0013.7227/92/1306-3453$03.00/O Endocrinology Copyright 0 1992 by The Endocrine

Dopamine Stimulated Expression Uterus*

Vol. 130, No. 6 Printed in U.S.A.

Society

Receptor Activation Inhibits EstrogenTransforming Growth Factor-a Gene and Growth in Anterior Pituitary, but not in

BJUG BORGUNDVAAGT, SUSAN R. GEORGES

JEFFREY

E. KUDLOWS,

SUSAN

G. MUELLER,

Departments of Medicine (J.E.K., S.R.G.), Pharmacology (B.B., S.R.G.), and Clinical S.G.M.), University of Toronto, Toronto, Ontario, Canada M5S lA8

ABSTRACT. Transforming growth factor-or (TGFa) has been localized to the anterior pituitary, specifically to the lactotroph and somatotroph cell populations, by our previous studies. Since pituitary lactotrophs are known to undergo growth in response to estrogens, we have used an estradiol-induced pituitary hyperplasia/adenoma model. Estradiol treatment resulted in induction of TGFa mRNA in anterior pituitary, evident by 48 h, preceding actual macroscopic growth, which attained a maximum greater than 500% by 12 weeks. This rapid effect of estradiol also enhanced PRL mRNA, but did not affect other species of mRNA encoding for proenkephalin, D2 receptor mRNA, or hexosaminidase-A. TGFo mRNA remained elevated for the duration of rapid pituitary growth. D2 receptor activation by its agonist bromocriptine resulted in marked attenuation of TGFa mRNA preceding regression of growth. Coadministration of bromocriptine with estradiol resulted in an involution of

AND

Biochemistry

(J.E.K.,

pituitary size, indicating the overriding influence of dopamine in spite of a continued estrogenic stimulus. Epidermal growth factor receptor mRNA was not affected by any of these manipulations, suggesting that the receptor was not coregulated in this tissue similarly to TGFa. Estradiol also induced uterine TGFa mRNA and marked growth of the organ, but TGFa in this location was not regulated by dopamine. These results indicate that TGFa in the anterior pituitary is rapidly induced by estrogen in a time course preceding the growth of the gland. Estrogen-induced TGFti is rapidly attenuated by D2 dopamine receptor activation and is accompanied by a regression of pituitary growth. Interaction between these opposing hormonal/transmitter responses will determine the growth potential of the anterior pituitary. (Endocrinology 130: 3453-3458, 1992)

T

RANSFORMING growth factor-a! (TGFcx), like its homolog epidermal growth factor (EGF), although originally described as an oncofetoprotein, has been reported in normal brain (1, 2) and pituitary (3, 4). The presence of TGFL~ in normal adult tissues implies a role for this peptide in normal physiological processes, which may include effects as a mitogen, in its capacity to stimulate epithelial and mesenchymal cell proliferation. TGFa in the pituitary gland has been localized to certain cell types within the anterior pituitary, most notably the lactotrophs and the somatotrophs (3). TGFa and EGF exert their effects through activation of the EGF receptor, which has also been localized to the same cells (5). Each peptide appears to bind different sites on

the receptor protein (6). These peptides have been shown to have nonmitogenic functional effects on the secretion of PRL and GH from pituitary cells (7-9). Other possible functions of TGFa within these cells have not yet been clarified. Growth of the normal adult pituitary occurs only under very specific circumstances, such as during pregnancy. However, pituitary cells do undergo turnover, and some shifts in population occur in response to physiological events (10). Under the influence of high circulating estrogen levels during pregnancy, the human pituitary undergoes hyperplasia, particularly of the lactotroph cell population (11). This effect of estrogen has also been confirmed in experimental animals (12,13). The normal function of the lactotroph is the secretion of PRL, which is maintained under tonic inhibitory control by dopamine from the hypothalamus (14, 15). The stimulatory action of estrogen on PRL synthesis and secretion is well characterized (16) and occurs by its antidopaminergic effects (17). Estrogen has been shown to affect dopamine receptor density and affinity states (18, 19) and the functioning of the receptor-linked adenylate cyclase system in

Received December 17, 1991. Address all correspondence and requests for reprints to: Dr. S. R. George, Department of Pharmacology, University of Toronto, MSB 4358, 1 Kings College Circle, Toronto, Ontario, Canada M5S lA8. * This work was supported by grants from the Medical Research Council of Canada (to S.R.G. and J.E.K.). t Suppported by a studentship from the Ontario Ministry of Health. $ Present address: Department of Medicine, University of Alabama, Birmingham, Alabama 35294. § Career investigator of the Ontario Ministry of Health.

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TGFa GENE

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pituitary (20,21). The effect of dopamine in the pituitary also mediates inhibition of growth, substantiated by the profound regression of growth by dopamine agonists observed in clinical and experimental lactotroph tumors (13,22-24). The present study reports changes in TGFa gene expression correlating with stimulation and inhibition of growth in the anterior pituitary. Concurrent studies were performed with uterine tissue as another example of an estrogen-responsive tissue. Materials

and Methods

Animals

Adult female Sprague-Dawleyrats (225-250 g; Charles River Canada, Montreal, Quebec,Canada) were housedin a temperature (20 C)- and humidity (43%)-controlled environmental room, with 12-h light, 12-h dark cycles and free accessto a pellet diet (Purina rat chow, Ralston-Purina, St. Louis, MO) and water. Animals were ovariectomized under halothane anaesthesia (5% for induction and 1.5-2.0% for maintenance), as described previously (13), and implanted SCwith sealed 3-cm Silastic cannulae (Dow-Corning Corp., Midland, MI; od, 0.125 in.; id, 0.078 in.) containing 2.5 mg 17P-estradiol-3-benzoate(Sigma ChemicalCo., St. Louis, MO) or left empty. This technique has been previously shown to releaseapproximately 7.5 pg estradial/day (25). For the short term study, animals were injected with 20 pg 17&estradiol in peanut oil SC. Rats were killed by cervical dislocation, and the pituitaries were rapidly dissectedand frozen on dry ice. All animal protocols were approved by the Animal Care and Ethics Committee of the University of Toronto. Bromocriptine

treatment

Bromocriptine (a generousgift of Sandoz Canada,Inc., Dorval, Quebec,Canada) was injected SCin a doseof 5 mg/kg.day, dissolvedin a vehicle of 0.025 M tartaric acid containing 25% ethanol and 50% distilled water. Control animals received vehicle injection. RNA blot analysis

Total cellular RNA was extracted as described by Chomczynski and Sacchi (26). Polyadenylated RNA waspurified by oligo(dT) column elution, as previously described (27). The RNA sampleswere separatedin a 1% agarose-6%formaldehyde gel and electrophoretically transferred onto a nylon membrane (GeneScreen,New England Nuclear, Boston, MA) (28). Each lane contained 2 Kg poly(A)+ RNA. The membrane was UV cross-linked (29), prehybridized for 16 h, and then hybridized for 24 h at 42 C. The cDNA 650-basepair(bp) probe, complementary to the entire coding sequenceof the rat TGFa peptide, was labeled by random primer extension, using [a-“2P]deoxyATP and the Klenow enzyme (30). Label incorporation was greater than 80% to yield a specific activity greater than 10’ cpm/pg. The blots were washed and exposedto Kodak XAR film (Eastman Kodak, Rochester,NY) for 8-24 h at -70 C. The other cDNA probes used were: a 769-bp fragment corresponding to the internal domain of the EGF receptor, a 1.6kilobasepair sequenceof the a-subunit of hexosaminidase-A,

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and a 935-bp coding sequencefor proenkephalin. For the D2 receptor mRNA, a 48-baseoligonucleotide, correspondingto the coding region of 16 N-terminal amino acids,wasused,with 3’-end labeling.

Results Estrogen treatment markedly increased anterior pituitary growth. Treatment with 17@-estradiol led to a substantial increment in pituitary size by 450% in 3 weeks, which increased further until 8 weeks and then plateaued by 12 weeks (Fig. 1). The estrogen-induced hyperplasia was effectively attenuated by subsequent treatment with the dopamine agonist bromocriptine (Fig. 2). This effect was also dependent on the duration of treatment. The effects of estradiol treatment on TGFa mRNA were examined after short term exposure, before any macroscopic increase in pituitary size had occurred. The effects of 12- and 24-h exposure to estradiol are shown in Fig. 3. By 24 h of estradiol treatment, there was a marked increase in TGFa and PRL mRNA. Levels of other species of mRNA encoding for the D2 dopamine receptor (Fig. 4), proenkephalin, and hexosaminidase-A (data not shown) were not affected. As shown in Fig. 1, maximal anterior pituitary weight was not achieved until at least 8 weeks of treatment, and the increase in TGFQ mRNA would be expected to be sustained over the rapid growth phase. After 3 weeks of estradiol treatment, an increase in TGFa mRNA was still clearly evident, as shown in Fig. 5. The effect of bromocriptine on TGFa mRNA levels was also examined at two treatment intervals. Administration of bromocriptine for 48 h after a 3week estrogen treatment period resulted in a significant decrease in the TGFa mRNA level to below the control

150 I s g100

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;

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ESTROGEN

1 i VEHICLE 15

TREATMENT

(wks)

FIG. 1. Time course of estrogen-induced anterior pituitary (AP) growth. Rats were administered 17P-estradiol by continuous SC infusion, using Silastic cannulae, for 3 (n = 25), 8 (n = 15), or 12 (n = 15) weeks. Control animals were implanted with empty cannulae for similar periodsof time (n = 6 at eachpoint). Significantdifferencesfrom control: *, P C 0.05; **, P < 0.0005.

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TGFa

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60 50

TGF-,

28s

40 30 20 10 0 0

2

BROMOCRIPTINE

7

18s PRL

TREATMENT (days)

FIG. 2. Regression of estradiol-induced pituitary hyperplasia by bromocriptine. Rats were implanted with 17j3-estradiol-containing Silastic cannulae. After 3 weeks of estradiol treatment, rats were treated with bromocriptine (5 mg/kg.day) or vehicle for 2 (n = 10) or 7 (n = 10) days. Significant differences from vehicle treatment: *, P < 0.001. AP, Anterior pituitary.

(vehicle-treated) value (Fig. 5). This decrease was apparent before any significant decrease in anterior pituitary size. Treatment with bromocriptine for 7 days also produced a marked attenuation of TGFa mRNA levels, as shown in Fig. 6. This effect appeared to be selective for TGFa mRNA, as hexosaminidase-A mRNA levels were unaffected. EGF receptor mRNA was also found not to change after bromocriptine treatment, as shown in Fig. 7. The effect of estradiol treatment on uterine growth was very pronounced, and near-maximal growth was achieved by the first week, as shown in Fig. 8. Treatment of estrogen-treated animals with bromocriptine for varying periods of time had no effect on uterine growth. Figure 9 shows the effect of estrogen treatment on uterine TGFa! mRNA. There was an increase in TGFa mRNA expression with estrogen, which was unaffected by treatment with bromocriptine.

FIG. 3. Estradiol induction of TGFcv and PRL mRNA. Northern blot showing effects of 12 and 24 h of estradiol treatment. Ovariectomized animals were injected with 17P-estradiol or vehicle (control). Each lane contains 2 pg anterior pituitary poly(A+) RNA, hybridized with “Plabeled rat TGFa and PRL cDNA probe sequentially. The relative positions of the 28s and 18s ribosomal RNAs are shown.

Discussion The results of the present study are consistent with the hypothesis that estrogen-induced lactotroph growth in anterior pituitary and bromocriptine-induced involution of such growth are mediated by alterations in TGFa gene expression. Although estrogen-induced uterine growth also appears to involve increased TGFcv gene expression, the lack of dopamine receptors in this tissue precluded a response to bromocriptine. The control of pituitary lactotroph secretory function is under sensitive tonic inhibitory regulation by dopamine (14, 15), and the most important signals received by these cells are transmitted via the D2 dopamine

c

12t

24t

FIG. 4. Lack of estradiol effect on D2 dopamine receptor mRNA. Northern blot showing effects of 12- and 24-h estradiol treatments. Ovariectomized rats were injected with estradiol (E) or vehicle (control; C). Each lane contains 2 pg anterior pituitary poly(A+) RNA, hybridized with a “P-labeled specific 48-base oligonucleotide complementary to rat D2 mRNA.

receptor. The D2 receptors located on lactotrophs play a critical role in regulating hormone secretion and receptor-linked effecters within these cells (31,32). The effects of dopamine in this tissue appear to be mediated through

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Endo. 1992 ~01130 * No 6

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: .,

Dopamine receptor activation inhibits estrogen-stimulated transforming growth factor-alpha gene expression and growth in anterior pituitary, but not in uterus.

Transforming growth factor-alpha (TGF alpha) has been localized to the anterior pituitary, specifically to the lactotroph and somatotroph cell populat...
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