K02288, ML347, and used LDN-193189, small molecule are known inhibitors. inhibitors Such of inhibitors the SMAD1 are /valuable5/8-pathway tools, via but BMP their type degree 1 receptors of specificity (e.g., ALK1,and sensitivity ALK2, ALK3 varies, and so ALK6) we chose [28– to30 ].use The six inhibitors different SB431542, inhibitors. RepSox, The inhibitors and ZC-47-C95 K02288, are ML347, described and asLDN-193189, inhibitors ofare the known SMAD2 inhibitors/3 pathway of the via SMAD1/5/8-pathway activin/TGF-β type via 1 receptorsBMP type (e.g.,1 receptors ALK4, (e.g., ALK5 ALK1, and ALK7)ALK2, ALK3 [24,31, 32and]. AALK6) table [28–30]. describing The relative inhibitors specificities SB431542, of RepSox, the inhibitors and ZC-47-C95 is shown (Supplementaryare described as Biomolecules 2020, 10, x 6 of 15

Biomolecules 2020, 10, 519 6 of 15 inhibitors of the SMAD2/3 pathway via activin/TGF-β type 1 receptors (e.g., ALK4, ALK5 and ALK7) [24,31,32]. A table describing relative specificities of the inhibitors is shown (Supplementary Table TableS2). We S2). treated We treated IH-1 cells IH-1 with cells withactivin activin A and A combined and combined activin activin A with A with the panel the panel of inhibitors of inhibitors for forcomparison. comparison. As expected, As expected, activation activation of SMAD1/5 of SMAD1 by /activin5 by activin A in these A in cells these was cells dependent was dependent on a BMP on atype BMP 1 receptor type 1receptor (Figure 3a). (Figure Activation3a). Activation of SMAD2 of was, SMAD2 also as was, expected, also as dep expected,endent on dependent an activin/TGF- on an activinβ type /1TGF- receptorβ type (Figure 1 receptor 3b). For (Figure comparison3b). For comparisonwe also tested we activin also tested B, and activin surprisingly, B, and surprisingly, the panel of theinhibitors panel ofshowed inhibitors less showed clear results less clear (Figure results 3c,d). (Figure For 3instance,c,d). For ML347 instance, was ML347 less wasefficient less etowardsﬃcient towardsactivin B-induced activin B-induced SMAD1/5 SMAD1 activation/5 activation and RepSox and RepSox showed showed some some inhibiting inhibiting activity activity (Figure (Figure 33c).c). Furthermore, inin addition addition to to the the expected expected inhibition inhibition by activin by activin/TGF-/TGF-β typeβ 1 type receptor 1 receptor inhibitors inhibitors towards activintowards B-induced activin B-induced SMAD2 activation,SMAD2 activation, there was there also signiﬁcantwas also significant inhibition inhibition by the BMP by type the BMP 1 receptor type inhibitors1 receptor inhibitors K02288 and K02288 LDN-193189. and LDN-193189. These results These could results indicate could indicate the dependency the dependency of a BMP of a type BMP 1 receptortype 1 receptor for activation for activation of SMAD2 of SMAD2 by activin by B. activin Thus, basedB. Thus, on based this experiment, on this experiment, activation activation of SMAD1 of/5 bySMAD1/5 activin Aby seems activin to A be seems entirely to be dependent entirely dependent on a BMP type on a 1 BMP receptor type in 1 IH-1receptor cells, in whereas IH-1 cells, there whereas might bethere an might interdependent be an interdependent relationship relationship between the between activation the of activation the two SMAD-branches of the two SMAD-branches for activin B. for activin B.

(a) (b)

Figure 3. Effect of small molecule inhibitors on activin-induced SMAD phosphorylation. IH-1 myeloma cells were treated for one hour with activin A (20 ng/mL) (a,b) or activin B (4 ng/mL) (c,d) with or Figure 3. Effect of small molecule inhibitors on activin-induced SMAD phosphorylation. IH-1 without different inhibitors. K02288, LDN-193189 and RepSox (100 nM), ML347 (200 nM), ZC-47-C95 (1myelomaµM) and cells SB431542 were treated (2 µM). for Then, one the hour phosphorylation with activin A status (20 ng/mL) of SMAD1 (a,b/)5 or and activin SMAD2 B (4 was ng/mL) measured (c,d) bywith western or without blot. different Representative inhibitors. experiments K02288, LD areN-193189 shown and (upper RepSox panels) (100 and nM), relative ML347 activation (200 nM), wasZC- calculated47-C95 (1 μ basedM) and on SB431542 signal intensities (2 μM). Then, of the the SMADs phosphorylation and GAPDH status for normalizationof SMAD1/5 and (lower SMAD2 panels). was Themeasured graphs by represent western meanblot. Representatives.e.m. of n = expe3 independentriments are experiments. shown (upper One-way panels) ANOVAand relative and ± Dunnett’sactivation multiplewas calculated comparisons based on test signal were inte performednsities of (* thep SMADs0.05, ** pand0.01, GAPDH **** p for 0.0001,normalization ns (not ≤ ≤ ≤ significant)(lower panels).p > 0.05).The graphs represent mean ± s.e.m. of n = 3 independent experiments. One-way Biomolecules 2020, 10, x 7 of 15

ANOVA and Dunnett’s multiple comparisons test were performed (*P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, Biomoleculesns (not2020 significant), 10, 519 P > 0.05). 7 of 15

3.4. Effect of the BMP Type 1 Receptor Inhibitor, K02288, on Activin-Induced SMAD Activity in HepG2 Cells3.4. Eﬀect of the BMP Type 1 Receptor Inhibitor, K02288, on Activin-Induced SMAD Activity in HepG2 Cells We also detected activation of both SMAD-branches in activin-treated HepG2 liver carcinoma cells. In In these these cells, cells, we we applied applied the the K02288 K02288 BMP BMP ty typepe 1 1 receptor receptor inhibitor inhibitor and and checked checked for for effects eﬀects of activin-induced SMAD1SMAD1/5-/5- andand SMAD2-activation.SMAD2-activation. In In comparison comparison to to the the IH-1 IH-1 cells, cells, the the results results were were as asexpected, expected, with with inhibition inhibition of SMAD1of SMAD1/5,/5, but but not not SMAD2 SMAD2 activation activation (Figure (Figure4). 4).

(a) (b)

Figure 4. Effect of the bone morphogenic protein (BMP) type 1 receptor inhibitor, K02288, on activin-induced SMAD activity in HepG2 cells. HepG2 cells were treated for one hour with activin FigureA, activin 4. Effect AB. Activinof the bone AC morphogenic (20 ng/mL), orprotein activin (BMP) B (50 type ng/ mL)1 receptor with orinhibitor, without K02288, K02288 on (100 activin- nM). inducedThen, the SMAD phosphorylation activity in HepG2 status cells. of SMAD1 HepG2/5 cells (a) andwere SMAD2 treated (forb) one was hour measured with activin by western A, activin blot. AB.Representative Activin AC experiments (20 ng/mL), are or shownactivin (upper B (50 panels)ng/mL) andwith relative or without activation K02288 was (100 calculated nM). Then, based the on phosphorylationsignal intensities ofstatus the SMADs of SMAD1/5 and GAPDH (a) and for normalizationSMAD2 (b) (lowerwas measured panels). The by graphs western represent blot. Representativemean s.e.m. experiments of n = 3 independent are shown experiments.(upper panels) Two-way and relative ANOVA activation and Bonferroni’swas calculated multiple based ± oncomparisons signal intensities test were of performed the SMADs (* p and0.05, GAPDH ** p 0.01, for ***normalizationp 0.001, ns (lower (not significant) panels). Thep > 0.05). graphs represent mean ± s.e.m. of n = 3 independent≤ experiments.≤ ≤ Two-way ANOVA and Bonferroni’s 3.5. ALK2multiple Knockdown comparisons Blunted test were Activin-Induced performed (*P SMAD1 ≤ 0.05,/ 5**P Activity ≤ 0.01, ***P ≤ 0.001, ns (not significant) P > 0.05).Since the results using inhibitors only partially explained type 1 receptor usage, we chose a more specific approach to further clarify the mechanism of SMAD1/5 activation. We performed ALK2 3.5. ALK2 Knockdown Blunted Activin-Induced SMAD1/5 Activity knockdown experiments in another multiple myeloma cell line, INA-6, which expresses ALK2, but not ALK3Since [16, 25the,33 results]. ALK2 using siRNA inhibitors or control only partially siRNA explained were transfected type 1 receptor into the usage, cells. we After chose two a more days, specificthe cells approach were treated to withfurther activin clarify A orthe activin mechanism B for 1 hof and SMAD1/5 SMAD activityactivation. was We measured performed by western ALK2 knockdownblots. Knockdown experiments of ALK2 in another significantly multiple blunted myelom botha cell activin line, A- INA-6, and activinwhich B-inducedexpresses ALK2, SMAD1 but/5 notphosphorylation ALK3 [16,25,33]. but hadALK2 no siRNA effect on or SMAD2 control/3 siRNA phosphorylation were transfected (Figure 5intoa,b). the The cells. knockdown After two e ffi days,cacy thewas cells just were above treated 50%, aswith measured activin A by or PCR activin (Figure B for5 c).1 h Forand comparison, SMAD activity we was also measured transiently by knocked western blots.down Knockdown the expression of ALK2 of the significantly other type 1 blunted receptors both ALK4, activin ALK7 A- andand ALK5.activin AsB-induced expected, SMAD1/5 none of phosphorylationthese siRNAs aff ectedbut had activin no A-effect and on activin SMAD2/3 B-induced phosphorylation SMAD1/5 phosphorylation (Figure 5a,b). The (Supplementary knockdown efficacyFigure S1).was Moreover,just above in50%, HepG2 as measured cells, knockdown by PCR (F ofigure ALK2 5c). counteracted For comparison, activation we also of transiently SMAD1/5, knockedbut not SMAD2, down the after expression treatment of with the activinother type A or 1 activin receptors B (Supplementary ALK4, ALK7 and Figure ALK5. S2). As expected, none Multipleof these myeloma siRNAs cellsaffect expressed activin high A- levels and of activin TGF-β mRNAB-induced and proteinSMAD1/5 [18 ].phosphorylation To exclude the (Supplementarypossibility that the Figure SMAD S1). activation Moreover, following in HepG2 activin cells, treatment knockdown wasnot of ALK2 caused counteracted by changes in activation autocrine ofTGF- SMAD1/5,β activity, but we not first SMAD2, measured afterTGFB1 treatmentmRNA with in ac IH-1tivin and A or INA-6 activin cells B (Su treatedpplementary with activin Figure A andS2). activinMultiple B for 4 myeloma h (Supplementary cells express Figure high S3a,b). levels There of TGF- wereβ no mRNA significant and changesprotein [18]. in TGFB1 To excludeexpression the possibilitylevels. Then, that we the treated SMAD IH-1 activation and INA-6 following cells with activin activin treatment A, activin was B ornot TGF- causedβ, with by changes or without in autocrinesoluble TGF TGF-βRII-Fc,β activity, an inhibitory we first TGF- measuredβ ligand TGFB1 trap and mRNA looked in at IH-1 activation and INA-6 of SMAD1 cells/ 5treated and SMAD2 with activin(Supplementary A and activin Figure B S3c–f).for 4 h The(Supplementary soluble TGF βFigureRII-Fc S3a,b). prevented There TGF- wereβ-induced no significant SMAD changes activation in TGFB1but failed expression to inhibit levels. activation Then, of we either treated of the IH-1 two and SMAD INA-6 branches cells with by activin activins. A, Similaractivin resultsB or TGF- wereβ, withseen withor without a neutralizing soluble TGF-TGFβRII-Fc,antibody an (datainhibitory not shown). TGF-β ligand trap and looked at activation of Biomolecules 2020, 10, x 8 of 15

SMAD1/5 and SMAD2 (Supplementary Figure S3c-f). The soluble TGFβRII-Fc prevented TGF-β- induced SMAD activation but failed to inhibit activation of either of the two SMAD branches by Biomolecules 2020, 10, 519 8 of 15 activins. Similar results were seen with a neutralizing TGF-β antibody (data not shown).

(a) (b) (c)

FigureFigure 5. 5. ALK2ALK2 knockdown knockdown blunted blunted activin-induced activin-induced SM SMAD1AD1/5/5 activity. activity. INA-6 INA-6 myeloma myeloma cells cells were were transfectedtransfected with siRNAsiRNA targetingtargeting ALK2 ALK2 or or Non-Targeting Non-Targeting Pool Pool as aas control. a control. The The cells cells were were treated treated 2 days 2 dayspost transfectionpost transfection with activin with Aactivin (50 ng A/mL) (50 or ng/m activinL) Bor (10 activin ng/mL) B for(10 2 h.ng/mL) Then, thefor phosphorylation2 h. Then, the phosphorylationstatus of SMAD1 /5status (a) and of SMAD2 SMAD1/5 (b) was (a) measured and SMAD2 by western (b) blot.was Representativemeasured by experimentswestern blot. are Representativeshown (upper panels)experiments and relative are shown activation (upper was panels calculated) and relative based on activation signal intensities was calculated of the SMADs based and GAPDH for normalization (lower panels). The graphs represent mean s.e.m. of n = 3 independent on signal intensities of the SMADs and GAPDH for normalization (lower± panels). The graphs experiments. Two-way ANOVA and Bonferroni’s multiple comparisons test were performed (* p 0.05, represent mean ± s.e.m. of n = 3 independent experiments. Two-way ANOVA and Bonferroni’s≤ multiplens (not signiﬁcant) comparisonsp > test0.05). were (c) ALK2performedmRNA (*P levels ≤ 0.05, were ns (not measured significant) by PCR P using> 0.05). the (c) comparative ALK2 mRNA Ct method and GAPDH as housekeeping gene. The graph represents mean s.e.m. of three independent levels were measured by PCR using the comparative Ct method and GAPDH± as housekeeping gene. experiments. A two-tailed, paired t-test was performed (**** p 0.0001). The graph represents mean ± s.e.m. of three independent experiments.≤ A two-tailed, paired t-test was 3.6. Antagonismperformed (**** of ActivinsP ≤ 0.0001). by Follistatin and Cerberus

3.6. AntagonismHaving shown of Activins that activins by Follistatin depend and on Cerberus. ALK2 for activation of the SMAD1/5 pathway, we wanted to compare extracellular activin antagonists to see if activation of both SMAD branches was inhibited. Having shown that activins depend on ALK2 for activation of the SMAD1/5 pathway, we Follistatin is an important activin antagonist, and the ratio of serum activin A/follistatin is commonly wanted to compare extracellular activin antagonists to see if activation of both SMAD branches was used as a measure of activin A bioavailability [34–36]. We treated IH-1 cells with activin dimers inhibited. Follistatin is an important activin antagonist, and the ratio of serum activin A/follistatin is together with follistatin for three days before measuring relative cell viability. As expected, the growth commonly used as a measure of activin A bioavailability [34–36]. We treated IH-1 cells with activin inhibitory effect of all the tested activin dimers was blunted in the presence of follistatin, except dimers together with follistatin for three days before measuring relative cell viability. As expected, for activin AC, where no significant effect was observed (Figure6a). Cerberus is recognized as an the growth inhibitory effect of all the tested activin dimers was blunted in the presence of follistatin, antagonist of the TGF-β family ligand nodal [23,37]. Interestingly, human cerberus also binds and except for activin AC, where no significant effect was observed (Figure 6a). Cerberus is recognized antagonizes activin B and, to a lesser extent, BMP6 and BMP7 [38]. As far as we know, activin AB and as an antagonist of the TGF-β family ligand nodal [23,37]. Interestingly, human cerberus also binds activin AC have never been tested for in vitro antagonism by human cerberus. We therefore treated and antagonizes activin B and, to a lesser extent, BMP6 and BMP7 [38]. As far as we know, activin IH-1 cells with activin dimers together with cerberus for three days before measuring relative cell AB and activin AC have never been tested for in vitro antagonism by human cerberus. We therefore viability. As expected, cerberus was found to specifically antagonize activin B, but did not antagonize treated IH-1 cells with activin dimers together with cerberus for three days before measuring relative activin A, activin AB or activin AC (Figure6b). We confirmed using western blots that the e ffect on cell viability. As expected, cerberus was found to specifically antagonize activin B, but did not cell viability correlated with effect on SMAD activity (Figure6c,d). Our results thus confirm and antagonize activin A, activin AB or activin AC (Figure 6b). We confirmed using western blots that supplement previous studies showing that follistatin is a general activin antagonist, whereas cerberus the effect on cell viability correlated with effect on SMAD activity (Figure 6c,d). Our results thus can distinguish between activin B and the other activins. confirm and supplement previous studies showing that follistatin is a general activin antagonist, whereas cerberus can distinguish between activin B and the other activins. Biomolecules 2020, 10, 519 9 of 15 Biomolecules 2020, 10, x 9 of 15

Medium Follistatin 150 *** *** 100 *** ns

0 Control Activin A Activin B Activin AB Activin AC

(a) (b)

Figure 6. Antagonism of activins by follistatin and cerberus. IH-1 myeloma cells were treated for three days with activin A or activin AC (20 ng/mL), activin B (4 ng/mL) or activin AB (5 ng/mL) in Figure 6. Antagonism of activins by follistatin and cerberus. IH-1 myeloma cells were treated for three combination with either (a) follistatin (1 µg/mL) or (b) cerberus (4 µg/mL) before cell viability was days with activin A or activin AC (20 ng/mL), activin B (4 ng/mL) or activin AB (5 ng/mL) in measured using CellTiter Glo. Results are plotted relative to the control. The phosphorylation status of combination with either (a) follistatin (1 μg/mL) or (b) cerberus (4 μg/mL) before cell viability was SMAD1/5 (c) and SMAD2/3 (d) was measured by western blot. IH-1 myeloma cells were treated for one measured using CellTiter Glo. Results are plotted relative to the control. The phosphorylation status hour with activin A (20 ng/mL) or activin B (50 ng/mL) with or without follistatin (1 µg/mL) or cerberus of SMAD1/5 (c) and SMAD2/3 (d) was measured by western blot. IH-1 myeloma cells were treated (4 µg/mL). Representative experiments are shown (upper panels) and relative activation was calculated for one hour with activin A (20 ng/mL) or activin B (50 ng/mL) with or without follistatin (1 μg/mL) based on signal intensities of the SMADs and GAPDH for normalization (lower panels). The graphs or cerberus (4 μg/mL). Representative experiments are shown (upper panels) and relative activation (a–c) represent mean s.e.m. of n = 3 independent experiments. Two-way ANOVA and Bonferroni’s was calculated based± on signal intensities of the SMADs and GAPDH for normalization (lower multiple comparisons test were performed (* p 0.05, ** p 0.01, *** p 0.001, **** p 0.0001, ns (not panels). The graphs (a–c) represent mean ± s.e.m.≤ of n ≤= 3 independent≤ experiments.≤ Two-way significant) p > 0.05). ANOVA and Bonferroni’s multiple comparisons test were performed (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 4. Discussion0.001, ****P ≤ 0.0001, ns (not significant) P > 0.05). Activin A usually signals through ALK4, whereas activin B and activin AB bind and signal either 4. Discussion through ALK4 or ALK7, resulting in activation of the SMAD2/3 pathway [5,39,40]. Activin C is thought to beActivin non-signaling, A usually and signals thereby through inhibitory, ALK4, by whereas forming ac activintivin B AC and heterodimers activin AB bind at the and expense signal of either more throughpotent activin ALK4 Aor homodimers ALK7, resulting [41,42 in]. activation Here, we showof the that SMAD2/3 activins pathway can activate [5,39,40]. signals Activin via the C two is thoughtmain branches to be non-signaling, of the SMAD signaling and thereby pathway, inhibito SMAD2ry, by/3 andforming SMAD1 activin/5/8, byAC binding heterodimers and activating at the expensedifferent of TGF- moreβ family potent type activin I receptors. A homodimers In addition, [41, we42]. directly Here, we compare show thethat activity activins of fourcan diactivatefferent signalsactivin via homo- the andtwo heterodimers.main branches We of showthe SMAD that the signaling signaling pathway, strength SMAD2/3 and dynamics and SMAD1/5/8, induced by theby bindingdifferent and ligands activating vary. Importantly,different TGF- thisβ family is to our type knowledge I receptors. the In first addition, time activin we directly AB and compare activin the AC activityhave been of four shown different to activate activin SMAD1 homo-/5 viaand the hetero wildtypedimers. ALK2 We show receptor. that the signaling strength and dynamicsInterestingly, induced by the the activin different AC heterodimerligands vary. activates Importantly, both this SMAD is to branches, our knowledge whereas the activin first time C is activininactive. AB Similarly, and activin activin AC have A is been a weak shown activator to activate of SMAD1 SMAD1/5/5, whereas via the thewildtype heterodimer ALK2 receptor. activin AB is approximatelyInterestingly, asthe potent activin as AC activin heterodimer B. Type activa I receptorstes both have SMAD been branches, shown to whereas bind at theactivin interface C is inactive.between Similarly, the two protomers activin A inis thea weak dimeric activator ligand of[ 43SMAD1/5,,44]. Thus, whereas our observations the heterodimer raisethe activin possibility AB is approximatelythat only one of as the potent ligand as protomers activin B. isType needed I receptors to activate have ALK2 been signaling. shown to bind at the interface betweenSequences the two bothprotomers in the in receptors the dimeric and ligand the ligand [43,44]. determine Thus, our bindingobservatio specificityns raise the and possibility strength. thatRecent only studies one of havethe ligand suggested protomers that the is needed finger2 to tip activate loops inALK2 activin signaling. A and GDF11 are important for theirSequences type I receptor both bindingin the receptors specificities and [ 44the,45 ligand]. Moreover, determine in some binding cell types, specificity only mutated and strength. ALK2, Recent studies have suggested that the finger 2 tip loops in activin A and GDF11 are important for their type I receptor binding specificities [44,45]. Moreover, in some cell types, only mutated ALK2, Biomolecules 2020, 10, 519 10 of 15 such as the variant encoded by ACVR1 (R206H), has been shown to relay a signal to SMAD1/5 after activin binding [11,12]. Thus, there has been a debate about whether activins can activate SMAD1/5 via the wildtype ALK2 type 1 receptor. For instance, overexpressed wildtype ALK2 could relay activin A induced SMAD1/5 activity in immortalized mouse embryonic fibroblasts [15], but this was not seen in HEK cells [11]. However, in systems with overexpression of ALK2, the balance between type I receptors, type II receptors and co-factors may be altered in a way that precludes proper signaling. It was shown that activin B activated SMAD1/5 and thereby increased the levels of the SMAD-regulated target gene HAMP (encoding hepcidin) in hepatocytes [13]. By using siRNA, induction of HAMP by activin B was found to be dependent on ALK2, ACVR2A, ACVR2B, and to a lesser extent ALK3 [14]. We showed that activin A and activin B likely signal through endogenous wildtype ALK2 to activate SMAD1/5 in myeloma and hepatocellular cell lines and the present study further supports this finding [16]. We speculate that the activin-mediated activation of SMAD1/5 via wildtype ALK2 that occurs in some cell types depends on a co-factor. In hepatocytes, hemojuvelin, encoded by HJV, may be such a co-factor [14]. In myeloma cells, on the other hand, HJV is not expressed. Thus, involvement of another, unidentified co-factor is more likely and may warrant further investigation. Interestingly, in cases where activin-induced activation of the SMAD1/5 pathway is not seen, and also in cases where the activation of SMADs via ALK2 is weak, the activins may act as BMP antagonists rather than agonists [8–11]. For example, activin A binds with high affinity to the type II receptors ACVR2A and ACVR2B, and since these receptors are shared with BMPs, activin A can act as an antagonist of BMP9 [9,10]. Ligand competition as a way of regulating SMAD activity is an interesting concept that may greatly impact signaling outcomes in cells. TGF-β is another ligand which can activate both SMAD branches; however, the mechanism is likely different. TGF-β induces SMAD2/3 signaling via ALK5 in complex with the type II TGF-β receptor [46]. However, TGF-β also induces SMAD1/5 signaling in endothelial cells, likely via lateral activation of ALK1, which was found in the same heteromeric signaling complexes as ALK5 [47,48]. In epithelial cells, similar lateral signaling was shown for TGF-β dependent activation of SMAD1/5 via ALK2 and ALK3 [49,50]. In these cases, activation of the SMAD1/5 pathway was dependent on the kinase activity of ALK5. This is not the case for the activins, as we have shown before and confirm here [16]. Therefore, the concept of lateral TGF-β signaling is likely largely dependent on context. There is currently no evidence of lateral TGF-β induced activation of SMAD1/5 via ALK2 in myeloma cells. We showed here that TGF-β activated SMAD1/5 in INA-6, but not IH-1 cells. In contrast, we found that activin-induced SMAD1/5 phosphorylation in both cell lines was independent of TGF-β and ALK5. There were no huge differences in SMAD1/5 and SMAD2/3 signaling dynamics in the IH-1 cells. However, for activin A and activin B there was a delay (peak at 2 h versus 1 h) in SMAD1/5 activation compared to SMAD2/3 activation, suggesting a more indirect effect of these ligands on SMAD1/5 activation. However, the different signaling dynamics between these pathways in myeloma cells could also suggest that there are different receptor complexes that relay the signal to each of the SMAD branches, i.e., activins do not activate ALK2 and ALK4/ALK7 via a common heteromeric receptor complex. It is not known if mixed SMAD complexes, which include SMADs from both branches, are formed by activin treatment in these cells. Another explanation for the delay in SMAD1/5 activation compared with SMAD2/3 could be that signaling dynamics are affected by variations in the localization and internalization of the two pathways’ respective receptors, as was suggested for BMP4 and activin A[51]. In that study, the authors suggested that BMP4 treatment caused transient turnover of receptors, which resulted in oscillatory signaling dynamics. By contrast, activin A induced a depletion of receptors matched with receptor renewal, which resulted in a more stable signal. Differences between BMP4 and activin A signaling dynamics also contrasted with TGF-β, which caused a refractory state in cells upon acute stimulation [52]. The refractory state was likely caused by a rapid depletion of TGF-β receptors from the cell surface. Finally, there is also a possibility that the activated SMAD molecules may be terminated by dephosphorylation at different rates for each SMAD branch [53]. Biomolecules 2020, 10, 519 11 of 15

In this study, we used six different small molecule inhibitors of varying specificities. Results obtained with the inhibitors should be interpreted with caution, as there is both a degree of uncertainty with regards to the actual specificities of each inhibitor, and the promiscuity in the ligand–receptor system is not fully known. For inhibition of activin A, the results were clear and supported the conclusion that activin A induces SMAD1/5 signaling via wildtype ALK2. For activin B, however, the results were less clear. Firstly, ML347, the most specific ALK2 inhibitor, less potently inhibited activin B-induced SMAD1/5 activation than other inhibitors for this pathway (K02288 and LDN-193189). Notably, both K02288 and LDN-193189 also inhibit ALK3 (Supplementary Table S2). This result could possibly be a matter of dose and a more careful titration of each inhibitor could potentially give clearer results. Secondly, the SMAD1/5 activation inhibitors K02288 and LDN-193189, also inhibited SMAD2 phosphorylation in IH-1 myeloma cells treated with activin B. It was puzzling that this effect was observed for activin B, but not activin A. The results could indicate that activin B-induced SMAD2/3 activation benefits from the kinase activity of a type I BMP receptor, which in IH-1 cells could be either ALK2 or ALK3, or that these inhibitors also block the activin B dependent kinase ALK7 [16,33]. However, the same trend was not replicated in HepG2 cells when these cells were treated with K02288 or siRNA targeting ALK2. Furthermore, INA-6 cells only express ALK2, and not ALK3 or ALK6, and in these cells, knocking down ALK2 with siRNA did not counteract activin B induced SMAD2 activation. Thus, when considering all results, we propose that activin B induced SMAD2 activation does not require ALK2 kinase activity. However, we cannot rule out the possibility that the kinase activity of another type I BMP receptor, such as ALK3, positively affects SMAD2 activation by activin B. Indeed, it was shown that ALK3 and ALK2 can form BMP-dependent heterodimers that induce hepcidin expression in hepatocytes in vitro [54]. Follistatin (FST) is a well-known antagonist of activins and it was therefore not surprising to see that FST inhibited the activity of all the tested activins, both via the SMAD2/3 and the SMAD1/5 pathway. Cerberus’ ability to bind and antagonize activin B, but not activin A, has also been clearly described before [23,38]. Notably, although cerberus antagonized activin B induced signaling via both the SMAD2/3 pathway and the SMAD1/5 pathway, it did not antagonize activin AB heterodimers. Notably, although both follistatin and cerberus completely inhibited activin B induced SMAD activation, only follistatin fully rescued the effects of activin B on cell viability. The most important difference between the SMAD activation assay (1 h treatment) and the viability assay (three days of treatment) is time. In the duration of the viability assay, it is possible that cerberus fails to have a long-lasting antagonistic activity towards activin B, or cerberus may have an effect on other non-SMAD signaling pathways. This is a matter for future studies.

5. Conclusions Activins act as dual-specificity TGF-β family molecules by engaging different type I receptors and activating both branches of the SMAD signaling pathway (Figure7). Activation likely occurs in different ligand–receptor complexes, as we show that the signaling dynamics are different between the two SMAD branches. The dual specificity is dependent on context and we speculate that co-factors that are so far not known may be key to this context dependence. Importantly, in cases where activins are not able to activate the SMAD1/5 pathway, they may still occupy receptors that are shared with BMPs, thereby acting as antagonists of the BMP pathway. Biomolecules 2020, 10, 519 12 of 15 Biomolecules 2020, 10, x 12 of 15

FigureFigure 7. Proposed7. Proposed dual dual specificity specificity of activins. of activins. Activins Activins belong belong to the activinto the/ TGF-activin/TGF-β branchβ of branch the TGF- ofβ the family,TGF- butβ family, share but the typeshare II the receptors type II receptors ACVR2A ACVR2A and ACVR2B, and ACVR2B, and in some and cases, in some BMPR2, cases, withBMPR2, BMPs. with TheBMPs. activins The can activins therefore can competetherefore with compete BMPs with for useBMPs of typefor use II receptors. of type II Usually,receptors. the Usually, type I receptor the type I inreceptor the complex in the is either complex ALK4 is oreither ALK7, ALK4 and activationor ALK7, of and these, activation lead to SMAD2of these,/3 phosphorylation.lead to SMAD2/3 Interestingly,phosphorylation. activins Interestingly, may also form activins ligand–receptor may also form complexes ligand–receptor where the typecomplexes I receptor where is the the BMP type I receptorreceptor ALK2. is the Activation BMP receptor of ALK2 ALK2. leads Activation to SMAD1 /of5 phosphorylation.ALK2 leads to SMAD1/5 The complex phosphorylation. of activins, type The II receptorscomplex andof activins, ALK2 can type either II bereceptors non-signaling and ALK2 (no SMAD1can either/5 phosphorylation), be non-signaling induce (no SMAD1/5 a weak activationphosphorylation), of SMAD1 /induce5, or potently a weak activateactivation SMAD1 of SM/5,AD1/5, depending or potently on which activate activin SMAD1/5, is in the complexdepending andon the which cellular activin context. is Thisin the image complex was created and the with ce Biorender.com.llular context. This image was created with Biorender.com. Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/4/519/s1, FigureSupplementary S1: Effects of Materials: knockdown The of following activin/TGF- areβ availabletype 1 receptors online at in www. INA-6mdpi.com/xxx/s1, myeloma cells, Figure Figure S2: S1: Eff Effectsects of of ALK2knockdown knockdown of activin/TGF- in HepG2 cells,β type Figure 1 receptors S3. Activin-induced in INA-6 myeloma SMAD cells, activity Figure was S2: not Effects caused of ALK2 by an autocrineknockdown TGF-in βHepG2loop, Tablecells, S1:Figure Approved S3. Activin-induced HUGO gene names SMAD for activity TGF-β wasfamily not receptors, caused by Table an autocrine S2. TGF- βTGF-/SMADβ loop, inhibitors; Table S1: reportedApproved inhibitory HUGO potential. gene names for TGF-β family receptors, Table S2. TGF-β/SMAD inhibitors; reported inhibitory Authorpotential. Contributions: Conceptualization, O.E.O. and T.H.; formal analysis, O.E.O.; investigation, O.E.O., H.H., S.E.; resources, C.J., and E.M.-H.; writing—original draft preparation, O.E.O and T.H.; writing—review and editing,Author O.E.O., Contributions: S.E., C.J., E.M.-H.,Conceptualization, and T.H.; supervision,O.E.O. and T.H.; project formal administration, analysis, O.E.O.; and fundinginvestigation, acquisition, O.E.O., T.H. H.H., AllS.E.; authors resources, have read C.J., and and agreed E.M.-H.; to the writing—original published version draft of thepreparation, manuscript. O.E.O and T.H.; writing—review and editing, O.E.O., S.E., C.J., E.M.-H., and T.H.; supervision, project administration, and funding acquisition, T.H. Funding: This research was funded by NIH R01 GM121499 (E.M.-H.), the Liaison Committee for education, researchAll authors and innovationhave read and in Central agreed Norwayto the published (O.E.O., version T.H.), the of the Joint manuscript. Research Committee between St. Olav’s HospitalFunding: and This Faculty research of Medicine was funded and Health by NIH Science, R01 GM121499 NTNU (T.H.). (E.M.H.), the Liaison Committee for education, Acknowledgments:research and innovationRecombinant in Central activin Norway A and activin(OEO,AB TH were), the kindly Joint providedResearch byCommittee Marko Hyvönen’s between groupSt. Olav’s at theHospital University and of Faculty Cambridge, of Medicine UK. and Health Science, NTNU (TH). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the Acknowledgments: Recombinant activin A and activin AB were kindly provided by Marko Hyvönen’s group study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publishat the theUniversity results. of Cambridge, UK. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References Biomolecules 2020, 10, 519 13 of 15

References

1. Morikawa, M.; Derynck, R.; Miyazono, K. TGF-beta and the TGF-beta Family: Context-Dependent Roles in Cell and Tissue Physiology. Cold Spring Harb. Perspect. Biol. 2016, 8, a021873. [CrossRef][PubMed] 2. Miyazono, K.; Kamiya, Y.; Morikawa, M. Bone morphogenetic protein receptors and signal transduction. J. Biochem. 2010, 147, 35–51. [CrossRef][PubMed] 3. Yadin, D.; Knaus, P.; Mueller, T.D. Structural insights into BMP receptors: Specificity, activation and inhibition. Cytokine Growth Factor Rev. 2016, 27, 13–34. [CrossRef][PubMed] 4. Ten Dijke, P.; Yamashita, H.; Ichijo, H.; Franzen, P.; Laiho, M.; Miyazono, K.; Heldin, C.H. Characterization of type I receptors for transforming growth factor-beta and activin. Science 1994, 264, 101–104. [CrossRef] [PubMed] 5. Tsuchida, K.; Nakatani, M.; Yamakawa, N.; Hashimoto, O.; Hasegawa, Y.; Sugino, H. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol. Cell. Endocrinol. 2004, 220, 59–65. [CrossRef] 6. Attisano, L.; Carcamo, J.; Ventura, F.; Weis, F.M.; Massague, J.; Wrana, J.L. Identification of human activin and TGF beta type I receptors that form heteromeric kinase complexes with type II receptors. Cell 1993, 75, 671–680. [CrossRef] 7. Ebner, R.; Chen, R.H.; Lawler, S.; Zioncheck, T.; Derynck, R. Determination of type I receptor specificity by the type II receptors for TGF-beta or activin. Science 1993, 262, 900–902. [CrossRef] 8. Piek, E.; Afrakhte, M.; Sampath, K.; van Zoelen, E.J.; Heldin, C.H.; ten Dijke, P. Functional antagonism between activin and osteogenic protein-1 in human embryonal carcinoma cells. J. Cell Physiol. 1999, 180, 141–149. [CrossRef] 9. Olsen, O.E.; Wader, K.F.; Hella, H.; Mylin, A.K.; Turesson, I.; Nesthus, I.; Waage, A.; Sundan, A.; Holien, T. Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B. Cell Commun. Signal. 2015, 13, 27. [CrossRef] 10. Aykul, S.; Martinez-Hackert, E. TransformingGrowth Factor-beta Family Ligands Can Function as Antagonists by Competing for Type II Receptor Binding. J. Biol. Chem. 2016, 291, 10792–10804. [CrossRef] 11. Hatsell, S.J.; Idone, V.; Wolken, D.M.; Huang, L.; Kim, H.J.; Wang, L.; Wen, X.; Nannuru, K.C.; Jimenez, J.; Xie, L.; et al. ACVR1R206H receptor mutation causes fibrodysplasia ossificans progressiva by imparting responsiveness to activin A. Sci. Transl. Med. 2015, 7, 303ra137. [CrossRef][PubMed] 12. Hino, K.; Ikeya, M.; Horigome, K.; Matsumoto, Y.; Ebise, H.; Nishio, M.; Sekiguchi, K.; Shibata, M.; Nagata, S.; Matsuda, S.; et al. Neofunction of ACVR1 in fibrodysplasia ossificans progressiva. Proc. Natl. Acad. Sci. USA 2015, 112, 15438–15443. [CrossRef][PubMed] 13. Besson-Fournier, C.; Latour, C.; Kautz, L.; Bertrand, J.; Ganz, T.; Roth, M.P.; Coppin, H. Induction of activin B by inflammatory stimuli up-regulates expression of the iron-regulatory peptide hepcidin through Smad1/5/8 signaling. Blood 2012, 120, 431–439. [CrossRef][PubMed] 14. Canali, S.; Core, A.B.; Zumbrennen-Bullough, K.B.; Merkulova, M.; Wang, C.Y.; Schneyer, A.L.; Pietrangelo, A.; Babitt, J.L. Activin B Induces Noncanonical SMAD1/5/8 Signaling via BMP Type I Receptors in Hepatocytes: Evidence for a Role in Hepcidin Induction by Inflammation in Male Mice. Endocrinology 2016, 157, 1146–1162. [CrossRef][PubMed] 15. Haupt, J.; Xu, M.; Shore, E.M. Variable signaling activity by FOP ACVR1 mutations. Bone 2017, 109, 232–240. [CrossRef][PubMed] 16. Olsen, O.E.; Sankar, M.; Elsaadi, S.; Hella, H.; Buene, G.; Darvekar, S.R.; Misund, K.; Katagiri, T.; Knaus, P.; Holien, T. BMPR2 inhibits activin- and BMP-signaling via wild type ALK2. J. Cell Sci. 2018, 131.[CrossRef] 17. Holien, T.; Vatsveen, T.K.; Hella, H.; Rampa, C.; Brede, G.; Groseth, L.A.; Rekvig, M.; Borset, M.; Standal, T.; Waage,A.; et al. Bone morphogenetic proteins induce apoptosis in multiple myeloma cells by Smad-dependent repression of MYC. Leuk. Off. J. Leuk. Soc. Am. Leuk. Res. Fund UK 2012, 26, 1073–1080. [CrossRef] 18. Holien, T.; Sundan, A. The role of bone morphogenetic proteins in myeloma cell survival. Cytokine Growth Factor Rev. 2014, 25, 343–350. [CrossRef] 19. Holien, T.; Vatsveen, T.K.; Hella, H.; Waage, A.; Sundan, A. Addiction to c-MYC in multiple myeloma. Blood 2012, 120, 2450–2453. [CrossRef] 20. Hjertner, O.; Hjorth-Hansen, H.; Borset, M.; Seidel, C.; Waage, A.; Sundan, A. Bone morphogenetic protein-4 inhibits proliferation and induces apoptosis of multiple myeloma cells. Blood 2001, 97, 516–522. [CrossRef] Biomolecules 2020, 10, 519 14 of 15

21. Burger, R.; Guenther, A.; Bakker, F.; Schmalzing, M.; Bernand, S.; Baum, W.; Duerr, B.; Hocke, G.M.; Steininger, H.; Gebhart, E.; et al. Gp130 and ras mediated signaling in human plasma cell line INA-6: A cytokine-regulated tumor model for plasmacytoma. Hematol. J. Off. J. Eur. Haematol. Assoc. 2001, 2, 42–53. [CrossRef][PubMed] 22. Harrington, A.E.; Morris-Triggs, S.A.; Ruotolo, B.T.; Robinson, C.V.; Ohnuma, S.; Hyvonen, M. Structural basis for the inhibition of activin signalling by follistatin. Embo J. 2006, 25, 1035–1045. [CrossRef][PubMed] 23. Aykul, S.; Ni, W.; Mutatu, W.; Martinez-Hackert, E. Human Cerberus prevents nodal-receptor binding, inhibits nodal signaling, and suppresses nodal-mediated phenotypes. PLoS ONE 2015, 10, e0114954. [CrossRef][PubMed] 24. Jin, C.H.; Sreenu, D.; Krishnaiah, M.; Subrahmanyam, V.B.; Rao, K.S.; Nagendra Mohan, A.V.; Park, C.Y.; Son, J.Y.; Son, D.H.; Park, H.J.; et al. Synthesis and biological evaluation of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles as transforming growth factor-beta type 1 receptor kinase inhibitors. Eur. J. Med. Chem. 2011, 46, 3917–3925. [CrossRef][PubMed] 25. Olsen, O.E.; Wader, K.F.; Misund, K.; Vatsveen, T.K.; Ro, T.B.; Mylin, A.K.; Turesson, I.; Stordal, B.F.; Moen, S.H.; Standal, T.; et al. Bone morphogenetic protein-9 suppresses growth of myeloma cells by signaling through ALK2 but is inhibited by endoglin. Blood Cancer J. 2014, 4, e196. [CrossRef][PubMed] 26. Fagerli, U.M.; Holt, R.U.; Holien, T.; Vaatsveen, T.K.; Zhan, F.; Egeberg, K.W.; Barlogie, B.; Waage, A.; Aarset, H.; Dai, H.Y.; et al. Overexpression and involvement in migration by the metastasis-associated phosphatase PRL-3 in human myeloma cells. Blood 2008, 111, 806–815. [CrossRef] 27. Baughn, L.B.; Di Liberto, M.; Niesvizky, R.; Cho, H.J.; Jayabalan, D.; Lane, J.; Liu, F.; Chen-Kiang, S. CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells. J. Immunol. 2009, 182, 1810–1817. [CrossRef] 28. Sanvitale, C.E.; Kerr, G.; Chaikuad, A.; Ramel, M.C.; Mohedas, A.H.; Reichert, S.; Wang, Y.; Triffitt, J.T.; Cuny, G.D.; Yu, P.B.; et al. A new class of small molecule inhibitor of BMP signaling. PLoS ONE 2013, 8, e62721. [CrossRef] 29. Engers, D.W.; Frist, A.Y.; Lindsley, C.W.; Hong, C.C.; Hopkins, C.R. Synthesis and structure-activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of dorsomorphin: The discovery of ML347 as an ALK2 versus ALK3 selective MLPCN probe. Bioorg. Med. Chem. Lett. 2013, 23, 3248–3252. [CrossRef] 30. Cuny, G.D.; Yu, P.B.; Laha, J.K.; Xing, X.; Liu, J.F.; Lai, C.S.; Deng, D.Y.; Sachidanandan, C.; Bloch, K.D.; Peterson, R.T. Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 4388–4392. [CrossRef] 31. Inman, G.J.; Nicolas, F.J.; Callahan, J.F.; Harling, J.D.; Gaster, L.M.; Reith, A.D.; Laping, N.J.; Hill, C.S. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 2002, 62, 65–74. [CrossRef] 32. Gellibert, F.; Woolven, J.; Fouchet, M.H.; Mathews, N.; Goodland, H.; Lovegrove, V.; Laroze, A.; Nguyen, V.L.; Sautet, S.; Wang, R.; et al. Identification of 1,5-naphthyridine derivatives as a novel series of potent and selective TGF-beta type I receptor inhibitors. J. Med. Chem. 2004, 47, 4494–4506. [CrossRef][PubMed] 33. Ro, T.B.; Holt, R.U.; Brenne, A.T.; Hjorth-Hansen, H.; Waage, A.; Hjertner, O.; Sundan, A.; Borset, M. Bone morphogenetic protein-5, -6 and -7 inhibit growth and induce apoptosis in human myeloma cells. Oncogene 2004, 23, 3024–3032. [CrossRef][PubMed] 34. Nakamura, T.; Takio, K.; Eto, Y.; Shibai, H.; Titani, K.; Sugino, H. Activin-binding protein from rat ovary is follistatin. Science 1990, 247, 836–838. [CrossRef][PubMed] 35. Schneyer, A.; Schoen, A.; Quigg, A.; Sidis, Y. Differential binding and neutralization of activins A and B by follistatin and follistatin like-3 (FSTL-3/FSRP/FLRG). Endocrinology 2003, 144, 1671–1674. [CrossRef] [PubMed] 36. Muenster, U.; Harrison, C.A.; Donaldson, C.; Vale, W.; Fischer, W.H. An activin-A/C chimera exhibits activin and myostatin antagonistic properties. J. Biol. Chem. 2005, 280, 36626–36632. [CrossRef][PubMed] 37. Piccolo, S.; Agius, E.; Leyns, L.; Bhattacharyya, S.; Grunz, H.; Bouwmeester, T.; De Robertis, E.M. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature 1999, 397, 707–710. [CrossRef] Biomolecules 2020, 10, 519 15 of 15

38. Aykul, S.; Martinez-Hackert, E. New Ligand Binding Function of Human Cerberus and Role of Proteolytic Processing in Regulating Ligand-Receptor Interactions and Antagonist Activity. J. Mol. Biol. 2016, 428, 590–602. [CrossRef] 39. Wakefield, L.M.; Hill, C.S. Beyond TGFbeta: Roles of other TGFbeta superfamily members in cancer. Nat. Rev. Cancer 2013, 13, 328–341. [CrossRef] 40. Attisano, L.; Wrana, J.L.; Montalvo, E.; Massague, J. Activation of signalling by the activin receptor complex. Mol. Cell. Biol. 1996, 16, 1066–1073. [CrossRef] 41. Mellor, S.L.; Cranfield, M.; Ries, R.; Pedersen, J.; Cancilla, B.; de Kretser, D.; Groome, N.P.; Mason, A.J.; Risbridger, G.P. Localization of activin beta(A)-, beta(B)-, and beta(C)-subunits in humanprostate and evidence for formation of new activin heterodimers of beta(C)-subunit. J. Clin. Endocrinol. Metab. 2000, 85, 4851–4858. [CrossRef] 42. Mellor, S.L.; Ball, E.M.; O’Connor, A.E.; Ethier, J.F.; Cranfield, M.; Schmitt, J.F.; Phillips, D.J.; Groome, N.P.; Risbridger, G.P. Activin betaC-subunit heterodimers provide a new mechanism of regulating activin levels in the prostate. Endocrinology 2003, 144, 4410–4419. [CrossRef][PubMed] 43. Kirsch, T.; Sebald, W.; Dreyer, M.K. Crystal structure of the BMP-2-BRIA ectodomain complex. Nat. Struct. Biol. 2000, 7, 492–496. [CrossRef][PubMed] 44. Goebel, E.J.; Corpina, R.A.; Hinck, C.S.; Czepnik, M.; Castonguay, R.; Grenha, R.; Boisvert, A.; Miklossy, G.; Fullerton, P.T.; Matzuk, M.M.; et al. Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity. Proc. Natl. Acad. Sci. USA 2019, 116, 15505–15513. [CrossRef][PubMed] 45. Idone, V.; Corpina, R.; Goebel, E.; Cunanan, C.; Dimitriou, A.; Kim, H.; Zhang, Q.; Rafique, A.; Leidich, R.; Wang, X.; et al. The finger 2 tip loop of Activin A is required for the formation of its non-signaling complex with ACVR1 and type II Bone Morphogenetic Protein receptors. bioRxiv 2019.[CrossRef] 46. Heldin, C.H.; Moustakas, A. Signaling Receptors for TGF-beta Family Members. Cold Spring Harb. Perspect. Biol. 2016, 8.[CrossRef] 47. Goumans, M.J.; Valdimarsdottir, G.; Itoh, S.; Rosendahl, A.; Sideras, P.; ten Dijke, P. Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors. Embo J. 2002, 21, 1743–1753. [CrossRef] [PubMed] 48. Goumans, M.J.; Valdimarsdottir, G.; Itoh, S.; Lebrin, F.; Larsson, J.; Mummery, C.; Karlsson, S.; ten Dijke, P. Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling. Mol. Cell 2003, 12, 817–828. [CrossRef] 49. Daly, A.C.; Randall, R.A.; Hill, C.S. Transforming growth factor beta-induced Smad1/5 phosphorylation in epithelial cells is mediated by novel receptor complexes and is essential for anchorage-independent growth. Mol. Cell. Biol. 2008, 28, 6889–6902. [CrossRef][PubMed] 50. Ramachandran, A.; Vizan, P.; Das, D.; Chakravarty, P.; Vogt, J.; Rogers, K.W.; Muller, P.; Hinck, A.P.; Sapkota, G.P.; Hill, C.S. TGF-beta uses a novel mode of receptor activation to phosphorylate SMAD1/5 and induce epithelial-to-mesenchymal transition. eLife 2018, 7, e31756. [CrossRef] 51. Miller, D.S.J.; Schmierer, B.; Hill, C.S. TGF-beta family ligands exhibit distinct signalling dynamics that are driven by receptor localisation. J. Cell Sci. 2019, 132.[CrossRef] 52. Vizan, P.; Miller, D.S.; Gori, I.; Das, D.; Schmierer, B.; Hill, C.S. Controlling long-term signaling: Receptor dynamics determine attenuation and refractory behavior of the TGF-beta pathway. Sci. Signal. 2013, 6, ra106. [CrossRef][PubMed] 53. Bruce, D.L.; Sapkota, G.P. Phosphatases in SMAD regulation. Febs Lett. 2012, 586, 1897–1905. [CrossRef] [PubMed] 54. Traeger, L.; Gallitz, I.; Sekhri, R.; Baumer, N.; Kuhlmann, T.; Kemming, C.; Holtkamp, M.; Muller, J.C.; Karst, U.; Canonne-Hergaux, F.; et al. ALK3 undergoes ligand-independent homodimerization and BMP-induced heterodimerization with ALK2. Free Radic. Biol. Med. 2018, 129, 127–137. [CrossRef][PubMed]

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