Androgen-dependent synthesis of a prostatic binding protein by rat prostate

Journul q/ Steroid Pergamon Press

Biochemisrr):

Vol.

I I. pp.209

to 213

Ltd 1979. PrInted in GreatBritain

ANDROGEN-DEPENDENT SYNTHESIS OF A PROSTATIC BINDING PROTEIN BY RAT PROSTATE W. Laboratorium Laboratorium

HEYNS,

B.

voor Experimentele voor Biochemie,

PEETERS. J. Mous,

W. ROMBAUTS

and P. DE

MCKIR

Geneeskunde. Katholieke Universiteit Leuven. Minderbroedersstraat IO. B-3000 Leuven. Belgium and Katholieke Universiteit Leuven. Gasthuisberg, B-3000 Leuven. Belgium SUMMARY

The non-specific binding of androgens in rat prostatic “cytosol” is due to a prostatic binding protein, which has been purified and characterised. This protein is secreted by the prostate. It contains about 37, of carbohydrate but no phosphate and consists of two subunits (MW 21.000 and 18,500). Each subunit dissociates further in the presence of dithiothreitol into two different components. One of these components is present in both subunits, the other one is specific for each subunit. In cytosol prepared from the ventral prostate of intact male rats prostatic binding protein contributes 46.4% of the total protein. Three weeks after castration this level decreases to 3.9% of the total protein. Androgens increase this concentration, whereas oestradiol, progesterone and cyproterone acetate lack this property. The in vitro synthesis of prostatic binding protein by prostatic slices and the corresponding mRNA activity of prostatic RNA are also regulated by androgens. Consequently. it is likely that androgens influence the level of this protein by a transcriptional regulation mechanism.

INTRODUCTION

steroids (e.g. See-dihydrotestosterone, androstenedione, pregnenolone) are bound to a similar degree. The binding increases very strongly after delipidation. Indeed, this procedure resulted in an increase of the apparent concentration of the binding site from 0.2 to 3.1 pmol per g of protein, while the apparent affinity went up from 0.3 to 1.7 PM- ‘. Moderate heating also has a favourable effect. In opposition to the low steroid specificity, the charcoal-resistant, high capacity type of binding appears to be a specific property of the ventral prostate, since no similar binding was found in a number of other organs. Furthermore, it could be shown that it was due to a component with well defined physicochemical characteristics (Table I) such as size, electrophoretic behaviour. ammonium sulphate precipitation and elution from DEAE cellulose columns. By use of these properties the binding protein has been purified and characterised further.

The rat ventral prostate constitutes an interesting model system for the study of the mechanism of action of androgenic hormones [I]. Indeed, this organ is strongly dependent on these hormones for its normal development and function. Androgen-receptor binding and nuclear uptake of the receptor have been well documented in this gland and there also is strong indirect evidence that the action of the hormone occurs, at least in part, by transcriptional regulation of mRNA’s coding for specific proteins [2,3]. Until recently, however, no major androgen-dependent proteins, which could offer a suitable endpoint for such studies, had been described. In a study of the nonreceptor binding of androgens in the rat prostate we found that this binding was due to a major prostatic binding protein [4,5]. We could show that this protein is androgen-dependent and that androgens also regulate the levels of the corresponding mRNA, measured by in vitro translation C6.73. In this communication we will summarize our data on this subject.

Purification and structure of prostatic binding protein

An efficient step for the purification of prostatic binding protein consists of DEAE cellulose chromatography with KCI gradient elution [8,9]. Electrophoretically pure prostatic binding protein is obtained by repeating this procedure in combination with gel filtration. Purified prostatic binding protein contains about 3% of carbohydrate but no sialic acid. Its phosphorus content is below 0.1%. Its amino acid composition has been determined [9]. Since 0.84 binding sites for pregnenolone were estimated per mole of purified protein, this probably indicates that each protein contains a single binding site. During polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. the protein dis-

RESULTS

Prostatic binding protein as a binding protein

In addition to the androgen receptor, the rat ventral prostate also contains androgen binding activity of a different nature, characterized by a high capacity and a low affinity. In a detailed study of this binding activity [4, S] we could show that, in spite of this low affinity, the bound steroid dissociates slowly at low temperature, providing a convenient method for its measurement. This binding is not specific for androgenic hormones, but a number of nonpolar 209

210

W. HEYNSet al. Table I. General properties of prostatic binding protein [5,9] Property

Method

Molecular weight Sedimentation coefficient Isoelectric point Salt precipitation Carbohydrate content

Gel filtration Ultracentrifugation Isoelectric focusing Ammonium sulphate Anthrone reaction

sociates in an F subunit (MW 18,500) and an S subunit (MW 21,000). These subunits have also been purified by hydroxylapatite chromatography and differ in amino acid composition and in isoelectric point [9]. Steroid binding appears to be localized on the S subunit. After reduction of disulphide bridges each subunit dissociates further in two different components. The F subunit dissociates into component 1 (MW 8500) and component 3 (MW 14,000) and the S subunit into component 2 (MW 10,500) and component 3 (MW 14,000). These components have been purified by hydroxylapatite chromatography and have different amino acid compositions and antigenic properties. Immunological techniques

Immunization of rabbits with purified prostatic binding protein by the Vaitukaitis technique [lo] of multiple intradermal injections yielded a specific antiserum [8]. which produced a simple immunoprecipitation band against total prostatic cytosol and against purified prostatic binding protein when using the Ouchterlony technique. This antiserum has been used for the quantitative assay of the protein by means of radial immunodiffusion [8,1 I], for the study of the localisation of prostatic binding protein by immunofluorescence and for quantitative and specific immunoprecipitation of the protein or related peptides after in vitro incorporation studies of C3H]leutine [7, 121. Antisera were also raised against the three purified components of prostatic binding protein. Each of the antisera reacted with its own antigen giving an immunoprecipitation line when using the Ouchterlony technique, but did not react with the other two antigens (unpublished results). Occurrence of prostatic binding protein

As already indicated by the binding studies, prostatic binding protein appears to be a specific component of the ventral prostate. This specific localisation is confirmed by immunofluorescent examination of tissue slices [6] and by immunological measurement of the binding protein in cytosol prepared from various organs [6,8]. Using these techniques prostatic binding protein could not be detected outside the prostate gland, results being negative for testes, epididymis, seminal vesicles, lung, submaxillary gland, uterus, and a number of other organs. On the other hand, this protein is present in prostatic fluid, extracts from the copulation plug and also in male rat urine.

Result 51,000 3.1 s 5.2 S(t70”/, saturation 3.2%

These observations are easily explained by an exocrine secretion of this protein by the prostate. This is confirmed by its typical localisation within the gland, with maximal immunofluorescence al the apical forder of the epithelial cells and within the acinar lumen. Effect of age on the concentration of prostatic binding protein[6]

Prostatic binding protein is present in low, but detectable, amounts (0.4 kg per prostate; 1.4% of cytosol protein) in the prostate of S-day-old rats. Its concentration starts to increase from day I5 on and approaches adult levels (7.9mg per prostate; 46.4% of protein) at day 88. Thereafter high concentrations of this protein are maintained during adult life at least to an age of 1 year. Effect of hormonal treatment on the concentration of prostatic binding protein [6]

The marked changes in the concentration of prostatic binding protein during the first months of life suggest that this parameter might be androgen-dependent. This was confirmed by castration of adult rats. Indeed, this procedure resulted in a marked decrease of the binding protein. During the first 4 days after castration this,decrease was proportional to the loss of total protein. Thereafter, prostatic binding protein disappeared more rapidly than the other proteins and its percentage dropped from 43.0% in intact adult rats to 10.1% 7 days after castration, 5.5% after 10 days and finally to 3.9% after 21 days. The effect of castration is abolished by administration of androgens (Table 2). For testosterone propionate a marked stimulation is observed at a daily dose of 5Opg, and maximal stimulation at 5OOpg daily. Oestradiol benzoate (100 pg daily). pfGgesterone (2 mg daily) and cyproterone acetate (10 mg/daily) do not increase the level of prostatic binding protein in castrated rats. Cyproterone acetate, however, antagonizes the stimulatory effect of androgens. There is some evidence that glucocorticoids have a less pronounced regulatory effect on prostatic binding protein. Indeed, 7 days after adrenalectomy the relative concentration of prostatic binding protein shows a moderate decrease from 29.6 f 3.1% to 18.4 f 5.7%. After administration of dexamethasone (SOpg daily for 7 days) the relative concentration returns to initial the levels (29.9 + 3.5”/,; mean f S.D.).

211

Synthesis of binding protein by rat prostate Table 2. Effect of castration and hormone treatment on the relative concentrations protein [6]

Dose Condition Control Castration

Treatment

Prostatic binding protein (% of cytosolic protein)

(mg/day)

Oil Testosterone propionate Testosterone propionate Oestradiol benzoate Progesterone Cyproterone acetate Testosterone propionate and cyproterone acetate

>

of prostatic binding

43.0 + 7.4 f 18.0 f 35.2 f 9.6 It 5.7 + 6.6 +

0.05 0.5 0.1 2.0 10.0 0.5 10.0 >

9.2 3.1 2.6 3.5 6.6 3.5 3.9

19.3 * 2.2

Adult male rats (98-105 days) were castrated and after IO days IO consecutive daily injections of the hormone were administered. Means and standard deviations are given for each treatment group (n = 4). Prostatic binding protein was measured immunologically and expressed as Lowry protein using purified prostatic binding protein as reference.

Effect of hormonal of prostatic

binding

treatment

on the

in vitro synthesis

protein

In order to evaluate the hypothesis that the observed changes in concentration reflect changes of synthesis, the incorporation of [3H]-leucine by prostate minces was studied during in vitro incubation. In these experiments the incorporation of C3H]-leutine into prostatic binding protein was measured by immunoprecipitation and expressed as y0 of total incorporation. This fraction decreased from 31.8% in intact rats to 6.9% seven days after castration. Administration of testosterone propionate produced a marked increase of this fraction after only 1 day; precastration levels (22.9%) were approached after 5 days of treatment. Translation

of prostatic

mRNA

in a cell free

germ system and in Xenopus’oocytes

wheat

[I21

Addition of RNA, extracted from ventral prostates of intact adult male rats, to a cell free system derived from wheat germ produced a marked stimulation of peptide synthesis [7]. A large fraction (29.7%) of the formed polypeptides was immunologically related to prostatic binding protein. Electrophoretic analysis of the immunoprecipitate revealed, however, that these polypeptides were somewhat different in size from those found in the native protein. A likely explanation for this discrepancy in size might be that the peptides

formed in the wheat germ system are precursor proteins, which need further processing into the native products. For this reason, the same mRNA was also injected into Xenopus’oocytes [12] which are known to perform processing in addition to protein synthesis. In this experiment the polypeptides formed in the immunoprecipitate were of exactly the same size as the native products. Furthermore, the oocytes not only produce the 3 peptide components of prostatic binding protein of exact size, they also assemble these components by means of disulphide bridges to form the S and F subunits observed in the native protein. Effect, of hormonal of prostatic

RNA

treatment

on the mRNA

As already mentioned, RNA extracted from the ventral prostate of adult male rats has a marked mRNA activity, and a large fraction of this mRNA codes for peptides immunologically related to prostatic binding protein (Table 3). After castration the total mRNA activity diminishes; moreover, there is a specific decrease of the mRNA for prostatic binding protein. Qpposite changes occur during treatment with androgens. This effect is already measurable after 1 day of androgen treatment, when both the total mRNA activity of prostatic RNA and the fraction of this mRNA which is related to prostatic binding protein have nearly doubled.

Table 3. Influence of androgens on the mRNA activity of prostatic RNA [7] mRNA Activity for prostatic binding protein Condition No treatment 6 Days castration 6 Days castration + 3 days of androgen

activity

[7-J

Total mRNA activity % Of control

% Of total activity

oA Of control

100 19.3

29.7 4.9

100 3.1

60.6

18.8

38.3

The mRNA activity of 3Opg RNA extracted from rat ventral prostate was measured in a cell free wheat germ system. The results obtained for intact rats served as controls (100%).

212

W. HEYNSrt (11. DISCUSSION

The nonspecific binding activity for androgens in cytosol of rat prostate has been known for a long time, but it has not been studied in great detail. Our investigations have shown that this binding is due to a characteristic component, which we have purified and called prostatic binding protein. On the basis of its binding properties, it is conceivable that this protein corresponds to the cc-protein described by Fang and Liao[ 131 and to the pregnenolone “receptor” described by Karsznia et a/.[14]. It also seems likely that the large increase of androgen “receptor” binding observed by Ichii[lS] upon delipidation is actually due to this prostatic binding protein. A closer look at the structure of this protein reveals that it is composed of two different subunits, which we have called subunit F and subunit S as a function of their elcctrophoretic mobility. Upon reduction of disulphide bridges each subunit dissociates further into two different components. One of these components (component 3) is present in both subunits and contains carbohydrate. The other component is different for each subunit. Consequently, prostatic binding protein contains three different components, which differ not only in size but also in amino acid composition and in immunological properties. Recently a few other reports have described the existence of major proteins in rat prostatic cytosol. Lea et a/,[163 described a prostatein, the tentative structure of which bears some likeness to that of prostatic binding protein. Although sufficient data are lacking for a conclusive comparison, both proteins may be identical. The androgen-dependent /I- and y-proteins, described by Parker et al.[17], on the other hand, probably correspond to components 3 and 1, respectively. As far as one may conclude from the investigations, prostatic binding protein appears to be a specific component of the rat ventral prostate [6]. Its localisation within the prostate and also its presence in prostatic fluid and in male rat urine indicate that it is secreted by this gland. This observation makes it unlikely that its primary function is intracellular. More probably it plays a role in the reproductive process. The binding properties of this protein, which are not restricted to hormonal steroids, suggest a transport function, but it is not certain that this binding is of functional importance. Other possibilities are that this protein favours fertilization or interferes with the process of the formation of the copulation plug. In the absence of any clear indication it may be difficult to establish its function. The marked effects of androgenic hormones on the rat ventral prostate are reflected in the variations of prostatic binding protein produced by these hormones. After castration the level of this protein decreases sharply and rises again after androgen administration. Since these changes are quantitatively much more important than the changes in total protein, androgens have a specific effect on the concentration

of prostatic binding protein. A similar specific effect is also observed during the study of its synthesis or of its mRNA activity [7]. The latter finding, which agrees well with the observation by Parker[18] that androgens influence an abundant class of mRNA in this organ, indicates that transcriptional regulation is involved. It cannot be excluded, however, that proliferation of certain epithelial cells, which is also promoted by androgens, amplifies these changes. It seems unlikely though that the marked changes of mRNA activity and protein synthesis observed after 1 day of androgen treatment, without a concomitant increase of prostatic weight and DNA content, are explained by cell proliferation. In the rat seminal vesicles, androgens also favour the synthesis of certain basic proteins which are clearly different from prostatic binding protein [ 191. In this case, however. the effect on these proteins is not specific since they are stimulated only to the same degree as the total protein [20]. Consequently, the action of androgens on rat prostate or seminal vesicles may be fundamentally different. One might then ask the question whether this difference is an intrinsic property of the gespective epithelial cells or whether it reflects the heterogeneity of the respective cell populations. Acknow/~~~e,#ents-The authors appreciate the valuable help of D. Bossyns, M. Hertogen, A. M. Ickroth and V. Feytons. This study was financed in part by FGWO grants 20486 and 20168 and by grant OT/V1/34 of the “Onderzoeksfonds K.U.Leuven”.

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16.