Jol,rnai of Neuroendocrinology, Vol. 2, NO. 5 0 1990 Oxford University Press, 0953-8194/90 $3.00

Regulation of the Rat Oxytocin Gene by Estradiol Examination of Promoter Activity in Transfected Cells and of Messenger Ribonucleic Acid and peptide Levels in the Hypothalamo-Neurohypophyseal System

J. Peter H. Burbach, Roger A. H. Adan, Hubert H. M. van Tol, Marleen A. E. Verbeeck, John F. Axelson?, Fred W. van Leeuwen;, Johanna M. Beekman f t and Geert A b t t Rudolf Magnus Institute, Medical Faculty, University of Utrecht, Vondellaan 6, Utrecht, The Netherlands. t Holy Cross College, Department of Psychology, Worcester, Massachusetts, USA. t Netherlands Institute for Brain Research, Meibergdreef 33, Amsterdam, The Netherlands. t i Biochemical Laboratory, University of Groningen, Nijenborgh 16, Groningen, The Netherlands.

Key words: oxytocin, estrogens, estrogen receptor, vasopressin, hypothalamo-neurohypophyseal system

Abstract

Oxytocin (OT) plays a role in reproduction at the level of the pituitary and mammary glands and uterus. This OT is synthesized in the hypothalamo-neurohypophyseal system (HNS). A number of observations have suggested that estrogens regulate the production of OT in the HNS. In this study the effect of 171-estradiol on the activity of the OT gene promoter was examined as well as the effect of 17/1-estradiol in vivoon OT messenger'ribonucleic acid (mRNA) and peptide tevels in the rat HNS. Vasopressin (VP) and its mRNA were also determined in the in vivo studies. The direct transcriptional stimulation of OT gene expression by 171-estradiol was studied in two different heterologous expression systems. When a plasmid having nucleotides - 363 to + 16 of the rat OT gene fused to the firefly luciferase reporter gene was co-transfected with an estrogen receptor expression vector in P19 embryonal carcinoma cells, luciferase activity was stimulated 80-fold by 171-estradiol. In estrogen receptor containing MCF-7 cells transfected with a plasmid having nucleotides - 188 to + 16 of the rat OT gene fused to the chloramphenicol acetyl transferase gene, 178-estradiol induced the expression of the chloramphenicol acetyl transferase gene through the cloned promoter element. After in vivo treatment of ovariectomized rats with 171-estradio1, levels of OT mRNA and VP mRNA were measured in microdissected supraoptic and paraventricular nuclei as well as VP and OT levels in these nuclei and the pituitary gland. As compared to non-treated ovariectomized rats there was no difference in contents of OT mRNA and VP mRNA in these hypothalamic nuclei and in levels of the peptides in paraventricular nuclei and the pituitary gland. A 30% reduction of the OT content of the supraoptic nuclei only was found, while the VP content did not change. To explain the results immunocytochemical analyses of the hypothalamus were performed, snowing that the estrogen receptor was absent in the magnocellular neurons of the supraoptic and paraventricular nuclei. The results demonstrate that the 5' flanking region of the OT gene confers estrogen-sensitivity to transcription of the OT gene. This potential to respond to estrogens is not used in the OT-producing neurons of supraoptic and paraventricular nuclei probably due to the absence of the estrogen receptor.

Oxytocin (OT) in the peripheral circulation originates from the classical hypothalamo-neurohypophyseal system (HNS) with the wpraoptic (SON) and paraventricular (PVN) nuclei as major sites of OT synthesis. OT plays a role in the control of reproduction in the female through its stimulation of prolactin release (1) and milk ejection ( 2 ) and its induction of uterus contraction (3). The role of cstrogens in the regulation of OT-producing neurons of the HNS has been indicated. In humans, estrogens markedly stimulate plasma levels of the OT-associated neurophysin (hence termed 'estrogen-stimulated neurophysin') (4). In the rat, a weak stimu-

lation of plasma OT and vasopressin (VP) levels by estrogen treatment of ovariectomized (OVX) animals has been reported (5), as well as variations in pituitary and plasma levels of OT and VP during the estrous cycle (6,7). At estrous an elevated level of OT mRNA, but not of VP mRNA, is present in the SON of the rat (8). Estrogen-related changes in neurosecretory activity of magno- cellular cells of the SON and PVN have been measured using a marker enzyme and electrophysiological parameters (9, 10). How- ever, very few estradiol-concentrating cells are present in the magnocellular neurons of the SON and PVN (1 1-14). A more

Correspondence 10: J. P. H. Burbach, Rudolf Magnus Institute, Vondellaan 6, 3521 GD Utrecht, The Netherlands.

634 Estradiol and oxytocin gene expression

outspoken sensitivity to estrogens has been found in the preoptic area with respect to the number of OT-immunoreactive neurons and contents of OT mRNA and immunoreactivity (12, 15-17). OT in this brain area has been implicated in the control of sexual behavior (1 8).

Here the question is addressed whether the OT gene is estrogen- sensitive and whether gene expression in the HNS is influenced by estrogens. Since the nucleotide sequence of the 5’ flanking region of the rat OT gene predicts an estrogen-responsive element (ERE) (8, 19,20), it was firstly tested whether 5’ flanking sequences of the rat OT gene confer estrogen-sensitivity to OT gene transcription in two heterologous expression systems. Secondly, it was investi- gated whether the estrogen sensitivity was functional in OT- producing neurons of the HNS. Therefore, the effect of 178- estradiol on OT mRNA levels in SON and PVN in vivo was evaluated. Since estrogen effects on VP together with OT have been reported (5, 6), VP mRNA and peptide levels were also determined in this study. Finally, immunocytochemistry of the estrogen receptor in the rat hypothalamus was performed to find an explanation for the results.

Results

OT promoter activity in transfected cells The estrogen sensitivity of OT gene transcription was tested in two different heterologous expression systems. First, the human estrogen receptor driven by the SV40 early promoter in the expression vector pHEO (21) was co-transfected with pROLUC, a 5’ flanking sequence (- 363 to + 16) of the rat OT gene linked to the firefly luciferase receptor gene (22) in P19 embryonal carci- noma (EC) cells. 17fl-estradiol (3 nM) caused an 80-fold increase in luciferase activity in PI9 EC cells which were co-transfected with the estrogen receptor expression vector and the OT pro-

moter/luciferase construct (Fig. 1). Co-transfection of pHEO and pROLUC without adding estrogen did not alter the basal lucifer- ase activity measured after transfection of pROLUC alone. These results, together with the lack of an estrogen effect on a promoter- less luciferase construct show that the 5’ flanking region of the OT gene confers the response to estrogen.

Since the estrogen receptor levels in pHEO transfected P19 EC cells may be abnormally high, the model of Seiler-Tuyns et al. (23) was also employed to test the estrogen sensitivity of the rat OT gene. This expression system employs the breast tumor cell line MCF-7 which has endogenous expression of the estrogen recep- tor. In these experiments, a shorter 5’ flanking region ( - 188 to + 16) and another reporter gene (chloramphenicol acetyl transfer- ase; CAT) were used to restrict the promoter region to the sequences which contain the putative ERE (- 168 to - 155) and to rule out a possible influence of the reporter gene. In the transfected MCF-7 cells again the 5’ flanking region of the OT gene conferred estrogen responsiveness, in this case to the CAT reporter gene (Fig. 2). No CAT activity was found in the absence of estrogen (143 dmp of product). In the presence of 0.2 pM 178- estradiol, CAT activity was stimulated to levels (4430 dpm) 30% of the constitutive expression obtained with the strong rous sarcoma virus promoter. Since expression with the pSCAT vector was virtually zero irrespective of the presence or absence of the hormone (143 and 93 dpm of product, respectively), the sequences responsible for the hormone effect must reside within the OT gene sequence.

Analyses of mRNA, peptide and estrogen receptor in the HNS

The OT mRNA levels in the SON and PVN of 178-estradiol- treated OVX rats were the same as in OVX controls receiving only vehicle (Fig. 3). In previous experiments no differences were found in OT mRNA levels of OVX, normal female rats and rats treated

50

pGEM4 pGEM4 +E pHEO PHEO +E

FIG. 1 . Stimulation of rat oxytocin promoter activity by 17B-estradiol in transfected P19 embryonal carcinoma cells. pROLUC has the - 363 to + 16 5’ flanking region of the rat oxytocin gene fused to the luciferase gene and was co-transfected with pHEO (the estrogen receptor expression plasmid) or pGEM4 as a control in P19 embryonal carcinoma cells. The cells were treated with 3 nM 17B-estradiol (+ E) for 24 h; controls received only vehicle. Luciferase activityi’was measured in cell extracts and expressed in light units. In pROLUC transfected cells, irrespective of co-transfection with pGEM4, pGEM4 plus estrogen treatment or pHEO, luciferase activity was 500 light units on average. The values are means of two experiments. The standard deviation is indicated.

Estradiol and oxytocin gene expression 635

- - + - + 0-estradiol

acetylated ['4C]-chloramphenicoI 1

- [ ''C]-chlorarnphenicol

- origin UU-

pRSVCAT pSCAT pROSCAT I

FIG. 2. The effect of 17b-estradiol on activity of the rat oxytocin promoter. Plasmids were transfected into MCF-7 cells. pROSCAT has the - 188 to + 16 5' flanking region of the rat oxytocin gene cloned in front of the chloramphenicol acetyl transferase (CAT) gene in pSCAT.pRSVCAT contains the rous sarcoma virus long terminal repeat. Transfected cells were treated with 0.2 pM 17j-estradiol(+) or vehicle ( - ) and CAT activity was determined using ['JC]-chloramphenicol as substrate. The 14C-acetylated forms of chloramphenicol were cut from TLC plates and counted. Counts and conversions were: pKSVCAT (-): 14160 dpm, 57% conversion; pSCAT ( - ): 92 dpm, 0.3% conversion; pSCAT (+ ): 140 dpm, 0.5% conversion; pROSCAT (-): 140 dpm, 0 5 % conversion; pROSCAT (+): 4430 dpm, 17% conversion.

rn RNA rn RNA

VASOPRESSIN OXY TOC IN 3 .. .. .. so[ .. .. .. .. .. .. .. .. .. ..

PVN

]I .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..

OVX OVX+E OVX C)VX+E Flc;. 3. Levels of oxytocin mRNA and vasopressin mRNA in the supraop- tlr (SON) and paraventricular (PVN) nuclei of placebo-treated OVX Li and 17b-estradiol-treated OVX rats El. Bars represent the mean k SEM. The number of animals is indicated. Statistical analysis was performed by the Student's t-test. No significant differences were found.

with high doses of 17S-estradiol (unpublished data). There was no significant difference in OT levels in the PVN and the pituitary gland of O W and estrogen-treated OVX rats but OT levels in the SON of estrogen-treated rats were significantly lower by about

30% than in control OVX animals (Fig. 4). Furthermore, VP mRNA and VP peptide levels were not different in O W and estrogen-treated OVX animals in both PVN and SON (Figs. 3 and 4). Since the scope of these experiments was the HNS, other brain areas were not included in this study.

Since the reason for the lack of an effect on OT m R N A in H N S neurons could be situated at the level of the estrogen receptor, the presence of the estrogen receptor was then investigated by immu- nocytochemistry in the same experimental protocol as used for mRNA and peptide analyses. The magnocellular neurons of the SON, PVN and hypothalamic islands of 7-day OVX rats did not display estrogen receptor immunoreactivity (Fig. 5B). whereas clear nuclear staining was obtained in the preoptic area (Fig. 5c) as reported previously (1 6). Several cells in the parvocellular division of the PVN, which did not belong to the HNS, contained nuclear staining by the estrogen receptor antibody (Fig. 5).

Discussion

These results obtained in two different heterologous expression systems demonstrate that promoter activity of the rat OT gene is stimulated by estrogen. We speculate that this estrogen sensitivity is conferred by an ERE-like sequence located - 168 to - 155 bp upstream of the transcription initiation site (numbered according to Ivell and Richter (19)). This sequence, GGTGACCTTGACC, differs in only one nucleotide from the ERE consensus sequence GGTCANNNTGACC (24). The sequence in the rat OT gene is not a perfect palindrome. However, it has been shown for the human pS2 gene that an imperfectly palindromic sequence can be a functional ERE (25). Richard and Zingg (26) recently reported the estrogen sensitivity of the thymidine kinase (TK) promoter fused to 5' flanking regions of the human OT gene and located a n ERE in a 5' flanking region which contains the same ERE-like element as the rat OT gene. Previously we have performed similar experiments with the 5' flanking region of the human OT gene

636 Estradiol and oxytocin gene expression

VASOPRESSIN OXYTOCIN

SON

60

.: LO- - e Q

20- - m

0-

-

.. .. ..

.. .. .. .. .. .. .. .. .. 3 .. .. ..

I PVN

.c 200 a c

g 100 .

m

0

04 -0 C U - - 0.2 m 1

O.O ovx 0VX.E ovx OW+€

12

B

L

O

GO

20

0

2

1

0

FIG. 4. Levels of oxytocin and vasopressin in the supraoptic (SON) and paraventricular (PVN) nuclei and the pituitary gland (PIT) of placebo- treated OVX 0 and 17p-estrodiol-treated OVX rats El. Bars represent the means+SEM. The number of animals in indicated. Statistical analysis was performed by the Student’s [-test (*P< 0.05).

fused in a TK-CAT vector (unpublished data). In these experi- ments a 4-fold increase in CAT activity was found in MCF-7 cells treated with 17D-estradiol. We here show that estrogen respon- siveness of the OT gene can be found on its own promoter. Interestingly, it has also been reported that the putative ERE alone is hardly able to stimulate CAT activity in MCF-7 cells transfected when cloned in front of the TK-CAT construct (27). Therefore, the context of the ERE in the rat OT gene and possibly the interaction with other cis-acting elements may be of import- ance for the estrogen regulation of the OT promoter. Further studies using deletion mutants are needed to prove the identity of the functional ERE in the rat OT gene.

Despite the effect of 17p-estradiol on OT promoter activity in gene transfer experiments, there appeared to be no effect of 178- estradiol on OT gene expression in the SON and PVN in vivo. The present results show that 178-estradiol given to OVX female rats does not alter OT mRNA levels of HNS neurons, while the same dose of 17fl-estradiol used is sufficient to induce marked effects on the brain, such as the increase of the density of OT receptors (28), and to alter OT mRNA levels in the preoptic area of the brain

The absence of estrogen receptor in the HNS of the OVX female rat, sh&n by the lack of immunostaining of the receptor protein may explain these results. This observation agrees with

(17).

data on the normal male rat (16). Furthermore, Rhodes et al. showed that magnocellular neurons which concentrate labelled 17a-estradiol are very rare in the rat HNS and that OT staining is not altered after estrogen treatment (1 3-15). 17a-Estradiol-con- centrating neurons were virtually absent in the SON, but in the PVN they were more abundant (13). However, these cells did not belong to the HNS, but to a system projecting to the hindbrain and brainstem. Variations in the concentrations of OT and VP in the PVN and brainstem during the estrous cycle of the rat (29, 30) and the excitability of OT neurons in the PVN (10, 31) may be due to estrogen-sensitivity of these cells. Previous studies of Axelson and van Leeuwen in the male rat (16) have shown estrogen receptor-immunoreactivity only in the parvocellular subdivision of the PVN and not in the posterior subnucleus as reported by Rhodes et al. (13). The difference may be explained by the sensitivity of the methods used.

Taken together, estrogens do not directly affect the regulation of OT gene expression in the HNS of the rat. Consequently, OT synthesis in the HNS for peripheral actions i.e. the pituitary lactotrophs (l), mammary gland and uterus (2, 3) is not directly modulated by estrogens. Estrogens rather could act on the OT targets by affecting the number of OT receptors (32). These data suggest that previously reported estrogen-related changes in vari- ous aspects of oxytocinergic neurons of the HNS (5-12) may be due to indirect effects on these cells. Changes in physiological status induced by estrogens, like fluid balance (33) or in electrical activity of neural inputs, might be responsible for the observed effects. The significant decrease in OT level in the SON may also be attributed to such indirect influences. Changes in transport rate or metabolic changes may underlie this effect. With respect to the magnocellular VP-producing neurons of the HNS the same conclusion can be drawn as for the OT neurons i.e. that gene expression in vivo is not directly regulated by estrogens. We have not tested the estrogen responsiveness of the rat VP promoter since the available sequence data of the 5’ flanking region does not indicate ERE-like nucleotide sequences (34). The reported, but conflicting, increase in VP plasma levels after estrogen treatment (5, 35) and changes during the estrous cycle (6, 29) may also have been established through indirect effects of the steroid.

It is of importance to note that several OT-expressing brain areas which were not included in this study reportedly respond to estrogen treatment. Data of Rhodes et al. (13-15) indicate that in the anterior commissural nucleus part of the OT neurons co- localize with estrogen-binding and that estrogens altered OT- immunoreactive staining, suggesting that in these neurons the estrogen responsiveness of the OT promoter could be functional. However, in the study of Axelson and van Leeuwen (16) it was found that the OT neurons of the anterior commissural nucleus did not contain any estogen receptor immunoreactivity, but that cells in the vicinity were immunopositive. Furthermore, the pre- optic area contains OT neurons which alter in number, OT content and OT mRNA content upon various estrogen treatments (1 1, 17). Again, no co-localization of OT immunoreactivity and estrogen binding was found in this area, but estrogen-concen- trating cells tended to surround OT neurons (17, 36). A similar observation has been made for the estrogen receptor and luteiniz- ing hormone-releasing hormone neurons (37). In all these cases the estrogen effect might be established through paracrine influ- ences from neighbouring estrogen-sensitive neurons.

It should be noted that in other species (mouse, guinea pig) the

Estradiol and oxytocin gene expression 637

Flci. 5. Immunocytochemical localization of the estrogen receptor in transverse sections of the hypothalamus of a 7-day OVX female Wistar rat. In a h - p o w e r micrograph of the mid-posterior hypothalamus (A) only weak nuclear reaction can be seen in the periventricular (Pe) nucleus and the Parvocellular part of the paraventricular nucleus (PaAP). Reaction is absent in the supraoptic nucleus (SON; see also 4 ~ ) . Bar: 200 pm. In (B), the SON is shown at a higher magnification. Due to the total absence of immunoreactivity, it was necessary to close the condensor diaphragm more than usual. This made it possible to visualize the contours of the SON cells. The spots (arrowheads) are erythrocytes showing pseudo-peroxidase activity. Bar: 50 pm. In (C), a dense nuclear reaction can be seen in the medial preoptic area (MPA) of the same animal. Bar: 100 pm (for details (16)). f-fornix; OC-optic chiasm; 3V - third ventricle. The micrographs are representative of data obtained from 12 animals in which the entire hypothalamo-neurohypophyseal

was examined.

638

estrogen receptor is present in the HNS (38, 39). This marked species difference could imply that estrogens may have a direct transcriptional effect o n the OT gene in the H N S of these species, if an ERE is present in the respective 5’ flanking regions. In conclusion, the promoter of the rat OT gene is sensitive to

estrogens, but in magnocellular HNS neurons of female OVX rats this potential is not used due to the absence of the estrogen receptor. Reported effects of estrogen o n OT-producing HNS neurons may therefore be established indirectly, i.e. independent of interaction of the estrogen receptor with the OT gene promoter. In other endocrine situations in which the estrogen receptor might be induced or in other organs which co-express the OT and estrogen receptor genes this potential could be of physiological significance.

Estradiol and oxytocin gene expression

Materials and Methods

Transfections, luciferase and CA T assays PI9 EC cells (40) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) without phenol red, and supplemented with stripped, steroid free, 7.5% fetal calf serum. The day before transfection the cells were plated to a 10% confluent density in 6 cm culture dishes. The cells were transfected with the calcium-phosphate precipitation method (41). Ten pg of pROLUC was co-transfected with 3 pg of pHEO or pGEM4. The cells were not glycerol shocked, but only medium was replaced 16 h after addition of the plasmids to the cells. Immediately after replacement of the medium, the cells received 3 nM 17P-estradiol with ethanol as vehicle; controls received ethanol only. After 24 h the cells were harvested in 350 pl 100 mM potassium phosphate, pH=7.8, and luciferase was measured in 60 p1 of extract according to the protocol of de Wet (22), using a Lumac/3m biocounter M20 10A luminometer.

The protocols of Seller-Tuyns et a/ . (23) were used to test the estrogen effects on OT promoter activity in MCF-7 cells, which contain endogenous estrogen receptors (42). Cells were cultured in DMEM without phenol red supplemented with 10% stripped, steroid free, fetal calf serum and the estrogen antagonist tamoxifen 1 pM and transfected with the calcium- phosphate precipitation method (41). In short, 10 pg of precipitated plasmid suspension was added dropwise to MCF-7 cells in 100 mm dishes (50% confluent), and cultures were glycerol shocked after 4 h. For induction 0.2 pM 17P-estradiol in ethanol as vehicle was added. Controls received ethanol only. Two days after the transfection the cells were harvested, lysed by three freeze and thaw cycles in 0.25 M Tris-HC1, pH 7.8, and centrifuged. Three hundred pg of cytosol protein was used for determination of CAT activity, as described elsewhere (43).

Plusmids A pl9LUC is a derivative of pSVOAL-AdS containing a multiple cloning site in front of the luciferase gene (44). The - 363/ + 16 rat OT 5‘ upstream region (taking the first A of the multiple transcription initiation sites as + 1 (19)) was cloned into pl9LUC as a Hind 111-Sau3A fragment to construct ROLUC. pRSVLUC has the RSV-long terminal repeat in front of the luciferase gene (22). pHEO is the human estrogen receptor coding region cloned in the eukaryotic expression vector pSG5 under the control of the SV40 early promoter (21). pSCAT is a pSVO-CAT derivative containing a multiple cloning site in front of the CAT gene (43,45). The - 188/ + 16 OT promoter fragment was subcloned as a 204 bp SacI-Sau3A fragment into the multiple cloning site of pSCAT to obtain pROSCAT. pRSV-CAT is a derivative of pSV2-CAT with the rous sarcoma virus long terminal repeat cloned in front of the CAT gene (45).

Animals and treatment Female Wistar rats (150 to 200g body wt) were OVX under ether anesthesia and treated with 17P-estradiol benzoate (10 pg/lOOg body wt in 0.5 ml oil) by daily sc injections for 7 days starting at the day of operation. Control rats were OVX and received daily sc injections of oil only. Animals were killed%y decapitation and brain and pituitary were dissected and frozen. The SON and PVN were punched from frozen sections by the punch techniques of Palkovits (46).

Quantitation of mRNAs and peptides OT mRNA and VP mRNA were determined in total RNA extracted from punches of single animals by filter hybridization to rat VP- and OT-specific DNA probes. Procedures have been extensively evaluated and detailed before (8, 47). Optical density of dot blots were measured in a micro-titer plate reader (EIA Reader 2550; Bio-Rad, Richmond, CA, USA). VP and OT were determined by RIAs (8, 48).

Imrnunocytochemistry Estrogen receptors were immunocytochemically localized in 50-pm thick vibratome sections of 7-day OVX rats using a monoclonal antibody (H222 Spy, 1.4 pg/ml) directed against the estrogen receptor of MCF-7 human breast cancer cells (49). The antibody was supplied by Abbott, Chicago, IL, USA. Biotinylated sheep anti-rat IgG (Amersham, UK) was used as second antibody diluted 1:200 and ABC-peroxidase (Vectastain) diluted 1: 100 as final reagent. Peroxidase was shown using 3,3’-diaminobenzidine tetrahydrochloride as chromogen and 0.2% nickel ammonium sulphate as intensifier. For details see Axelson and van Leeuwen (16).

Acknowledgements

H. H. M. Van To1 and R. A. H. Adan have been supported by the NWO- foundation for Medical and Health Research Medigon (grants no. 900- 546-044 and 900-546-065). M. A. E. Verbeeck has been supported by the Programme of Medical Biotechnology, Institute for Molecular Biology and Medical Biotechnology, University of Utrecht, The Netherlands. Drs R. Ivell and D. Richter (Hamburg) are gratefully acknowledged for providing the rat OT clone POAHjS, Dr Loskutoff (La Jolla) for pl9LUC, Dr S. Subramani (San Diego) for pRSVLUC and Professor P. Chambon (Strasbourg) for pHEO. We are also grateful to colleagues in the Hubrecht Laboratory, Utrecht, for helpful discussions, gifts of cell lines and use of the luminometer.

Accepted 17 March 1990

Note added in proof. After acceptance of this paper, Richard and Zingg reported a detailed study on the estrogen regulation of the human oxytocin promoter (J Biol Chem. 265: 6098-6103).

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