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Mammary gland 1, 25-dihydroxyvitamin D3 receptor content during pregnancy and lactation
Molecular and Cellular Endocrinology, 60 (1988) 15-22 Elsevier Scientific Publishers Ireland. Ltd.
15
MCE 01930
Mammary
gland 1,25dihydroxyvitamin D, receptor during pregnancy and lactation
K.W. Colston I, U. Berger 2, P. Wilson 2, L. Hadcocks and R.C. Coombes ’
content
I, I. Naeem ‘, H.M. Earl 3
’ Department of Chemrcal Pathology and ’ Ludwig Instituie for Cancer Research (London - St. George’s Group), St. George’s Hospital MedIcal School, London, U.K., and -’ Department of Radiotherapy and Oncology, University College Hospital, London, U.K. (Received
Key words: Mammary
gland;
23 February
1,25_Dihydroxyvitamin
1988; accepted
D, receptor:
Casein;
21 June 1988)
Alkaline
phosphatase;
Pregnancy;
Lactation;
(Rat)
Summary The purpose of this study was to establish the time course and magnitude of changes in 1,25_dihydroxyvitamin D receptor activity in rat mammary gland during pregnancy and lactation and to correlate these changes with casein production and alkaline phosphatase activity. Marked increases in both 1,25dihydroxyvitamin D, receptor and alkaline phosphatase activities were seen towards the end of pregnancy but the time course of these changes was not synchronous. Receptor activity was first detectable at 11 days of pregnancy with a marked rise in receptor levels at 3 days post-partum. Changes in alkaline phosphatase activity more closely correlated with casein production and peak activity was observed at the time of parturition. We conclude that 1,25-dihydroxyvitamin D, receptor content increases during pregnancy and lactation and may be involved in maintaining milk calcium concentration.
Introduction During gestation and lactation, the mammary gland proliferates and mammary cells undergo differentiation which includes the acquisition of enzymes necessary for the formation of milk. The synthesis of milk proteins by the mammary gland is controlled by multiple interactions of several peptide and steroid hormones (Cowie et al., 1980). Caseins are a major group of secretory phosphoproteins synthesised during lactation in
Address for correspondence: Dr. K.W. Colston, ment of Chemical Pathology, St. George’s Hospital School, London, SW17 ORE, U.K. 0303-7207/88/$03.50
0 1988 Elsevier Scientific
DepartMedical
Publishers
Ireland,
mammals and are stored and secreted as stable calcium phosphate complexes (McMeekin, 1970). Lactation represents a physiological state of calcium stress: in the rat the equivalent of approximately 40% of maternal skeletal calcium is transported through the mammary secretory cells into the milk by the time of weaning (Robinson et al., 1982). This increased calcium requirement is met by a facultative increase in intestinal calcium absorption, (Fournier and Susbielle, 1952) mediated by 1,25-dihydroxyvitamin D, (1,25(OH),D,) (Boass et al., 1977). This steroid hormone may also play a role in transporting calcium into milk (Fry et al., 1983) and it previously has been demonstrated that lactating mammary gland contains specific receptor for 1,25(OH),D, (Colston et al., Ltd
16
1980: Reinhardt and Conrad, 1984) while the concentration of cytosolic 1,25(OH),D, binding activity is negligible in non-lactating mammary gland (Eisman et al., 1980; Colston et al., 1986). Alkaline phosphatase activity changes quantitatively during pregnancy and lactation in the rat mammal gland (Munford, 1963) but the basis for this regulation is not known. However, vitamin D status has been shown to influence alkaline phosphatase activity in a number of rat tissues (Ghan and Atkins, 1983; Colston and Cleeve, 1986). In vitro, 1,25(OH),D, stimulates alkaline phosphatase activity in rat osteogenic sarcoma cells by a receptor-mediated process (Manalogas et al., 1983). Since 1,25(OH),D, also has been reported to stimulate alkaline phosphatase in cultured breast cancer cells (Mu&ins and Sussman. 1985) this enzyme activity may be a useful marker for 1,25(OH),D, responsiveness in mammary tissue. The purpose of our study was 2 fold; firstly to establish the time course and magnitude of changes in 1,25(OH),D, receptor activity in rat mammary gland during pregnancy and lactation. Secondly we wished to determine if a temporal relationship exists between these changes in receptor concentration and alterations in both casein production (an index of mammary cell differentiation) and alkaline phosphatase activity. Materials and methods Chemicals 1,25-Dihyroxy-[23,24-N-3H]cholecalciferol (85 and 100 Ci/mmol) was obtained from Amersham International (Amersham, U.K.). Crystalline 1,25(OH),D, and 25-hydroxyvitamin D, (25(OH)D3) were gifts from Dr. W. Meier, Hoffmann-La Roche, Basle, Switzerland. Dithiothreitol was obtained from Sigma Chemical Co. (London, U.K.). Pregnant and lactating rats Weight- (250-300 g) and age-matched female Wistar albino rats (Charles Rivers) were time mated. Virgin rats of the same strain were used as controls. All animals had free access to food and water. Animals were killed at various times during pregnancy and lactation, and blood and tissues obtained. Five adult animals were used at each
time point studied. Following parturition, sizes ranged from ten to 13 pups.
litter
Mammary tissue (-‘HJ1,25(OH),D, binding activity Mammary tissue was rapidly removed and weighed. All subsequent steps were performed at O-4” C. Tissue was minced and washed three times in calciumand magnesium-free phosphatebuffered saline (CMF-PBS, 6.6 mM Na,HPO, and 1.5 mM KH,PO,). A tissue homogenate was prepared (15520%, w/v) in hypertonic buffer containing 300 mM KCl, 10 mM Tris-HCl (pH 7.4) 1 mM EDTA, 10 mM sodium molybdate, 4 mM dithiothreitol plus 50 PI/ml Trasylol solution. High speed supernatants were obtained and designated KTEDM extracts. Portions (200 ~1) of KTEDM extracts were incubated with 1.0 nM ~~H]l,25(OH)~D~ at 0°C for 3 h. Non-specific binding was assessed by parallel incubations containing a 250-fold molar excess of unlabelled 1,25(OH),D,. Bound [3H]1,25(OH),D, was separated from free hormone by charcoal adsorption and binding macromolecules for [ ‘H]l,25fOH),D:, were analysed by sucrose density analysis as previously described (Colston et al., 1980). Bovine serum albumin ( 14C radiolabelled) from Amersham International (4.4 S), was used as gradient marker. Total dpm comprising the 3.2 S sedimentation peak was determined. Specific binding in the 3.2 *S region was calculated by the difference between the sedimentation patterns in the absence and presence of a 250-fold molar excess of unlabelled 1,25(OH),D,. DNA was determined on the washed sediment from the tissue homogenate using the method of Burton (1956) and protein content of the KTEDM extracts measured by the method of Bradford (1976). All results were expressed in three ways: first as fmol of specifically bound [ “H]1,25(OH),D3 per g wet weight of tissue, second as fmol bound per mg cytosol protein and last as fmol bound per 100 pg DNA. Mammary tissue alkaline phosphatase activity Samples of mammary tissue from virgin, pregnant and lactating rats were stored at - 20°C until analysed. Tissue was weighed, minced and homogenates (lo%, w/v) prepared in Tris buffer (pH 7.4, 10 mmol/l) using a Potter-Elvehjem ho-
17
mogeniser. An equal volume of n-butanol was added and the tube shaken gently for 2 h at room temperature. Tubes were then centrifuged at 1200 rpm for 5 min, and the aqueous layer removed and filtered through a Sartorius syringe filter with membrane pore size of 0.2 pm. Extracts were stored at 4’ C until analysed. Alkaline phosphatase activity in the tissue extracts was measured on an LKB reaction rate analyser at 37 o C using p-nitrophenyl phosphate ad diethanolamine (Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, 1974).
Immunocytochemical method for detection of casein We utilised the LICR-LON 32.2 antiserum. This is an IgG mouse monoclonal antibody raised to a protein band from preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis of human milk fat globule membrane. It recognises human P-casein, rat band 2 casein and bovine casein. In human tissues this antibody shows a positive reaction only in lactating breast tissue and in occasional cells in a few benign breast lesions (Earl and McIlhinney, 1985). Mammary tissues from virgin, pregnant and lactating rats were snap frozen and stored in liquid nitrogen until analysed. Two 5-7 pm cryostat sections were cut at - 30 o C, thaw mounted onto poly-L-lysine-coated slides and air dried. Fixation was in acetone/chloroform for 5 min at 4” C. Slides were then washed twice in PBS and incubated with primary antibody for 1 h. After washing, slides were incubated with goat antimouse alkaline phosphatase conjugate (Sigma) diluted 1 to 100 in PBS/BSA for 1 h. After washing again the casein was visualised by incubating for 1 h with fast red/naphthol substrate solution containing levamisole to block endogenous alkaline phosphatase activity. Slides were then washed with counter-stained with Mayer’s distilled water, haemalum for 30 s, ‘blued’ in lithium carbonate solution and mounted in polyvinylalcohol (PVA). The reaction demonstrates casein in mammary epithelial cells with a weak to strong red staining. In addition, the secreted material present in the lumina of the alveolae and ducts shows a positive reaction.
Results Binding of [3H]1,25(OH), D, in lactating mammary gland In our preliminary experiments, sedimentation analysis on sucrose density gradients was used to compare the binding macromolecules for [3H]1,25(OH),D, present in KTEDM extracts prepared from lactating and non-lactating rat mammary gland. KTEDM extracts from lactating rat mammary gland demonstrated two binding proteins for [3H]1,25(OH),D3; one sedimenting at 5-6 S representing binding of [‘H]1,25(OH),D, to the 25(OH)D, binding protein described by Van Baelen (1977) and a second moiety sedimenting, like the rat intestinal receptor, at approximately 3.2 S. Binding of [3H]1,25(0H)2D3 to the mammary gland 3.2 S component but not the 5-6 S moiety was eliminated in the presence of a 250-fold excess of non-radioactive 1,25(OH),D,. However, a 50-fold molar excess of radioinert 25(OH)D, failed to eliminate binding of [3H]1,25(OH)zD3 to the 5-6 S binder (data not shown). KTEDM extracts from non-lactating rat mammary gland obtained from a retired breeder demonstrated only the 5-6 S binder. Changes in rat mammary gland [3HJ1,25(OH),D, binding activity during pregnancy and lactation It was concluded that the large and possibly variable amount of this contaminating 5-6 S protein in mammary gland KTEDM extracts precluded the accurate determination of receptor concentration by Scatchard analysis using conventional ligand binding techniques. For this reason the technique of sucrose density gradient analysis was adopted to quantitate the content of receptorlike [3H]1,25(OH),D, binding protein in rat mammary gland during pregnancy and lactation. Fig. 1 shows typical sedimentation patterns of KTEDM extracts of mammary glands at 7 and 11 days of pregnancy (a and 6) and at 6 and 17 days of lactation (c and d). The earliest time at which a specific peak of binding was observed in the 3.2 S region was 11 days of gestation. Specific binding in the 3.2 S region was determined and mammary gland content of the receptor-like protein was quantitated in three ways: fmol [3H]1,25(OH),D3 bound per mg cytosol protein, per 100 pg DNA
IIOr
a
b 0
b *
2 0 ._ c U
e
‘=
S-
z B
OL
to‘P
IO
C
5
IO
15
20
25
15
20
25
d
?I
'0 x
z0
.-
?J
F 4.45
q5
c
3 n
n
.Op
5
IO
15
20
25
fraction
top
5
IO
number
Fig. 1. Sucrose density gradient analysis of [3H]1,25(OH),D, binding in KTEDM extracts of rat mammary tissue at various stages of pregnancy and lactation. Aliquots (0.2 M) were incubated for 3 h at 0 o C with (0) 1.0 nM [ 3H]1,25(OH)ZD3 alone: (0) plus 250 nM BSA (4.4 S). (a) 7 days pregnant; (b) 11 days pregnant; (c) 6 days lactation; unlabelled 1,25(OH) 2 D,. The marker was “C-labelled (d) 17 days lactation.
and per g wet weight of mammary tissue. All three denominators were used in an attempt to overcome the problems both of differences in cellularity between quiescent and proliferating mammary tissue and variation in protein components of cytosols from lactating and non-lactating glands. Fig. 2 demonstrates that changes in mammary
gland [3H]1,25(OH)zD, binding activity with time of gestation and lactation are similar when expressed in terms of cytosol protein or DNA content and essentially the same results were seen when wet weight of tissue was used as the denominator. Thus the observed variation is unlikely to be attributable to changes in cellularity of the
19
b
25 -
10 pregnant
lactating
pregnant
20
lactating
IO -
10
pregnant
v
20: *v
i
0,
20
10
10 pregnant
lactating
20
lactating
days
Fig. 2. = and b: Changes in [3H]1,25(OH),D3 binding activity in KTEDM extracts of rat mammary gland during pregnancy and lactation. Results are expressed both as fmol bound per mg cytosol protein (a) and per 100 pg DNA (b). c and d: Changes in alkaline phosphatase activity in rat mammary gland during pregnancy and lactation. Results are expressed as units per mg protein (c) and per g wet weight of tissue (d). Results are means (f SEM). Dashed line indicates time of parturition.
tissues or changes in protein content of cytosols with the onset of lactation. Receptor-like activity was absent at 7 days and was first detected at 11
days gestation. There was little change in binding activity from 11 days gestation up to the time of parturition but a marked increase was observed at
20
3 days post-partum. Binding activity declined from about 6 days of lactation but appreciable activity was still present at 17 days post-partum. Alkaline phosphatase activity Mammary gland alkaline phosphatase activity also increased during pregnancy with highest levels seen at about the time of parturition. Results were similar whether expressed as activity per 100 pg DNA, per mg of extract protein or per g of tissue. Peak alkaline phosphatase activity was thus achieved some days before the marked increase in [3H]1,25(OH),D, binding activity which was apparent at 3 days post-partum. Fig. 2 compares the time course of change in alkaline phosphatase activity with that of [3H]1,25(OH),D, binding activity. Casein immunochemistry The histological and immunocytochemical studies showed that the mammary tissue of pregnant rats changes towards the time of lactation with mammary tissue showing an increase in develop-
TABLE
ment of glandular structures, lobules and alveolar structures by day 19 of pregnancy. Specimens from the lactating rats showed further changes with the appearance of vesicular cytoplasmic structures and basal location of the nuclei. The production of casein demonstrated by immunocytochemistry was low in the pregnant rats at days 7 and 11 of gestation. Only a small proportion of single cells gave a positive reaction. By day 19 of the pregnancy a strong and diffuse reaction was detected in all glandular structures. During the lactation period, although the intensity of reaction was somewhat reduced, immunoreactivity was seen in virtually all of the alveolar structures as a pink or slightly red cytoplasmic staining reaction. In specimens of mammary tissue at days 6, 12 and 17 of lactation the reactivity was more epithelial-membrane related (Table 1). Discussion The significance of specific receptors 1,25(OH),D, in normal and malignant breast
for tis-
1
CHANGES IN RAT MAMMARY CASEIN DURING PREGNANCY
GLAND [3H]1,25(OH),D, AND LACTATION
These results are mean + SEM. The number Days of
13HlLWOW,D,
pregnancy/ lactation
binding
of animals
(fmoI/mg)
BINDING
ACTIVITY,
at each time point is shown
ALKALINE
PHOSPHATASE,
in parentheses.
Alkaline phosphatase activity (units/ mg protein)
Casein staining intensity and pattern
Serum calcium (mmol/l) a
Pregnant 7 (3) 11 (5) 13 (3) 19 (5)
ND 0.82 f 0.29 0.78 * 0.03 2.51 f 0.94
1.00 f 0.21 1.14 f 0.27 1.48kO.12 4.94 f 0.57
+ single ( + ) single ++ +++
2.32 2.57 2.67 2.51
Lactating l(5) 3 (5) 6 (5) 12 (5) 17 (5)
2.11 f 0.75 8.7Ok1.7 10.42 + 2.99 7.53 f 1.52 4.87 f 0.25
3.05 &-0.61 2.72 +0.36 2.72 f 0.41 1.59+0.21 1.05 *0.34
++ + +/+ + (Membrane + / + + (Membrane + / + + (Membrane
2.76 _+0.04 2.54kO.17 2.42 f 0.20 2.21 f 0.09 2.06 + 0.28
Virgin (4)
ND=
0.25 f 0.08
ND
a (+) single cells or very weak staining; b Staining associated with membrane. ’ ND, not detectable.
+ weak;
+ + moderate;
+ + + strong.
b, b, b,
f 0.44 k 0.10 & 0.17 i 0.09
2.46 f 0.05
AND
sue is not known. Specific binding proteins for 1,25(OH),D, which display properties similar to the receptor in classical target organs have been reported in mammary glands from pregnant and lactating animals (Colston et al., 1980; Eisman et al., 1980). Cytosol from non-lactating bovine mammary glands showed significantly less binding of 1,25(OH) z D, than cytosol prepared from lactating tissue (Reinhardt and Conrad, 1980). Our previous findings have demonstrated the presence of receptors for 1,25(OH),D, in extracts of nitrosomethylurea-induced rat mammary tumours (Colston et al., 1986). Furthermore the receptor content of lactating mammary gland is of the same order as that in this tumour tissue. Negligible binding of 1,25(OH),D, was detected in non-lactating mammary tissue. Thus possession of substantial amounts of 1,25(OH),D, receptor activity would appear to be a property shared only by neoplastic and lactating rat mammary gland. Using the sucrose density gradient technique we were unable to detect the presence of the 3.2 S receptor in non-pregnant mammary gland. However, we cannot exclude the possibility that normal mammary gland contains a small proportion of receptor-containing cells which proliferate as the tissue undergoes differentiation. Indeed we have been able to demonstrate immunoreactive 1,25(OH),D, receptor protein in epithelial cells of lobules and ducts of normal (non-lactating) human breast tissue using the more sensitive technique of immunocytochemistry (Berger et al., 1987). In the present study the time course of change in rat mammary tissue receptor activity demonstrates a sharp increase at approximately 3 days post-partum. Although the mitotic rate of mammary cells remains constant into the early period of lactation in the rat (Knight, 1984) it is unlikely that this large increase in activity is solely due to a proliferation of receptor-containing cells but may, in part, be attributable to a regulation of cellular receptor content dependent upon changes in the hormonal status of the animal with onset of lactation. Without further data it is only possible to speculate as to the hormonal factors involved in this regulation. Several hormones have been implicated as regulators of the 1,25(OH),D, receptor in other tissues including oestrogen
(Walters, 1981) glucocorticoids (Hirst and Feldman, 1982) and insulin (Seino et al., 1983) three hormones which are known to be of importance in the proliferation and differentiation of mammary cells. 1,25(OH),D, itself is also reported to upregulate the concentration of its own receptor (Costa et al., 1985) and circulating 1,25(OH),D, concentrations are elevated in lactating animals (Boass et al., 1977). Furthermore, 1,25(OH),D, is an inducer of cellular differentiation in certain cell types (Abe et al., 1981). In addition, effects of prolactin on regulation of 1,25(OH),D, receptor levels have recently been reported (Mezzeth et al., 1987). Histological examination of mammary glands showed that the expected changes in development of glandular structures was virtually complete by day 19 of pregnancy which is the time when alkaline phosphatase activity was found to be maximal. However, during the early lactation period little further change was seen in the histological appearance of the gland whereas alkaline phosphatase activity declined following parturition. The observed changes in casein production quite closely mirrored the measured changes in alkaline phosphatase activity in mammary tissue during the per-i-partum and post-partum period. However, appreciable casein immunoreactivity persisted from the mid to late lactation period whereas there was a more marked decrease in enzyme activity during this period. Peak casein production and alkaline phosphatase activity were both seen prior to the marked increase seen in 1,25(OH)2D, receptor activity post-partum. Interestingly, the time course of change of receptor activity appears better to correlate with reported rat milk calcium levels which rise sharply from the third day of lactation (Bassler, 1978) and thus the presence of this receptor may be of importance in regulating the calcium concentration of milk. In summary, our results show marked changes in both 1,25(OH),D, binding activity and alkaline phosphatase activity during pregnancy and lactation in the rat mammary gland. Little t3H]1,25(OH),D3 binding activity was seen in mammary glands of non-pregnant rats with increasing amounts observed as the mammary gland differentiates. The time course of change of these two activities was found not to be synchronous, and
22
alkaline phosphatase activity more closely correlated with change in casein production. The hormonal basis for the regulation of 1,25(OH),D, receptors and alkaline phosphatase activity during pregnancy and lactation remains to be established. Acknowledgements This study was supported by a grant from the Cancer Research Campaign. Preliminary results from this study were presented at the 7th Workshop on Vitamin D, Ranch0 Mirage, California, 24-29 April 1988. References Abe, E., Miyuara, C., Sakagami, H., Takeda, M., Kanno, K., Yamazaki, T., Yoshiki, S. and Suda, T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4990-4994. Blssler, R. (1978) in Pathologie der Bustdruse in spezielle pathologische Anatomie (Doerr, W., Seirfert, G. and Uehlinger, E., eds.), Vol 2, pp. 156-157, Springer-Verlag, Berlin. Berger, U., Wilson, P., McClelland, R.A., Colston, K., Haussler, M.R., Pike, J.W. and Coombes, R.C. (1987) Cancer Res. 47, 6793-6799. Boass, A., Toverud, S.U., McCain, T.A., Pike, J.W. and Haussler, M.R. (1977) Nature 267, 630-632. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Burton, K. (1956) Biochem. J. 62, 315-323. Chan, D.S. and Atkins, D. (1983) Gut 24, 886-892. Colston, K. and Cleeve, H.J.W. (1986) Comp. Biochem. Physiol. 839, 681-684. Colston, K., Hirst, M. and Feldman, D. (1980) Endocrinology 107, 1916-1922. Colston, K., Wilkinson, J.R. and Coombes, R.C. (1986) Endocrinology 119, 397-403. Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974) Stand. J. Clin. Invest. 33, 290-306.
Costa, E.M., Hirst, M.A. and Feldman, D. (1985) Endocrinology 117, 2203-2210. Cowie, A.T., Forsyth, LA. and Hart, I.C. (1980) Hormonal Control of Lactation, Springer-Verlag, Berlin. Earl, H.M. and McIlhinney. R.A.J. (1985) Mol. Immunol. 22. 981-991. Eisman, J.A., MacIntyre, I., Martin, T.J., Frampton, R.J. and King, R.J.B. (1980) Clin. Endocrinol. 13, 267-272. Folley, S.J. and Greenbaum, A.L. (1947) Biochem. J. 41, 261-266. Fournier, P. and Susbielle. H. (1952) J. Physiol. 44, 123-134. Fry, J.M., Cumow, D.H., Gutteridge, D.H. and Retallack, R.W. (1983) Life Sci. 27, 1255-1263. Hirst, M. and Feldman, D. (1982) Endocrinology 111. 1400-1402. Knight, C.H. (1984) in Physiological Strategies in Lactation (Symposium of the Zoological Society. London. No. 51) (Peaker, M., Vernon, R.G. and Knight, C.H., eds.), pp. 147-170, Academic Press, London.. Manolagas, SC., Spiess, Y.H., Burton, D.W. and Deftos, L.J. (1983) Mol. Cell Endocrinol. 33, 27-36. McMeekin, T.L. (1970) in Milk Proteins: Chemistry and Molecular Biology (McKenzie. H.A.. ed.). Vol 1, pp. 3-15. Academic Press, New York. Mezzehi, G., Barbiroli, G. and Oka, T. (1987) Endocrinology 120, 2488-2493. Mulkins, M.A. and Sussman, H.H. (1985) in Vitamin D. Chemical, Biochemical and Clinical Update (Norman, A.W., Schafer, K., Grigoleit, H.G. and v. Herrath, D.. eds.), pp. 889-890, Walter de Gruyter, Berlin. Munford, R.E. (1963) J. Endocrinol. 28, 17-34. Reinhardt, T.A. and Conrad, H.R. (1980) Arch. Biochem. Biophys. 203, 1088116. Robinson. C.J., Spanos, E., James, M.F., Pike, J.W.. Haussler, M.R., Makeen, A.M., Hillyard, C.J. and MacIntyre. 1. (1982) J. Endocrinol. 94. 443-453. Seino, Y., Sierra, R.I., Sonn, Y.M., Jafari, A., Birge, S.J. and Avioli, L.V. (1983) Endocrinology 113, 1721-1725. Van Baelen, H., Bouillon, R. and DeMoor, P. (1977) J. Biol. Chem. 252, 251552518. Walters, M. (1981) Biochem. Biophys. Res. Commun. 103, 721-726.
15
MCE 01930
Mammary
gland 1,25dihydroxyvitamin D, receptor during pregnancy and lactation
K.W. Colston I, U. Berger 2, P. Wilson 2, L. Hadcocks and R.C. Coombes ’
content
I, I. Naeem ‘, H.M. Earl 3
’ Department of Chemrcal Pathology and ’ Ludwig Instituie for Cancer Research (London - St. George’s Group), St. George’s Hospital MedIcal School, London, U.K., and -’ Department of Radiotherapy and Oncology, University College Hospital, London, U.K. (Received
Key words: Mammary
gland;
23 February
1,25_Dihydroxyvitamin
1988; accepted
D, receptor:
Casein;
21 June 1988)
Alkaline
phosphatase;
Pregnancy;
Lactation;
(Rat)
Summary The purpose of this study was to establish the time course and magnitude of changes in 1,25_dihydroxyvitamin D receptor activity in rat mammary gland during pregnancy and lactation and to correlate these changes with casein production and alkaline phosphatase activity. Marked increases in both 1,25dihydroxyvitamin D, receptor and alkaline phosphatase activities were seen towards the end of pregnancy but the time course of these changes was not synchronous. Receptor activity was first detectable at 11 days of pregnancy with a marked rise in receptor levels at 3 days post-partum. Changes in alkaline phosphatase activity more closely correlated with casein production and peak activity was observed at the time of parturition. We conclude that 1,25-dihydroxyvitamin D, receptor content increases during pregnancy and lactation and may be involved in maintaining milk calcium concentration.
Introduction During gestation and lactation, the mammary gland proliferates and mammary cells undergo differentiation which includes the acquisition of enzymes necessary for the formation of milk. The synthesis of milk proteins by the mammary gland is controlled by multiple interactions of several peptide and steroid hormones (Cowie et al., 1980). Caseins are a major group of secretory phosphoproteins synthesised during lactation in
Address for correspondence: Dr. K.W. Colston, ment of Chemical Pathology, St. George’s Hospital School, London, SW17 ORE, U.K. 0303-7207/88/$03.50
0 1988 Elsevier Scientific
DepartMedical
Publishers
Ireland,
mammals and are stored and secreted as stable calcium phosphate complexes (McMeekin, 1970). Lactation represents a physiological state of calcium stress: in the rat the equivalent of approximately 40% of maternal skeletal calcium is transported through the mammary secretory cells into the milk by the time of weaning (Robinson et al., 1982). This increased calcium requirement is met by a facultative increase in intestinal calcium absorption, (Fournier and Susbielle, 1952) mediated by 1,25-dihydroxyvitamin D, (1,25(OH),D,) (Boass et al., 1977). This steroid hormone may also play a role in transporting calcium into milk (Fry et al., 1983) and it previously has been demonstrated that lactating mammary gland contains specific receptor for 1,25(OH),D, (Colston et al., Ltd
16
1980: Reinhardt and Conrad, 1984) while the concentration of cytosolic 1,25(OH),D, binding activity is negligible in non-lactating mammary gland (Eisman et al., 1980; Colston et al., 1986). Alkaline phosphatase activity changes quantitatively during pregnancy and lactation in the rat mammal gland (Munford, 1963) but the basis for this regulation is not known. However, vitamin D status has been shown to influence alkaline phosphatase activity in a number of rat tissues (Ghan and Atkins, 1983; Colston and Cleeve, 1986). In vitro, 1,25(OH),D, stimulates alkaline phosphatase activity in rat osteogenic sarcoma cells by a receptor-mediated process (Manalogas et al., 1983). Since 1,25(OH),D, also has been reported to stimulate alkaline phosphatase in cultured breast cancer cells (Mu&ins and Sussman. 1985) this enzyme activity may be a useful marker for 1,25(OH),D, responsiveness in mammary tissue. The purpose of our study was 2 fold; firstly to establish the time course and magnitude of changes in 1,25(OH),D, receptor activity in rat mammary gland during pregnancy and lactation. Secondly we wished to determine if a temporal relationship exists between these changes in receptor concentration and alterations in both casein production (an index of mammary cell differentiation) and alkaline phosphatase activity. Materials and methods Chemicals 1,25-Dihyroxy-[23,24-N-3H]cholecalciferol (85 and 100 Ci/mmol) was obtained from Amersham International (Amersham, U.K.). Crystalline 1,25(OH),D, and 25-hydroxyvitamin D, (25(OH)D3) were gifts from Dr. W. Meier, Hoffmann-La Roche, Basle, Switzerland. Dithiothreitol was obtained from Sigma Chemical Co. (London, U.K.). Pregnant and lactating rats Weight- (250-300 g) and age-matched female Wistar albino rats (Charles Rivers) were time mated. Virgin rats of the same strain were used as controls. All animals had free access to food and water. Animals were killed at various times during pregnancy and lactation, and blood and tissues obtained. Five adult animals were used at each
time point studied. Following parturition, sizes ranged from ten to 13 pups.
litter
Mammary tissue (-‘HJ1,25(OH),D, binding activity Mammary tissue was rapidly removed and weighed. All subsequent steps were performed at O-4” C. Tissue was minced and washed three times in calciumand magnesium-free phosphatebuffered saline (CMF-PBS, 6.6 mM Na,HPO, and 1.5 mM KH,PO,). A tissue homogenate was prepared (15520%, w/v) in hypertonic buffer containing 300 mM KCl, 10 mM Tris-HCl (pH 7.4) 1 mM EDTA, 10 mM sodium molybdate, 4 mM dithiothreitol plus 50 PI/ml Trasylol solution. High speed supernatants were obtained and designated KTEDM extracts. Portions (200 ~1) of KTEDM extracts were incubated with 1.0 nM ~~H]l,25(OH)~D~ at 0°C for 3 h. Non-specific binding was assessed by parallel incubations containing a 250-fold molar excess of unlabelled 1,25(OH),D,. Bound [3H]1,25(OH),D, was separated from free hormone by charcoal adsorption and binding macromolecules for [ ‘H]l,25fOH),D:, were analysed by sucrose density analysis as previously described (Colston et al., 1980). Bovine serum albumin ( 14C radiolabelled) from Amersham International (4.4 S), was used as gradient marker. Total dpm comprising the 3.2 S sedimentation peak was determined. Specific binding in the 3.2 *S region was calculated by the difference between the sedimentation patterns in the absence and presence of a 250-fold molar excess of unlabelled 1,25(OH),D,. DNA was determined on the washed sediment from the tissue homogenate using the method of Burton (1956) and protein content of the KTEDM extracts measured by the method of Bradford (1976). All results were expressed in three ways: first as fmol of specifically bound [ “H]1,25(OH),D3 per g wet weight of tissue, second as fmol bound per mg cytosol protein and last as fmol bound per 100 pg DNA. Mammary tissue alkaline phosphatase activity Samples of mammary tissue from virgin, pregnant and lactating rats were stored at - 20°C until analysed. Tissue was weighed, minced and homogenates (lo%, w/v) prepared in Tris buffer (pH 7.4, 10 mmol/l) using a Potter-Elvehjem ho-
17
mogeniser. An equal volume of n-butanol was added and the tube shaken gently for 2 h at room temperature. Tubes were then centrifuged at 1200 rpm for 5 min, and the aqueous layer removed and filtered through a Sartorius syringe filter with membrane pore size of 0.2 pm. Extracts were stored at 4’ C until analysed. Alkaline phosphatase activity in the tissue extracts was measured on an LKB reaction rate analyser at 37 o C using p-nitrophenyl phosphate ad diethanolamine (Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, 1974).
Immunocytochemical method for detection of casein We utilised the LICR-LON 32.2 antiserum. This is an IgG mouse monoclonal antibody raised to a protein band from preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis of human milk fat globule membrane. It recognises human P-casein, rat band 2 casein and bovine casein. In human tissues this antibody shows a positive reaction only in lactating breast tissue and in occasional cells in a few benign breast lesions (Earl and McIlhinney, 1985). Mammary tissues from virgin, pregnant and lactating rats were snap frozen and stored in liquid nitrogen until analysed. Two 5-7 pm cryostat sections were cut at - 30 o C, thaw mounted onto poly-L-lysine-coated slides and air dried. Fixation was in acetone/chloroform for 5 min at 4” C. Slides were then washed twice in PBS and incubated with primary antibody for 1 h. After washing, slides were incubated with goat antimouse alkaline phosphatase conjugate (Sigma) diluted 1 to 100 in PBS/BSA for 1 h. After washing again the casein was visualised by incubating for 1 h with fast red/naphthol substrate solution containing levamisole to block endogenous alkaline phosphatase activity. Slides were then washed with counter-stained with Mayer’s distilled water, haemalum for 30 s, ‘blued’ in lithium carbonate solution and mounted in polyvinylalcohol (PVA). The reaction demonstrates casein in mammary epithelial cells with a weak to strong red staining. In addition, the secreted material present in the lumina of the alveolae and ducts shows a positive reaction.
Results Binding of [3H]1,25(OH), D, in lactating mammary gland In our preliminary experiments, sedimentation analysis on sucrose density gradients was used to compare the binding macromolecules for [3H]1,25(OH),D, present in KTEDM extracts prepared from lactating and non-lactating rat mammary gland. KTEDM extracts from lactating rat mammary gland demonstrated two binding proteins for [3H]1,25(OH),D3; one sedimenting at 5-6 S representing binding of [‘H]1,25(OH),D, to the 25(OH)D, binding protein described by Van Baelen (1977) and a second moiety sedimenting, like the rat intestinal receptor, at approximately 3.2 S. Binding of [3H]1,25(0H)2D3 to the mammary gland 3.2 S component but not the 5-6 S moiety was eliminated in the presence of a 250-fold excess of non-radioactive 1,25(OH),D,. However, a 50-fold molar excess of radioinert 25(OH)D, failed to eliminate binding of [3H]1,25(OH)zD3 to the 5-6 S binder (data not shown). KTEDM extracts from non-lactating rat mammary gland obtained from a retired breeder demonstrated only the 5-6 S binder. Changes in rat mammary gland [3HJ1,25(OH),D, binding activity during pregnancy and lactation It was concluded that the large and possibly variable amount of this contaminating 5-6 S protein in mammary gland KTEDM extracts precluded the accurate determination of receptor concentration by Scatchard analysis using conventional ligand binding techniques. For this reason the technique of sucrose density gradient analysis was adopted to quantitate the content of receptorlike [3H]1,25(OH),D, binding protein in rat mammary gland during pregnancy and lactation. Fig. 1 shows typical sedimentation patterns of KTEDM extracts of mammary glands at 7 and 11 days of pregnancy (a and 6) and at 6 and 17 days of lactation (c and d). The earliest time at which a specific peak of binding was observed in the 3.2 S region was 11 days of gestation. Specific binding in the 3.2 S region was determined and mammary gland content of the receptor-like protein was quantitated in three ways: fmol [3H]1,25(OH),D3 bound per mg cytosol protein, per 100 pg DNA
IIOr
a
b 0
b *
2 0 ._ c U
e
‘=
S-
z B
OL
to‘P
IO
C
5
IO
15
20
25
15
20
25
d
?I
'0 x
z0
.-
?J
F 4.45
q5
c
3 n
n
.Op
5
IO
15
20
25
fraction
top
5
IO
number
Fig. 1. Sucrose density gradient analysis of [3H]1,25(OH),D, binding in KTEDM extracts of rat mammary tissue at various stages of pregnancy and lactation. Aliquots (0.2 M) were incubated for 3 h at 0 o C with (0) 1.0 nM [ 3H]1,25(OH)ZD3 alone: (0) plus 250 nM BSA (4.4 S). (a) 7 days pregnant; (b) 11 days pregnant; (c) 6 days lactation; unlabelled 1,25(OH) 2 D,. The marker was “C-labelled (d) 17 days lactation.
and per g wet weight of mammary tissue. All three denominators were used in an attempt to overcome the problems both of differences in cellularity between quiescent and proliferating mammary tissue and variation in protein components of cytosols from lactating and non-lactating glands. Fig. 2 demonstrates that changes in mammary
gland [3H]1,25(OH)zD, binding activity with time of gestation and lactation are similar when expressed in terms of cytosol protein or DNA content and essentially the same results were seen when wet weight of tissue was used as the denominator. Thus the observed variation is unlikely to be attributable to changes in cellularity of the
19
b
25 -
10 pregnant
lactating
pregnant
20
lactating
IO -
10
pregnant
v
20: *v
i
0,
20
10
10 pregnant
lactating
20
lactating
days
Fig. 2. = and b: Changes in [3H]1,25(OH),D3 binding activity in KTEDM extracts of rat mammary gland during pregnancy and lactation. Results are expressed both as fmol bound per mg cytosol protein (a) and per 100 pg DNA (b). c and d: Changes in alkaline phosphatase activity in rat mammary gland during pregnancy and lactation. Results are expressed as units per mg protein (c) and per g wet weight of tissue (d). Results are means (f SEM). Dashed line indicates time of parturition.
tissues or changes in protein content of cytosols with the onset of lactation. Receptor-like activity was absent at 7 days and was first detected at 11
days gestation. There was little change in binding activity from 11 days gestation up to the time of parturition but a marked increase was observed at
20
3 days post-partum. Binding activity declined from about 6 days of lactation but appreciable activity was still present at 17 days post-partum. Alkaline phosphatase activity Mammary gland alkaline phosphatase activity also increased during pregnancy with highest levels seen at about the time of parturition. Results were similar whether expressed as activity per 100 pg DNA, per mg of extract protein or per g of tissue. Peak alkaline phosphatase activity was thus achieved some days before the marked increase in [3H]1,25(OH),D, binding activity which was apparent at 3 days post-partum. Fig. 2 compares the time course of change in alkaline phosphatase activity with that of [3H]1,25(OH),D, binding activity. Casein immunochemistry The histological and immunocytochemical studies showed that the mammary tissue of pregnant rats changes towards the time of lactation with mammary tissue showing an increase in develop-
TABLE
ment of glandular structures, lobules and alveolar structures by day 19 of pregnancy. Specimens from the lactating rats showed further changes with the appearance of vesicular cytoplasmic structures and basal location of the nuclei. The production of casein demonstrated by immunocytochemistry was low in the pregnant rats at days 7 and 11 of gestation. Only a small proportion of single cells gave a positive reaction. By day 19 of the pregnancy a strong and diffuse reaction was detected in all glandular structures. During the lactation period, although the intensity of reaction was somewhat reduced, immunoreactivity was seen in virtually all of the alveolar structures as a pink or slightly red cytoplasmic staining reaction. In specimens of mammary tissue at days 6, 12 and 17 of lactation the reactivity was more epithelial-membrane related (Table 1). Discussion The significance of specific receptors 1,25(OH),D, in normal and malignant breast
for tis-
1
CHANGES IN RAT MAMMARY CASEIN DURING PREGNANCY
GLAND [3H]1,25(OH),D, AND LACTATION
These results are mean + SEM. The number Days of
13HlLWOW,D,
pregnancy/ lactation
binding
of animals
(fmoI/mg)
BINDING
ACTIVITY,
at each time point is shown
ALKALINE
PHOSPHATASE,
in parentheses.
Alkaline phosphatase activity (units/ mg protein)
Casein staining intensity and pattern
Serum calcium (mmol/l) a
Pregnant 7 (3) 11 (5) 13 (3) 19 (5)
ND 0.82 f 0.29 0.78 * 0.03 2.51 f 0.94
1.00 f 0.21 1.14 f 0.27 1.48kO.12 4.94 f 0.57
+ single ( + ) single ++ +++
2.32 2.57 2.67 2.51
Lactating l(5) 3 (5) 6 (5) 12 (5) 17 (5)
2.11 f 0.75 8.7Ok1.7 10.42 + 2.99 7.53 f 1.52 4.87 f 0.25
3.05 &-0.61 2.72 +0.36 2.72 f 0.41 1.59+0.21 1.05 *0.34
++ + +/+ + (Membrane + / + + (Membrane + / + + (Membrane
2.76 _+0.04 2.54kO.17 2.42 f 0.20 2.21 f 0.09 2.06 + 0.28
Virgin (4)
ND=
0.25 f 0.08
ND
a (+) single cells or very weak staining; b Staining associated with membrane. ’ ND, not detectable.
+ weak;
+ + moderate;
+ + + strong.
b, b, b,
f 0.44 k 0.10 & 0.17 i 0.09
2.46 f 0.05
AND
sue is not known. Specific binding proteins for 1,25(OH),D, which display properties similar to the receptor in classical target organs have been reported in mammary glands from pregnant and lactating animals (Colston et al., 1980; Eisman et al., 1980). Cytosol from non-lactating bovine mammary glands showed significantly less binding of 1,25(OH) z D, than cytosol prepared from lactating tissue (Reinhardt and Conrad, 1980). Our previous findings have demonstrated the presence of receptors for 1,25(OH),D, in extracts of nitrosomethylurea-induced rat mammary tumours (Colston et al., 1986). Furthermore the receptor content of lactating mammary gland is of the same order as that in this tumour tissue. Negligible binding of 1,25(OH),D, was detected in non-lactating mammary tissue. Thus possession of substantial amounts of 1,25(OH),D, receptor activity would appear to be a property shared only by neoplastic and lactating rat mammary gland. Using the sucrose density gradient technique we were unable to detect the presence of the 3.2 S receptor in non-pregnant mammary gland. However, we cannot exclude the possibility that normal mammary gland contains a small proportion of receptor-containing cells which proliferate as the tissue undergoes differentiation. Indeed we have been able to demonstrate immunoreactive 1,25(OH),D, receptor protein in epithelial cells of lobules and ducts of normal (non-lactating) human breast tissue using the more sensitive technique of immunocytochemistry (Berger et al., 1987). In the present study the time course of change in rat mammary tissue receptor activity demonstrates a sharp increase at approximately 3 days post-partum. Although the mitotic rate of mammary cells remains constant into the early period of lactation in the rat (Knight, 1984) it is unlikely that this large increase in activity is solely due to a proliferation of receptor-containing cells but may, in part, be attributable to a regulation of cellular receptor content dependent upon changes in the hormonal status of the animal with onset of lactation. Without further data it is only possible to speculate as to the hormonal factors involved in this regulation. Several hormones have been implicated as regulators of the 1,25(OH),D, receptor in other tissues including oestrogen
(Walters, 1981) glucocorticoids (Hirst and Feldman, 1982) and insulin (Seino et al., 1983) three hormones which are known to be of importance in the proliferation and differentiation of mammary cells. 1,25(OH),D, itself is also reported to upregulate the concentration of its own receptor (Costa et al., 1985) and circulating 1,25(OH),D, concentrations are elevated in lactating animals (Boass et al., 1977). Furthermore, 1,25(OH),D, is an inducer of cellular differentiation in certain cell types (Abe et al., 1981). In addition, effects of prolactin on regulation of 1,25(OH),D, receptor levels have recently been reported (Mezzeth et al., 1987). Histological examination of mammary glands showed that the expected changes in development of glandular structures was virtually complete by day 19 of pregnancy which is the time when alkaline phosphatase activity was found to be maximal. However, during the early lactation period little further change was seen in the histological appearance of the gland whereas alkaline phosphatase activity declined following parturition. The observed changes in casein production quite closely mirrored the measured changes in alkaline phosphatase activity in mammary tissue during the per-i-partum and post-partum period. However, appreciable casein immunoreactivity persisted from the mid to late lactation period whereas there was a more marked decrease in enzyme activity during this period. Peak casein production and alkaline phosphatase activity were both seen prior to the marked increase seen in 1,25(OH)2D, receptor activity post-partum. Interestingly, the time course of change of receptor activity appears better to correlate with reported rat milk calcium levels which rise sharply from the third day of lactation (Bassler, 1978) and thus the presence of this receptor may be of importance in regulating the calcium concentration of milk. In summary, our results show marked changes in both 1,25(OH),D, binding activity and alkaline phosphatase activity during pregnancy and lactation in the rat mammary gland. Little t3H]1,25(OH),D3 binding activity was seen in mammary glands of non-pregnant rats with increasing amounts observed as the mammary gland differentiates. The time course of change of these two activities was found not to be synchronous, and
22
alkaline phosphatase activity more closely correlated with change in casein production. The hormonal basis for the regulation of 1,25(OH),D, receptors and alkaline phosphatase activity during pregnancy and lactation remains to be established. Acknowledgements This study was supported by a grant from the Cancer Research Campaign. Preliminary results from this study were presented at the 7th Workshop on Vitamin D, Ranch0 Mirage, California, 24-29 April 1988. References Abe, E., Miyuara, C., Sakagami, H., Takeda, M., Kanno, K., Yamazaki, T., Yoshiki, S. and Suda, T. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4990-4994. Blssler, R. (1978) in Pathologie der Bustdruse in spezielle pathologische Anatomie (Doerr, W., Seirfert, G. and Uehlinger, E., eds.), Vol 2, pp. 156-157, Springer-Verlag, Berlin. Berger, U., Wilson, P., McClelland, R.A., Colston, K., Haussler, M.R., Pike, J.W. and Coombes, R.C. (1987) Cancer Res. 47, 6793-6799. Boass, A., Toverud, S.U., McCain, T.A., Pike, J.W. and Haussler, M.R. (1977) Nature 267, 630-632. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Burton, K. (1956) Biochem. J. 62, 315-323. Chan, D.S. and Atkins, D. (1983) Gut 24, 886-892. Colston, K. and Cleeve, H.J.W. (1986) Comp. Biochem. Physiol. 839, 681-684. Colston, K., Hirst, M. and Feldman, D. (1980) Endocrinology 107, 1916-1922. Colston, K., Wilkinson, J.R. and Coombes, R.C. (1986) Endocrinology 119, 397-403. Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974) Stand. J. Clin. Invest. 33, 290-306.
Costa, E.M., Hirst, M.A. and Feldman, D. (1985) Endocrinology 117, 2203-2210. Cowie, A.T., Forsyth, LA. and Hart, I.C. (1980) Hormonal Control of Lactation, Springer-Verlag, Berlin. Earl, H.M. and McIlhinney. R.A.J. (1985) Mol. Immunol. 22. 981-991. Eisman, J.A., MacIntyre, I., Martin, T.J., Frampton, R.J. and King, R.J.B. (1980) Clin. Endocrinol. 13, 267-272. Folley, S.J. and Greenbaum, A.L. (1947) Biochem. J. 41, 261-266. Fournier, P. and Susbielle. H. (1952) J. Physiol. 44, 123-134. Fry, J.M., Cumow, D.H., Gutteridge, D.H. and Retallack, R.W. (1983) Life Sci. 27, 1255-1263. Hirst, M. and Feldman, D. (1982) Endocrinology 111. 1400-1402. Knight, C.H. (1984) in Physiological Strategies in Lactation (Symposium of the Zoological Society. London. No. 51) (Peaker, M., Vernon, R.G. and Knight, C.H., eds.), pp. 147-170, Academic Press, London.. Manolagas, SC., Spiess, Y.H., Burton, D.W. and Deftos, L.J. (1983) Mol. Cell Endocrinol. 33, 27-36. McMeekin, T.L. (1970) in Milk Proteins: Chemistry and Molecular Biology (McKenzie. H.A.. ed.). Vol 1, pp. 3-15. Academic Press, New York. Mezzehi, G., Barbiroli, G. and Oka, T. (1987) Endocrinology 120, 2488-2493. Mulkins, M.A. and Sussman, H.H. (1985) in Vitamin D. Chemical, Biochemical and Clinical Update (Norman, A.W., Schafer, K., Grigoleit, H.G. and v. Herrath, D.. eds.), pp. 889-890, Walter de Gruyter, Berlin. Munford, R.E. (1963) J. Endocrinol. 28, 17-34. Reinhardt, T.A. and Conrad, H.R. (1980) Arch. Biochem. Biophys. 203, 1088116. Robinson. C.J., Spanos, E., James, M.F., Pike, J.W.. Haussler, M.R., Makeen, A.M., Hillyard, C.J. and MacIntyre. 1. (1982) J. Endocrinol. 94. 443-453. Seino, Y., Sierra, R.I., Sonn, Y.M., Jafari, A., Birge, S.J. and Avioli, L.V. (1983) Endocrinology 113, 1721-1725. Van Baelen, H., Bouillon, R. and DeMoor, P. (1977) J. Biol. Chem. 252, 251552518. Walters, M. (1981) Biochem. Biophys. Res. Commun. 103, 721-726.