estrogen and progesterone augment growth responsiveness of mammary tissue to cholera toxin

PRODUCTION RESEARCH PAPERS

Estrogen and Progesterone Augment Growth Responsiveness of Mammary Tissue to Cholera Toxin

LEWIS G. SHEFFIELD Dairy Science Department

University of Wisconsin Madison 53706

ABSTRACT

Second (thoracic) mammary glands of endocrine intact mice were removed intact and incubated in Dulbecco's Modi- fied Eagle's Medium supplemented with insulin, aldosterone, and cholera toxin. Insulin and aldosterone resulted in relatively little mammary development. How- ever, insulin, aldosterone, and cholera toxin substantially increased mammary development, as assessed by development scores and DNA after 6 d of culture. Ovariectomy abolished the ability of cholera toxin to augment mammary development in vitro. Estradiol and progesterone injections for 3 d partly restored responsiveness of mammary tissue to cholera toxin, whereas responsiveness was greater after 6 d of injection than in endocrine-intact mice. Additionally, cyclic AMP-dependent protein kinase (kinase A) activity of fourth (inguinal) mammae was increased after as little as 3 d of estradiol and progesterone treatment. Cholera toxin induced phosphorylation of at least one protein was also increased by estradiol and progesterone. Because cholera toxin is a potent activator of adenylate cyclase, these findings suggest that estradiol and progesterone interact with cyclic AMP active agents to promote mammary development. This interaction may be mediated, at least in part, by increased kinase A activity and increased kinase A substrate availability.

Received May 31, 1988. Accepted October 28, 1988. lSupported by University of Wisconsin College of

Agricultural and Life Science and Hatch Grant 3108.

INTRODUCTION

Cyclic AMP concentration in mammary tissue increases during pregnancy in rats (10), mice (9), and guinea pigs (7). Additionally, estradiol and progesterone increase intramammary cyclic AMP concentrations in mice (12) and in bovine mammary tissue main- mined in athymic nude mice (15). These findings lead to the suggestion that cyclic AMP may be causally related to mammogenesis. In vitro studies found that agents that increase in- traceUular cyclic AMP (e.g., cholera toxin) augment mammary gland development (17, 18, 20). Similarly, cholera toxin, administered lo- cally with plastic implants or systemically via injections, increases growth of mouse mammary tissue in vivo (13, 16) and bovine mammary tissue maintained in athymic nude mice (14).

Cyclic AMP is thought to exert its effects by increasing activity of a cyclic AMP-dependent protein kinase (kinase A). Activity of kinase A increases in mammary tissue during pregnancy, at least in rodent mammary tissue (8, 11). The kinase A activity of mouse mammary tissue is also increased by in vivo treatment with estradiol and progesterone (12). Furthermore, similar results have also been observed in bovine mammary tissue transplanted subcutaneously to athymic nude mice (15). Whether these changes in kinase A activity are causally related to mammary epithelial proliferation is not known at this time.

There is currently some disagreement con- cerning whether ovarian steroid hormones are necessary for cyclic AMP active agents to increase mammary development. End bud forma- tion and ductal development was increased by cholera toxin administered by local release implants (16). However, Sheffield et al. (13) found that cholera toxin injections were not effective in increasing mammary development in ovariectomized mice treated with saline, but

1989 J Dairy Sci 72:892-899 892

CYCLIC NUCLEOTIDES AND STEROIDS IN MAMMARY DEVELOPMENT 893

dramatically increased mammary development in endocrine-intact or estradiol plus progesterone-treated mice.

The objective of this study was to examine the effect of estradiol plus progesterone injections on growth responsiveness of mouse mammary tissue to cholera toxin in vitro. In addition, these studies also determined the effect of estradiol plus progesterone injections on cholera toxin induced protein phosphorylation in mouse mammary tissue.

MATERIALS AND METHODS

Cholera Toxin in Organ Cultures

Female ND/4 mice (Harlan Sprague-Daw- ley, Madison, WI) were killed at 10 wk of age and second' (thoracic) mammary glands removed. Glands were spread on Dacron rafts and cultured in Dulbecco's Modified Eagle's Medium supplemented with insulin (1 ktg/ml), aldosterone (.5 ~tg/ml), and cholera toxin (0,. 1, 1, 10, 100, or 1000 ng/ml) (Sigma Chemical Co., St. Louis, MO). Media were changed ev- ery other day, and cultures were maintained for 6 d. Mammary glands were fixed in glacial acetic acid:ethanol (1:3), stained with carmine- alum, and examined as whole mounts. Mam- mary development was assessed on a scale of 1 to 6 as previously described (19). Mammary glands were then homogenized, extracted with 5% perchloric acid (70°C), and DNA determined by the diphenylamine reaction (2).

Ovariectomy on Cholera Toxin Response

Mice were ovariectomized at 7 wk of age and allowed 2 wk recovery. Mice were then injected for 9 d with saline or estradiol plus progesterone (1 p.g plus 1 mg/d). In addition, mice were also injected for 6 or 3 d with saline, followed by 3 or 6 d injection with estradiol plus progesterone. Treatment groups will be referred to by the duration of estradiol plus progesterone treatment (e.g., 0, 3, 6, or 9 d). On the 10th d, mice were killed and second (thoracic) mammae removed and placed in organ culture as described, except that cholera toxin treatments were 0 or 100 ng/ml culture media. One mammary gland from each mouse received 0 ng/ml cholera toxin and the other 100 ng/ml

cholera toxin. After 6 d of culture, mammary development was assessed as described.

Estradiol plus Progesterone on Protein Kinase

Fourth (inguinal) mammary glands were removed from mice treated with estradiol plus progesterone for 0, 3, 6, or 9 d (above) and homogenized in Tris:HCl buffer (50 mM, pH 7.0) containing 5 mM phenylmethylsulphonyl- fluoride and 150 mM NaC1. Homogenates were centrifuged (1000 x g, 15 min) and protein content determined by dye binding (1). Protein kinase A activity was determined as previously described (13) with the exception that [~sS]thio-ATP was substituted for ~-[32p]ATP (3). Briefly, mammary homogenate (10 ~tl) was incubated with assay buffer containing 30 mM phosphate (pH 6.8), 1 mg/ml histone (Sigma type VII), 10 mM MgCI 2, 5 mM dithiothreitol, and 10 -4 M 7-[35S]thio-ATP (250,000 dpm/ tube) in a volume of 60 l.tl. For kinase A activity, 10 -5 M cyclic AMP was added to each tube. Basal kinase activity was determined in the absence of cyclic AMP. Assays were started by adding ATP and continued for 10 min at 300C. Assays were stopped by removing 30 ktl, spotting it onto Whatman 3MM filter paper (Whatman, Clifton, N J) and placing the filter paper into 10% TCA. Filter paper was washed three times with 5% TCA, twice with ethanol, dried, and counted by liquid scintillation. Ki- nase A was determined by subtracting basal thiophosphate incorporation from cyclic AMP stimulated thiophosphate incorporation. One unit of activity was defined as 1 pmol of phosphate or thiophosphate incorporated into histone per minute per milligram homogenate protein.

Cholera Toxin on Protein Phosphoryletion

Fourth (inguinal) mammary glands from mice injected 0, 3, 6, or 9 d with estradiol plus progesterone were minced into small (2 to 3 mm 3) pieces and placed in phosphate- free Ea- gle's Minimum Essential Medium for 2 h. Me- dium was changed and 25 ktCi/ml 32i) (as ortho- phosphoric acid) (DuPont, Boston, MA) added. Tissue was incubated an additional 2 h to equil- ibrate phosphate pools with medium, then chol-

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era toxin (100 ng/ml) was added. Incubations were continued an additional 15, 30, or 60 min, then stopped by removing medium and adding SDS-PAGE loading buffer (.05 M Tris, pH 6.8, .2% SDS, .05% 13- mercaptoethanol, .001% bromophenol blue, and 10% sucrose). Tissue was boiled for 30 min, then allowed to set at room temperature overnight. Proteins were then separated by SDS- PAGE (5% stacking gel, 12. 5 or 7.5% separating gels) (6) and 32p detected by autoradiography using Kodak X-AR film.

Statistical Analysis

In the first experiment (using endocrine intact mice), in vitro treatments were applied to tissue from six mice per treatment. Treatments were not blocked by mouse, but mice served as sampling units. Data were analyzed by ANOVA and regression analysis used to de- scribe response of mammary tissue development to cholera toxin.

In subsequent experiments, a split plot design was used in which mice served as whole units (treated with estradiol plus progesterone for various times) and tissue within mice (treated with or without cholera toxin) served as subunits. Data were analyzed by ANOVA and the effect of estradiol plus progesterone treatment duration on cholera toxin responsiveness described by regression analysis. Statisti- cal tests were two sided with P<.05 (4).

RESULTS

Initial studies, using tissue from endocrine- intact mice, indicated that mammary gland development in vitro could be induced by a combination of insulin, aldosterone, and cholera toxin. Insulin and aldosterone alone were not sufficient to induce substantial mammary development (Figure 1). The combination of insulin, aldosterone, and cholera toxin resulted in greatly increased mammary epithelial proliferation (relative to the absence of cholera toxin), easily discernible in mammary whole mount preparations. In addition, DNA content of the mammary glands was increased by cholera toxin in Vitro (P<.05). Maximum development was observed with 10 to 100 lxg/ml cholera toxin (Figure 2). Mammary development and DNA were not different among 10, 100, or 1000 ng/ml cholera toxin (P>.05).

Journal of Dairy Science Vol. 72, No. 4, 1989

Subsequent studies, using 100 ng/ml cholera toxin, found that ovariectomy abolished or greatly inhibited cholera toxin induced mammary development in vitro (Figure 3) (P>.05 for cholera toxin vs. no cholera toxin in ovariectomized saline-primed mice). Responsiveness to cholera toxin was partly restored by 3 or 6 d treatment with estradiol plus progesterone. In- jection of estradiol plus progesterone for 9 d completely restored responsiveness of mouse mammary tissue to cholera toxin, using either development score or DNA as a criteria for mammary development.

The ability of estradiol plus progesterone treatment to restore cholera toxin responsiveness of mouse mammary tissue was associated with changes in kinase A activity of mouse mammary tissue (Figure 4). Although kinase A was detectable in mammary tissue of mice treated with saline only, the activity was fairly low. Treatment with estradiol plus progesterone for 3, 6, or 9 d increased kinase A activity in a linear fashion (P<.05). Of the times examined, maximum kinase A activity was observed after 9 d estradiol plus progesterone treatment. In addition, the phosphorylation of at least one protein, with an estimated molecular weight of 29 kdal, was increased by short-term cholera toxin treatment. Scanning densitometry indicated that the amount of phosphate incorporated into the 29-kdal band was increased by estradiol plus progesterone and the increase was linear over duration of estradiol plus progesterone treatment (P<.05) (Figure 5).

DISCUSSION

Results of these studies clearly indicate that cholera toxin increased mouse mammary gland development in vitro. These results are in agreement with previous studies indicating that cholera toxin increases proliferation of mammary epithelia in vitro under a variety of conditions (13, 14, 16, 17, 18, 20). Presumably, this increased proliferation is due to increased intra- cellular cyclic AMP induced by cholera toxin (5). The ability of cholera toxin to increase mammary development in organ cultures, at least under the conditions used in these studies, is dependent on the mammary tissue having been exposed to ovarian steroids. These results are in agreement with studies by Sheffield et al.


Figure 1. Typical whole mounts of mouse mammary glands incubated for 6 d with insulin (5 I~ghnl) plus aldosterone (1 ktghnl) upper photograph) or insulin plus aldosterone plus cholera toxin (100 ng/ml) (lower photograph). Note alveolar development in the presence of cholera toxin.

(13), showing that cholera toxin injections increased mammary development in endocrine- intact mice or mice treated with estradiol plus

progesterone, but not in ovariectomized mice treated with saline. However, other researchers (16) have found that cholera toxin, adminis-


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Cholera Toxin (ng/ml) Figure 2. Whole mount development scores and DNA content of mouse mammary glands incubated for 6 d with insulin

(5 gg/ml) plus aldosterorte (1 ~g/ml) or insulin plus aldosterone plus cholera toxin (dosage indicated). Tissue from endocrine treated mice.

tered by plastic local release implants, was capable of increasing mammary development in ovariectomized mice. The exact reasons for this discrepancy are not clear, but reasons could be differences in cholera toxin dose, strain of

Journal of Dairy Science Voi. 72, No. 4, 1989

mice, age of mice, or other differences in ex- perimental protocols.

These studies indicate that differences in cholera toxin responsiveness of mouse mammary tissue are associated with differences in


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Figure 3. Whole mount development scores and DNA content of mouse mammary glands removed from ovariectomized mice injected for 0, 3, 6, or 9 d with estradiol (1 ktg/day) plus progesterone (1 mg/day) and incubated for 6 d with insulin (5 }.tg/ml) plus aldosterone (1 I.tg/ml) or insulin plus aldosterone plus cholera toxin (100 p.g/ml).

kinase A activity and kinase A substrate amount or availabili ty in mammary tissue. This association was expected, because cyclic AMP is considered to act by increasing protein kinase A activity. Kinase A activity was increased after as little as 3 d estradiol plus progesterone

treatment. However, kinase A activity was greatest after 9 d treatment, which corre- sponded to the greatest developmental responsiveness of mammary tissue to cholera toxin. Furthermore, concentrations of kinase A observed after 9 d of estradiol plus progesterone

Jottmal of Dairy Science Vol. 72, No. 4, 1989

898 SHEFFIELD

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Figure 4. Effect of daily injections of estradiol (1 }.tg/d) plus progesterone (1 nag/d) on kinase A activity of mouse mammary glands. One unit kinase A activity = 1 pmol phosphate incorporated into histidine per minute per mg tissue protein in response to cyclic AMP.

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Figure 5. Relative cholera toxin induced phosphorylation of 29 kdal mouse mammary protein in tissue from mice treated for 0, 3, 6, or 9 d with estradiol (1 I.tg/d) plus progesterone (1 rag/d).

treatment were similar to that observed in mid- pregnant rats (11) and in mice injected for 18 d with estradiol plus progesterone (12).

Because kinase A alters cellular function by phosphorylating protein substrates, an objective of this study was to determine the effects of cholera toxin on protein phosphorylation in mouse mammary tissue. Cholera toxin increased phosphorylation of at least one protein band, concentration of which was increased by estradiol plus progesterone. Because kinase A phosphorylates a number of substrates, the presence of a single protein phosphorylated in response to cholera toxin was surprising. How- ever, protein phosphorylation patterns in intact mammary tissue are complex, and cholera toxin-induced phosphorylation of substrates pre- sent in relatively small amounts could easily be obscured by the complexity of the autoradio- grams.

Whether the effect of estradiol plus progesterone on cholera toxin induced protein phosphorylation was due to increased kinase A or increased kinase A substrate cannot be determined unequivocally, However, the magnitude of the increase in substrate phosphorylation was greater than the magnitude of the increase in kinase A, suggesting that estradiol plus progesterone increased the amount of kinase A substrate in the mammary gland.

In conclusion, these results support a model of mammary development in which steroid hormones interact with cyclic nucleotide active agents to increase amount of kinase A and kinase A substrates.

REFERENCES

1 Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248.

2 Burton, K. 1956. A study of the conditions and mecha- nisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315.

3 Cassel, D., and L. Glaser. 1982. Resistance to phospha- tase of thiophosphorylated epidermal growth factor receptor in A431 membranes. Proc. Natl. Acad. Sci. 79:2231.

4 Gill, J. L. 1978. Design and analysis of experiments in the animal and medical sciences. Vol. 1. Iowa State Univ. Press, Ames.

5 Holmgreen, J. 1981. Actions of cholera toxin and the prevention and treatment of cholera. Nature 292:413.

6 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.

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Med. 151:748. 10 Sapag-Hagar, M., and A. L. Greenbaum. 1974. The role

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12 Sheffield, L. G., C. F. Aylsworth, and C. W. Welsh. 1987. Cyclic nucteotides and protein phosphorylation m mouse mammary glands: effects of estrogen and progesterone administered in vivo. Proc. Soc. Exp. Biol. Med. 185:283.

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16 Silberstein, G. B., P. Strickland, V. Trumpboar, S. Coleman, and C. W. Daniel. 1984. In vivo, cAMP stimulates growth and morphogenesis of mouse mammary ducts. Proc. Natl. Acad. Sci. 81:4950.

17 Stampfer, M. R. 1982. Cholera toxin stimulation of human mammary epithelial cells in culture. In Vitro 18:531.

18 Taylor-Papadimitriou, J., P. Purkis, and I. S. Fentiman. 1980. Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells. J. Cell Physiol. 102:317.

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estrogen and progesterone augment growth responsiveness of mammary tissue to cholera toxin

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