Homologous up-regulation of the prolactin receptor in rat prostatic expiants

Moleculur and Cellular Endocrinolo~, Elsevier Scientific Publishers Ireland.

46 (1986) 53-51 Ltd.

MCE 01482

Homologous

up-regulation

of the prolactin

receptor in rat prostatic

H. Rui ‘, I. Brekke I, P.A. Torjesen

explants

2 and K. Purvis ’

’ Institute of Pathology, The Natronal Hospital, and ’ Hormone Laboratory, Aker iJmuersit_v Hospital, Oslo (Norwa_y) (Received

Key words: prolactin;

prolactin

receptor;

29 November

rat ventral

1985; accepted

prostate;

28 February

Leydig cell tumour;

explant;

1986)

up-regulation;

down-regulation

Summary Exposure of explants of rat ventral prostates and a rat Leydig cell tumour to ovine prolactin for 20 h prolactin to an extent and in a caused alterations of the subsequent membrane binding of “‘I-human direction dependent on the dose of hormone used. Low prolactin concentrations (l-10 pg/2 ml) were associated with an increase in binding (up-regulation) which was 75% in the case of the prostatic tissue and 500% in the case of the tumour tissue above control levels. Higher concentrations caused a dose-dependent decrease in binding to below control levels (down-regulation), alterations which could not be explained by receptor occupancy. Time studies with an up-regulatory dose of hormone (3 pg/2 ml) indicated that the effects of prolactin on its receptor did not begin to become manifest until after 6-12 h of culture. The results suggest that homologous up-regulation of prolactin binding may be a general feature of prolactin target organs and that explant cultures of prostatic tissue may provide a convenient model for exploring its mechanisms.

Introduction The existence of prolactin receptors on rat prostatic membranes is well documented (Aragona and Friesen, 1975). Although a great deal of evidence is available supporting a role for prolactin in prostatic physiology (e.g. Rui et al., 1985), the intracellular mechanisms which are activated by hormone binding and which mediate this influence on prostatic function are still largely hypothetical (Kelly et al., 1984). An additional source of controversy concerns the influence of prolactin on its membrane receptors subsequent to the binding process. A large number of protein hormones (see review Tell et al., 1978) precipitate a net loss of their respective receptors after binding (downCorrespondence to: Dr. Ken Purvis, Institute Rikshospitalet, 0027 Oslo 1 (Norway).

of Pathology,

0303.7207/86/$03.50

Publishers

0 1986 Elsevier Scientific

Ireland,

regulation). In contrast, evidence for a clear down-regulatory influence of prolactin is less forthcoming, although one research group has reported the phenomenon in rat liver and rabbit mammary glands (Dijane et al., 1979). On the other hand, an increase in the binding of prolactin to rat liver membranes and mammary gland membranes (up-regulation) has been observed after in vivo exposure to the hormone (Dijane and Durand, 1977; Manni et al., 1978). Moreover, although at least 3 reports could neither detect a down-regulatory nor an up-regulatory influence of prolactin on the rat prostate receptor (Kledzik et al., 1976; Barkey et al., 1979; Amit et al., 1983) others have subsequently testified that an up-regulation could be demonstrated under conditions of chronic hyperprolactinaemia or after treatment with large doses of ovine prolactin in vivo (Dave and Witorsch, 1985). One explanation for the Ltd.

54

difficulty in clearly establishing such regulatory mechanisms in the prostate may be related to the confounding effects of androgen on the organ. To rule out this possibility, studies of prolactin-receptor interaction were performed on prostatic explants cultured in medium up to 24 h after removal. For comparison, similar studies were performed on Leydig cell tumour cells which have also been shown to contain prolactin receptors (Erichsen et al., 1984). Materials and methods Preparation of the explants Adult male Sprague-Dawley rats (Mollegaard Breeding Centre, Skenved, Denmark) aged 140160 days were sacrificed by cervical dislocation under ether anaesthesia. The ventral prostate lobes from each animal were divided into 6-8 culture dishes (Costar, MA, U.S.A.; 3.9 cm diameter) prefilled with medium (2 ml; Eagle’s minimum essential, Flow Laboratories, U.K.) containing ovine prolactin (31 IU/mg; Sigma Chemical Co., MO, U.S.A.), and minced immediately. This procedure was then repeated with the ventral lobes of a second animal which were divided into the same culture dishes. Each culture dish thus contained prostatic tissue (approximately 200 mg) from 2 animals equivalent to approximately one-sixth or one-eighth of the total ventral prostate. This enabled 6 or 8 observations to be made on the same prostatic pools. In the prostate studies all of the culture series were carried out in quadruplicate, i.e. using the prostates from 8 rats. This procedure was carried out under sterile conditions at room temperature. 5 ml of an antibiotic-antimycotic mixture (Gibco Laboratories, Ohio, U.S.A.) containing penicillin (10000 units/ml), streptomycin base (10 mg/ml) and fungizone (25 pg/ml) were added per litre medium as a preservative. In one study, explants from a transplantable Leydig cell tumour (Erichsen et al., 1984) were also exposed to various prolactin concentrations, in triplicate cultures. After mincing, the tissues were incubated at 37°C in a 5% CO,/95% 0, atmosphere. At the end of the incubation period the tissue and medium were transferred to tubes and centrifuged at 1000 x g for 5 min at room temperature. The supernatant was discarded and the pellet frozen in an

ethanol-solid CO, bath. The tissue was stored at -70°C and prolactin binding was carried out within 2 days. Prolactin binding assay After thawing, the tissue was homogenized on ice in 5 ~01s. (5 ml/g tissue) of 4 M MgCl and incubated for 5 min to facilitate dissociation of bound prolactin from the receptors, as described elsewhere (Kelly et al., 1979). It was then diluted with 20 ~01s. of assay buffer (phosphate-buffered saline, pH 7.4 with 1 mM Ca2+ and 1 mM Mg’+). filtered through a nylon filter (pore size 100 pm) and centrifuged at 20000 x g for 30 min. The pellet was resuspended in assay buffer and centrifuged once more. The final pellet was resuspended in 5 ~01s. of assay buffer containing 0.1% bovine serum albumin and 0.1% neomycin. Triplicate aliquots (50 ~1; 150-200 pg membrane protein) of this suspension were then equilibrated with 15-20000 cpm of ‘251-human prolactin (spec. act. 50-90 pCci/pg) for 16 h at 22’C in the presence or absence of a lOOO-fold concentration of unlabelled hormone (ovine prolactin, Sigma Chemical Co.). The final incubation volume of 215 ~1 included 15 ~1 human serum. After the incubation, the tubes were centrifuged at 5000 X g at 2-4°C for 30 min, washed twice with the same buffer, and the radioactivity in the remaining pellets was determined in a gamma spectrometer with an efficiency of 70%. Details of the validation of the receptor assay for the rat prostate have been presented elsewhere (Charreau et al., 1977). Dose-response studies Prostatic explants were exposed to various concentrations of ovine prolactin (0, 1, 10. 50, 100 and 500 pg/2 ml) for 20 h at 37°C. On another occasion explants of Leydig cell tumour were incubated with approximately the same concentrations of hormone (0, 1, 3, 10, 100 and 500 pg/2 ml) for the same length of time. Time-response studies In a preliminary study prostatic explants were incubated in the absence or presence of a constant amount of ovine prolactin (3 pg/2 ml) for different times (0, 6, 12 and 24 h). On the basis of the results from this study a second experiment was

55

carried out with the same dose of prolactin but with incubation periods between 12 and 24 h (12, 16, 20 and 24 h). In the latter case control dishes were incubated for 12 and 24 h without added hormone. Results Fig. 1 shows the effect of a 20 h incubation period with increasing concentrations of ovine prolactin on ventral prostate and Leydig cell tumour explants in vitro. An up-regulatory effect was associated with concentrations of prolactin l-10 pg/2 ml. In the prostatic tissue this increase represented 75% of the control binding expressed per mg membrane protein. However, as judged from the shape of the curve a greater increase could have been anticipated with doses between 1 and 10 pg/2 ml. The apparently large, within-dose

PROSTATE

f

2.0

LEYDIG

P ‘0

variation of the prostatic explants shown in Fig. 1 reflected differences in the general levels of binding and the degree of up-regulation between the various animals. Nevertheless, in all cases a clear biphasic response could be demonstrated. A twoway analysis of variance confirmed a significant increase in binding over control levels at the 1 pg (P < 0.01) and 10 pg/2 ml (P < 0.05) doses of hormone, respectively. In the case of the Leydig cell tumour prolactin significantly elevated binding above control levels with 1 pg (P < O.OOl), 3 pg (P < 0.01) and 10 pg/2 ml (P < 0.05) doses, respectively. In the case of the Leydig cell tumour, the recorded increase in binding was more marked (up to 500% of the control level) and appeared to be more sensitive to the prolactin concentration than the prostatic explants. Raising the concentrations of prolactin beyond the levels causing upregulation caused a dose-dependent reduction in binding to below controls in both prostate and Leydig cell tumour tissues. The time-response profile of prolactin binding in prostatic explants incubated with and without an up-regulatory dose of ovine prolactin (3 pg/2

CELL

TUMOUR 1.5

p”

oL

;

6 INCUBATION

PROLACTIN

CONCENTRATION

(pgl’bl)

Fig. 1. Effects of exposure of prostatic and Leydig cell tumour explants to various concentrations of ovine prolactin for 20 h. The values represent geometric means of prolactin binding obtained from 4 individual tissue cultures, each consisting of one-sixth of a ventral prostate from 2 animals. Vertical bars indicate SE. In the case of the Leydig cell tumours, values represent geometric means from binding analysed on triplicate explant cultures, and vertical bars represent SE. The prolactin concentrations are expressed as pg/2 ml incubation medium.

1;

16 TIME

2b

2;

(hrs)

Fig. 2. Prolactin binding in prostatic explants during a 24 h incubation period with (O -0) and without (O0) a constant dose of ovine prolactin (3 pg/2 ml). Values represent geometric means of 4 individual explant cultures, each consisting of one-eighth of a ventral prostate from 2 animals. Tissues from each animal served as control for the effects of hormone treatment. Vertical bars indicate SE. Inset shows the results of a second experiment with measurements after 12, 16, 20 and 24 h. Values represent geometric means of prolactin binding in 4 individual explant cultures, and vertical bars SE.

ml) is shown in Fig. 2. In control tissues a gradual loss of receptors occurred over the 24 h observation period to a level approximately 40% of the preincubation value. In contrast, exposure to prolactin appeared to prevent this loss by stimulating prolactin binding after 6 h to a level after 24 h which was approximately double that of the control value (P < 0.05by one-way analysis of variance). A confirmatory study was conducted which focused on the period between 12 and 24 h after hormone exposure. On this occasion, a similar pattern of activation of prolactin binding sites was observed (Fig. 2, inset). In this case the 24 h prolactin binding at 24 h was 50% higher than control (P < 0.01). Discussion

Exposure of rat prostatic explants to ovine prolactin in vitro caused alterations in the memprolactin to an exbrane binding of “‘I-human tent and in a direction which was dependent on the hormone concentration: an up-regulation associated with low doses and a down-regulation at high concentrations. Since these changes could be demonstrated after treatment of the membranes with 4 M MgCl, it suggested that they reflected a net loss of receptors from the surface and not simply occupancy of the receptors by non-labelled prolactin. Further support for this conclusion came with the observation that additional washing of the cell membranes after MgCl, treatment could not improve the binding any further (data not shown). The fact that a similar biphasic response could also be demonstrated with Leydig cell tumour explants implies that this may be a capacity possessed by all prolactin target cells. Interestingly, the tumour cells exhibited a greater up-regulatory response to the hormone and appeared to be more sensitive to its effects than prostatic cells, the significance of which is as yet unknown. The doses of hormone necessary to cause upregulation of the prolactin receptor were high (500-5000 ng). More recent studies (data not published) indicate that the first dose eliciting a significant elevation above control levels is approximately 150 ng/ml, which is still in excess of the normal range of plasma concentration of the hormone encountered in vivo (Rui et al., 1985).

On the other hand, the effects of purified rat prolactin on its receptor have yet to be tested. Furthermore, the use of relatively high concentrations of hormone, which are still only 3-4 times greater than that observed in vivo, may simply accelerate and exaggerate a physiological effect which under normal conditions may involve relatively minor elevators in endogenous hormone but over long periods. The prolactin-induced increase in the number of its receptors began to be manifested after a latent period of approximately 6-12 h after the start of exposure to the hormone, implying de novo receptor synthesis. In contrast, prostatic explants cultured in the absence of hormone exhibited a gradual loss of their membrane receptor complement over the 24 h observation period. Although it is tempting to assume that this may reflect cell death, the fact that exposure to prolactin reversed this tendency, presumably by stimulating protein synthesis, would imply that it reflects a biochemical adjustment to the in vitro conditions, possibly due to the absence of endogenous hormones such as testosterone or prolactin itself. The opposing effects of prolactin on its membrane receptor concentration may explain the paucity of information which is available on this subject in the literature. No net change in the receptor content could be anticipated using doses intermediate between those up-regulating and those down-regulating the receptor, whereas a choice of concentrations lower or higher than this level would stimulate a response in opposing directions. In recent experiments using rats rendered hyperprolactinaemic with pituitary implants an up-regulatory effect of the hormone on its prostatic receptor could be clearly demonstrated (Blankenstein et al., 1985) suggesting an additional factor of importance may be the chronicity of the hormonal change. A further complication of such in vivo studies is the acknowledged positive interaction between androgens and the prostatic prolactin receptor (Charreau et al., 1977). Prolactin treatment is known to stimulate Leydig cell function in the rat (Purvis et al., 1979) and an increased androgenic influence on the prostate with its consequent effects on prolactin receptors may mask any evidence for down-regulation of the receptors by prolactin. The use of prostatic ex-

57

plants avoids many of the above problems encountered in vivo. Future studies using this model will focus on the mechanisms of prolactin-receptor interaction in the rat prostate and determine whether these biphasic effects can also be demonstrated in human prostatic explants. Acknowledgements These studies have been supported by ‘Norsk Forening til Kreftens Bekjempelse’ and ‘Landsforeningen mot Kreft’. H.R. is in receipt of a NAVF scholarship. We thank Dr. Aa. Erichsen for the Leydig cell tumour tissue. References Amit. T., Barkey, R.J. and Youdim. M.B.H. (1983) Mol. Cell. Endocrinol. 30, 179-187. Aragona, A. and Friesen, H.G. (1975) Endocrinology 97, 677-684. Barkey, R.J., Shani, J. and Barzilai, D. (1979) J. Endocrinol. 81. 11-18.

Blankenstein, M.A., Bolt-de Vries, J., Coert, A., Nievelstein. H. and Schroder, F.H. (1985) Prostate 6, 277-283. Charreau, E.H., Attramadal, A. Torjesen, P.A., Purvis, K., Calandra, R. and Hansson, V. (1977) Mol. Cell. Endocrinol. 6, 3033307. Dave, J.R. and Witorsch, R.J. (1985) Am. J. Physiol. 248, 687-693. Dijane, J. and Durand, P. (1977) Nature 266, 641-643. Dijane, J.. Clauser, H. and Kelly, P.A. (1979) Biochem. Biophys. Res. Commun. 90, 87-98. Erichsen, Aa., Jahnsen, T., Andersen, D., Torjesen, P. and Hansson, V. (1984) J. Steroid B&hem. 21, 539-543. Kelly, P.A., Leblanc, G. and Dijane, J. (1979) Endocrinology 104, 1631-1638. Kelly, P.A., Dijane, J., Katoh, M.. Ferland, L.H., Houdehne, L.-M., Teyssct, B. and Dusanter-Fourt, I. (1984) Recent Prog. Horm. Res. 40, 379-439. Kledzik, G.S., Marshall, S., Campbell, G.A., Gelato. M. and Meites, J. (1976) Endocrinology 98, 373-379. Manni, A., Chambers, M.J. and Pearson, O.H. (1978) Endocrinology 103, 2168-2171. Purvis, K., Clausen, O.P.F., Olsen, A., Haug, E. and Hansson, V. (1979) Arch. Androl. 3, 219-230. Rui, H., Haug. E., MevPg, B., Thomassen, Y. and Purvis, K. (1985) J. Reprod. Fertil. 75. 421-432. Tell, G.P.E., Haour, F. and Saez, J.M. (1978) Metabolism 27, 156661592.