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Vincent Center for Reproductive Biology, Massachusetts General Hospital and Harvard Medical School, Thier 913, 55 Fruit Street, Boston, Massachusetts 02114, USA
1 Pediatric Surgical Research Laboratories and
2 Reproductive Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA
(Correspondence should be addressed to J Teixeira; Email: teixeira{at}helix.mgh.harvard.edu)
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Abstract |
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 Top Abstract IntroductionExperimental proceduresResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionExperimental proceduresResultsDiscussionReferences |
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How cells respond specifically to a family of greater than 30 secreted members with only five type II receptors and seven type I receptors is still not well understood. Some of the type I receptors have shown a remarkable degree of promiscuity for both ligand and type II receptor, which suggests a mechanism whereby their combination provides an additional level of specificity (Piek et al. 1999). For example, activin receptor-like kinase-2 (Alk2) was originally thought to be an activin type I receptor because it could bind activin in the presence of the activin type II receptors (Attisano et al. 1993, Tsuchida et al. 1993). Subsequent studies showed that Alk2 could not transduce activin-mediated signaling but could transduce both BMP and MIS signaling (ten Dijke et al. 1994, Clarke et al. 2001, Visser et al. 2001). However, the functional significance of Alk2-binding activin type II receptors was not well resolved.
Both MIS and activin can inhibit steroidogenesis in testicular Leydig cells, which are the predominant source of testosterone in males (Lin et al. 1989, Mauduit et al. 1991, Laurich et al. 2002). Using the MA-10 mouse Leydig tumor cell line, we investigated the functional significance of Alk2 in activin-mediated inhibition of testosterone synthesis. Recent studies by others have led to speculation that the differential expression of Alk1 and Alk2 may represent a mechanism for specifying cell-specific responses to TGFß (Oh et al. 2000, Desgrosellier et al. 2005). Here, we show evidence that Alk2 inhibits activin signaling by preventing activin binding to its type II receptor.
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Experimental procedures |
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TopAbstractIntroductionExperimental proceduresResultsDiscussionReferences |
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Waymouth’s MB 752/1, horse serum, and gentamicin were obtained from Invitrogen Life Technologies Inc. Protein G Sepharose 4 Fast Flow was purchased from Amersham Biosciences. Monoclonal myc-tagged and HA-tagged antibodies were purchased from Covance Inc. (Berkeley, CA, USA). His-tagged polyclonal antibody, phospho-Smad2, and Smad2 antibody were obtained from Cell Signaling (Danvers, MA, USA). Radionuclides were purchased from Perkin–Elmer (Boston, MA, USA). Recombinant human MIS was prepared from Chinese hamster ovary (CHO) cells stably transfected with a construct of the human MIS gene and purified from serum-free media by serial carbohydrate affinity and ion exchange chromatography (Lorenzo et al. 2002). Activin A and the BMPs were purchased from R&D Systems (Minneapolis, MN, USA). The CAGA-Luc and BMP-responsive element (BR)-Luc reporter plasmids were provided by Dr ten Dijke (The Netherlands Cancer Institute, The Netherlands).
Cell culture
MA-10 mouse Leydig tumor cell line (Ascoli 1981), originally a gift from Dr Mario Ascoli (University of Iowa, Iowa City, IA, USA), was cloned and cultured in Waymouth’s MB 752/1 supplemented with 15% horse serum, 20 mM HEPES (pH 7.4), penicillin/streptomycin, and 50 µg/ml gentamicin (Laurich et al. 2002). Cells were maintained at 37 °C with 5% CO2 in a humidified incubator. Transfections were performed using either Fugene6 (Roche Molecular Biochemicals) or TransIT-LT1 (Mirus Bio Corporation, Madison, WI, USA) lipid reagents the day after plating, and the experiments were performed 2 days post-transfection. MA-10 cells were normally transfected with 30–50% efficiency.
Western blotting
Eluates were run on precast 4–12% NuPAGE Bis–Tris gels with 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (Invitrogen) followed by transfer to nitrocellulose membrane. Membranes were blocked for 1 h in 5% non-fat dry milk in tris-buffered saline (TBS)–0.1% Tween 20, incubated with a phospho-Smad2-specific antibody or total Smad2 overnight at 4 °C according to the manufacturer’s instructions and detected with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG followed by enhanced chemiluminescence (Pierce Biotechnology Inc., Rockford, IL, USA).
Northern blotting
Total RNA was collected from treated cells using TRIZol (Invitrogen) reagent according to the manufacturer’s instructions and quantitated by absorbance at 260 nm. RNA was separated by electrophoresis, blotted onto nylon membranes, and probed with a cytochrome P450 (Cyp17) antisense riboprobe as described (Laurich et al. 2002). Blots were subsequently reprobed with a human actin cDNA probe. Quantitation of the signal intensity was performed with a Fuji BAS1800 Phosphorimager. Northern results were analyzed by ANOVA on repeated measurements followed by the Bonferroni multiple comparison test with GraphPad Software, San Diego, CA, USA.
Ligand binding and cross-linking
MA-10 cells cultured in 100 mm dishes and transfected with HA-tagged type I receptors and myc-tagged activin type II receptor were washed before addition of Krebs–Ringer buffer (20 mM HEPES (pH 7.5) and 5 mM MgSO4) with 0.5% BSA and treated with activin labeled with 125I (kindly provided by Dr Alan Schneyer, Masachusetts General Hospital) for 1 h followed by cross-linking using 1 mM disuccinimidyl suberate (Pierce Biotechnology Inc.) for an additional 2 h at 4 °C. The reaction was stopped by adding 1 M Tris (pH 7.5) to a final concentration of 20 mM. Cells were washed in cold PBS and protein extracts were collected in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 1 mM ß-glycerol phosphate) containing protease inhibitor cocktail (Roche). Protein was quantified using the Bradford method according to the manufacturer’s instructions (Bio-Rad Laboratories). One milligram of total protein was incubated with a mouse monoclonal myc antibody overnight at 4 °C. The following day, immunoprecipitates were incubated with protein G-sepharose for 2 h at 4 °C, washed three times, and eluted in loading buffer containing dithiothreitol by heating at 100 °C for 5 min. Eluates were run on precast 4–12% NuPAGE Bis–Tris gels with MOPS buffer (Invitrogen) and dried. Results were detected by autoradiography using intensifying screens.
Co-immunoprecipitations
COS-7 cells cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin were transiently transfected with tagged receptor constructs and enhanced green fluorescent protein (EGFP)-N1 using TransIT-LT1 transfection reagent (Mirus). After 24-h incubation, the cells were washed with PBS and proteins were isolated in 400 µl mammalian protein extraction reagent (Pierce). For immunoprecipitation, 200 µl protein lysate was incubated with a myc-tagged polyclonal (Abcam, Cambridge, MA, USA) or His-tagged polyclonal antibody (Cell Signaling) at 4 °C overnight with mixing. Immune complexes were collected by addition of 20 µl Protein G Sepharose 4 Fast Flow (Amersham Biosciences) and mixed for 1 h at 4 °C. The pellet was washed thrice in PBS and the complex eluted by boiling in 20 µl NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) and separated on a 4–12% NuPAGE Bis–Tris gel in MOPS buffer (Invitrogen) under non-reducing conditions. Proteins were transferred to a nitrocellulose membrane and blocked in 5% milk in TBS–T before incubation with mouse monoclonal HA-antibody 1:1000 (Covance). The membranes were washed before incubation with HRP-conjugated secondary antibody and the proteins were visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce).
Luciferase experiments
Cells were plated on day 0 in 24-well plates and transfected with constant total concentration of DNA in a given experiment on day 1. Cells were transfected with an activin/TGFß-responsive promoter, firefly luciferase reporter construct, CAGA-luc (Dennler et al. 1998), or the BMP-responsive promoter/ reporter, BRE-luc (Korchynskyi & ten Dijke 2002), in conjunction with a thymidine kinase (TK) promoter–Renilla luciferase reporter to control for transfection efficiency. The following day, cells were either untreated or treated overnight as indicated. Cell lysates were harvested on day 3 and luciferase activity was measured using the Dual Luciferase system from Promega. Experiments were performed in triplicate and repeated at least twice more. In the type II receptor-transfection experiments, CAGA and BRE promoter-driven luciferase activity was normalized to the Renilla luciferase activity, averaged, and plotted. Luciferase results were analyzed by one-way or two-way ANOVA, as appropriate, on the repeated measurements followed by the Bonferroni post hoc test with GraphPad Software. In the Alk-transfected experiments where Renilla luciferase was also regulated, a representative of at least three experiments is shown.
siRNA experiments
MA-10 cells were plated in 96-well plates at a density of 10 000 cells/well. The following day, cells were transfected with constructs for the activin type II receptor A (ActRIIA), CAGA-luc, and phRL-cytomegalovirus (CMV) Renilla luciferase reporter using TransIT-LT1 and SiControl Non-targeting siRNA #3 D-001210-03-05 (Dharmacon Inc., Lafayette, CO, USA), or siGENOME SMARTpool M-042047-00-0005, mACVR1, NM_007394 (Dharmacon) using TransIT-TKO. On the day after transfection, cells were treated with activin for 24 h and the luciferase activity was recorded using the Dual Luciferase system (Promega). Significance was assigned at P < 0.05 using the Student t-test.
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Results |
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TopAbstractIntroductionExperimental proceduresResultsDiscussionReferences |
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Both activin and MIS have been shown to inhibit testosterone synthesis in primary cell cultures and in vivo respectively (Lin et al. 1989, Trbovich et al. 2001). In MA-10 Leydig cells, we have previously shown that MIS inhibits testosterone synthesis, at least in part, by inhibiting the expression of cytochrome P450 C17 lyase/17
-hydroxylase (Cyp17; Laurich et al. 2002). In order to determine whether these cells could also be used to study endogenous activin signaling, MA-10 cells were treated with activin both in the absence and in the presence of 8Br-cAMP, an inducer of Cyp17 expression (Fig. 1
).We show in this representative northern blot experiment that 5 ng/ml activin A inhibits Cyp17 mRNA expression in resting MA-10 cells and significantly reduces Cyp17 expression in cAMP-stimulated cells. The degree of inhibition was consistently less than that observed with MIS. However, MIS was added at a concentration of 5 µg/ml, the concentration that we normally need to use to observe complete regression of the Müllerian duct in organ culture (Donahoe et al. 1977), which is 1000-fold higher than the concentration of activin used. Quantitative analyses of several northern blots with a phosphorimager show that the inhibition of Cyp17 mRNA expression by activin is best observed when the cells have been stimulated with cAMP. These results indicate that MA-10 Leydig cells are responsive to activin signaling.
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Activin and MIS are thought to activate the Smad2/3 and Smad1/5/8 pathways respectively. However, in light of the observation that both activin and MIS inhibit the expression of Cyp17 in MA-10 cells, we speculated whether activin and MIS might share a common downstream signaling component. Both activin type II receptors and MISRII bind Alk2, which was once thought to be a functional type I receptor for activin and is considered one of the putative MIS type I receptors (Clarke et al. 2001, Visser et al. 2001). Therefore, we investigated whether Alk2 might be playing a role in activin signaling in MA-10 cells using the activin-responsive CAGA-luc reporter (Fig. 2A). Transfected Alk2 inhibited CAGA-luc activity to a level that was approximately half that of control-transfected cells. In contrast, transfection of Alk4, the activin type I receptor, resulted in a 15-fold increase in CAGA-Luc activity. Similar results were observed with cells stimulated with activin A (Fig. 2B). Addition of 5 ng/ml activin A to control-transfected cells caused a nearly 14-fold increase in CAGA-Luc activity over untreated cells. CAGA-Luc reporter activity after activin treatment of Alk2- and Alk4-transfected cells was reduced to one-third and induced by nearly threefold respectively, when compared with that observed in activin-treated, control-transfected cells. In contrast, cotransfection of Alk2 with the MIS/BMP-responsive BRE-Luc reporter induced luciferase activity approximately threefold (Fig. 2C and D). Overexpression of Alk2 enhanced BRE-Luc expression (Fig. 2D), as well as that of control TK and CMV Renilla luciferase reporters (data not shown), in both untreated and BMP2-treated cells, ensuring that the Alk2-mediated transcriptional inhibition was not general but likely particular to CAGA-Luc. These results indicate that in MA-10 cells, Alk2 acts as a BMP type I receptor and can inhibit downstream signaling by activin, while enhancing downstream signaling by MIS. Therefore, activin and MIS signaling through Alk2 could not be a common mechanism for inhibition of Cyp17 expression. This left the possibility that, while Alk2 might be involved in MIS signaling, with activin, Alk2 plays a non-signaling role.
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To better examine the role of Alk2 in activin signaling, we used an siRNA for Alk2 to inhibit expression of endogenous Alk2 expression. Transfection of siAlk2 into MA-10 cells resulted in almost a doubling of activin-induced luciferase activity compared with when a control siRNA was used (Fig. 3). These results indicate that there might be a constant inhibitory effect of endogenous Alk2 on activin signaling.
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Activin binding to its heteromeric receptor complex induces a conformational change that triggers the activation of the latent kinase domain of the type I receptor for subsequent downstream signaling via the Smad2/3. Therefore, we investigated whether overexpression of Alk2 affected the phosphorylation of Smad2. Because we expected that high levels of overexpression would be needed to observe an effect on endogenous Smad2 phosphorylation, we used COS cells transfected with either control plasmid or an Alk2 expression construct. The amount of phosphorylated Smad2 was determined by western blot analysis with an antibody specific to phosphorylated Smad2 (pSmad2; Fig. 4A). Overexpression of Alk2 inhibited the basal phosphorylation in these cells, suggesting that Alk2 inhibited activin signaling upstream of Smad2 phosphorylation. Addition of activin did not affect the level of pSmad2 phosphorylation above basal levels in COS cells, precluding our ability to assay the effect of Alk2 overexpression on pSmad2 in the presence of added activin. These basal levels of pSmad2 are presumably due to endogenous expression of either activin or TGFß. Western blot analyses were also preformed to ensure that overexpression of Alk2 was not having an indirect effect on activin signaling by inducing expression of Smad7, the inhibitory Smad (data not shown). We have also performed these experiments in MA-10 cells (Fig. 4B). Although expression of the transgene is not nearly as high as it is in COS cells, we were still able to observe a noticeable decrease in pSmad2 phosphorylation.
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MISRII has previously been shown to be specific for the MIS ligand (Mishina et al. 1996) and that downstream signaling by the MISRII occurs through the BMP group of type I receptors and Smads, including Alk2 (Gouedard et al. 2000, Clarke et al. 2001, Visser et al. 2001, Jamin et al. 2002). Therefore, we attempted to exploit the ability of MISRII to interact and sequester Alk2 as a mechanism to induce activin signaling. Although overexpression of the MISRII in MA-10 cells raised the basal level of BRE reporter activity by approximately twofold (data not shown), we show that it had no significant effect on the activin-, MIS-, or BMP-induced activation of the BRE-Luc over the control plasmid (Fig. 5A). With either the control plasmid or the MISRII expression construct, activin inhibited BRE reporter activity by at least 50%, while addition of MIS or BMP2 increased reporter activity two- to threefold respectively. In contrast, MISRII overexpression enhanced the activin-induced CAGA-Luc activation by approximately fivefold enhancement of luciferase activity (Fig. 5B). Neither MIS nor BMP had any effect on this reporter.
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). Cotransfection of MA-10 cells with the MISRII increased CAGA reporter activity approximately twofold at 2 ng/ml activin A over cotransfection with a control plasmid; 25 ng/ml caused more variable increases that averaged approximately threefold higher than with the control. MISRII-induced activin signaling was also observed in HepG2 cells, which were tested to assess whether the phenomenon was specific to MA-10 cells (data not shown). To determine whether MISRII receptor kinase activity was required for the induced CAGA reporter activity, MA-10 cells were also transfected with a mutated form of the receptor with an inactive kinase domain (K228R; Clarke et al. 2001). Similar experiments with BMPRIIA-transfected cells did not significantly induce CAGA-luciferase activity over that of control-transfected cell, suggesting that the phenomenon was specific to MISRII (data not shown). Although the mean induction was lower in cells treated with 25 ng/ml activin than in cells transfected with the plasmid expressing the native MISRII, the induction of CAGA reporter activity was still significantly higher than that of control-transfected cells (Fig. 5C). These results suggested that MISRII expression in MA-10 Leydig cells enhances activin signaling in the absence of MISRII signaling. In contrast, transfection of MA-10 cells with the activin type II receptor expression construct (ActRIIA) showed that, although the basal level of CAGA reporter activity was sixfold higher than that of control-transfected cells and 1 ng/ml activin A did increase the response by twofold, further increasing concentrations of activin were not able to induce the relative luciferase activity significantly (Fig. 5C). Similar results were observed with ActRIIB (data not shown). Alk2 blocks the binding of activin to its type II receptor
One of the possible mechanisms that may be used by Alk2 to downregulate activin signaling prior to Smad2 phosphorylation would be by interfering with the productive binding of activin to the activin type II receptor. We investigated this possibility using binding assays with radiolabeled activin A (Fig. 6). MA-10 cells were transfected with ActRIIA and either with a control plasmid or with expression constructs for the TGFß type I receptor, Alk5; the activin type I receptor, Alk4; and Alk2. Cells that were transfected with ActRIIA alone or together with Alk5 showed two cross-linked bands with the radiolabeled activin representing what is believed to be two different glycosylated forms of the activin type II receptor (Harrison et al. 2003). Cotransfection with Alk4 showed an additional band that represents activin cross-linked to the shorter type I receptor. Faint cross-linked activin bands were observed in cells that were cotransfected with Alk2, suggesting that Alk2 inhibited activin signaling by blocking activin binding to its receptor. The highest molecular weight band likely represents activin cross-linked to a complex of ActRII and either endogenous or transfected Alk4.
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We have shown above that Alk2 inhibits activin signaling and transfection of the MISRII cDNA enhanced activin signaling by a mechanism that we speculate involves the sequestering of Alk2 away from activin. Furthermore, in Fig. 6, we showed that Alk2 cotransfection inhibited binding of iodinated activin to the ActRIIA receptor. These results have led us to hypothesize that Alk2 inhibits activin signaling by binding the activin type II receptor and blocking access of the ligand to its binding site. Therefore, Alk2 would have to bind ActRIIA in the absence of ligand. In order to test this possibility, we cotransfected COS cells with ActRIIA or MISRII and either Alk2 or green fluorescent protein (GFP) as a negative control and co-immunoprecipitated the complexes (Fig. 7). Western blot analysis of the immunoprecipitated complexes shows that Alk2 is co-immunoprecipitated with both ActRIIA and MISRII but not GFP, and that the immunoprecipitated complexes occur in the absence of ligand. Additional experiments were performed with added MIS and BMP, but these ligands did not interfere with the ActRIIA/Alk2 co-immunoprecipitations (data not shown).
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Discussion |
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TopAbstractIntroductionExperimental proceduresResultsDiscussionReferences |
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In the presence of MIS ligand, Alk2 and MISRII initiate BMP pathway-specific signaling (Clarke et al. 2001, Visser et al. 2001), but the interaction of Alk2 with MISRII and its subsequent sequestration in the absence of ligand might also prove to be a physiological mechanism to induce activin signaling. The molecular details of such a mechanism remain to be elucidated. MISRII can also signal through Alk3 (Jamin et al. 2002) and Alk6 (Gouedard et al. 2000); our studies with Alk3, however, show that overexpression of Alk3 does not appear to affect activin signaling. Additionally, increasing the amount of activin added to cells transfected with the MISRII expression construct but not the ActRIIA expression construct led to increasing CAGA-luc expression, suggesting that overexpression of ActRIIA is not sufficient to overcome the inhibitory effect of Alk2 and/or other type I receptors.
MISRII has previously been shown to interact in a ligand-independent manner with Alk5 in transfected CHO cells (Gouedard et al. 2000). In that report, MISRII interaction with Alk6 was enhanced with MIS ligand and neither Alk2 nor 3 could be immunoprecipitated with MISRII in the presence or absence of MIS ligand. This raises the possibility that another factor that might be absent in CHO cells but present in MA-10 may be facilitating MISRII interaction with Alk2 or conversely, that CHO cells express an inhibitor of MISRII and type I heterodimerization. The latter situation would be similar to that shown with the pseudoreceptor, BAMBI (BMP and activin membrane-bound inhibitor), which binds to type I receptors and inhibits downstream signaling by preventing formation of active receptor complexes (Onichtchouk et al. 1999). Early studies to identify the type I receptor partner for MISRII using yeast two-hybrid assays (Wang et al. 1996) showed that the cytoplasmic domain of the rat MISRII interacted with those of all the type I receptors tested (data not shown). In support of these studies, we have recently found that transfected MISRII co-immunoprecipitates with all the type I receptors as well (data not shown). Together, these results indicate that MISRII can interact with multiple type I receptors, and that in cells that express endogenous MISRII, these interactions may be of physiological significance. The finding that inhibition of endogenous Alk2 or its sequestration by another type II receptor induces activin signaling might indicate that differential expression of Alk2 during different times of development could be an important mechanism of regulating activin signaling in steroidogenic and other cells expressing these receptors. Another possibility is that MISRII expression alone may be a mechanism to regulate activin signaling by sequestering Alk2. Additionally, while it would be difficult to assess quantitatively, in the co-immunoprecipitation experiments in Fig. 7, it does appear that MISRII has a greater affinity for Alk2 than does ActRIIA, suggesting that the type I and type II receptors have differing affinities for each other, which would give the cell another mechanism for modulating TGFß family signal transduction. We could also speculate that the relative expression levels and affinities of ActRIIA for Alk2 and Alk4, in either the absence or presence of ligand, may affect the degree of inhibition of activin signaling in a given context.
In summary, we have shown that Alk2 is an inhibitor of activin signaling. A similar role for other TGFß receptor family members may provide yet another mechanism for determining cell-specific responses to individual ligands with a limited repertoire of receptors.
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Acknowledgements |
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References |
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TopAbstractIntroductionExperimental proceduresResultsDiscussionReferences |
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Received in final form 20 July 2007
Accepted 30 July 2007
Made available online as an Accepted Preprint 7 August 2007
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