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J. Biol. Chem., Vol. 280, Issue 44, 36626-36632, November 4, 2005

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An Activin-A/C Chimera Exhibits Activin and Myostatin Antagonistic Properties^*

Uwe Muenster, Craig A. Harrison, Cynthia Donaldson, Wylie Vale, and Wolfgang H. Fischer1

From the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037 and Prince Henry's Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia

Received for publication, July 5, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Activins are involved in many physiological and pathological processes and, like other members of the transforming growth factor- superfamily, signal via type II and I receptor serine kinases. Ligand residues involved in type II receptor binding are located in the two anti-parallel strands of the TGF- proteins, also known as the fingers. Activin-A mutants able to bind ActRII but unable to bind the activin type I receptor ALK4 define ligand residues involved in ALK4 binding and could potentially act as antagonists. Therefore, a series of FLAG-tagged activin-A/C chimeras were constructed, in each of which eight residues in the wrist loop and helix region (A/C 46–53, 54–61, 62–69, and 70–78) were replaced. Additionally, a chimera was generated in which the entire wrist region (A/C 46–78) was changed from activin-A to activin-C. The chimeras were assessed for ActRII binding, activin bioactivity, as well as antagonistic properties. All five chimeras retained high affinity for mouse ActRII. Of these, only A/C 46–78 was devoid of significant activin bioactivity in an A3 Lux reporter assay in 293T cells at concentrations up to 40 nM. A/C 46–53, 54–61, 62–69, and 70–78 showed activity comparable with wild type activin-A. When tested for the ability to antagonize ligands that signal via activin type II receptors, such as activin-A and myostatin, only the A/C 46–78 chimera showed antagonism (IC₅₀, 1–10 nM). Additionally, A/C 46–78 decreased follicle-stimulating hormone release from the LT2 cell line and rat anterior pituitary cells in primary culture in a concentration-dependent manner. These data indicate that activin residues in the wrist are involved in ALK4-mediated signaling. The activin antagonist A/C 46–78 may be useful for the study and modulation of activin-dependent processes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Activins belong to the TGF-² superfamily of growth factors, which control a variety of physiological functions such as cell growth, differentiation and apoptosis, endocrine function, metabolism, wound repair, immune responses, homeostasis, mesoderm induction, bone growth, and many others (1–5). The TGF- family comprises at least 42 members in human (6) including activin, TGF-, bone morphogenetic protein, growth and differentiation factor, and nodal proteins, which are all characterized by a distinct structural feature, namely a cysteine knot scaffold (7).

Activins are homo- or heterodimers consisting of two subunits, which are linked by a single covalent disulfide bond. In each monomer, two pairs of antiparallel strands form short and long "fingers" that stretch out from the cysteine core of the dimer. The base of the fingers is formed by an -helix together with a preceding loop and is also known as the "wrist" epitope (8, 9).

In human, genes encoding for four different subunits have been identified (A, B, C, and E), which, in theory, offers a variety of different activin dimers. However, only activin-A (AA), activin-B (BB), and activin-AB (AB) have been proven to be biologically active (10). The expression of activins C (CC), E (EE), AC (AC), AE (AE), CE (CE) (11), as well as BC (BC) (12) has been demonstrated, but little is known on their function and stability. The C subunit appears to be able to attenuate activin activity by forming a heterodimer with a A subunit and thereby decreasing the source for the formation of biologically active activins (13). Results on the functional activity of activin-C were inconsistent. Lau et al. (14) found that genes encoding for activin C and E were not essential for mouse liver growth, differentiation, and regeneration. Chabicovsky et al. (15) observed that the overexpression of activin C or activin E in the mouse liver inhibited regenerative DNA synthesis of hepatic cells, and Vejda et al. (16) demonstrated that the expression of activins C and E induces apoptosis in human and rat hepatoma cells. Wada et al. (17), however, showed that activin-C is unable to bind to ActRII, although they observe an increase in [³H]thymidine incorporation in AML12 hepatocytes after activin-C treatment, suggesting a pathway different from that known for biologically active activins.

Activin signaling involves binding of the activin dimer to two types of cell surface transmembrane serine threonine kinase receptors (18). First, activin binds to its type II receptor (ActRII/ActRIIB), allowing the recruitment, phosphorylation, and activation of the type 1 receptor, ALK4, which then leads to intracellular phosphorylation of Smad2 and Smad3 (19, 20).

Crystal structures of activin bound to ActRIIB (8, 9) as well as the related BMP7 bound to ActRII (21) revealed that ligand residues in the fingers' "knuckles" are involved in type II receptor binding. Especially Lys¹⁰² in activin appears to be highly critical, because when changed to Glu, the mutant completely loses its ability to bind ActRII (22). Less is known of activin residues participating in ALK4 binding. So far, knowledge can be deduced from the crystal structure of BMP2 bound to its type I receptor ALK3, which suggests that BMP2 wrist and finger residues are involved (23). Mutagenesis studies showed that mutation of several of these residues induces a decrease in BMP2 activity. Of these, the BMP2 variants F49A, P50A (24), and L51P (25) almost completely lost the ability to bind ALK3 as well as to induce alkaline phosphatase secretion from C₂C1₂ cells, indicating that these residues are of great importance for ALK3 binding.

Interruption of type I receptor binding in activin has the potential of causing antagonistic effects. Despite the fact that many residues in the BMP2 wrist are involved in ALK3 binding, mutation of corresponding activin residues has not yielded an antagonist so far. However, a point mutation in the finger region (M108A) of activin yields a ligand that binds the type II receptor but exhibits a biological activity that is 3 orders of magnitude lower than wild type. This point mutant acted as an antagonist in activin-responsive cell culture systems (26). Here we focus on investigating mutations in the wrist region by introducing multiple residues of the biologically less active activin-C into the activin-A structure. The activin-A/C chimeras presented in this study were characterized with respect to their binding affinities for ActRII and follistatin, their ability to disrupt activin signaling, as well as their antagonistic properties. Antagonists of the TGF- family members offer a potential treatment of diseases such as muscular dystrophy, liver cirrhosis, and fibrosis or may be used to improve wound healing.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PCR Mutagenesis—A/C 46–53, 54–61, 62–69, and 70–78 chimeras were generated by "long PCR" (initial denaturation, 5 min at 94 °C; 12 cycles of 1 min at 94 °C, 2 min at 58 °C, and 3 min at 72 °C; and final extension, 10 min at 72 °C) using a pGem vector containing the wt activin-A sequence with a FLAG tag inserted at the N terminus of the mature activin region as template and primers introducing base pairs encoding for the respective homologous activin-C residues. Blunt linear products were ligated overnight at 16 °C using T4 ligase (Invitrogen). To subclone chimeras from pGem into pCDNA, another PCR was performed (initial denaturation, 5 min at 94 °C; 25 cycles of 1 min at 94 °C, 1 min at 58 °C, and 2 min at 72 °C; and final extension, 10 min at 72 °C) using the pGem plasmids containing the DNA of the chimeras as a template, and a forward primer containing an NheI site 169 bp upstream of the N-terminal FLAG sequence (NheI pr), together with a reverse primer containing an XhoI site annealing to the C terminus of the chimeras (XhoI pr). The resulting products were cut with NheI and XhoI and then ligated overnight at 16 °C into an NheI-XhoI cut pCDNA cassette containing the remaining wt activin-A sequence. To obtain the cassette, an NheI site was introduced by silent mutation 169 bp upstream of the mature activin-A in pCDNA. The A/C 46–78 chimera was constructed by "overlapping PCR." First, in two separate PCRs (initial denaturation, 5 min at 94 °C; 25 cycles of 1 min at 94 °C, 1 min at 58 °C, and 2 min at 72 °C; and final extension, 10 min at 72 °C) using pCDNA containing the sequence for either A/C 46–53 or A/C 70–78 as template together with NheI pr or XhoI pr, respectively, as well as primers introducing base pairs encoding for activin-C residues 54–69, two pieces of overlapping DNA were generated. In a second PCR (initial denaturation, 5 min at 94 °C; 25 cycles of 1 min at 94 °C, 1 min at 58 °C, and 2 min at 72 °C; and final extension, 10 min at 72 °C), the overlapping DNAs were combined, and NheI pr as well as XhoI pr were used as primers. The resulting product was cut with NheI and XhoI and ligated into the pCDNA cassette described above. All of the PCRs were performed using 2.5 units of Takara DNA polymerase (Takara, Madison, WI) along with 0.2 unit of Pfu Polymerase (Stratagene, La Jolla, CA). PCR products were separated on 1% agarose gels (Bio-Rad). To amplify the constructs, the plasmids were transformed into Top 10 competent bacteria by chemical transformation. Mini- and maxipreps as well as gel purifications were carried out using Qiagen kits.

Transfection—For protein expression, plasmids were transfected into 293T cells using polyethylenimine as described (27). Briefly, 293T cells were grown in 15-cm cell culture plates coated with polylysine to 70–80% confluence (Dulbecco's modified Eagle's medium, 10% FCS, 200 mM L-Glu). FCS-containing medium was removed, the cells were washed with serum-free medium, and 11 ml of serum-free medium was added to the cells. A solution of 36 µg of polyethylenimine and 24 µgof plasmid DNA in 1.2 ml of serum-free medium was prepared and let sit for 10 min at room temperature was then added to each cell culture dish. The cells were incubated for 3 h at 37°C, 5% CO₂. Finally, FCS was added up to a final concentration of 10%, the media were harvested after 72 h, and the expressed proteins were purified.

Western Blot—Crude medium, Anti-FLAG column eluate and HPLC fractions were checked for activin-A/C chimera expression by Western blot. The samples were run under reducing and nonreducing conditions on 12.5% SDS-polyacrylamide gels (Bio-Rad) along with known amounts of wt activin as well as multi-marker (Sigma). Affinity column-purified primary antibodies raised in rabbit against activin residues 81–113 (kindly provided by Joan Vaughan, Peptide Biology Lab, Salk Institute, La Jolla, CA) were used in combination with an alkaline phosphatase-conjugated secondary goat anti-rabbit antibody (Bio-Rad). The proteins were visualized by the addition of the alkaline phosphatase substrates 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.

Silver Staining—A protein silver staining method was used for the visualization of impurities not detectable by the A antibody. The samples were run on SDS gels, which were then dried twice for 5 min with 50% methanol, followed by a 10-min 50 mM dithiothreitol treatment and a 20-min 0.1% silver nitrate solution treatment. The excess of silver nitrate was sequestered by the addition of 25 ml of a 0.1% formaldehyde, 3% Na₂CO₃ solution for 30 s, which was discarded. Protein bands on the gel were then visualized by the addition of another 50 ml of the 0.1% formaldehyde, 3% Na₂CO₃ solution.

Protein Purification—Crude media from 293T cells transfected with DNAs encoding for the activin-A/C chimeras were filtered through a 5-µm nylon filter to separate cell debris. 1 M MES buffer, pH 6.2, was added to the filtrate up to a final concentration of 50 mM, along with 0.5 ml of M2 anti-FLAG-agarose bead suspension (Sigma). To allow protein binding to the beads, the media were shaken overnight at 4 °C. Then the suspension was poured into columns (10 cm x 1 cm; Bio-Rad) equipped with a one-way stop cock, the flow-through and an additionally added 15 ml of 50 mM MES washing buffer were discarded, and activin chimeras bound to the M2 anti-FLAG agarose beads remaining in the column were eluted in five fractions of each 1 ml of glycine-HCl buffer, pH 2.8. Fractions were neutralized with 100 µl of Tris-HCl, pH 8, and then subjected to HPLC purification. Chimeras were separated in a single gradient run using a C4 column (2.1x 150 mm, particle size, 5 µm; pore size, 300 Å; Vydac, Hesperia, CA) on an HP1100 HPLC machine (HP1100; Hewlett Packard). 0.05% trifluoroacetic acid (solvent A) as well as 0.05% trifluoroacetic acid dissolved in 90% acetonitril, 10% water (solvent B) were used as solvents. The gradient was set up as follows: min 0, 20% solvent B; min 40, 50% solvent B; min 41, 100% solvent B; min 45, 20% B; followed by a 12-min post-run with 20% solvent B at a flow rate of 0.2 ml/min. Chromatograms showed single peaks at 29.5–32.5 min for A/C 46–53, 54–61, 62–69, and 70–78. The respective fractions were collected, quantified by comparing the peak areas of the chimera with those of known activin amounts, dried down in the presence of bovine serum albumin, and redissolved in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 200 mM glutamine to a final chimera concentration of 10 mg/ml and 0.01% bovine serum albumin. Because the A/C 46–78 chimera did not come off of the column as a single protein, it was subjected to a further, isocratic HPLC sizing purification step. The fraction of the first HPLC step was dried down, redissolved in 50 µl of a 6 M guanidinium chloride, 0.1 M sodium acetate solution, injected onto a Superose 12 column (Amersham Biosciences), and eluted with 6 M guanidinium chloride, 0.1 M sodium acetate at a flow rate of 0.04 ml/min. A single peak fraction at 35 min was collected and directly subjected to a desalting HPLC run using the same C4 column as it was used for the first purification step. Conditions were as follows: min 0, 0% solvent B; min 5, 20% solvent B; min 30, 50% solvent B; min 31, 100% solvent B; min 35, 0% solvent B; followed by a 10-min post-run with 0% solvent B at a flow rate of 0.2 ml/min. A single peak fraction at 24 min was collected, dried down, and redissolved as described above for the other chimeras. The proteins were stored at –80 °C.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Sequence alignment of activin-A, activin-C, and BMP2. Activin-A residues changed to activin-C are boxed, activin-A residues implicated in ActRII binding are shaded gray, and BMP2 residues involved in ALK3 binding are in white letters with black shading.

A3Lux Assay—293T cells were seeded into 24-well plates coated with polylysine at a density of 150,000 cells/well. The next day, the cells of each well were transfected overnight with a mixture containing A3 Lux (25 ng) and -galactosidase (25 ng) reporter plasmids, the transcription factor FAST2 (50 ng), and empty pCDNA3 vector (400 ng) using Perfectin transfection reagent. Then the cells were treated with wt activin-A and the different activin-A/C chimeras in concentrations from 0–40 nM for 16–24 h. The media were removed; the cells were lysed with ice-cold lyses buffer containing 25 mM glycylglycine, 4 nM EGTA, 15 mM MgSO₄, 1 mM dithiothreitol, as well as 1% Triton X-100 for 20 min; and the lysates were assayed for luciferase and -galactosidase activities. To assess antagonistic properties of the A/C 46–78 chimera, the cells were treated alternatively either with 100 pM wt activin, 500 pM myostatin, or 50 pM TGF- in the presence of increasing concentrations of the chimera for 16–24 h.

Inhibition of Follicle-stimulating Hormone (FSH) Release from LT2 Cells—LT2 cells were seeded into 48-well plates and cultured in Dulbecco's modified Eagle's medium supplemented with 2% FCS and glutamine. At 50% confluence, the cells were washed three times with Hepes dissociation buffer and then treated with increasing concentrations of the A/C 46–78 chimera (0–40 nM) in the presence of 100 pM wt activin-A. 72 h later the media were harvested, and the concentration of the secreted FSH was determined by radioimmunoassay.

Inhibition of FSH Release from Rat Interior Pituitary Cells—The assay was performed as described (28). In brief, freshly isolated cells from male Sprague-Dawley rat interior pituitaries of several animals were combined and seeded into 96-well plates at a density of 50,000 cells/well in PJ medium supplemented with 2% bovine serum albumin and appropriate growth factors (29). The next day, the cells were treated with increasing concentrations of the A/C 46–78 chimera (0–40 nM) with or without the addition of 100 pM wt activin-A. 72 h later the media were harvested, and the concentration of the secreted FSH was determined by radioimmunoassay.

Follistatin Binding—Follistatin binding was carried out in a cell-free assay system as described (28). Briefly, 2 µg of follistatin 288 were incubated with increasing concentrations of the chimeras (0–40 nM) in the presence of a constant amount of radiolabeled wt activin-A along with a primary antibody against follistatin 288 raised in sheep for 90 min at room temperature in a final volume of 100 µl. Then 100 µl of a premix containing 1% normal rabbit serum, 1% sheep anti-rabbit secondary antibody, and 10% polyethyleneglycol was added and then incubated on ice for 30 min to precipitate the follistatin antibody-follistatin-activin-chimera complexes. Precipitates were spun down, the supernatant was removed, and the radioactivity in the pellet was determined using a counter (APEX Micromedic Systems).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chimera Construction—Because the majority of the BMP2 residues involved in ALK3 binding are located in the BMP2 wrist, (23) we focused on this region in activin as well. However, with respect to a recent finding showing that the introduction of single point mutations in the activin wrist epitope does not have any significant effect on activin activity (26), we decided to generate activin chimeras with more severe mutations in the wrist. The biologically less active activin-C only shares 11 out 32 amino acids in the wrist with activin-A, which might contribute to its low activity and also to a lower affinity for ALK4 when compared with activin-A. Therefore, we constructed four mutants in which in each of them eight residues are changed from activin-A to activin-C (A/C 46–53, A/C 54–61, A/C 62–69, and A/C 70–78) and one chimera with the entire wrist changed from activin-A to activin-C (A/C 46–78) (Fig. 1). Activin mutants retaining high affinity for the type 2 receptor ActRII but losing the ability to signal in an activin-like manner would be potential activin antagonists and might reveal a general principle for the generation of desired antagonists of TGF- superfamily proteins.

Expression—The Western blots of single peak HPLC fractions (for details see "Materials and Methods") of the A/C 46–53, A/C 54–61, A/C 62–69, and A/C 70–78 chimeras showed bands at 14 and 28 kDa, whereas the wt activin-A standard appeared at about 13 and 26 kDa when run under reduced and nonreduced conditions, respectively (Fig. 2). This indicates that the activin-A/C chimeras produced and secreted by 293T cells are, like wt activin-A, able to form dimers. The difference in size observed between wt activin and the chimeras is due to the FLAG tag introduced at the N terminus of the chimeras. As for the chimera with the entire wrist changed from activin-A to activin-C (A/C 46–78 chimera), one HPLC step on a C4 column was not sufficient for its complete purification. To obtain a pure A/C 46–78 chimera protein, the peak fraction collected at 34 min in the first HPLC run was subjected to a further sizing step followed by a desalting run as described under "Materials and Methods." A single peak fraction eluting off of the C4 column in the desalting run at 24 min resulted in a single band when analyzed by Western blotting (Fig. 2). Like the four chimeras with only eight residues changed from activin-A to activin-C, the chimera A/C 46–78 is able to form a dimer as indicated by a 14-kDa band under reducing conditions and a 28-kDa band under nonreducing conditions.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Protein expression. Collected HPLC samples run on 12.5% SDS-polyacrylamide gels under reducing and nonreducing conditions and blotted onto polyvinylidene difluoride membranes were treated overnight with a primary antibody raised in rabbit against activin residues 81–113. The bands were then visualized by incubating the blot with a secondary alkaline phosphatase-conjugated antibody for 1 h followed by the addition of the alkaline phosphatase substrates 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Lane 1, wt activin-A (reduced); lanes 2–6, reduced samples of A/C 46–53, A/C 54–61, A/C 62–69, A/C 70–78, and A/C 46–78, respectively; lanes 7–11, nonreduced samples of A/C 46–53, A/C 54–61, A/C 62–69, A/C 70–78, and A/C 46–78, respectively; lane 12, wt activin-A (nonreduced). The figure is composed from two different blots.

ActRII Binding—To test the ability of the chimeras to bind the activin type II receptor, a binding assay was performed using 293T cells transiently transfected with mouse ActRII. Displacement curves were generated by incubating the cells with ¹²⁵I-activin in the presence of increasing concentrations of wt activin-A or activin-A/C chimeras. The EC₅₀ value of wt activin-A for ActRII was 200 pM, and the EC₅₀ values of the FLAG-tagged chimeras for ActRII ranged from 262 to 545 pM (Fig. 3). Thus, the binding data indicate that the affinity of all five chimeras for ActRII is only slightly reduced when compared with wt activin-A. This result is in line with predictions made based on the crystal structures of activin bound to ActRII (8, 9) and the related BMP7 bound to ActRIIB (21) as well as the mutagenesis study carried out by Wuytens et al. (22) demonstrating residues in the fingers to be involved in ActRII binding. Additionally, because the chimeras bind ActRII with wt-like affinity, it is likely that the overall structure of the chimeras does not significantly differ from wt activin.

A3Lux Assay—To determine whether the chimeras retained activin-like bioactivity, their ability to activate an A3 Lux reporter gene was assessed. HEK 293T cells transiently transfected with the transcription factor FAST2 as well as the A3 Lux and -galactosidase reporter genes were incubated with increasing concentrations of wt activin-A or activin-A/C chimeras. After a 16–24-h incubation time, luciferase activities were measured and normalized to -galactosidase activities. Of the five chimeras tested, only A/C 46–78 was devoid of significant activin-like bioactivity in concentrations of up to 40 nM. A/C 46–53, 54–61, 62–69, and 70–78 (EC₅₀ values ranging from 113–862 pM) showed activities comparable with wt activin-A (EC₅₀ ranging from 70 to 320 pmol; Fig. 4A); however, the maximum activity of A/C 54–61, 62–69, and 70–78 was somewhat reduced when compared with wt activin-A.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. ActRII binding. HEK 293T transfected with ActRII were incubated with wt activin-A or the different activin-A/C chimeras in the presence of a constant amount of 125I-radiolabeled wt activin-A for 2 h. Displacement curves for wt activin (closed squares), A/C 46–53 (open triangles), A/C 54–61 (closed triangles), A/C 62–69 (open diamonds), A/C 70–78 (closed circles), and A/C 46–78 (open squares) were generated as described under "Materials and Methods." The amount of bound 125I-activin was determined in triplicate for each experiment, and the values are the means ± S.D. The experiment was repeated at least three times. conc., concentration.

The A/C 46–78 chimera presented in this study and M108A-activin (26) revealed ALK4-binding sites in the wrist and the second finger of activin, respectively. However, when we combined the two different mutant epitopes (A/C 46–78, M108A-activin), enhanced antagonistic effects could not be observed (data not shown).

To test whether or not the A/C 46–78 chimera is also able to antagonize activin-induced FSH release from the mouse gonadotrope cell line LT2, we incubated LT2 cells with increasing concentrations of the chimera in the presence of 100 pM activin for 72 h. Fig. 5 shows that 100 pM activin alone increased the concentration of FSH released from LT2 cells into the medium from a basal 9.1 ng/ml up to 16.2 ng/ml. This activin-induced increase in FSH release was completely inhibited at chimera concentrations of 13 nM. Significant reduction of FSH release could be achieved at concentrations of 1.5 nM (Fig. 5).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. A, dose response curves in 293T cells for wt activin and the activin-A/C chimeras. HEK 293T cells transfected with A3 Lux and -galactosidase reporter plasmids as well as the transcription factor FAST2 were incubated with increasing concentrations of wt activin or the different activin-A/C chimeras for 24 h. The cells were then lysed, luciferase and -galactosidase activities were determined, and dose response curves were generated for wt activin (closed squares), A/C 46–53 (open triangles), A/C 54–61 (closed triangles), A/C 62–69 (open diamonds), A/C 70–78 (closed circles), and A/C 46–78 (open squares). The curves represent luciferase activities normalized to -galactosidase activities. Each point of the curve is the mean ± S.D. of three measurements. The experiment was repeated at least three times. B, activin and myostatin antagonism by A/C 46–78. HEK 293T cells transfected with A3 Lux and -galactosidase reporter plasmids as well as the transcription factor FAST2 were incubated with increasing concentrations of the A/C 46–78 chimera in the presence of 100 pM wt activin, 500 pM myostatin, or 50 pM TGF- for 24 h. The cells were then lysed, the luciferase and -galactosidase activities were determined, and the curves were generated for A/C 46–78 + 100 pM wt activin (closed squares), A/C 46–78 + 500 pM myostatin (open triangles), and A/C 46–78 + 50 pM TGF- (open squares). The curves represent luciferase activities normalized to -galactosidase activities. Each point of the curve is the mean ± S.D. of three measurements. The experiment was repeated at least three times. conc., concentration.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Inhibition of FSH release from LT2 cells by A/C 46–78. LT2 cells were treated with increasing concentrations of A/C 46–78 chimera in the presence of 100 pM wt activin for 72 h. The media were harvested and assayed for released FSH by radioimmunoassay. Each point of the curve is the mean ± S.D. of three measurements. The experiment was repeated twice. *, p = 0.05 (Wilcoxon).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Inhibition of FSH release from rat anterior pituitary cells by A/C 46–78. Rat anterior pituitary cells were treated with increasing concentrations of A/C 46–78 chimera without (A) or with (B) 100 pM wt activin for 72 h. The media were harvested and assayed for released FSH by radioimmunoassay. Each point of the curve is the mean ± S.D. of three measurements. The experiment was repeated twice. *, p = 0.05 (Wilcoxon). conc., concentration.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Follistatin binding. Displacement of 125I-activin-A by wt activin-A (closed squares), A/C 46–78 (open triangles), A/C 54–61 (closed triangles), A/C 62–69 (open diamonds), A/C 70–78 (closed circles), and A/C 46–78 (open squares) was determined. Follistatin-activin complexes were immunoprecipitated to separate bound from free ligand. Radioactivity in the precipitate was determined using a counter. Each point of the curve is the mean ± S.D. of three measurements. The experiment was repeated at least three times. conc., concentration.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The characterization of the activin-A/C chimeras presented in this study reveals an insight into the activin wrist epitope with respect to its role in ActRII and follistatin binding, biological activity as well as how its manipulation can be used as a tool for the construction of TGF- protein receptor antagonists.

With inconsistent data on the activity and physiological function of activin-C (for review see Ref. 32), its role in the complex interaction between ligands and receptors within the TGF- superfamily remains unresolved. Here we demonstrate that exchanging multiple activin-A wrist residues to residues of the homologous activin-C does not alter the affinity of the proteins for ActRII and follistatin. These results are in agreement with predictions made based on the crystal structures of activin bound to ActRII (8, 9) and BMP7 bound to ActRIIB (21) as well as mutagenesis studies (22, 28) demonstrating that the outer surface of the fingers form the binding interface with the type II receptors. As for follistatin binding, a deletion mutant of activin (activin 85–109) has been generated that does not bind follistatin, indicating that the second activin finger is of great importance for the activin/follistatin interaction (28).

As for type I receptor binding, the crystal structure of BMP2 bound to ALK3 (23) revealed several residues in the BMP2 wrist (Phe⁴⁹, Pro⁵⁰, Leu⁵¹, Ala⁵², Asp⁵³, His⁵⁴, Ser⁵⁷, and Ser⁶⁹ of one monomer and Asn⁵⁹, Ile⁶², Val⁶³, and Leu⁶⁶ of the other monomer) as well as finger residues (Lys¹⁵ of one monomer and Trp²⁸, Trp³¹, Met⁸⁹, Tyr¹⁰³, and Met¹⁰⁶ of the other monomer) to be on the binding interface to ALK3. Several of these residues were confirmed to participate in ALK3 binding by mutagenesis studies (24, 25) showing especially the BMP2 mutants F49A, P50A, L51P, and H54E and the double mutant F49A/P50A to possess a significantly decreased affinity for ALK3 as well as a reduced ability to induce alkaline phosphatase activity in C₂C1₂ cells. Those mutants, however, did not show any BMP2 antagonism because BMP2, unlike activin, first binds with high affinity to its type I receptor ALK3 followed by the recruitment of the type II receptors ActRII or BMPRII (33, 34). Therefore, BMP2 mutants with a disrupted ability to bind to the type II receptor were found to possess BMP2 antagonistic properties (24). With activin first binding with high affinity to its type II receptor ActRII/ActRIIB and then to the type I receptor ALK4, mutants retaining wt-like ActRII/ActRIIB affinity in which the ALK4-binding site is disrupted should be activin antagonists. Indeed, the A/C 46–78 chimera possessing wt-like affinity for ActRII and at the same time being devoid of significant bioactivity turned out to be an antagonist for ligands signaling via ActRII. Chimeras with eight residues changed at a time (A/C 46–53, 54–61, 62–69, and 70–78) retained their signaling ability, which is in line with previous findings for activin-A/C chimeras (26). The fact that it takes larger parts of the wrist to be exchanged from activin-A to activin-C to observe disrupted signaling suggests several weak rather than a few strong interactions between activin and ALK4 in this region. Therefore, the quality of the activin wrist/ALK4 interaction seems to be different from that observed for BMP2 binding to ALK3 because for BMP2 single point mutations (F49A, P50A, L51P, and H54E) in the wrist led to a significant decrease in ALK3 binding and activity. The antagonistic potential of the A/C 46–78 chimera appeared similar to the one reported for activin-M108A (26). As with M108A, there is a discrepancy in the affinity of the chimera for ActRII (262–545 pM) on one hand and the relatively lower EC₅₀ values for the ability of the chimera to block activin (1–10 nM) and myostatin (1–8 nM) signaling on the other hand. A similar effect has been described for BMP2 antagonists (24). It has been suggested that the low affinity receptors, e.g. ALK4 for activin and BMPRII for BMP2, may contribute to the stability of the receptor complex (26). A weaker ability of M108A-activin to compete with wt activin-A for ActRII binding was observed when ALK4 was cotransfected into the cells. We observed a similar effect for the A/C 46–78 chimera. Its EC₅₀ for ActRII was not significantly altered when ALK4 was cotransfected into 293T cells, whereas the EC₅₀ value of activin for ActRII decreased about 7-fold in the presence of ALK4 (data not shown).

A combination of the two mutant epitopes (A/C 46–78, M108A-activin) did not enhance antagonistic effects (data not shown), indicating that for the A/C 46–78 chimera, activin-like signaling is probably already disrupted to a maximal degree. Therefore, a further improvement of antagonistic properties might be achievable by increasing the affinity of the chimera for ActRII (22). With respect to blocking activin and myostatin signaling, the A/C 46–78 chimera as well as second generation antagonists could potentially be used for the treatment of diseases such as cancer, muscular dystrophy, liver cirrhosis, and fibrosis and to improve wound healing. Whether changing the entire wrist between different members of the TGF- superfamily might allow the construction of desired type II receptor antagonists remains to be elucidated further.

	FOOTNOTES

 
^* This work was supported by the Foundation for Medical Research, California Division, and by National Institutes of Health Grant HD-13527. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.:858-453-4100; Fax: 858-552-1546; E-mail: fischer{at}salk.edu.

² The abbreviations used are: TGF, transforming growth factor; FSH, follicle-stimulating hormone; wt, wild type; FCS, fetal calf serum; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.

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