Evidence of estrogen receptors in the trigone area of human urinary bladder

IS.pp. I? to320,

Journal of Steroid Bi0rhsmitr.t Vol. 3 Printed in Great Britain. All rights reserved

198

I

00?2-4731~81/010317-o4u)z.oo1o Copyright 0 1981 Pergamon Press Ltd

EVIDENCE OF ESTROGEN RECEPTORS IN THE TRIGONE AREA OF HUMAN URINARY BLADDER

tFacultt

SIMONESARA* and PIERRE M. MARTR~ *Centre L&n Bernard, 28, rue Laennec, 69373 Lyon cedex 2, and de Mtdecine Marseille Nerd, Bd Pierre Dramard, 13015 Marseille, France

SUMMARY The induction by estrogens of the maturation of vaginal epithelial cells has been one of the first steroid effects to be evidenced on a target tissue in women. It has been shown that the trigone, a limited area of the urinary bladder formed from the same embryonic bud as the anterior vagina, represents a distinct anatomic zone of the bladder and demonstrates similar cytological modifications under hormone stimulation to vaginal cells. Estrogen receptors were determined by incubating cytosol fraction with [3H]estradiol ([“HI-ES) 30nM + 10-O M inert estradiol, in the presence of IOOnM Sa dihydrotestosterone (DHT) at 2O’C for 5 hr and unbound radioactive hormone was removed by adsorption on Dextran coated charcoal. We found that only the cytosol from the trigone mucosa contained estradiol receptors: in ten biopsies from females, nine contained receptors in variable amounts from 12 to 120 fmol/mg protein. In eight male cases, only one was positive. On the contrary, no specific binding was found in 20 biopsies from non trigone area, either in. male or female. Specific binding protein had a limited capacity; labeling at 5 nM [‘HI-E2 concentration was completely inhibited by addition of lOO-fold excess of inert E2, R-2858 and DES. Addition of estradiol and estrone at higher concentration was less effective. R-5020. testosterone, DHT, dexamethasone did not compete. Labeled bound protein analysed on sucrose gradient presented a labeled peak with a 8s sedimentation coefficient, displaced by an excess of inert ligand. These data indicate that an anatomically well defined area of the urinary bladder presents biochemical properties of a steroid target tissue and account for its cytological modifications induced by hormone stimulation.

TISSUE MATERIAL AND METHODS

INTRODUCTION

Several lines of data suggest that all or part of the mucosa of the human urinary bladder is hormone dependent. The embryonic development of the bladder is double: dome, fundus and lateral parts derive from the urogenital sinus. The trigone is formed from the same embryonic bud as the anterior vagina in women, and remains able to respond to steroid hormones. It has been observed that the maturation of vaginal epithelial cell induced by estrogens is simultaneously reproduced in trigonal cells. Vaginal and urinary smears are both used for evaluating hormonal status [4]. In the case of experimental animals bladder carcinoma has been induced by hormonal stimulation in rats, under the same conditions as those used for mammary carcinogenesis [l, 4,5]. Furthermore, chemical induction of bladder carcinoma may be modulated by hormones [2,9, lo]. It is likely that steroids per se are not responsible for carcinogenesis but may bring target tissue to a stage more sensitive to carcinogenic events [3]. The aim of this study was to investigate if bladder epithelial cells contained steroid receptors required to mediate hormonal effects on target tissue.

Mucosa samples were obtained from male or female patients operated upon for non-neoplastic urologic disease. After pathological examination, the epithelial layer was carefully peeled from the underlying connective tissue before being frozen in liquid nitrogen. Hormonal compounds 17jLestradiol. diethylstilboestrol, Sa-dihydrotestosterone, estrone, estradiol, dexamethasone and progesterone were purchased from Roussel Uclaf (Romainville, France). The non radioactive compounds R2858 and R5020 were gifts from J. P. Raynaud (Roussel Uclaf). The labeled [2,4(n j3H]-estradiol (45 to 50 Ci/mmol SA) was purchased from CEA (Gif-sur-Yvette, France) and the tritiated compound C3H]-R2858 and [3H]-R5020 (45_50Ci/mmol SA) were gifts from J. P. Raynaud (Roussel Uclaf). Labeled steroids were freshly purified by paper chromatography before use. Frozen tissue was pulverized with a Thermovac apparatus (Thermovac Industries Corp., Copiague. NY) and allowed to thaw on ice in TEG buffer (0.01 M Tris-HCl, 1.5 mM HCl, 1.5 mM EDTA and 0.5 mM dithiothreitol, glycerol 10% v/v; pH 7.4). The homogenization was completed with a Polytron

317

SIMONE

318

SAEZ

and PIERREM.

MARTIN

Table I. [‘HI-R2858 and [‘HI-R5020 binding assay of bladder mucosa cytosol from various area. in females and males Bound Estradiol Case No. Fern&s (A) Trigone area

(B) Limit of the trigone (0 Dome

(fmoles/mg prot.)

1 2 3 4 5 6 7 8 9 10 11 12 10 Cases

84 24 120 21 18 39 20 12 0 0 0 0 0

Sucrose gradient (S)

Bound R5020 (fmolesimg prot.1

8 Displaced 4 Non displaced 8 Displaced 8 Displaced 8 Displaced 4-8 Displaced 8 Displaced 4 Displaced 0 8 Displaced ND ND

22 0 42 12 0 12 0 0 0 0 0 0 0

8 ND

ND 0 0 0

Males

(A) Trigone area (B) Dome Tissue underlying the mucosa

I Case 7 Cases 10 Cases 10 Cases

30 0 0 0

Estradiol and progesterone receptor data on bladder mucosa.

PT-10. Cytosol was obtained by ultracentrifugation of 106,OOOg for 60 mn and its protein concentration was determined by the method of Lowry. the homogenate

DETERMINATION

OF ESTROGEN AND

PROGESTERONE BINDING

For estrogen receptor (ER) measurement, two sets of cytosoi aiiquots were incubated in triplicate with 30nM labeled estradiol and 100 nM DHT. One set received lob6 M inert estradiol for the evaluation of non-specific binding. Incubation was carried out at 20°C for 5 h according to the technique of Katzenellenbogen[6,21]. Samples were then chilled and stripped of unbound hormone and of hormone bound with low affinity, by the addition of dextrancoated charcoal (2.5 vol. of 0.5% charcoal, 0.05% dextran T70 in TEG buffer), and were incubated for 30 min. and centrifuged at 2000 g for 15 mn. Bound radioactivity was counted in the supernatant. This method measured total cytosol estradiol binding sites. Sucrose gradients (5-20:(A) were prepared in homogenization buffer. Aliquots of cytosol (2OOgl) were incubated with saturating amount of [‘HI-estradiol, in the absence or presence of inert ligand. treated with charcoal as above. [“Cl-ovalbumin prepared according to the method of Rice and Means[12] was used as a marker of sedimentation coefficient. Specificity of estrogen binding was assessed by using 5 nM labeled R2858 in the absence or presence of various inert steroids, to test their competing capacity for estradiol binding sites.

For progesterone receptor (PGR) measurement two sets of triplicate samples were incubated with 10 nM labeled R5020 in the presence of 10e6 M cortisol. One set received 10e6 M inert R5020 for evaluation of non-specific binding. They were incubated at 4°C for 12 h. Radioactivity bound to receptor sites was counted in the supernatant after charcoal treatment of cytosol(2.5 vol. of a suspension of 0.25% w/v charcoal and 0.025% w/v dextran T-70) and immediate centrifugation at 4°C. This method measured free and endogenously occupied progesterone receptor sites. RESULTS

Ten specimens were obtained from female subjects. Results of receptor determinations are indicated on Table 1: nine contained specific binding protein in various amount from 12 to 120 fmol/mg protein. The gradient elution profile of these samples shows a radioactive peak which is displaced by the addition of a 100 excess of inert hormone. The sedimentation coefficient of this peak was 8S (Fig. 1A) in six of the nine cases. In one case, the profile indicated on Figure 1B was obtained: one 8s peak is small, and coexists with a 4s peak. While both are displaced by inert hormone. In two cases, bound radioactive hormone migrated with a 4s sedimentation coefficient and was not inhibited by competing inert hormone. Eight specimens taken in the trigone area of male subjects were precessed. Only one showed specific binding for estradiol and exhibited an 8s peak in sucrose gradient specimens. Figure 2 shows the values obtained from competition experiments using trigone mucosa incubated at

319

Steroid receptors in btadder mucosa

Fig. 1. Sucrose gradient uhracentrifugation of bladder mucosa cytosol after in vitro incubation with [‘HI-estradiol. (A) elution profile shows a major 8S labeled peak inhibited in the presence of an excess inert hormone. (B) elution profile shows two peaks inhibited in the presence of an excess inert hormone.

[3H]-R2858 5 nM in the presence of various steroids and synthetic compounds. The maximum competitive effect is obtained with estradiol and diethyl stilbestrol (DES). R5020 and DHT exhibit a small effect when added at very high concentratjon. Dexamethasone does not compete. In the nine female specimens which contained ER, four were positive for FGR. RSOZObinding was negative in all cases negative for ER. None of the male samples contained PGR. Binding determination performed on samples not taken from the trigone area were all negative for estradiol and progesterone. The underlying muscle and connective tissue remaining after peeling off the mucosa was saved in four cases and processed in the same way as the other samples.

Fig. ?. Ability of various inert hormones

None untied one.

receptors for estradiol or progester-

Dl!%XJSSION

Data from different approaches confirm that normal epithehal mucosa of a restricted zone of the human urinary bladder contains estradiol receptors. Specific binding sites for progesterone also have been found in some of the specimens which contained estradiol receptor. In other tissues which are known targets for estrogens, a strong relationship has been estab~ish~ between estrogen activity and the presence of progesterone receptor sites.

to compete with [“HI-R2858 for specific binding sites in trigone mucosa.

SIMONESAEZ and PIERREM. MARTIN

320

Both the ontogenesis of the trigone area and the biological effects of estrogen and progesterone on other tissue arising in the same embryonic bud. sup port the demonstration of the existence of steroid receptor in this epithelial tissue. Almost all the female specimens coming from this anatomical area contained measurable amounts of ER while only one in nine male samples was positive. This discrepancy is likely to be due to different development of ER during the adult age: stimulated by estrogenic hormones, inhibited by androgen influence. The modification of epithelial cell structure and growth which express the endocrine variations during the menstrual cycle are known to be mediated by steroid receptors. In the bladder mucosa two characteristics are associated. It is target for steroid hormones and is also the site of carcinoma of various origins. In man, most of its tumors are likely to be stimulated by chemicals and experimental tumors evidenced the role of hormones. The association of these two characters is widely documented in other organs like breast. The role of chemicals in the epidemiology of bladder cancer and the existence of steroid receptors in this epithelial tissue are similar to observation of pharyngo-larynx tumors [ 123. Acknowledgement-This work has been supported in part by a grant from the Federation Francaise des Centres de Lutte Contre le Cancer. REFERENCES 1. Angrist A., Capurro P. and Moumgis B.: Studies on squamous metaplasia in rat bladder. II effects of estradiol and estradiol and hexoestrol. Cancer Res. 20 (1960) 568-572.

2. Capurro P., Angrist A., Black J. and Moungis B.: Studies in squamous metaplasia in rat bladder: I effects of hypovitaminosis A. foreign bodies and methyl cholanthrene. Comer Res. 20 (19601 563-567. 3. Dao T. L.: Inhibition of tumor induction in chemical carcinogenesis in the mammary gland. Prog. hp. Tumor Res. 14, (1971) 58-88. 4. Dunning W. F.. Curtis M. R. and Segaloff A.: Strain differences in response to diethylstilboestrol and the induction of mammary gland and bladder cancer in the rat. Cancer Res. 5 (1947) 511-521. 5. Dunning W. F.. Curtis M. R. and Segaloff A.: Strain differences in response to estrone, and the induction of mammary gland adrenal, and bladder cancer in rats. Cancer Res. 13 (1953) 147-152. 6. Katsenellenbogen J. A., Johnson H. J. and Carlson K.: Studies on the uterine, cytoplasmic estrogen binding protein. Thermal stability and ligand dissociation rate. kn essay of empty and-filled &es by exchange. Biochemistry 12 (1973) 40924099. 7. Lencioni L. i.: i’lirocytogramme. (Edited by S. A. Malone) Paris. 1975. 8. Milgrom E., Thi L., Atger M., and Baulieu E. E.: Mechanism regulating the concentration and the conformation of $ogest&one receptor(s) in the uterus. J. biol. Chem. 248 (1977) 6366-6374. 9. Noble R. L., H’&hedhka B. C. and King D.: Spontaneous and estrogen-produced tumors in Nb rats and their behaviour after transplantation. Cancer Res. 35 (1975) 766-777. 10. Noble R. L.: Progressive hyperplastic lesions of the bladder uro-epithelium after hormone stimulation in Nb rats. Incest. Ural. 18, 387-391. 11. Pavlik E. J. and Coulson P. B.: Modulation of estrogen receptors in four different target tissues: differential effects of estrogen versus progesterone. J. steroid Biohem. 7 (1976) 369-376. 12. Rice J. and Means A.: Radioactive labeling of proteins in citro. J. biol. Chem. 246 (1971) 831-836. 13. Saez S. and Sakai F.: Androgen receptors in human pharyngo-laryngeal mucosa and pharyngo-laryngeal epithemioma. J. steroid Biochem. 7 (1976) 919-921.