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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 12 4851-4858
Copyright © 2000 by The Endocrine Society

Original Studies

Localization of Activin ßA-, ßB-, and ßC-Subunits in Human Prostate and Evidence for Formation of New Activin Heterodimers of ßC-Subunit1

Sally L. Mellor, Mark Cranfield, Rainer Ries, John Pedersen, Belinda Cancilla, David de Kretser, Nigel P. Groome, Anthony J. Mason and Gail P. Risbridger

Monash Institute of Reproduction and Development (S.L.M., B.C., D.d.K., G.P.R.), Monash University, Melbourne, Victoria, Australia; School of Biological and Molecular Sciences, Oxford Brookes University (M.C., N.P.G.), Headington, Oxford, United Kingdom; BIOPHARM (R.R.), Heidelberg, Germany; Melbourne Pathology, Collingwood, Victoria, Australia (J.P.); and Department of Microbiology, Monash University (A.J.M.), Clayton, Victoria, Australia

Address all correspondence and requests for reprints to: Dr. Gail P. Risbridger, Monash Institute of Reproduction and Development, 27–31 Wright Street, Clayton 3168, Victoria, Australia. E-mail: gail.risbridger{at}med.monash.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activin ligands are formed by dimerization of activin ßA- and/or ßB-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ß superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ßC-, ßD-, and ßE-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ßC can form dimers with activin ßA and ßB in vitro, but not with the inhibin {alpha}-subunit. Using a specific antibody, activin ßC protein was localized to human liver and prostate and colocalized with ßA- and ßB-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTIVINS ARE members of the transforming growth factor-ß (TGFß) superfamily of proteins (1), which consist of homo- and heterodimers of activin ßA- or ßB-subunits that combine to form activin A (ßA-ßA), activin AB (ßA-ßB), and activin B (ßB-ßB) (2, 3, 4). Activins were originally identified in ovarian follicular fluid as regulators of FSH secretion from the pituitary gland. However, activins are regulators of growth and differentiation in a range of tissues and cells, where they act in autocrine or paracrine pathways. Activins (mainly activin A) have been implicated in diverse processes, including mesoderm induction (5), mammalian embryogenesis (6), erythropoiesis (7), nerve cell survival (8), release of insulin (9), and wound repair (10).

Newer members of the activin family were identified, and ßC- and ßE-subunits were cloned from mouse (11, 12) and human liver (13). The activin ßD-subunit has been cloned from Xenopus, but no mammalian equivalent has yet been described (14). In contrast to activins formed from ßA- and ßB-subunits, those from ßC, ßD, and ßE appear to have limited actions in liver and mesoderm induction (14, 15, 16) and are thought to be a subset of related sequences (17). In the liver it was suggested that activin ßC was a putative chalone (15, 16) due to the temporal expression of ßA- and ßC-subunit messenger ribonucleic acids (mRNAs) after partial rat hepatectomy, but there is no evidence for biological activity of activin ßC in a dimeric or monomeric form in the liver. It is possible that heterodimers of activin ßC and ßA or ßB are produced, and if so, this may affect the level of production of activin A, B, or AB ligands. Furthermore, the putative heterodimers ßA-ßC (activin AC) and ßB-ßC (activin BC) may be inactive or have biological activity different from that of activins A, B, or AB. The idea that heterodimers of ßC and ßA or ßB exist would require at least the colocalization of subunit proteins to the same cell/tissue type, but coexpression and localization of subunit proteins have not been possible to date, as no specific antibodies are available, nor are there any data to show that activin ßC is able to dimerize with ßA- or ßB-subunits.

As we have identified activin ßC-subunit mRNA in human prostate (18) and human prostate tumor cell lines LNCaP, DU145, and PC3 (19), the first aim of this investigation was to clone activin ßC from the DUI45 human prostate tumor cell line. To determine whether the ßC-subunit could form homodimers or heterodimers with activin ßA- or ßB- or inhibin {alpha}-subunits, we cotransfected the corresponding cDNAs into human kidney 293 cell lines (20). To examine the expression of ßC-subunit protein in human prostate and liver, we developed a specific monoclonal antibody to the activin ßC-subunit and described the localization of activin ßC in tissue sections from men with benign prostatic hyperplasia (BPH) and high grade prostate cancer. ßC-Subunit in nonmalignant and malignant prostate tissues was compared with that of ßA- and ßB-subunits (21) to determine the potential cellular site of novel activin synthesis in the human prostate. Finally, to determine possible biological activity of activin C, the effect of activin C on the proliferation of prostate and liver tumor cell lines in culture was investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ßC expression studies

Construction of expression vectors. ßC complementary DNA (cDNA) was obtained by RT-PCR using RNA purified from the human prostate tumor cell line DU145. RNA was isolated using the method of Chomczynski and Sacchi (22). Total RNA was reverse transcribed to cDNA using oligo(deoxythymidine) and AMV reverse transcriptase (Promega Corp., Madison, WI).

PCR reactions to amplify the cDNA included the equivalent of 0.3 µg reverse transcribed DU145 RNA, 2.5 U Pfu polymerase (Stratagene, La Jolla, CA), 15 pmol of each of the following primer pairs: 1, 5'-CCAGCCATGGCCTCCTCATTGCTTCTGGCCTT-3'; and 2, 5'-GTAGTCGAAACGACTCTGTCCGGAG-3' (denaturation temperature of 95 C for 1 min, annealing temperature of 60 C for 30 s, extended at 72 C for 2 min for 35 cycles); 3, 5'-GCCCTGTGTCCAGAGCTGCTTTGA-3' and 4, 5'-CGTTTGTGGTCTAAGTGGCTGCTCC-3' (denaturation temperature of 95 C for 1 min, annealing temperature of 55 C for 45 s, extended at 72 C for 2 min for 40 cycles); and 5, 5'-CTGGAGCTGGTACTTGAAGGCCAGG-3' and 6, 5'-GGACACCCACGTCAATCAGATTCGAACCATA-3' (denaturation temperature of 95 C for 1 min, annealing temperature of 72 C for 1 min, extended at 72 C for 2 min for 35 cycles). In separate reactions, 1 x Pfu buffer, 2 mmol/L MgCl2, and 0.2 mmol/L deoxy-NTP (Pharmacia Biotech, Piscataway, NJ) were used in a final volume of 50 µL. These PCR primers (Integrated DNA Technologies, Coralville, IA) were based on the published sequence for human ßC-inhibin (13). The 319-bp product (fragment A) from primer pair 1 and 2 was digested with XbaI/NcoI and gel purified, the 516-bp product (fragment B) from primer pair 3 and 4 was digested with SacI and XbaI, and the 489-bp product (fragment C) from primer pair 5 and 6 was digested with HindIII and SacI. Fragment A (NcoI/XbaI) was ligated to the linkers 5'-AATTCCAGCCAG-3' and 5'-CATGGTGGCTGG-3' to generate an EcoRI site at the 5'-end. Full-length ßC cDNA was obtained by sequential ligation of three cDNA fragments (fragments A, B, and C) into pUC19 (New England Biolabs, Inc., Beverley, MA). The total fragment was excised with EcoRI and HindIII. A suitable full-length cDNA was subcloned into the pRK5 expression vector as an EcoRI/HindIII fragment. This pRK5 plasmid contains a cytomegalovirus promoter, a polylinker region, a simian virus 40 polyadenylase addition signal, and a simian virus 40 origin of replication as described by Mason et al. (20).

Cotransfection of cDNAs encoding {alpha}-, ßA-, and ßB-subunits was performed using plasmids previously described (20, 24, 25). Activin/inhibin cDNAs were transfected, either alone or as cotransfections, into the human embryonic kidney cell line 293 as described by Mason et al. (20). The pRK5 expression plasmid was used as a control plasmid for mock transfections. A total of 5 µg DNA/60-mm dish was used for transfection, and cells were cultured for 48–72 h in serum-free medium and metabolically labeled by culture for 5 h in serum-free, cysteine/methionine-free medium containing 140 µCi [35S]Translabel (NEN Life Science Products). The supernatant was stored at -20 C.

SDS-PAGE, immunoprecipitation, and Western blotting. Protein samples (10 µL of a total of 500 µL supernatant) were loaded and electrophoresed under reducing or nonreducing conditions using the minigel system (Bio-Rad Laboratories, Inc., Hercules, CA) in either 10% or 12% SDS-PAGE. Gels were fixed in 7% acetic acid/30% methanol for 5 min, treated with Enhance (NEN Life Science Products) for 1 h, and vacuum-dried before exposure to x-ray film at -70 C for 1–5 days.

Immunoprecipitation of {alpha}-subunit-containing complexes was performed as described previously (20) using rabbit antiserum 29A provided by Genentech, Inc. (South San Francisco, CA). The detection limit was based on visual observation compared with control lanes and was considered the background.

Antibody preparation. A synthetic peptide of sequence (VPTARRPLSLLYYDRDSNIVKTDIPDMVVEAC) corresponding to amino acids 82–113 of the deduced mature human activin ßC-subunit (13) was synthesized by fluorenylmethoxycarbonyl chemistry (27). The ßC peptide was made corresponding to homologous regions of ßA- and ßB-subunits that have been used to generate ßA and ßB monoclonal antibodies. Outbred female mice of strain TO were immunized with ßC peptide as described by Groome et al. (28). The housing and care of the animals were in accordance with Medical Research Council guidelines. Tail bleeds were obtained at monthly intervals and screened using a standard enzyme-linked immunosorbent assay (ELISA) procedure (29, 30) for reactivity to ßC peptide. After a booster immunization the mice were killed, their spleens were removed, and splenocytes were fused to SP2/0 myeloma cells using a standard fusion protocol with polyethylene glycol (31). Ninety-six positive clones were chosen and expanded to provide supernatant for further testing by immunohistochemistry.

Recloning, isotyping, and purification. Selected cell populations secreting ßC antibody were recloned in methylcellulose (32). The subsequently chosen antibody, clone 1, was isotyped using a Sigma ImmunoType kit (St. Louis, MO) and was found to secrete a mouse IgG1 antibody. Clone 1 antibody was then purified using protein G affinity chromatography (Prosep-G Bioprocessing, Consett, UK).

Specificity testing of activin ßC antibody

Cross-reactivity test of the ßC antibody with ßA, ßB, ßC, and ßE peptides. Ninety-six-well plates were coated with synthetic peptides to ßA, ßB, ßC, and ßE in bicarbonate buffer. The top row was coated with 1 µg/ml ßA peptide, the second row with 1 µg/ml ßB peptide, the third row with 1 µg/ml ßC peptide, the fourth row with 1 µg/ml ßE peptide, and the last row was an uncoated control. After coating, plates were blocked with 100 µL 1% (wt/vol) BSA in phosphate-buffered saline (PBS) containing 1% (vol/vol) H2O2 for 30 min. After blocking, purified ßC antibody (1.6 mg/ml) was serially diluted from 10-2–10-8 and added to the plate. Dilutions were made in Tris conjugate buffer [25 mmol/L Tris-HCl buffer, pH 7.5, containing 0.15 mol/L NaCl, 1% (wt/vol) BSA, and 1% (vol/vol) Tween-20]. After 60 min the plates were washed, and rabbit antimouse immunoglobulin/peroxidase conjugate (DAKO Corp., High Wycombe, UK) was added at a dilution of 1:2000 in Tris conjugate buffer and incubated at room temperature for 30 min. After washing, the plate was developed by the addition of 50 µL/well tetramethylbenzidine peroxidase substrate (Dynatech Corp., Billinghurst, UK). The reaction was stopped after 30 min by the addition of 50 µL/well 6% (vol/vol) phosphoric acid. Absorbances were read at 450 nm using a standard microplate reader.

Western blot to determine whether clone 1 recognizes both mono- and dimeric activin ßC. Western analysis was performed to determine the cross-reactivity of clone 1 with monomeric and dimeric activin ßC and monomeric activin ßE (obtained from Biopharm, Heidelberg, Germany). SDS-PAGE was performed under nonreducing conditions using 15% polyacrylamide gel. Protein samples of 10 and 20 ng recombinant human monomeric activin ßC produced in Escherichia coli, 10 and 20 ng recombinant human dimeric activin ßC produced in CHO cells, and 100 and 200 ng recombinant mouse monomeric activin ßE produced in E. coli were diluted in sample buffer consisting of 7 mol/L urea, 0.1% NaH2PO4·H2O, 1% SDS, and 0.01% bromophenol blue, pH 7.2. The proteins in the gel were transferred to Biotrace polyvinylidene difluoride membrane using blotting buffer (0.7 mol/L glycine, 0.3 mol/L Tris, and 15.6% ethanol). To reduce dimeric proteins during transfer, 2.31 g dithiothreitol were added to 10 mmol/L to blotting buffer. The Western Light chemiluminescent detection system (Tropix) was used throughout this procedure and was performed according to the manufacturer’s instructions. Clone 1 ßC antibody was added at a dilution of 1:5000 in I block (from kit) overnight at 4 C. Goat antimouse IgG secondary antibody conjugated with alkaline phosphatase was diluted in 1:5000 in I block (from kit) for 1 h at room temperature.

Immunohistochemistry

Tissues. Human prostate needle biopsy specimens from 22 men were obtained from Melbourne Pathology (Collingwood, Australia) and consisted of 12 diagnosed with BPH and 10 with high grade prostate cancer (each having a Gleason score of 7–10). Human liver specimens were obtained from the John Radcliffe Hospital (Oxford, UK). Tissue were fixed in buffered formalin and processed to paraffin. Signed consent forms were obtained from patients, and the specimens were used in accordance with the requirements and approval of the standing committees for human and animal ethics and experimentation at Monash Medical Center and Monash University.

Screening of ßC antibodies on human liver. Tissue culture supernatants were screened by immunohistochemistry on sections of human liver tissue. The protocol followed was identical to that described by Thomas et al. (21) except for the following steps. Sections were dewaxed and placed in 0.01 mol/L glycine buffer solution (pH 4.4). Antigen retrieval involved exposing sections to microwaves at 2.25 watts/mL·min for 3 min followed by 0.3 watts/mL·min for 3 min. All tissue culture supernatants were tested on liver sections and incubated overnight at 4 C. Sections were washed with PBS and incubated for 50 min with biotinylated horse antimouse secondary antibody (DAKO Corp., Botany, Australia) at a dilution of 1:200 in PBS. Sections were washed with PBS and incubated with ABC reagent from the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Peterborough, UK) for 40 min. Peroxidase activity was detected using 3'3'-diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc.). The reaction was terminated by immersion in distilled water, and the sections were counterstained with Mayers’ hematoxylin (Sigma), washed with tap water, dehydrated, and permanently mounted with DPX (BDH, Poole, UK).

Immunohistochemistry using monoclonal antibody to the ßC-subunit on human liver and ßA, ßB, and ßC monoclonal antibodies on human prostate sections. Immunohistochemistry demonstrating ßA-, ßB-, and ßC-subunit localization in BPH patients was performed on serial 3-µm tissue sections. Clone 1 was added to sections of human liver, BPH, and prostate cancer at 5.8 µg/ml and incubated overnight at 4 C. To test the specificity of immunohistochemical staining, the antibody was preabsorbed with the synthetic ßC peptide mentioned above at 800 µg/ml for 4 h before addition to sections.

ßA immunolocalization was reinvestigated using monoclonal E4 antibody, which was used to measure activin A with ELISA (33). For immunostaining, E4 was used at 2 µg/ml and incubated overnight at 4 C on tissue sections that had undergone antigen retrieval with 0.01 mol/L Tris buffer (pH 9.7).

The C5 monoclonal antibody was previously used to detect ßB immunoreactivity (21), and this antibody was used to measure inhibin B with ELISA (34). However, it was reported to have a 1% cross-reaction with the ßA-subunit (23). Thus, after antigen retrieval with 0.01 mol/L citrate buffer (pH 6.0), immunolocalization of ßB-subunit using biotinylated C5 antibody was performed on tissue sections that were swamped with 2 µg/ml E4 for 1 h. Sections were washed with PBS, and ßB-subunit was detected using 20 µg/ml biotinylated C5 added overnight at 4 C. Sections were then directly detected with ABC (Vector Laboratories, Inc.) for 50 min.

Nerve cells were identified in tissue from patients with BPH after antigen retrieval in 0.01 mol/L citrate buffer (pH 6.0), using a monoclonal anti-pan neurofilament antibody (Zymed Laboratories, Inc., San Francisco, CA) at a 1:50 dilution added overnight at 4 C. Blood vessels were detected in BPH patients with a monoclonal anti-{alpha}-smooth muscle actin IgG (Sigma) added at 6.9 µg/ml for 30 min at room temperature.

Double immunofluorescence. Double immunofluorescence was used to investigate whether the stromal staining observed with antibodies to activin ßB and ßC was localized to smooth muscle cells. After ßB- and ßC-subunits had been localized in BPH patients, sections were incubated with double staining enhancer (Zymed Laboratories, Inc.) for 30 min, washed with PBS, and blocked with CAS (CAS-Block, Zymed Laboratories, Inc.) for 30 min. Monoclonal anti-{alpha}-smooth muscle actin IgG (Sigma) was used at 13.8 µg/ml for 2 h. Sections were washed with PBS and incubated with goat antimouse fluorescein isothiocyanate-conjugated antibody (Zymed Laboratories, Inc.) at a concentration of 15 µg/ml for 1 h.

In vitro studies with activin A and activin C

Prostate epithelial tumor cell lines. The human prostate tumor cell line, LNCaP, was obtained from American Type Culture Collection (Rockville, MD). The human liver tumor cell line, HepG2, was provided by Dr. David Phillips, Monash Institute of Reproduction and Development. Cell lines were routinely cultured in DMEM (Life Technologies, Inc., Grand Island, NY) with 10% heat-inactivated FCS (CSL Ltd., Parkville, Australia) and antibiotics (100 IU/ml penicillin and 10 µg/ml streptomycin; CSL Ltd.) in 75-cm2 culture flasks (Falcon; Becton Dickinson and Co., Franklin Lakes, NJ) at 37 C in a humidified atmosphere of 5% CO2 in air.

Growth factors and other reagents. Human recombinant activin A was provided by Biotech Australia Pty. Ltd. (East Roseville, Australia). Human recombinant activin C was provided by Biopharm GmbH (Heidelberg, Germany). Activin A was stored in 0.1% trifluoroacetic acid/25% acetonitrile/0.2% BSA and freeze-dried. Activin C lyophilized protein was reconstituted in 0.1% trifluoroacetic acid/50% acetonitrile and freeze-dried. All proteins were diluted in DMEM and 5% FCS for cell culture. Tritiated thymidine ([3H]thymidine) was obtained from NEN Life Science Products (Boston, MA).

[3H]Thymidine incorporation assay/DNA synthesis. LNCaP and HepG2 cells were plated at a density of 5000 cells/well in DMEM and 5% FCS into 96-well plates (Falcon, Becton Dickinson and Co.). After 72 h medium was replaced, and activin A, activin C, or vehicle buffer controls were added to the cell cultures and incubated for 2 days. [3H]Thymidine (0.5 µCi/ml) was added to the cells for 20 h, after which cells were harvested using a micromate 196 Cell Harvester (Packard Instrument Co., Meriden, CT), and levels of incorporated [3H]thymidine were determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of ßC from human prostate tumor cell lines and evidence for formation of ßA-ßC and ßB-ßC heterodimers

The subunit of ßC cloned from DU145 cells was identical to that cloned from human liver (13) with the exception of amino acid 19 (in the signaling sequence), where cytosine was replaced by thymine as designed in the cloning strategy. As shown in Fig. 1AGo, transfection of ßA- or ßB-subunits alone confirmed the formation of homodimers of approximately 24- and 22-kDa dimeric activin A or B, respectively (lanes 1 and 2). Transfection of ßC-subunits formed ßC homodimers, i.e. activin C, with an apparent molecular mass of 20 kDa (lane 3). Cotransfection of ßA and ßC (lane 4) or ßB and ßC (lane 5) subunits demonstrates the capacity of ßC to heterodimerize with ßA or ßB and form putative activin AC (~23 kDa) or activin BC (~21 kDa), respectively. The molecular masses of proteins within complexes was confirmed by running the gel under reducing conditions (data not shown). ßA- and {alpha}-subunits dimerize to form mono- and diglycosylated molecular mass forms of inhibin A, and in cells cotransfected with ßA- and {alpha}-subunits, inhibin A was detected as well as ßA-ßA and pro-ßA proteins (lane 6). Similarly, cotransfection of cells with the ßB- and {alpha}-subunit proteins formed mono- and diglycosylated inhibin B and ßB-ßB (lane 7). In contrast, cotransfection of the ßC- and {alpha}-subunits did not form heterodimers, and only the ßC-ßC complex was formed (lane 8). Control lanes consisted of transfection of the {alpha}-subunit alone (lane 9), transfection of plasmid pRK5 alone (lane 10), and transfection of pro-{alpha} inhibin subunit (lane 11). These results suggested that the ßC-subunit forms homodimers or heterodimers with ßA and ßB, but not inhibin {alpha}-subunit.



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  		Figure 1. A. Formation of activins comprised of ßA-, ßB-, and ßC-subunit proteins. Autoradiograph of supernatants from transfected and 35S-labeled 293 cells run under nonreducing conditions on a 12% polyacrylamide gel (Fig. 1AGo). Cells transfected with ßA alone produced approximately 24-kDa activin ßA-ßA (<-) complexes. A 43- to 46-kDa high molecular mass band corresponds to the pro-ßA protein (lane 1). Cells transfected with ßB alone produced approximately 22-kDa activin ßB-ßB (<-) complexes (lane 2). Transfection of ßC alone produced approximately 20-kDa activin ßC-ßC (<-) complexes (lane 3). Cells cotransfected with ßA- and ßC-subunits produced an activin dimer ßA-ßC of about 23 kDa ({blacktriangleleft}). A significant amount of ßA-ßA (<-) complexes were also formed; ßC-ß C (<-) was formed in low amounts (lane 4). Cells cotransfected with ßB- and ßC-subunits produced activin dimer ßB-ßC complexes of about 21 kDa ({blacktriangleleft}). ßB-ßB (<-) complexes were also formed in a higher amount compared with ßC-ßC (<-) complexes (lane 5). Cells cotransfected with {alpha}- and ßA-subunits produced pro-ßA, both mono- (~30 kDa) and diglycosylated (~32 kDa) forms of {alpha}-ßA complexes (*), and pro-{alpha}C and ßA-ßA complexes (<-; lane 6). Cells cotransfected with {alpha}- and ßB-subunits produced both mono-(~29 kDa) and diglycosylated (~31 kDa) forms of {alpha}-ßB complexes (*) and pro-{alpha}C and ßB-ßB (<-) complexes (lane 7). Cells cotransfected with {alpha}- and ßC-subunits produced only ßC-ßC (<-) complexes (lane 8). Control lanes consisted of cells transfected with {alpha} alone (lane 9), the pRK5 plasmid alone (lane 10), and pro-{alpha} inhibin subunit (lane 11). B, Analysis of inhibin {alpha} and ß dimers by immunoprecipitation. Supernatants from transfected and 35S-labeled cells were immunoprecipitated using {alpha}C-subunit antiserum 29A and analyzed on a 10% SDS-PAGE gel under nonreducing conditions. Cells cotransfected with the {alpha}- and ßA-subunits produced mono- and diglycosylated {alpha}-ßA with molecular masses of approximately 30 and 32 kDa, respectively. High molecular mass bands of pro-{alpha}N-{alpha}C-ßA (~60 kDa) and {alpha}N-{alpha}C-ßA (~55 kDa) were also formed (lane 1). Cells cotransfected with {alpha}- and ßB-subunits produced mono- and diglycosylated {alpha}-ßB with molecular massess of 29 and 31 kDa, respectively (lane 2). Cells transfected with {alpha}- and ßC-subunits did not produce any {alpha}-subunit-containing complexes (lane 3). Control lanes consisted of cells transfected with the {alpha}-subunit (lane 4) and pRK5-transfected cells (lane 5).

 
The inability of ßC to dimerize with the {alpha}-subunit was confirmed by immunoprecipitation. As shown in Fig. 1BGo complexes of {alpha}-ßA (lane 1) and {alpha}-ßB (lane 2) were immunoprecipitated using {alpha}-subunit antiserum, but no band corresponding to an {alpha}-ßC complex was observed (lane 3).

Specificity of ßC antibody

Cross-reactivities of all ßC supernatants with ßA, ßB, ßC, and ßE peptides were tested by ELISA. Clone 1 ßC antibody showed minimal cross-reactivity (0.1%) with ßA, ßB, or ßE peptides by ELISA, as shown in Fig. 2AGo.



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  		Figure 2. A, Cross-reactivity of purified clone 1 ßC antibody with ßA, ßB, ßC, and ßE peptides using ELISA. Graph demonstrating the dilution factor of the ßC antibody reacted against 1 µg/ml ßA, ßB, ßC, and ßE peptide or uncoated control. Absorbance decreased with decreasing concentrations of ßC antibody. B, Determination of specificity of ßC clone 1 antibody for mono- and dimeric activin ßC by Western blot. To test the specificity of the ßC antibody, Western analysis was performed as described in Materials and Methods. The ßC antibody reacted with human recombinant monomeric activin ßC (lanes 1 and 2) and human recombinant dimeric activin ßC (lanes 3 and 4). The ßC antibody did not react with recombinant mouse monomeric activin ßE (lanes 5 and 6). The positions of molecular mass markers are indicated.

 
Western analysis was used to determine the cross-reactivity of the purified ßC clone 1 antibody with monomeric and dimeric activin ßC and monomeric activin ßE. The data shown in Fig. 2BGo indicate that purified ßC clone 1 antibody recognized both human monomeric activin ßC (lanes 1 and 2) and dimeric activin ßC (lanes 3 and 4); no cross-reaction was observed with mouse monomeric activin ßE (lanes 5 and 6).

Immunohistochemical screening of ßC antibody supernatants and purified ßC clone 1 antibody was compared on human liver tissue sections. ßC-Subunit immunoreactivity was localized to hepatocytes using the ßC clone 1 supernatant (arrow, Fig. 3AGo) and purified clone 1 antibody (Fig. 3BGo). Specificity of staining was shown by preabsorption with ßC synthetic peptide, which abolished immunostaining (Fig. 3CGo).



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  		Figure 3. Immunolocalization of ßC- subunit protein to human liver. ßC-Subunit immunoreactivity was detected in hepatocytes in human liver sections with ßC clone 1 antibody supernatant (A; <-) and purified ßC clone 1 antibody (B). Immunoreactivity was abolished in the control section preabsorbed with ßC peptide (C). Bar, 50 µm.

 
Immunolocalization of the activin ßC subunit in human prostate tissues

Localization of ßC-subunit protein relative to that of ßA- and ßB-subunits was compared in tissue from patients with BPH (Fig. 4Go). As previously reported, ßA subunit was localized to the basal and secretory epithelial cells (Fig. 4AGo), whereas ßB-subunit was localized to the basal epithelial cells only (Fig. 4BGo). ßC-Subunit immunoreactivity was present in basal epithelial cells (Fig. 4CGo). No immunoreactivity was detected when the ßC antibody was preabsorbed with ßC peptide (Fig. 4DGo).



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  		Figure 4. A–H, Localization of activin ßA-, ßB-, and ßC-subunit protein in tissue from men with BPH and prostate cancer. Using tissues from men with BPH (A–D) or high grade cancer prostate cancer (E–H), this figure shows the cellular localization of ßA (A and E), ßB (B and F), and ßC (C and G) immunoreactivity. ßA-Subunit immunoreactivity was detected in both basal (<-) and secretory ({blacktriangleleft}) cells of prostate epithelium (A). Basal epithelial cells displayed ßB-subunit immunoreactivity (B; <-) and ßC-subunit immunoreactivity (C; <-). All three (ßA, ßB, and ßC) subunit proteins were localized to malignant epithelial cells in tumor tissue (E, F, and G, respectively). Preabsorbed controls for activin ßC in both BPH (D) and prostate cancer patients (H) were negative. Bar, 100 µm. ßA and ßB preabsorbed controls were negative, as previously described (21 ), and are not shown. I–W, Localization of activin ß-subunits in serial BPH tissue sections and in compartments of the prostatic stroma. Serial sections of tissue from men with BPH were used to localize ßA-, ßB-, and ßC-subunit proteins to basal epithelial cells (I, J, and K, respectively). ßA protein (I), but not ßB or ßC proteins, was localized to secretory epithelial cells (I). The same adjacent regions displayed positive localization of ßA (I; <-), ßB (J; <-), and ßC (K; <-) activin protein in basal epithelial cells only (I, J, and K; bar, 50 µm). Immunolocalization of ßB-subunit was observed in prostatic stroma (L; <-). Double staining identified the cells as {alpha}-actin positive (M; <-); similarly, ßC-subunit protein was detected in stroma (N; <-), which was {alpha}-actin positive (O; <-; bar, 50 µm). Positive immunolocalization of ßA (P) and ßC (R) activin subunits was detected in nerve cells identified using a neurofilament marker antibody (S). No immunoreactivity was observed using the ßB antibody (Q; bar, 50 µm) or in control tissue (inset to S; bar, 25 µm). ßA (T; <-), ßB (U; <-; bar, 50 µm), and ßC (V; <-) subunit proteins were immunolocalized to blood vessels identified by an {alpha}-actin smooth muscle antibody (W; <-). No immunoreactivity was observed in control sections (inset to W; bar, 10 µm).

 
Localization of ßC-subunit relative to ßA- and ßB-subunits in tumor tissue from patients with high grade cancer is shown in Fig. 4Go. In 10 patients with poorly differentiated prostate cancer, immunoreactivity for ßA (Fig. 4EGo), ßB (Fig. 4FGo), and ßC (Fig. 4GGo) subunits was detected in tumor cells in all patients. Preabsorption of ßC antibody with ßC peptide abolished staining (Fig. 4HGo).

These patterns of staining suggested that the same cell types contain ßA, ßB, and ßC, and to expand this further, serial tissue sections from BPH patients were used. All ßA-, ßB-, and ßC-subunits were colocalized to basal epithelial cells (Fig. 4Go, I, J, and K, respectively). Because the total thickness of the three serial sections examined was large (~9 µm) relative to the cell diameter, the boundary pattern of the cells within the focus plane appeared different.

In addition, activin ßB was localized to stromal cells (Fig. 4LGo), which were identified as a subset of smooth muscle cells (Fig. 4MGo). Stromal staining for activin ßC (Fig. 4NGo) was localized to a subset of smooth muscle cells in the stroma (Fig. 4OGo), consistent with ßB and ßC colocalized in {alpha}-actin-positive stroma. In serial tissue sections, ßA (Fig. 4PGo) and ßC (Fig. 4RGo) subunit proteins were localized to nerve cells, which were identified by neurofilament immunoreactivity (Fig. 4SGo). No immunoreactivity for ßB-subunit protein (Fig. 4QGo) or control mouse IgG (inset) was detected. Using serial sections of prostate tissue, blood vessel smooth muscle was identified by {alpha}-smooth muscle actin staining (Fig. 4WGo). ßA (Fig. 4TGo), ßB (Fig. 4UGo), and ßC (Fig. 4VGo) activin subunits were localized to the cells of blood vessels. No immunoreactivity was detected in the control section (inset).

Effect of activin C on the growth of LNCaP and HepG2 tumor cell lines

Activin C had no effect on [3H]thymidine uptake by LNCaP cells (Fig. 5AGo) or HepG2 cells (Fig. 5BGo) when added alone at a dose of 40 or 200 ng/ml. In contrast, activin A significantly inhibited the incorporation of [3H]thymidine by LNCaP cells (P < 0.001) and HepG2 cells (P < 0.004) compared with controls when added at a dose of 40 ng/ml. To determine whether activin C interferes with the inhibitory effect of activin A on LNCaP cells, activin C was added to the cells for 1 h before the addition of activin A. Activin C (40 or 200 ng/ml) had no effect on the inhibitory effect of activin A (40 ng/ml) on the uptake of [3H]thymidine by LNCaP or HepG2 cells.



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  		Figure 5. The effect of activin A and activin C, alone or in combination, on DNA synthesis by LNCaP and HepG2 tumor cells. LNCaP (A) and HepG2 (B) cells were plated and cultured in DMEM and 5% FCS, and the medium was replenished on day 3 with activin A, activin C, or a combination of these treatments. Activin A (40 ng/ml; A) or activin C (40 or 200 ng/ml) and matching vehicle control buffers were added alone. Activin C (40 or 200 ng/ml) or matching vehicle buffer controls were added 1 h before addition of activin A. Each value represents the mean ± SD from five replicate wells. *, Significance between P < 0.001 and P < 0.006.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides new data to indicate that the activin ßC-subunit can dimerize with ßA- and ßB-subunits to form heterodimers of activin AC and BC in vitro. Dimerization between inhibin {alpha}-subunit and activin ßC was not observed and provides evidence indicating that inhibin C is not formed. As dimerization of ßA and ßB is an intracellular event, and liver and prostate cells have the capacity to synthesize these subunit proteins, we concluded that the localization of activin ßA-, ßB-, and ßC-subunit proteins within the same cells serves as an indication that dimerization may occur to form activin dimers. Although there has been speculation about the role of activin C in the regulation of liver growth, our data raise several questions because we failed to demonstrate changes in the proliferation of prostate or liver tumor cell lines. Therefore, it is possible that the formation of activin heterodimers may have significant consequences on regulation of the levels and/or bioactivity of other forms of activin, A, B, and AB.

We have used specific monoclonal antibodies to demonstrate that ßA, ßB, and ßC proteins localize to a specific cell type, basal epithelial cells, in the human prostate. The prostatic epithelium consists of secretory and basal epithelial cells and neuroendocrine cells. In nonmalignant epithelium, ßA-subunit protein was expressed in secretory and basal epithelial cells, whereas ßB-subunit protein was expressed predominantly in basal epithelial cells (21). ßC localization was restricted to basal, but not secretory, epithelial cells. Similarly, in the liver, ßA- and ßB-subunits are localized to hepatocytes (35), and we found ßC-subunit immunoreactivity in these cells. Previous studies have shown the expression of activin subunits mRNAs in liver and human prostate cells (19, 37). These data demonstrate that the liver and prostate cells have the capacity to synthesize a full repertoire of activin dimers, i.e. activin A, B, and AB, as well as new forms of activin C and the heterodimers AC and BC. The immunolocalization of activin subunits does not demonstrate whether the subunits are in monomeric or dimeric form. At present, dimers of the known activin proteins are detected by specific assays, but the development of new assays is required to measure novel activins and discriminate between bound and free activin ligands in tissues and fluid.

The localization of activin subunit proteins in different components of the prostatic stroma is a novel observation that warrants further investigation, as most studies have focused on luminal secretory epithelial cells and human prostate epithelial tumor cell lines. Although the inhibin {alpha}-subunit was localized to basal cells (38), it is unlikely that inhibin C is formed, as our data indicate that ßC did not dimerize with the {alpha}-subunit in vitro. In malignancy, inhibin {alpha}-subunit expression is down-regulated (38), but tumor cells retain the capacity to synthesize activins, as ßA-, ßB-, and ßC-subunit proteins were localized to tumor cells from men with high grade cancer.

Activin A (like TGFß) inhibits the growth of hepatocytes in primary culture (26, 39) and induces apoptosis (40, 41). Similarly, the effect of activin A and activin B on LNCaP cells and primary cultures of human prostate epithelial cells is to inhibit proliferation and/or induce apoptosis (19, 36, 42, 43, 44, 45). In contrast, activin C failed to exert an effect on the proliferation of the human prostate tumor cell line, LNCaP, or the human liver tumor cell line, HepG2, when added alone or before activin A. This indicates that activin C does not interfere with the activin A receptor and/or its signaling pathway after binding to the receptor. We hypothesize that the formation of heterodimers of ßC with either ßA or ßB may be important in regulating the intracellular levels and/or biological activity of other activins by the following proposed mechanism. The heterodimerization of ßC may result in the formation of activin AC and BC that are biologically inactive, or it may alter the levels of dimeric activin A, B, and AB. These hypotheses remain to be tested; the bioactivity of activin AC or activin BC is not known, and the formation of activin AC and BC heterodimers requires the development of new assay methods to reveal the consequence on the level and activity of activin A, B, or AB in liver and prostate.

Using partial hepatectomy as a model system to study liver growth and regeneration, a change in the temporal expression of activin ßA and ßC mRNAs has been described in mouse and rat liver. The basal expression of activin ßA mRNA in liver is low, and activin ßC mRNA is abundantly expressed in intact liver (45). Activin ßA mRNA was up-regulated at 12–24 h after partial hepatectomy (16), whereas levels of ßC mRNA expression are down-regulated at approximately 12 h (15, 16). The reciprocal expression of ßA- and ßC-subunit mRNAs led to the hypothesis that activin ßC was a putative liver chalone, which was transiently down-regulated within hours of hepatectomy to allow the proliferation of hepatocytes. To restrict further growth of the liver beyond the regeneration phase, mRNA for ßA increased, whereas that for ßC decreased to restore the growth inhibitory effects of activin A on newly formed hepatocytes (15). A more recent study describes the initiation of DNA synthesis in the intact rat liver after the infusion of follistatin, an activin antagonist. In this study, after the addition of follistatin, ßA-subunit expression was reduced at 24 h and increased up to 120 h (the time when liver weight returned to control levels), whereas ßC-subunit mRNA expression was abundant and increased 120 h after follistatin infusion. Collectively, these results suggested that ßc-activin dimers have a role in the tonic inhibition of cell growth of hepatocytes (45). However, our results failed to show any specific biological activity in vitro of activin C on HepG2 cells. Similar to the scenario in prostate tissues, the role or action of novel activin ßC heterodimers and the consequence of ßC-subunit on the levels of dimerized activin A in the liver remain to be established.

In summary, these data demonstrate the localization of ßA-, ßB-, and ßC-subunits to specific cell types in human liver and benign and malignant prostate. Activin A is a potent regulator of apoptosis in liver and prostate, but there is no known biological role for activin C. However, there is a relationship among ßA, ßB, and ßC subunits, whereby heterodimers can be formed in vitro. The immunohistochemical localization of activin subunits in specific cell types suggests that activin C and its heterodimers are formed in these cells, and this deserves further investigation. The consequences of the formation of new activin heterodimers may have a significant impact in regulating the levels or the biological activity of activins A, B, and AB. Therefore, our data demonstrate a need for reevaluation of the known roles of activin A in liver and prostate.


   Acknowledgments
 
The authors thank Petra Niclasen for her excellent technical assistance, Ghanim Almahbobi for help with preparation of the manuscript, and Moira O’Bryan, Julie Brauman, Jens Pohl, and Jeffrey Bonadio.


   Footnotes
 
1 This work was supported by a Program Grant from the National Health and Medical Research Council of Australia and by the Medical Research Council and the European Commission [Contract BMH4-CT98–9574 (DG12-SSMI); to N.P.G.). Back

Received December 8, 1999.

Revised June 16, 2000.

Accepted September 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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