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Original Article |
Production
Austrian Academy of Sciences (H.R., G.U., K.M., P.B.), Institute for Biomedical Aging Research, Innsbruck A6020, Austria; Ludwig Boltzmann Institute for Andrology and Urology (E.P.), Hospital Lainz, Vienna A1130, Austria; and Institute of Medical Chemistry and Biochemistry (M.H.), Leopold-Franzens-University of Innsbruck, Innsbruck A6020, Austria
Address all correspondence and requests for reprints to: Peter Berger, Ph.D., Austrian Academy of Sciences, Institute for Biomedical Aging Research, Peter Mayr Strasse 4b, Innsbruck, A6020, Austria. E-mail: peter.berger{at}oeaw.ac.at.
Abstract
Age-related development of benign prostatic hyperplasia is an important health issue in developed countries. The histopathogenetic hallmark of this disease is the increase in relative and absolute numbers of smooth muscle cells (SMC). Glycoprotein hormone 
-subunit (GPH) is expressed in the human prostate, and, because of its structural similarities to other cystine knot growth factors, it has been considered to have growth regulatory functions of its own. Primary cell cultures allowing for selective cultivation of prostatic epithelial cells, fibroblasts, and SMC were established. Directed trans-differentiation and cellular homogeneity was pursued by confocal scanning laser microscopy with cell type-specific markers. GPH production by these cells was assessed by immunofluorimetric assays. Its predominant source was young fibroblasts, whereas replicative senescent fibroblasts, SMC, and control fibroblasts from foreskin did not produce significant amounts. Functionally, GPH reduced growth of stromal cells at concentrations of 10 and 100 nmol/liter as shown by cell viability assays. It is concluded that histogenetic reorganization over the adult lifetime, guided by endocrine factors like steroid hormones together with senescence of fibroblasts, leads to a decreased production of growth inhibitors, such as GPH, favoring proliferation and the development of benign prostatic hyperplasia.
BENIGN PROSTATIC HYPERPLASIA (BPH) occurs in more than 70% of men aged 70 yr or older. However, not all afflicted men show signs of urinary flow obstruction because the presence of such clinical symptoms is not necessarily related to the size of the individual prostate, but rather to the proportion and composition of its volume occupied by stromal tissue (1, 2, 3). In asymptomatic hyperplasia, the average ratio of stroma to epithelium is 2.7 vs. 4.6 in cases of obstruction (4), an observation indicating a significant contribution of the prostatic stroma in developing BPH accompanied by infravesical obstruction. Nodular hyperplasia, as observed in BPH, results from focal proliferation of the stromal and glandular-epithelial compartments whereby the stromal proliferation growth precedes the glandular epithelial enlargement.
Of the two predominant cell types in the stroma of the prostate, which are smooth muscle cells (SMC) and fibroblasts, SMC are suggested to play a key role in this pathomorphogenetic process (5). Significant age-related increases in the ratio of SMC organelles to cytoplasmatic volume (1) and increases in rough endoplasmatic reticulum and free ribosomes (6) suggest higher SMC cellular metabolic activity with age and in BPH compared with young and normal prostates. Furthermore, the relative numbers of fibroblasts to SMC vary with age, especially with the development of BPH (7). The density of SMC in prostatic stroma increases in the first year of life, followed by a progressive decline in childhood and puberty and an increase after puberty (8). A possible explanation for increased numbers of SMC in BPH is the increased level of TGF-ß in BPH tissue (9), which was shown to trans-differentiate prostatic fibroblasts into SMC (10).
The prostatic stroma and the epithelial cells are sources of growth factors that form a network responsible for local growth regulation complementing androgens of systemic origin. A possible member of this network is the glycoprotein hormone -subunit (GPH), the common glycopeptide chain to the heterodimeric glycoprotein hormone family, which comprises human (h) chorionic gonadotropin (CG), hFSH, hTSH, and hLH. Their functions as heterodimers are well known, but there is a paucity of data regarding the functions of the uncombined subunits. These are assumed to influence cell growth, because they are highly similar in structure to growth factors such as TGF-ß1, platelet-derived growth factor BB, and nerve growth factor, all of which possess a characteristic cystine knot in the center of the molecule (11). Such a function has been shown for the ß subunit of hCG (hCGß), which stimulates growth of the bladder cancer cell line T24 (12), very likely due to an antiapoptotic effect (13). However, signal transduction pathways are still unclear. The implication in local growth regulation is further underlined by their expression in a variety of organs (14, 15, 16, 17).
The stroma of a variety of organs plays a key role in growth regulation of both stromal and epithelial cells. With respect to the pathogenesis of prostatic proliferative diseases, we investigated which stromal cells produce GPH and whether growth of stromal cells is influenced by this molecule. Because age is one of the most important risk factors for the development of BPH and prostate cancer (PCa), expression of GPH in young and senescent fibroblast was studied as well, and tissue specificity was assessed by comparing fibroblasts from the prostate and the foreskin.
Materials and Methods
Selective cell culture of prostatic cells
Human prostate epithelial cells were established from patients with hormonally untreated prostate cancer. Informed consent was obtained from all patients before surgery. After radical prostatectomy, two tissue wedges were removed from areas showing no histological signs of malignancy. These explants were minced into organoids of 1 mm3 and seeded on collagen I-coated plates (Becton Dickinson and Co., Vienna, Austria) in RPMI 1640 (BioWhittaker, Inc., Verviers, Belgium) containing 10% bovine calf serum (A-2151, Hyclone Laboratories, Inc., Logan, UT), 10 mg/ml penicillin, 100 U/ml streptomycin, and 10 mg/ml L-glutamine (PSG, PAA Laboratories, Vienna, Austria).
Prostatic stromal cells were obtained by seeding the organoids on uncoated plastic cell culture plates in the same medium described above. Log-phase growing fibroblasts were differentiated into SMC by graded amounts of TGF-ß1 (1, 0.1, or 0.01 ng/ml; Sigma, St. Louis, MO) in RPMI 1640 containing PSG and 1% BSA for 72 h to obtain SMC. In parallel, cells were treated with 1 ng/ml basic fibroblast growth factor (Sigma) to maintain their fibroblastic phenotype.
Foreskin fibroblasts were obtained by mincing samples from circumsections and seeding these on plastic cell culture plates in the medium described above.
Immunofluorescence and confocal laser scanning microscopy
Cultured cells were fixed in 4% paraformaldehyde on ice for 10 min and permeabilized in 0.2% Triton X-100 (Sigma) for another 10 min on ice. Nonspecific binding was blocked by 1% BSA in PBS for 1 h at room temperature. Primary antibodies were diluted to 1 µg/ml in 1% BSA/PBS and added to the cells for 90 min at room temperature. Those antibodies were directed against either cytokeratin 8/18 (diluted 1:25; Autogen-Bioclear, Wiltshire, UK) to identify epithelial cells or prolyl-4-hydroxylase (diluted 1:50; DAKO Corp., Vienna, Austria), SMC--actin, or SMC-myosin (both diluted 1:50; both from Sigma) to distinguish fibroblasts from SMC. Secondary antibodies, i.e. tetramethyl-isothiocyanate [TRITC-conjugated F(ab')2] and fluorescein-isothiocyanate [FITC-conjugated F(ab')2]-labeled rabbit antimouse Ig (DAKO Corp.), diluted 1:100 in 1% BSA/PBS, were added for another hour at room temperature. Nuclei were counterstained with SYTOX nucleic stain (1:4000) or with TO-PRO-3 iodide nucleic acid stain (1:400; both from Molecular Probes, Inc. Leiden, The Netherlands). After several washing steps with PBS, cells were embedded in 50% glycerol and viewed in a confocal laser scanning microscope.
Production of GPH by prostatic cells
Both SMC and fibroblasts were seeded at densities of 60,000 cells per well on 24-well plates (Becton Dickinson and Co.). Duplicate wells were stimulated with 1 µmol/liter 8-bromo-cAMP (Sigma) for 4 d. For analysis of GPH in conditioned media, ultrasensitive time-resolved immunofluorometric assays (IFMAs) were used, as described elsewhere (18, 19). Briefly, 96-well microtiter Maxi Sorb Surface plates (Nunc, Roskilde, Denmark) were coated with a monoclonal antibody (mAb) specific for free GPH (code: INN-hCG-72). Nonspecific binding sites were blocked with 1% BSA/PBS. After several washing steps, hormone standards (first World Health Organization reference reagent for immunoassay of hCG; 99/720; Ref. 20) or supernatants diluted 1:2 in assay buffer were added and allowed to react on an orbit shaker (300 rpm, 90 min, 20 C). Thereafter, europium-labeled detection mAb (code: INN-hFSH-158) was incubated for 60 min, and after intensive washing, enhancement solution was added (Pharmacia, Upsala, Sweden). Time-resolved fluorescence was measured for 1 sec in a fluorometer (1232 Delfia-fluorometer; Wallac, Inc., Turku, Finland). GPH concentrations were correlated to cell numbers. The experiments were repeated with cells from six different prostates.
Staining of senescence-associated ß-galactosidase (SA-ß-Gal)
Fibroblasts were passaged 5 or 35 times (P5 or P35), corresponding to approximately 10 or 60 population doublings, respectively. After reaching 80% confluence, cells were fixed (2% formaldehyde, 0.2% glutaraldehyde in PBS) for 5 min at room temperature and thereafter rinsed several times in PBS. To measure SA-ß-Gal activity, cells were incubated in staining solution [4.2 mmol/liter citric acid, 12.5 mmol/liter sodium phosphate, 158 mmol/liter sodium chloride, 0.21 mmol/liter magnesium chloride, 2.21 mg/ml potassium ferrocyanid, 1.68 mg/ml potassium ferricyanide, 1 mg/ml X-Gal (pH 6.0)] for 24 h at 37 C. For control purposes, cells were stained at pH 4.0. Cells were washed and embedded in PBS, viewed in an inverted transmission-microscope, and documented by a digital imaging system (Nikon-Coolpix 990, Nikon, Melville, NY).
Cell proliferation assay
A total of 4,000 prostatic stromal cells were seeded into each well of a 96-well plate in RPMI 1640, containing PSG and 3% bovine calf serum, and left for adherence overnight before the tissue culture medium was changed to medium containing GPH (NIH CR 123) at concentrations ranging from 0.1–100 nmol/liter. Specificity was shown by preimmunoabsorption of 100 nmol/liter GPH with a specific mAb recognizing uncombined subunit only (code: INN-hCG-80). This process was controlled by mock absorption with an isotype-matched mAb directed against the ß core-fragment of hCG (hCGßcf; code: INN-hCG-104). After further incubation for 48 h, a tetrazolium salt (WST-1, Roche Molecular Biochemicals, Mannheim, Germany) was added to the cells at a 1:10 dilution. After 2 h, OD was determined at 450 nm in a colorimeter.
Western blot
SMC were harvested after 72 h of incubation with TGF-ß1 (Sigma) at concentrations of 0.01, 0.1, and 1 ng/ml and lysed in a lysis buffer [10 mM Tris-Cl, 0.2% Triton X-100 (pH 7)]. Ten micrograms of total protein were boiled for 10 min in denaturing sample buffer consisting of 10% glycerol, 1% SDS, 1% ß-mercaptoethanol, 10 mmol/liter Tris-HCl (pH 6.8), and 0.01% bromphenol-blue; separated on a 4–20% gradient Tris-glycin gel; and transferred on an Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA). After the membrane was blocked in 3% skimmed milk powder dissolved in PBS, it was probed with a mouse-mAb directed against SMC--actin (0.1 µg/ml, Sigma) for 1 h. The membrane was then incubated with a 1:10,000 dilution of a horseradish peroxidase-conjugated goat-antimouse IgG1 (Pierce Chemical Co., Rockford, IL) for 1 h. After several washing steps, a chemiluminescent substrate (SuperSignal, Pierce Chemical Co.) was added to the membrane, which was then exposed to the ECL Hyperfilm (Amersham, Buckinghamshire, UK).
Results
Prostatic cell types
Cells growing out on collagen I-coated plates stained positive for K 8/18 (Fig. 1B
), which is typical for prostatic luminal cells. Stromal cells were obtained by seeding organoids on uncoated plastic cell culture plates. All cells expressed prolyl-4-hydroxylase (Fig. 1A) and very low amounts of SMC-specific--actin (Fig. 1C), identifying them as collagen-producing fibroblasts.
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production between fibroblasts and SMC, fibroblasts were converted into SMC by stimulation with various concentrations of TGF-ß1 for 72 h (Fig. 1
, C and E; Refs. 10 and 21). This trans-differentiation was controlled by the up-regulation of SMC-specific--actin in a Western blot (Fig. 1D). Marker expression of the cells is summarized in Table 1.
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production in prostatic cell compartments
As assessed by IFMA (assay measuring range from 0.009–40 ng/ml), stromal cells proved to be the main source of GPH, whereas K 8/18-positive epithelial cells did not produce detectable amounts (Fig. 2). This was confirmed by stimulating GPH production with 8-bromo-cAMP at a concentration of 10-6 mol/liter, thereby provoking transcription of GPH through phosphorylation-by protein kinase A-and homodimerization of the cAMP-responding element binding protein, as described earlier (22).
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production in prostatic stroma: fibroblasts vs. SMC
The stroma of the prostate consists mainly of fibroblasts and SMC. Because these cell types play different roles in the pathogenesis of BPH, GPH production was assessed in both (Fig. 3). Basal production of GPH (4.1 ± 1.6 ng/ml) was well detected in fibroblasts, but minimally so in SMC (0.2 ± 0.027 ng/ml). Furthermore, induced production was significantly increased in fibroblasts (32 ± 2.16 ng/ml) and was still low in SMC (0.65 ± 0.069 ng/ml).
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production by fibroblasts during in vitro senescence
To investigate changes of GPH production in young and old prostatic fibroblasts, cells were cultured for either P5 or P35. Cellular senescence was controlled by SA-ß-Gal staining at pH 6, which is known to be a marker for senescence in fibroblasts (23). Because proliferation of aging fibroblasts slows down, GPH production was correlated to cell number. SA-ß-Gal-negative young fibroblasts from P5 produced more GPH (basal, 410 ng/ml x cells; induced, 1300 ng/ml x cells) than SA-ß-Gal-positive aged cells from P35 (basal, 38 ng/ml cells-1; induced, 54 ng/ml cells-1; Fig. 4, A and B). Interestingly, in aged fibroblasts, GPH expression could not be induced to the same extent as in young fibroblasts.
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Growth of SMC was assessed by WST-1 tetrazolium reduction assay after incubation with GPH for 48 h (Fig. 5). The result showed a 20% reduction in growth at a concentration of 100 nmol/liter GPH. For specificity control, added GPH was removed from the medium by GPH-specific immunoabsorption, the effectiveness of which was controlled by IFMA. This restored normal growth of the cells, whereas mock absorption with an isotype-matched nonsense antibody (anti-hCGßcf) did not. This experiment was repeated with cells from three different prostates.
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production
To assess whether GPH regulation and production are limited to prostatic fibroblasts, we compared them to human foreskin fibroblasts. Parallel to prostatic fibroblasts, duplicate wells were left either unstimulated or stimulated with cAMP for 72 h, and GPH was measured by IFMA. Basal production of GPH by prostatic fibroblasts was basal 4.1 ± 1.6 ng/ml, and induced production was 32 ± 2.16 ng/ml. In contrast, basal production of human foreskin fibroblasts was only 0.2 ± 0.03 ng/ml, and induced production was only 0.6 ± 0.04 ng/ml (Fig. 6).
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BPH is a disease characterized by an initial stromal proliferation. Histologically, this proliferation is accompanied by an increase of fibromuscular stroma, including SMC, which are considered to play a key role in the pathogenesis of BPH.
Mechanisms increasing SMC-to-fibroblast ratios with age could be increasing trans-differentiation of fibroblasts into SMC or selective proliferation of SMC.
In this context, we investigated the role of GPH, which is expressed in the human prostate (15) and has been assumed to have growth regulatory functions due to its 3-D similarities to other known growth factors (11). Such functions have already been shown for the free hCGß (12, 13). Our study reported herein indicates that GPH is produced in a cell- and tissue-specific manner, preferentially by young, and to a reduced degree by old, prostatic fibroblasts. GPH specifically inhibits growth of stromal cells of the prostate, an age-related decrease in production that leads to waning of inhibitory capacity and stromal proliferation.
As shown herein, GPH is produced in the stroma of the prostate. Highly differentiated secretory epithelial cells, positive for luminal cell markers K 8/18, did not produce significant amounts. The latter is in agreement with the literature in which GPH expression in the epithelial compartment of the prostate has been shown by immunohistochemistry to be limited to neuroendocrine cells (24). The finding that GPH is also produced and differentially regulated in prostatic stroma is new. The stroma of the prostate consists mainly of two cell types, fibroblasts and SMC; basal and induced GPH expression was thus investigated in both. Fibroblasts were trans-differentiated into SMC by stimulation with TGF-ß1 (10, 21). This process of trans-differentiation could be traced by markers, such as SMC--actin (Fig. 1), prolyl-4-hydroxylase or SMC-myosin (Table 1; Refs. 10 and 25). Measurement of GPH in the supernatant of unstimulated and cAMP-stimulated fibroblasts and SMC clearly showed that fibroblasts are the main source of stromal-derived GPH in both basal and induced expression. However, whether GPH is directly linked to or is just accompanying the trans-differentiation process cannot be answered. Because aging is the most important independent risk factor for developing BPH, changes in GPH expression were investigated during replicative senescence of prostatic fibroblasts. It appeared that, during the replicative active life period of the fibroblast, GPH expression declined when comparing young, i.e. SA-ß-Gal-negative, to old, i.e. SA-ß-Gal-positive, fibroblasts of three different prostates (Fig. 4).
SMC play a key role in the pathogenesis of BPH (5). The mechanism that increases the number of these cells, therefore, might be crucial for the development of this proliferative disease with age and may be indirectly dependent on the age of the fibroblast. We therefore speculate that the decrease in GPH production could, in part, explain the increased numbers of SMC observed in the prostate with age (7). The growth inhibitory effect of GPH on SMC could be lost due to fibroblast aging resulting in increased numbers of SMC. Unfortunately, as with hCGß, the signal transduction pathway of GPH remains unclear, because neither cAMP metabolism nor tyrosine phosphorylation could be unequivocally observed (data not shown). This also makes the binding of GPH to the G protein-coupled LH/hCG receptor, activating adenylyl cyclase resulting in an increased production of intracellular cAMP concentration upon ligand binding. Possible interactions with other proteins are currently being investigated by yeast two hybrid assays.
The different expression patterns of young and old stromal cells could account not only for proliferation of SMC but also for the concomitant basal cell hyperplasia. Such differences have been shown in mammary glands in which old stromal cells accelerated epithelial cell growth, whereas young stromal cells did not (26, 27). The proliferation of prostatic cells could further be supported by prolactin (PRL), which is known to stimulate growth of prostatic cells (28, 29). As shown previously, SMCs are the main source of PRL in the prostate (30). The increase in numbers of SMC with age would also favor growth of prostatic cells via this hormone.
To gain insight into tissue specificity, GPH production was compared between prostatic and foreskin fibroblasts. It appeared that the latter did not produce comparable amounts of GPH, suggesting that GPH-dependent growth regulation is not a general mechanism, but rather specific for some, or at least one organ. Such tissue-specific differentiation of fibroblasts is not surprising because in mammary glands, for example, only mammary-derived fibroblasts can induce mammary morphogenesis, whereas fibroblasts from other tissues cannot (27).
It is concluded that GPH plays a role as a growth inhibitory factor in the local growth regulation in the stroma of the prostate. Its loss during aging due to decreased numbers of fibroblasts and replicative senescence might contribute to the age-associated proliferative disease, BPH. The role of the endocrine system could be the initiation of histological reorganization over life. Several studies showed differentiation of prostatic fibroblasts into SMC upon stimulation with steroid hormones, especially estradiol and cortisol and, to a lesser extent, androgens (25, 31, 32). The importance of estradiol is underlined by its relative age-related increase compared with androgens (33). Thus, a histogenetic reorganization favoring the development of SMC results in decreased fibroblast-derived growth inhibitors, such as GPH. Due to the subsequent increase in numbers of SMC, the production of SMCderived stimulators of growth, such as PRL, is also markedly increased. The resulting imbalance within the microenvironment favors growth and does not directly depend on the endocrine system, but on the altered histological composition of the stroma (Fig. 7). The conservation of these histological changes in prostatic stroma could also account for incomplete efficacy of BPH treatment at the endocrine level.
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Acknowledgments
We thank Dr. Evelin Hütter for providing human foreskin fibroblasts and Ms. Barbara Witting for her excellent technical assistance.
Footnotes
This project was supported in part by the Austrian Science Fund (Project 13652-GEN). H.R. is the recipient of a Hans and Blanca Moser Foundation scholarship.
Abbreviations: BPH, Benign prostatic hyperplasia; CG, chorionic gonadotropin; GPH, glycoprotein hormone -subunit; h, human; hCGß, ß subunit of hCG; hCGßcf, ß core-fragment of hCG; IFMA, immunofluorometric assay; mAb, monoclonal antibody; P5, passage 5; P35, passage 35; PCa, prostate cancer; PRL, prolactin; PSG, penicillin-streptomycin-L-glutamine; SA-ß-Gal, senescence-associated ß-galactosidase; SMC, smooth muscle cells.
Received April 16, 2002.
Accepted August 7, 2002.
References
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-subunit in a healthy elderly population selected according to the SENIEUR protocol. Analyses with ultrasensitive time resolved fluoroimmunoassays. Mech Ageing Dev 71:223–233[CrossRef][Medline]
-chain-immunoreactive endocrine cells in prostate and cloacal-derived tissues. Arch Pathol Lab Med 111:836–840[Medline]
1-adrenoceptor antagonists on cultured prostatic smooth muscle cells. Prostate Suppl 9:34–41[Medline]
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