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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 2 508-513
Copyright © 1997 by The Endocrine Society

Reproductive Endocrinology

In Vitro Expression of Endothelin-1 (ET-1) and the ETA and ETB ET Receptors by the Prostatic Epithelium and Stroma1

Ewan S. Grant, Tom Brown, Alan Roach, Brent C. Williams and Fouad K. Habib

University Department of Surgery (E.S.G., F.K.H.)/Medicine (B.C.W.), University of Edinburgh, Western General Hospital, Edinburgh, Scotland EH4 2XU; and Rhône-Poulenc Rorer Limited (T.B., A.R.), Dagenham Research Centre, Dagenham, Essex, United Kingdom RM10 7XS

Address all correspondence and requests for reprints to: Dr. E. S. Grant, University Department of Surgery, Western General Hospital, Crewe Road South, Edinburgh, United Kingdom EH4 2XU.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RT-PCR analysis of total RNA prepared from the prostate cancer cell lines DU145 and PC3 and from primary epithelial cells indicated the presence of endothelin-1 (ET-1) messenger RNA (mRNA). Neither the LNCaP cell line nor primary prostatic stromal cells possess ET-1 mRNA transcripts. Seventy-two-hour-conditioned media derived from DU145, PC3, and primary epithelia contain immunoreactive ET concentrations equivalent to 0.814 ± 0.048, 0.330 ± 0.050, and 0.856 ± 0.055 fmol/mL/106 cells after 72 h, respectively. Basal immunoreactive ET secretion was exhibited by LNCaP (0.029 ± 0.009 fmol/mL/106 cells after 72 h) and stromal cells (0.067 ± 0.007 fmol/mL/106 cells after 72 h). Examination of ETA and ETB gene expression by RT-PCR demonstrates that ET receptor mRNA is almost completely undetectable in the prostate cancer cell lines. Both ETA and ETB mRNAs are detectable in primary cultures of prostatic epithelia and stroma. Competitive binding studies demonstrate a single class of binding site in both primary benign epithelia (dissociation constant = 1.85 x 10-10 mol/L; maximal binding capacity = 2.7 x 104 binding sites/cell), and stroma (dissociation constant = 1.93 x 10-10 mol/L; maximal binding capacity = 3.7 x 105 binding sites/cell). Use of selective ET receptor antagonists confirmed that the predominant stromal receptor subtype expressed in vitro is ETB. This receptor seems not to be coupled to mitogenic pathways because no growth response to exogenous ET-1 or cooperation between ET-1 and bFGF could be observed. Similarly, no effect of ET-1 or the ET-converting enzyme inhibitor, phosphoramidon, on benign epithelial cells could be observed over a 4-day period.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ENDOTHELIN (ET) family comprises three structurally related peptides, ET-1, ET-2, and ET-3 (potentially derived from a common ancestral gene), whose primary action is as potent vasoconstrictors but which display a multitude of functions in a diverse array of tissues (1, 2, 3, 4, 5). All of the ETs are initially synthesized as larger propeptides (big ETs), which are converted to mature active peptides by the action of the ET-converting enzymes (ECEs) (6). Cellular responses to the activated ETs are mediated by two cell-surface ET receptors that have been cloned and sequenced and designated ETA and ETB (7, 8, 9). ETA has high and equivalent affinity for ET-1 and ET-2 but displays little cross-reactivity with ET-3, whereas the ETB receptor is nonselective and exhibits similar affinities for the three ET subtypes. Both receptor subtypes are believed to be coupled to phosphatidylinositol (4, 5)-bisphosphate hydrolysis via G protein-coupled phospholipase C and to the generation of inositol phosphates and diacylglycerol, ultimately resulting in increases of intracellular Ca2+.

Recently, much attention has focused upon the role of the ETs within the human prostate with particular reference to benign enlargement. Previous studies in the prostate suggest that ET-1-like immunoreactivity is associated primarily with the glandular epithelium, with ETA and ETB receptors predominating in the stromal and glandular components, respectively (5, 10). This ET receptor distribution produces potent contraction of isolated prostatic tissues in response to exogenous ET-1 that cannot be attributed to {alpha}1-adrenergic pathways, to PG synthesis, or to dihydropyridine-sensitive calcium channels (5). These observations, coupled with significant increases in both ETA and ETB within benign prostatic hyperplasia (BPH) (11), have earmarked ET-1 as potential mediator of the muscle tension symptomatic of BPH.

Observed mitogenic responses to ET-1 in vitro within a number of systems, including human carcinoma (12), rat vascular smooth muscle cells (13), and mouse embryonic fibroblasts (14), have prompted conjecture that prostatic ETs may possess similar properties. It was the purpose of the present study to assess the growth factor potential of ET-1 within the prostate and, in particular, whether it is involved in the stromal expansion associated with BPH. Established prostatic cancer cell lines and primary prostate cultures were characterized with respect to the expression of ET-1 and the ETA and ETB ET receptors before examination of growth response to exogenous ET-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Establishment of primary prostatic epithelial and stromal cultures

Prostatic tissues were obtained from patients undergoing transurethral resection of the prostate to relieve urethral obstruction. Randomly selected prostatic chippings from each specimen were evaluated histopathologically for verification of their benign status.

Primary cultures of prostatic epithelium and stroma were established as previously described (15). Verification of the cultures as epithelial or stromal was accomplished by means of both immunohistochemistry and phase contrast microscopy (15).

Cell lines

The DU145, LNCaP (clone FGC [fast growing colony]), and PC3 prostatic carcinoma cell lines were obtained from the American Type Culture Collection. The DU145 cell line was cultured in DMEM supplemented with 10% FBS. LNCaP were maintained in RPMI1640 containing 10% FBS. The PC3 cell line was maintained in Ham’s F12 medium supplemented with 7% FBS.

Preparation of total RNA

Total cellular RNA was extracted using the acid-guanidium-phenol-chloroform method of Chomczynski and Sacchi (16).

RT-PCR amplification

Single-stranded complementary DNA (cDNA) was synthesized from 5 µg total RNA using a commercial Reverse Transcription kit (Promega Corporation, Madison, WI) and the manufacturer’s protocol. Twenty percent of the cDNA was removed for PCR.

The PCR reactions were performed in 1x Reaction Buffer [Promega Corporation; 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH8.8)], 1.5 mmol/L MgCl2 and 0.1% Triton X-100 containing 0.125 mmol/L each deoxyribonucleotide triphosphate, 0.5 µg of each primer, and 1 U Taq polymerase (Promega Corporation). The primers used in the detection of human ET-1, ETA, and ETB cDNA sequences were as published by Winkles et al (17). PCR reactions also were performed using primers specific to the gene for the housekeeping protein, hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The HGPRT sense and antisense primers have the sequences 5'CTT GCT CGA GAT GTG ATG AAG3' and 5'GTC TGC ATT GTT TTG CCA GTG3', respectively. All samples were subjected to 35 cycles of PCR reaction, each cycle comprising denaturation at 94 C for 1 min, annealing at 62 C for 1 min, and primer extension at 72 C for 1 min. The ET-1 and ETA/ETB PCR products were electrophoresed on 1.2% and 2% ethidium bromide agarose gels, respectively, and visualized under ultraviolet transillumination. No DNA and no RT reactions were included in each set of PCRs to control for DNA contamination of reagents and cDNA specimens. The authenticity of all PCR products was verified by restriction analysis (data not shown).

ET RIA

ET-like immunoreactivity was determined using the ET 1–21 specific [125I] Biotrak assay system (Amersham International plc, Little Chalfont, UK). The rabbit anti-ET antibody cross-reacts fully with ET-2 (144%) and ET-3 (52%) but not with big ET (0.4%).

Cells were grown in monolayer culture to 60–70% confluence before replacing the normal growth medium with 12 mL serum-free medium. The cells were subsequently grown for 72 h at 37 C and the medium was harvested and centrifuged at 1000 x g for 10 min to remove suspended cells. The number of cells in each flask at 72 h was determined. Lyophilized medium was resuspended in PBS and acidified with an equal volume of 20% acetic acid. ET was purified by solid-phase sorbent extraction through AmprepTM C2 columns (500 mg; Amersham International plc) and the eluant dried under nitrogen and resuspended in assay buffer before RIA following the manufacturers protocol. The concentrations of immunoreactive ET (iET) are quoted in fmol/mL/106 cells after 72 h and represent the mean of three independent assays ± SE.

Specific binding of 125I-ET-1 to primary prostate cells

Primary prostatic epithelial and stromal cells were plated into 24 well plates at a density of 0.75 x 105 cells/well and 1.0 x 105 cells/well, respectively. The cells were allowed to adhere before a 20-min preincubation with serum-free medium containing 0.1% BSA. Subsequently, the cells were supplemented with 100 µL 125I-ET-1 (0.4 nmol/L) and 50 µL of increasing concentrations of unlabeled ET-1. Nonspecific binding was assessed as binding in the presence of 10-7 mol/L ET-1. The cells were incubated at 37 C for 1 h before aspirating the medium and washing the cells three times with ice-cold PBS. The plates were blotted briefly on absorbent paper before adding 0.5 mL 1% (vol/vol) Triton X-100 to each well to solubilize the cells. Cell-bound radioactivity was assessed in triplicate by {gamma} counting. Receptor subtyping in primary stromal cells was performed by including either the ETA antagonist BQ123 (1 µmol/L) or the ETB antagonist BQ788 (1 µmol/L) in the binding assay. The apparent dissociation constant (Kd) and maximal binding capacity (Bmax) were calculated by Scatchard analysis (18).

Cell proliferation

Prostatic stromal cells and epithelial cells were seeded into 96 well plates in their normal growth medium at a density of 500-1000 cells/well and 3000–3500 cells/well, respectively, and incubated at 37 C for 24 h. Stromal cell medium was replaced with phenol red-free RPMI1640 supplemented with 2.5% dextran-coated charcoal-treated FBS. Epithelial cells were supplemented with their normal medium containing transferrin (10 mg/L-1) and sodium selenite (4 ng/L-1). After a further 24-h incubation, the dose response of the cells to 0.01–100.00 nmol/L ET-1 over a 4-day period was assessed. Potential cooperation between ET-1 and polypeptide growth factors in the stromal cells was determined by incubation of the cells with 1 nmol/L ET-1 and 5.0 ng/mL-1 bFGF. The significance of endogenously produced ET-1 on benign epithelial cells was determined by treating the cells with the ECE inhibitor, phosphoramidon (1 µmol/L or 100 µmol/L). Cell proliferation was measured spectrophotometrically using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (19). Each data point represents the mean OD540 ± SE.

Statistical analysis

Statistical significance was determined using Student’s t test for comparison of two means and the Bonferroni method for comparison of a number of means against a control mean.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of ET-1 gene expression in cultured prostatic cells

Examination of total RNA isolated from prostate cancer cell lines, benign epithelial cells, and stromal cells demonstrates striking differences in the extent of ET-1 gene expression assessed by RT-PCR (Fig. 1Go). Primary epithelial cells derived from BPH specimens display high level ET-1 gene expression (Fig. 1DGo). In contrast, RT-PCR of total RNA isolated from stromal cell cultures of the same tissue sample did not generate a discernable ET-1 signal on an agarose gel (Fig. 1EGo). The DU145 and PC3 cell lines (Fig. 1Go, A and C.) produced the expected fragment of size 1097 bp. This product was undetectable in the case of the LNCaP cell line (Fig. 1BGo).



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  		Figure 1. Total RNA prepared from DU145 (A), LNCaP (B), PC3 (C), benign epithelial cells [BE] (D), and stromal cells [ST] (E) was subjected to RT-PCR using ET-1-specific oligonucleotide primers. Control PCR reactions were performed using primers for the housekeeping gene HGPRT. Amplification products were separated on 1.2% agarose gels and visualized by ethidium bromide staining. One-kilobase DNA ladders (MW) were run in conjunction.

 
Production of iET in prostatic cells

Fig. 2Go shows the levels of iET present within 72-h-conditioned medium. Benign epithelial cells release iET at a rate of 0.856 ± 0.055 fmol/mL/106 cells after 72 h. This is significantly (P < 0.05) greater than the rate of secretion observed in the stromal cell cultures, which is equivalent to 0.067 ± 0.055 fmol/mL/106 cells after 72 h. DU145- and PC3-conditioned medium contains concentrations of iET equivalent to 0.814 ± 0.048 and 0.33 ± 0.050 fmol/mL/106 cells after 72 h. This is in contrast with the relatively low rate of iET secretion observed in the LNCaP cell line (0.029 ± 0.009 fmol/mL/106 cells after 72 h).



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  		Figure 2. The amount of iET (fmol/mL-1) in the conditioned media of human prostate cells, after 72 h per million cells was determined by RIA. The values represent the mean ± SE.

 
Analysis of ET receptor expression in cultured prostatic cells

Amplification of cDNA prepared from benign epithelial cells and stromal cells, using primer pairs specific to the ETA and ETB ET receptors, generates PCR fragments of sizes 714 bp and 701 bp, respectively (Fig. 3DGo). In the DU145, LNCaP, and PC3 cell lines, a 35-cycle PCR reaction is insufficient to elicit signals corresponding to ETA and ETB messenger RNA (mRNA) on ethidium bromide gels (Fig. 3Go, A–C).



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  		Figure 3. Total RNA was prepared from DU145 (A), LNCaP (B), PC3 (C), benign epithelial cells [BE] and stromal cells [ST] (D) and was subjected to RT-PCR using ETA receptor- or ETB receptor-specific oligonucleotide primers. Control PCR reactions were performed using primers for the housekeeping gene HGPRT. The amplification products were separated on 2% agarose gels and visualized by ethidium bromide staining. One hundred-base pair DNA ladders (MW) were electrophoresed alongside the PCR products.

 
Competitive binding of 125I-ET-1 to primary prostatic cells

Fig. 4AGo demonstrates competitive inhibition of 125I-ET-1 binding in benign epithelial and stromal cells. Scatchard analysis of specific binding sites for ET-1 indicated the presence of a single class of noninteracting binding site in both cell types with apparent Kd and Bmax of 1.85 x 10-10 mol/L and 2.7 x 104 sites/cell for the epithelial cells and of 1.93 x 10-10 mol/L and 3.7 x 105 sites/cell for stromal cells. Inclusion of selective ETA and ETB antagonists at saturating concentrations within the competition studies demonstrated marked inhibition of binding by ETB-specific BQ788 in stromal cells (Fig. 4BGo). BQ123, a specific ETA antagonist, produces no such inhibition, suggesting that the receptor subtype expressed by prostatic stroma in vitro is primarily ETB.



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  		Figure 4. The ET binding characteristics of prostatic epithelia and stroma in vitro were assessed by competition of 125I-ET-1 binding by ET-1. Cultured benign epithelia (0.75 x 105 cells per well) and stroma (1.00 x 105 cells per well) were plated into 24 well plates and incubated with 0.4 nmol/L 125I-ET-1 plus unlabeled ET-1 at 1 pmol/L to 0.1 µmol/L (nonspecific binding) for 1 h at 37 C (A). The ET receptor subtype predominating on stromal cells was identified by incubation with 1 µmol/L BQ123 (ETA antagonist) or with 1 µmol/L BQ788 (ETB antagonist) (B). Bound radioligand was assessed by {gamma} counting. Each data point represents the mean ± SE of triplicate determinations.

 
Effect of ET-1 on prostatic stromal and epithelial growth in vitro

Stromal and epithelial cells supplemented with ET-1 at 10 pmol/L and 1 nmol/L over 4 days show no significant increase (P > 0.05) in cell number compared with controls (Fig 5AGo and 6AGo). The addition of 1 nmol/L ET-1 does not potentiate the stimulatory effects of 50 ng/mL-1 bFGF on stromal cells (Fig 5BGo). This lack of synergy is consistent over bFGF concentrations of 0.01 ng/mL-1 to 10 ng/mL-1 (data not shown). Phosphoramidon inhibits both the conversion of exogenously administered big ET-1 and the de novo production of ET-1 through direct inhibition of the ECE (6). Phosphoramidon at concentrations of 1 µmol/L and 100 µmol/L does not significantly alter the growth rate of benign epithelia in vitro (Fig. 6BGo).



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  		Figure 5. The effects of exogenous ET-1 on the growth characteristics of stromal cells in vitro. A, Growth response of stromal cells to ET-1 (0.01 nmol/L + 1.00 nmol/L). B, Growth profile of stromal cells exposed to ET-1 (1.0 nmol/L) plus bFGF (5.00 ng/mL-1) over a 4-day period. Proliferation was measured spectrophotometrically using the MTT assay. Each data point represents the mean OD540 ± SE of four replications.

 


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  		Figure 6. The effects of ET-1 and phosphoramidon on benign epithelial cells in vitro. A, Growth response of epithelial cells to ET-1 (0.01 nmol/L + 1.00 nmol/L). B, Growth profile of epithelial cells exposed to phosphoramidon (1.0 µmol/L and 100 µmol/L) over a 4-day period. Proliferation was measured using the MTT assay. Each data point represents the mean OD540 ± SE of four replications.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ET family latterly has received much attention from individuals studying prostatic pathology since ET-1 was first measured in extremely high amounts within the seminal fluid of vasectomized men (20). This group of 21 amino acid peptides act primarily as vasoconstrictors but have been observed to induce potent contraction of smooth muscle of nonvascular origin (3, 4, 5). These findings, coupled with proliferative responses in fibroblasts and smooth muscle cells (13, 14), have implicated ET-1 as a potential contractile agent/mitogen in BPH.

RT/PCR amplification of primary epithelial RNA using an ET-1-specific primer pair resulted in the generation of a diagnostic 1.1-kilobase DNA product clearly visible on agarose gels. In contrast, attempts to amplify ET-1 sequences from stromal cell RNA by RT-PCR failed to produce a detectable signal. Furthermore, examination of conditioned medium demonstrated that epithelial cells secrete in excess of 10 times more iET than their stromal counterparts over a 72-h period. The present study indicates that, in keeping with previous findings in vivo, expression of the ET-1 gene in vitro is primarily an epithelial phenomenon (5).

RT/PCR analysis of RNA isolated from the DU145 and PC3 prostate cancer cell lines elicited ET-1-specific signals, albeit a weak signal in the case of the latter, on agarose gels. This level of gene expression corresponded to concentrations of secreted iET equivalent to 0.814 ± 0.048 and 0.33 ± 0.050 fmol/mL/106 cells after 72 h for DU145 and PC3, respectively. Clearly, these models of malignant epithelia retain the ET secretory function of their benign counterparts. The exception to this rule is, of course, the LNCaP cell line, which, of all the prostatic epithelial cells studied, is the only one demonstrating near-basal ET-1 secretion. Indeed, in a previous study, Nelson et al (21) claim that the amount of iET secreted by LNCaP after 24 h was approximately 600 times less than that of DU145 as measured by enzyme-linked immunosorbent assay. Interestingly, LNCaP is the only cell that presents a truly androgen-responsive phenotype. DU145, PC3, and primary benign epithelial cells either exhibit decreased or no expression of the androgen receptor gene with the consequence that they do not possess functionally significant levels of the receptor protein and do not display growth response to dihydrotestosterone (22).

Both the ETA and ETB ET receptors have been identified within prostate tissues, the former predominating in the fibro-muscular stroma, the latter in the epithelium (10, 11, 21). In cultures of benign stroma and epithelia, we have observed mRNA transcripts for both the ETA and ETB receptors by RT-PCR. None of the prostate cancer cell lines used in this study express the ET receptor genes to the same extent as the primary cells, based upon a complete inability to detect any receptor-specific PCR products on agarose gels. Competitive binding studies demonstrated a single class of saturable ET-1-binding site of almost identical dissociation constant on both epithelial (nH = 0.999; Kd = 1.85 x 10-10 mol/L) and stromal cells (nH = 0.995; Kd = 1.93 x 10-10 mol/L). The number of binding sites per cell type is, however, very different. The stromal cells employed in this study exhibit a Bmax of 3.7 x 105 sites/cell, which is more than 10 times the level of maximal binding in the epithelial cells (Bmax = 2.7 x 104 sites/cell). Closer examination of stromal cells, using receptor-specific antagonists, showed that the receptor subtype characterizing these cells and, by extrapolation, the epithelial cells is ETB. Obviously, the ETA mRNA transcripts detected by RT/PCR do not contribute to the receptor pool of either cell type.

Because both cultured epithelial and stromal cells, in keeping with cells in vivo, possess receptors for the ETs, is it possible that epithelially produced ET-1 behaves in a paracrine or autocrine fashion within the prostate gland? The finding presented here would not tend to support this notion. Benign epithelial cells supplemented with ET-1 do not exhibit any alteration of normal growth patterns. The influence of endogenously produced ET-1 on epithelial growth was examined also through treatment of the cells with pharmacological concentrations of the ECE inhibitor phosphoramidon, and it was found that the removal of activated ET-1 again does not change the inherent growth characteristics. Primary prostatic stromal cells grown on collagen gels have been shown to induce contraction of the gel support when supplemented with ET-1 (23). This contractility, which is presumably attributable to the smooth muscle cells present within these cultures (24), is not paralleled by mitogenic responses. ET-1 administration, whether alone or in combination with a known mitogen in bFGF (24, 25), does not significantly stimulate stromal cell growth.

In summary, we have demonstrated that primary cultures of the prostate mimic the prostate in vivo, in that both benign epithelial cell cultures and stromal cells possess receptors for ETs; however, production of ET-1 is exclusively epithelial. The ETB receptor expressed by both stroma and epithelium does not seem to be coupled to intracellular mitogenic pathways, suggesting that the role of the ETs in the prostate is not as autocrine or paracrine growth factors.


   Footnotes
 
1 This work was supported by Rhône-Poulenc Rorer Ltd. and CaPCURE Foundation. Back

Received May 23, 1996.

Revised August 30, 1996.

Accepted October 9, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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