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EFFECTS OF 1α,25-DIHYDROXYCHOLECALCIFEROL AND CORTISOL ON THE GROWTH AND DIFFERENTIATION OF PRIMARY CULTURES OF MOUSE MAMMARY EPITHELIAL CELLS IN COLLAGEN GEL
Cell Biology International 1999, Vol. 23, No. 7, 481–487 Article No. cbir.1999.0406, available online at http://www.idealibrary.com on
EFFECTS OF 1,25-DIHYDROXYCHOLECALCIFEROL AND CORTISOL ON THE GROWTH AND DIFFERENTIATION OF PRIMARY CULTURES OF MOUSE MAMMARY EPITHELIAL CELLS IN COLLAGEN GEL TAKUYA KANAZAWA1*, JUMPEI ENAMI2 and KAORU KOHMOTO1 1
Department of Animal Breeding, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; 2 Research Laboratory, Zenyaku Kogyo Co., Ltd, 2-33-7 Oizumi, Nerima-ku, Tokyo 178-0062, Japan Received 3 September 1998; accepted 12 April 1999
We examined the effects of 1,25-dihydroxycholecalciferol (1,25-DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10 11–10 9 ) of 1,25-DHCC stimulated growth of the cells in a collagen gel matrix culture in serum-free DMEM+Ham’s F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8-fold the control after 6 d in culture. In contrast, supraphysiological concentrations (10 8–10 7 ) of 1,25-DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose-dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25-DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25-DHCC also affected casein synthesis by cells cultured in a serum-free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10 11–10 9 ) and inhibiting it above 10 8 . In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25-DHCC. These results suggest a physiological role for 1,25-DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25-DHCC does not substitute for 1999 Academic Press glucocorticoids in the differentiation of the cells. K: vitamin D3; cortisol; primary mammary epithelial cell; collagen gel culture; mouse.
INTRODUCTION Steroid hormones are involved in the regulation of growth and differentiation of cells of a variety of tissues. Glucocorticoids are stimulatory for both differentiation (Oka and Topper, 1971) and growth (Mori et al., 1983) of mammary epithelial cells of the mouse in organ culture systems. Previously, several reports have shown that 1,25dihydroxycholecalciferol (1,25-DHCC), the most potent form among vitamin D3 derivatives, stimulates differentiation of normal and malignant myelocytes (Abe et al., 1981; McCarthy et al., 1983), and normal epidermal cells (Hosomi et al., 1983), more effectively than do glucocorticoids. These *Present address: Laboratory of Animal Breeding, Division of Biotechnology, Faculty of Agriculture, Ibaraki University, Ami-machi, Ibaraki 300-0393, Japan, and to whom correspondence should be addressed. 1065–6995/99/070481+07 $30.00/0
1,25-DHCC-responsive cells have specific receptors, and biological effects of 1,25-DHCC are exerted through binding the ligand to its receptor. Mammary glands of mice also have vitamin D3 receptor (Colston et al., 1980). However, little is known about the physiological role of 1,25-DHCC in mammary epithelial cells. Besides, in the previous reports, cell differentiation stimulated by 1,25-DHCC was accompanied by inhibition of cell growth. It is, however, not clear whether this applies to other types of cells, including mammary epithelial cells. We have therefore attempted to clarify the following equations: (1) does 1,25DHCC affect growth and/or differentiation of mouse mammary epithelial cells? (2) can 1,25DHCC substitute for glucocorticoids in inducing differentiation of the cells? (3) is inhibition of cell growth by 1,25-DHCC associated with differentiation of the cells? In the present study, we have 1999 Academic Press
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used two types of serum-free collagen gel culture systems, each of which is appropriate for growth (Yang et al., 1979) and differentiation (Enami et al., 1979) of the cells, and demonstrated that 1,25-DHCC is stimulatory for both growth and differentiation of the cells at physiological concentrations. MATERIALS AND METHODS Primary mouse mammary epithelial cells Female 2- to 4-month-old KA strain mice bred in a temperature- (222C) and light- (14 h light:10 h dark) controlled room were used as the source of primary mammary epithelial cells. Mammary epithelial cells were prepared by the method described by Enami et al. (1979). Briefly, mammary glands dissected from mice on day 7 of their first pregnancies (presence of a vaginal plug was defined as Day 0) were serially digested with collagenase (1 mg/ml; Sigma, St Louis, MO) for 90 min and actinase E (0.5 mg/ml; Kaken Pharmaceutical, Tokyo) for 30 min in a 1:1 mixture of Dulbecco’s modified Eagle’s (DME) medium and Ham’s F-12 (F12) medium (hereafter referred to as DF medium; both from Nissui, Tokyo). The liberated cells were washed five times with DF medium containing no supplements, and epithelial cell clusters were collected by a low-speed centrifugation. Proportion of epithelial cells in the collected cell fraction, estimated by keratin by an indirect immunocytochemistry (Kanazawa and Hosick, 1992), was more than 96%. The viability of collected cells determined by the trypan blue exclusion method was more than 95%. Collagen gel culture Collagen solution (Cellmatrix type IA; Nitta Gelatine, Osaka) containing F12 medium with 20 mM HEPES was prepared at ice-cold temperature (Enami and Tsukada, 1994). For examination of cell growth, mammary epithelial cells were suspended in chilled collagen mixture (1.2 mg/ml collagen) at a density of 2–3105 cells/ml and 0.5 ml was pipetted into 16-mm diameter wells of plastic multi-well culture plates (NUNC, Roskilde, Denmark). After gelling at 37C for about 30 min, 0.5 ml of growth medium was overlaid on the gel. Two types of growth medium were used; one was serum-containing DF medium supplemented with 10% fetal bovine serum (FBS), and the other was serum-free growth medium consisting of DF medium supplemented with bovine serum albumin
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(BSA, Fraction V; Boehringer Mannheim GmbH, Germany, 5 mg/ml), transferrin (Tf; Green Cross, Osaka, 10 g/ml), insulin (Organon, Oss, The Netherlands, 10 g/ml), purified mouse epidermal growth factor (EGF; Toyobo, Osaka, 0–100 ng/ ml), and cholera toxin (CT; Sanko Junyaku, Tokyo, 0–100 ng/ml). Cortisol (Sigma) or 1,25DHCC (a gift from Chugai Pharmaceutical, Japan) was added to the growth medium at concentrations ranging from 6.410 12 to 1.010 7 using 0.1% ethanol as vehicle. Medium was changed the next day and thereafter every other day. For the induction of casein secretion, 1.5–2106 cells suspended in 1 ml DF medium were seeded on collagen gel membrane (0.3 ml) which had previously been prepared in 16-mm diameter wells, and incubated at 37C. After incubation for 1 day, cells attached and formed monolayers on the collagen gel. The medium was then changed to 0.3 ml of DF medium supplemented with following hormones: 5 g/ml insulin, 5 g/ml ovine prolactin (PRL; NIADDK-P-S17, Bethesda, MD), and cortisol or 1,25-DHCC at 10 12–10 6 . The gel was detached from the well and allowed to float in the medium. Culture medium was collected 72 h after the addition of hormones. Quantification of cell DNA After culture, gels were digested with 0.05% collagenase, and the liberated cells were washed twice with phosphate-buffered saline (pH 7.4), fixed in 95% ethanol and air-dried. DNA was determined according to the method of Hinegardner (1971) with modification by Enami et al. (1984), using herring sperm DNA as a standard. Data from growth studies and immunoblotting analysis were expressed as mean.. from triplicate cultures. Preparation of antiserum against mouse whole casein Mouse milk was collected from lactating KA mice by aspiration as described by Nagasawa (1979), and mouse whole casein was prepared by precipitation at pH 4.6 (Enami and Nandi, 1977). Neutralized mouse whole casein (5 mg) treated with glutaraldehyde (Reichlin et al., 1970; Enami and Nandi, 1977) was emulsified with Freund’s complete adjuvant, and injected into popliteal lymph nodes of a male Japanese White rabbit under pentobarbital anaesthesia. Intradermal booster injections were repeated five times at 2-week intervals with 2 mg mouse whole casein mixed with Freund’s incomplete adjuvant. The rabbit was bled
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through a marginal ear vein one week after the booster injections. The blood was clotted and serum was separated by centrifugation.
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Denatured sample solutions (60 l) consisting of 30 l of culture medium or mouse whole casein and an equal volume of denaturing agents (5% 2-mercaptoethanol, 20 mg/ml sodium dodecyl sulfate, 8 urea and 125 m Tris-HCl) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide containing 4 urea). Blotting and immunodetection procedures were essentially the same as the method of Rittenhause and Marcus (1984). After electrophoresis, proteins were electrophoretically transferred on to a nitrocellulose filter (Schleicher and Schuell, BA 85, pore size 0.45 m, Dassel, Germany) at 40 mA at 4C overnight in transfer buffer (25 m Tris, 192 mM glycine and 20% methanol by volume). Mouse casein components on nitrocellulose filter were detected using rabbit anti-mouse whole casein serum, and visualized by the reaction of 4-chloro1-naphthol and peroxidase-conjugated goat antirabbit IgG serum (Cappel, Malvern, PA). Rabbit anti-mouse whole casein serum was used at 1:500 dilution with 10 m phosphate-buffered saline (pH 7.4). Goat serum was added at 10% by volume to antiserum or quenching solutions as a blocking agent. EDTA and Triton X-100 were added to wash solutions (10 m phosphate-buffered saline, pH 7.4) at concentrations of 0.05 m and 0.05% (v/v), respectively. After colour reaction, the membrane filters were rinsed in distilled water, and air-dried. Then optical densities of coloured bands were scanned on an Imaging Densitometer (Model GS-670, Bio-Rad) using Molecular Analysist Software (ver. 2.1). After subtraction of background values, optical densities of each band were converted into percentage of the maximal values. Data were expressed as mean.. from three independent experiments.
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Fig. 1. Time course of growth of mammary epithelial cells cultured in collagen gel matrix. Mammary epithelial cells dissociated from mice at day 7 of their first pregnancies were cultured in collagen gel matrix at the density of 1.5105 cells/ well in serum-free DF medium containing BSA (5 mg/ml), Tf (10 g/ml), insulin (10 g/ml), EGF (10 ng/ml) and CT (10 ng/ ml; serum-free), or in medium supplemented with 10% FBS (10% FBS). On the indicated days, cultures were terminated and cell DNA was quantified, as described in Materials and Methods. All values are expressed as mean.. of triplicate cultures. ( ), serum free; ( ), 10% FBS.
RESULTS
culture in the presence of BSA, Tf and insulin. Among combinations of EGF and CT at concentrations of 0, 1, 10 and 100 ng/ml, a maximal stimulatory effect was observed at a combination of 10 ng/ml each (16.050.56-fold increase over culture without EGF and CT after 11 days in culture, data not shown), confirming a previous report (Imagawa et al., 1982). The cells grew slower at the beginning in serum-free medium, but continued to grow for a longer period than in FBSsupplemented medium (Fig. 1).
Growth of mammary epithelial cells under serum-free conditions
Effects of cortisol and 1,25-DHCC on growth of mammary epithelial cells
Effects of EGF and CT on the growth of mammary epithelial cells were examined in collagen gel matrix
Effects of cortisol and 1,25-DHCC on growth of mammary epithelial cells in collagen-gel matrix
Statistic analysis Data from both growth and differentiation studies were analysed statistically based on ANOVA followed by Dunnett’s multiple range test.
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Effects of 1,25-DHCC and cortisol on casein secretion by mammary epithelial cells Three major bands corresponding to casein components, i.e., s1-, -, and -casein, were detected by the immunoblotting method in the medium after mammary epithelial cells were cultured in the presence of insulin, PRL and cortisol for 3 days (Fig. 3, lane 2). 1,25-DHCC affected casein synthesis with a biphasic dose-dependency when added with insulin, PRL and cortisol. All the components tended to be augmented at 10 11–10 9 , and reduced by the addition at 10 8 and 10 7 (Fig. 3, lanes 3–7). Relative values of optical densities of immunostained casein bands collected from three independent experiments are shown in Figure 4. Significant augmentation was detected for s1- and -casein at low concentrations of 1,25-DHCC, but not for -casein, while reductions at the high concentrations were significant for all the casein components. In the absence of cortisol, however, 1,25-DHCC (10 11–10 7 ) did not stimulate casein synthesis (data not shown). Cell growth was not affected significantly by concentrations of
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culture are summarized in Figure 2. In serum-free medium containing BSA, Tf, insulin, EGF (10 ng/ ml) and CT (10 ng/ml), cortisol stimulated the growth of mammary epithelial cells at concentrations 6.410 12 and 3.210 11 . A maximal value, 1.450.07-fold increase over control, was observed at 3.210 11 . On the contrary, cell growth was decreased to 0.760.02- and 0.840.09-fold of that of control at 210 8 and 110 7 , respectively. These decreases were, however, not statistically significant. 1,25-DHCC also biphasically influenced the growth of mammary epithelial cells in serum-free medium, with more drastic action. Cell growth was stimulated significantly at 3.210 11 and 1.610 10 of 1,25-DHCC, declined around 4.010 9 , and was inhibited significantly at 2.010 8 and 1.010 7 . Maximal growth (1.810.10-fold increase over the control) was observed at 3.210 11 (Fig. 2a). Biphasic effects of both steroids were not observed in FBS-supplemented medium. A supressive effect only of 1,25-DHCC was observed at concentrations over 8.010 10 (Fig. 2b). The effect of cortisol on the growth of mammary epithelial cells was not obvious in FBS-supplemented medium (Fig. 2b). These effects of 1,25-DHCC and cortisol were reproducibly observed at least twice with similar magnitude of influence.
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Fig. 2. Dose-dependent effects of 1,25-DHCC ( ) and cortisol ( ) on growth of primary mouse mammary epithelial cells cultured in collagen gel matrix. The cells, seeded in collagen gels at the density of 1105 cells/well, were cultured with serum-free DF medium containing BSA, Tf, insulin, EGF and CT (a), or with DF medium supplemented with FBS (b). Concentrations of supplements in the media were the same as in Fig. 1. The indicated concentrations of 1,25-DHCC or cortisol were added to the medium. After culture for 6 days in each medium, the cells were recovered, fixed and assayed for DNA. The amount of DNA at the beginning of culture (1.760.13 g/well) is indicated by the arrowhead. Data were expressed as mean.. of triplicate cultures from a set of experiments. Asterisks were added to groups significantly different from control without 1,25-DHCC in Dunnet’s multiple range test using data from three independent experiments (P<0.05).
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αs1 β γ
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Fig. 3. Immunoblotting of mouse casein components secreted from cultured primary mouse mammary epithelial cells. The cells (1.5106/well) were cultured for 72 h with hormones, and culture media were subjected to Western blotting as described in Materials and Methods. The final concentrations of hormones were 5 g/ml for insulin and PRL, 310 6 for cortisol and 110 11–110 7 for 1,25-DHCC. Lane 1, 0.5 g mouse whole casein; Lanes 2–7, media from mammary cell cultures in the presence of insulin+PRL+cortisol. Concentrations of 1,25-DHCC were Lane 2, 0; Lane 3, 10 11 ; Lane 4, 10 10 ; Lane 5, 10 9 ; Lane 6, 10 8 ; Lane 7, 10 7 .
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Fig. 5. Dose dependency of immunoblotting for mouse casein components. The immuno-stained bands were scanned on a densitometer. The maximal value of each component was made as 100. Data are expressed as mean.. from three independent experiments. ( ), ; ( ); ; (), .
casein for s1- and -casein, and 0.04 and 1 for -casein. *
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Fig. 4. Dose-dependent effect of 1,25-DHCC on casein secretion by primary mouse mammary epithelial cells. Immunostained casein bands after Immunoblotting were densitometrically analysed as described in Materials and Methods. Data are expressed as mean.. from three independent experiments. Means with asterisks are significantly different from each control by Dunnet’s multiple range test (P<0.05). ( ), ; ( ), ; (), .
1,25-DHCC during 3-day culture (data not shown). Dose dependency of immunoblotting for casein components is shown in Figure 5. A correlation between the amount of whole casein and density of bands was observed between 0.1 and 1.4 g whole
The present results obtained by using a serum-free culture system clearly show that 1,25-DHCC stimulates growth and casein synthesis of mammary epithelial cells at low concentrations (10 12– 10 10 ) and inhibits them at high concentrations (over 10 8 ). FBS masks the stimulatory effect but not the inhibitory effect of 1,25-DHCC on cell growth. Most reports on several other types of cells agree well with this general feature of the regulation of cell growth by 1,25-DHCC. Inhibition of proliferation by 1,25-DHCC, mostly at concentrations higher than 10 9 , was reported for various cell lines cultured in the presence of FBS or calf serum (Abe et al., 1981; Miyaura et al., 1981; Hosomi et al., 1983; Kuribayashi et al., 1983; Dokoh et al., 1984; Chouvet et al., 1986). In contrast, stimulation of growth by 1,25-DHCC was not observed in serum-containing cultures, as observed in the present study. These observations suggest the possibility that serum contains some factors which may mask the stimulatory effect by 1,25-DHCC. In fact, serum contains steroids, including glucocorticoids (Frampton et al., 1983), and forms of vitamin D3 derivatives (Hosomi et al., 1983) and their binding proteins. It is highly probable that these factors interfere with or diminish the effects of added steroids. Indeed, treatment of serum with charcoal removes interfering factors,
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allowed the stimulatory effect of 1,25-DHCC to be revealed (Freak et al., 1981). The inhibition of cell growth by higher concentration of 1,25-DHCC may be considered as a pharmacological effect in terms of serum concentrations and dissociation constants (Kd) of these steroids. In normal mouse mammary glands, binding of 1,25DHCC has a Kd of 210 9 (Colston et al., 1980), and glucocorticoids a Kd of 3.910 9 (Chomczynski and Zwierzchowski, 1976), respectively. The minimum concentration of cortisol required for stimulating casein synthesis by mammary epithelial cells is 110 9 (T. Kanazawa, unpublished data), which is in good agreement with the Kd value. In the present study, the inhibitory effect of 1,25-DHCC was observed at concentrations over 210 8 , substantially higher than the Kd value, while the stimulatory effect was observed at concentrations around the Kd value. The concentration of 1,25-DHCC in rats during pregnancy and lactation is reported to range from 25–158 pg/ml (6.510 11–4.110 10 ) (Halloran et al., 1979). From these facts, it is likely that the inhibitory effect of 1,25-DHCC at high concentrations on growth of normal mammary epithelial cells is not physiological, but pharmacological. Scatchard analysis of 1,25-DHCC binding to its receptor from various cells showed high binding affinity Kd =1– 2010 11 (Colston et al., 1980; Eisman et al., 1980; Fry et al., 1980; Reinhardt and Conrad, 1980; Freak et al., 1981; Hosomi et al., 1983), whereas most of the inhibitory effects of 1,25-DHCC have been observed at higher concentrations (Abe et al., 1981; Miyaura et al., 1981; Hosomi et al., 1983; Kuribayashi et al., 1983; Dokoh et al., 1984; Chouvet et al., 1986). Taking together with the generally accepted relationship between physiologically effective dose and Kd values of steroids, 1,25DHCC may normally exert its physiological effect within the concentration range of the Kd value. 1,25-DHCC has been shown to act more effectively than glucocorticoids in inducing differentiation of myeloid leukemia cells (Abe et al., 1981) and normal myelocytes (McCarthy et al., 1983). In the present study, however, 1,25-DHCC did not substitute for cortisol in the differentiation of mammary epithelial cells. Glucocorticoids induce a number of cellular changes, leading directly to lactogenesis. Effects of glucocorticoids include increase in ribosomal binding to the endoplasmic reticulum (Oka and Topper, 1971), increase in the number of prolactin binding sites in mammary glands of mice ovariectomized and adrenalectomized during pregnancy (Harigaya et al., 1982), and increased synthesis of casein messenger RNA
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(Ganguly et al., 1980). However, glucocorticoids cannot stimulate synthesis of casein without prolactin and insulin. These findings suggest that glucocorticoids are essential for induction of differentiation in mammary epithelial cells. On the other hand, 1,25-DHCC does not seem to be essential for differentiation of mammary epithelial cells, since physiological and superphysiological concentrations (10 11–10 7 ) of 1,25-DHCC did not induce casein synthesis in the absence of cortisol. Physiological low concentration of 1,25-DHCC, however, did not inhibit the differentiation of primary mammary epithelial cells induced by the presence of cortisol. Rather casein secretion, a marker of differentiated mammary epithelial cells, was enhanced by 1,25-DHCC at lower than 110 8 , in the presence of insulin, prolactin and cortisol, in the present study. Although the present immunoblotting is not sufficiently quantitative and the observed augmentation in casein secretion is small, this result suggests that 1,25-DHCC synergizes with these hormones which are essential for differentiation of normal mouse mammary epithelial cells. This finding does not contradict the earlier report that PRL increases specific binding of 1,25-DHCC in cultured mammary glands while 1,25-DHCC augments uptake of calcium by cultured mammary glands in the presence of insulin, cortisol and PRL (Mezzetti et al., 1988). It is not clear how 1,25DHCC synergizes in the synthesis of casein. However, it is noteworthy that 1,25-DHCC enhances elongation of chromogranin-A peptide chain by increasing recruitment of its mRNA into the polyribosomes in bovine parathyroid cells (Mouland and Hendy, 1992). This mode of action of 1,25-DHCC may account for the increase in casein synthesis in the present study. From these experiments, we conclude that: (1) 1,25-DHCC and cortisol both affect the growth and differentiation of mammary epithelial cells of the mouse in dose-dependent ways though 1,25DHCC does not substitute for cortisol in inducing differentiation of the cells; (2) 1,25-DHCC synergizes with cortisol for the differentiation; and (3) inhibition of cell growth by 1,25-DHCC is not directly associated with induction of differentiation in the cells.
ACKNOWLEDGEMENT This work was supported in part by a Grant-inAid for Cancer Research from the Ministry of Education, Science and Culture of Japan.
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EFFECTS OF 1,25-DIHYDROXYCHOLECALCIFEROL AND CORTISOL ON THE GROWTH AND DIFFERENTIATION OF PRIMARY CULTURES OF MOUSE MAMMARY EPITHELIAL CELLS IN COLLAGEN GEL TAKUYA KANAZAWA1*, JUMPEI ENAMI2 and KAORU KOHMOTO1 1
Department of Animal Breeding, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; 2 Research Laboratory, Zenyaku Kogyo Co., Ltd, 2-33-7 Oizumi, Nerima-ku, Tokyo 178-0062, Japan Received 3 September 1998; accepted 12 April 1999
We examined the effects of 1,25-dihydroxycholecalciferol (1,25-DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10 11–10 9 ) of 1,25-DHCC stimulated growth of the cells in a collagen gel matrix culture in serum-free DMEM+Ham’s F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8-fold the control after 6 d in culture. In contrast, supraphysiological concentrations (10 8–10 7 ) of 1,25-DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose-dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25-DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25-DHCC also affected casein synthesis by cells cultured in a serum-free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10 11–10 9 ) and inhibiting it above 10 8 . In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25-DHCC. These results suggest a physiological role for 1,25-DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25-DHCC does not substitute for 1999 Academic Press glucocorticoids in the differentiation of the cells. K: vitamin D3; cortisol; primary mammary epithelial cell; collagen gel culture; mouse.
INTRODUCTION Steroid hormones are involved in the regulation of growth and differentiation of cells of a variety of tissues. Glucocorticoids are stimulatory for both differentiation (Oka and Topper, 1971) and growth (Mori et al., 1983) of mammary epithelial cells of the mouse in organ culture systems. Previously, several reports have shown that 1,25dihydroxycholecalciferol (1,25-DHCC), the most potent form among vitamin D3 derivatives, stimulates differentiation of normal and malignant myelocytes (Abe et al., 1981; McCarthy et al., 1983), and normal epidermal cells (Hosomi et al., 1983), more effectively than do glucocorticoids. These *Present address: Laboratory of Animal Breeding, Division of Biotechnology, Faculty of Agriculture, Ibaraki University, Ami-machi, Ibaraki 300-0393, Japan, and to whom correspondence should be addressed. 1065–6995/99/070481+07 $30.00/0
1,25-DHCC-responsive cells have specific receptors, and biological effects of 1,25-DHCC are exerted through binding the ligand to its receptor. Mammary glands of mice also have vitamin D3 receptor (Colston et al., 1980). However, little is known about the physiological role of 1,25-DHCC in mammary epithelial cells. Besides, in the previous reports, cell differentiation stimulated by 1,25-DHCC was accompanied by inhibition of cell growth. It is, however, not clear whether this applies to other types of cells, including mammary epithelial cells. We have therefore attempted to clarify the following equations: (1) does 1,25DHCC affect growth and/or differentiation of mouse mammary epithelial cells? (2) can 1,25DHCC substitute for glucocorticoids in inducing differentiation of the cells? (3) is inhibition of cell growth by 1,25-DHCC associated with differentiation of the cells? In the present study, we have 1999 Academic Press
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used two types of serum-free collagen gel culture systems, each of which is appropriate for growth (Yang et al., 1979) and differentiation (Enami et al., 1979) of the cells, and demonstrated that 1,25-DHCC is stimulatory for both growth and differentiation of the cells at physiological concentrations. MATERIALS AND METHODS Primary mouse mammary epithelial cells Female 2- to 4-month-old KA strain mice bred in a temperature- (222C) and light- (14 h light:10 h dark) controlled room were used as the source of primary mammary epithelial cells. Mammary epithelial cells were prepared by the method described by Enami et al. (1979). Briefly, mammary glands dissected from mice on day 7 of their first pregnancies (presence of a vaginal plug was defined as Day 0) were serially digested with collagenase (1 mg/ml; Sigma, St Louis, MO) for 90 min and actinase E (0.5 mg/ml; Kaken Pharmaceutical, Tokyo) for 30 min in a 1:1 mixture of Dulbecco’s modified Eagle’s (DME) medium and Ham’s F-12 (F12) medium (hereafter referred to as DF medium; both from Nissui, Tokyo). The liberated cells were washed five times with DF medium containing no supplements, and epithelial cell clusters were collected by a low-speed centrifugation. Proportion of epithelial cells in the collected cell fraction, estimated by keratin by an indirect immunocytochemistry (Kanazawa and Hosick, 1992), was more than 96%. The viability of collected cells determined by the trypan blue exclusion method was more than 95%. Collagen gel culture Collagen solution (Cellmatrix type IA; Nitta Gelatine, Osaka) containing F12 medium with 20 mM HEPES was prepared at ice-cold temperature (Enami and Tsukada, 1994). For examination of cell growth, mammary epithelial cells were suspended in chilled collagen mixture (1.2 mg/ml collagen) at a density of 2–3105 cells/ml and 0.5 ml was pipetted into 16-mm diameter wells of plastic multi-well culture plates (NUNC, Roskilde, Denmark). After gelling at 37C for about 30 min, 0.5 ml of growth medium was overlaid on the gel. Two types of growth medium were used; one was serum-containing DF medium supplemented with 10% fetal bovine serum (FBS), and the other was serum-free growth medium consisting of DF medium supplemented with bovine serum albumin
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(BSA, Fraction V; Boehringer Mannheim GmbH, Germany, 5 mg/ml), transferrin (Tf; Green Cross, Osaka, 10 g/ml), insulin (Organon, Oss, The Netherlands, 10 g/ml), purified mouse epidermal growth factor (EGF; Toyobo, Osaka, 0–100 ng/ ml), and cholera toxin (CT; Sanko Junyaku, Tokyo, 0–100 ng/ml). Cortisol (Sigma) or 1,25DHCC (a gift from Chugai Pharmaceutical, Japan) was added to the growth medium at concentrations ranging from 6.410 12 to 1.010 7 using 0.1% ethanol as vehicle. Medium was changed the next day and thereafter every other day. For the induction of casein secretion, 1.5–2106 cells suspended in 1 ml DF medium were seeded on collagen gel membrane (0.3 ml) which had previously been prepared in 16-mm diameter wells, and incubated at 37C. After incubation for 1 day, cells attached and formed monolayers on the collagen gel. The medium was then changed to 0.3 ml of DF medium supplemented with following hormones: 5 g/ml insulin, 5 g/ml ovine prolactin (PRL; NIADDK-P-S17, Bethesda, MD), and cortisol or 1,25-DHCC at 10 12–10 6 . The gel was detached from the well and allowed to float in the medium. Culture medium was collected 72 h after the addition of hormones. Quantification of cell DNA After culture, gels were digested with 0.05% collagenase, and the liberated cells were washed twice with phosphate-buffered saline (pH 7.4), fixed in 95% ethanol and air-dried. DNA was determined according to the method of Hinegardner (1971) with modification by Enami et al. (1984), using herring sperm DNA as a standard. Data from growth studies and immunoblotting analysis were expressed as mean.. from triplicate cultures. Preparation of antiserum against mouse whole casein Mouse milk was collected from lactating KA mice by aspiration as described by Nagasawa (1979), and mouse whole casein was prepared by precipitation at pH 4.6 (Enami and Nandi, 1977). Neutralized mouse whole casein (5 mg) treated with glutaraldehyde (Reichlin et al., 1970; Enami and Nandi, 1977) was emulsified with Freund’s complete adjuvant, and injected into popliteal lymph nodes of a male Japanese White rabbit under pentobarbital anaesthesia. Intradermal booster injections were repeated five times at 2-week intervals with 2 mg mouse whole casein mixed with Freund’s incomplete adjuvant. The rabbit was bled
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through a marginal ear vein one week after the booster injections. The blood was clotted and serum was separated by centrifugation.
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Detection of casein components by immunoblotting 20
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Denatured sample solutions (60 l) consisting of 30 l of culture medium or mouse whole casein and an equal volume of denaturing agents (5% 2-mercaptoethanol, 20 mg/ml sodium dodecyl sulfate, 8 urea and 125 m Tris-HCl) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide containing 4 urea). Blotting and immunodetection procedures were essentially the same as the method of Rittenhause and Marcus (1984). After electrophoresis, proteins were electrophoretically transferred on to a nitrocellulose filter (Schleicher and Schuell, BA 85, pore size 0.45 m, Dassel, Germany) at 40 mA at 4C overnight in transfer buffer (25 m Tris, 192 mM glycine and 20% methanol by volume). Mouse casein components on nitrocellulose filter were detected using rabbit anti-mouse whole casein serum, and visualized by the reaction of 4-chloro1-naphthol and peroxidase-conjugated goat antirabbit IgG serum (Cappel, Malvern, PA). Rabbit anti-mouse whole casein serum was used at 1:500 dilution with 10 m phosphate-buffered saline (pH 7.4). Goat serum was added at 10% by volume to antiserum or quenching solutions as a blocking agent. EDTA and Triton X-100 were added to wash solutions (10 m phosphate-buffered saline, pH 7.4) at concentrations of 0.05 m and 0.05% (v/v), respectively. After colour reaction, the membrane filters were rinsed in distilled water, and air-dried. Then optical densities of coloured bands were scanned on an Imaging Densitometer (Model GS-670, Bio-Rad) using Molecular Analysist Software (ver. 2.1). After subtraction of background values, optical densities of each band were converted into percentage of the maximal values. Data were expressed as mean.. from three independent experiments.
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Fig. 1. Time course of growth of mammary epithelial cells cultured in collagen gel matrix. Mammary epithelial cells dissociated from mice at day 7 of their first pregnancies were cultured in collagen gel matrix at the density of 1.5105 cells/ well in serum-free DF medium containing BSA (5 mg/ml), Tf (10 g/ml), insulin (10 g/ml), EGF (10 ng/ml) and CT (10 ng/ ml; serum-free), or in medium supplemented with 10% FBS (10% FBS). On the indicated days, cultures were terminated and cell DNA was quantified, as described in Materials and Methods. All values are expressed as mean.. of triplicate cultures. ( ), serum free; ( ), 10% FBS.
RESULTS
culture in the presence of BSA, Tf and insulin. Among combinations of EGF and CT at concentrations of 0, 1, 10 and 100 ng/ml, a maximal stimulatory effect was observed at a combination of 10 ng/ml each (16.050.56-fold increase over culture without EGF and CT after 11 days in culture, data not shown), confirming a previous report (Imagawa et al., 1982). The cells grew slower at the beginning in serum-free medium, but continued to grow for a longer period than in FBSsupplemented medium (Fig. 1).
Growth of mammary epithelial cells under serum-free conditions
Effects of cortisol and 1,25-DHCC on growth of mammary epithelial cells
Effects of EGF and CT on the growth of mammary epithelial cells were examined in collagen gel matrix
Effects of cortisol and 1,25-DHCC on growth of mammary epithelial cells in collagen-gel matrix
Statistic analysis Data from both growth and differentiation studies were analysed statistically based on ANOVA followed by Dunnett’s multiple range test.
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Effects of 1,25-DHCC and cortisol on casein secretion by mammary epithelial cells Three major bands corresponding to casein components, i.e., s1-, -, and -casein, were detected by the immunoblotting method in the medium after mammary epithelial cells were cultured in the presence of insulin, PRL and cortisol for 3 days (Fig. 3, lane 2). 1,25-DHCC affected casein synthesis with a biphasic dose-dependency when added with insulin, PRL and cortisol. All the components tended to be augmented at 10 11–10 9 , and reduced by the addition at 10 8 and 10 7 (Fig. 3, lanes 3–7). Relative values of optical densities of immunostained casein bands collected from three independent experiments are shown in Figure 4. Significant augmentation was detected for s1- and -casein at low concentrations of 1,25-DHCC, but not for -casein, while reductions at the high concentrations were significant for all the casein components. In the absence of cortisol, however, 1,25-DHCC (10 11–10 7 ) did not stimulate casein synthesis (data not shown). Cell growth was not affected significantly by concentrations of
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culture are summarized in Figure 2. In serum-free medium containing BSA, Tf, insulin, EGF (10 ng/ ml) and CT (10 ng/ml), cortisol stimulated the growth of mammary epithelial cells at concentrations 6.410 12 and 3.210 11 . A maximal value, 1.450.07-fold increase over control, was observed at 3.210 11 . On the contrary, cell growth was decreased to 0.760.02- and 0.840.09-fold of that of control at 210 8 and 110 7 , respectively. These decreases were, however, not statistically significant. 1,25-DHCC also biphasically influenced the growth of mammary epithelial cells in serum-free medium, with more drastic action. Cell growth was stimulated significantly at 3.210 11 and 1.610 10 of 1,25-DHCC, declined around 4.010 9 , and was inhibited significantly at 2.010 8 and 1.010 7 . Maximal growth (1.810.10-fold increase over the control) was observed at 3.210 11 (Fig. 2a). Biphasic effects of both steroids were not observed in FBS-supplemented medium. A supressive effect only of 1,25-DHCC was observed at concentrations over 8.010 10 (Fig. 2b). The effect of cortisol on the growth of mammary epithelial cells was not obvious in FBS-supplemented medium (Fig. 2b). These effects of 1,25-DHCC and cortisol were reproducibly observed at least twice with similar magnitude of influence.
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Fig. 2. Dose-dependent effects of 1,25-DHCC ( ) and cortisol ( ) on growth of primary mouse mammary epithelial cells cultured in collagen gel matrix. The cells, seeded in collagen gels at the density of 1105 cells/well, were cultured with serum-free DF medium containing BSA, Tf, insulin, EGF and CT (a), or with DF medium supplemented with FBS (b). Concentrations of supplements in the media were the same as in Fig. 1. The indicated concentrations of 1,25-DHCC or cortisol were added to the medium. After culture for 6 days in each medium, the cells were recovered, fixed and assayed for DNA. The amount of DNA at the beginning of culture (1.760.13 g/well) is indicated by the arrowhead. Data were expressed as mean.. of triplicate cultures from a set of experiments. Asterisks were added to groups significantly different from control without 1,25-DHCC in Dunnet’s multiple range test using data from three independent experiments (P<0.05).
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αs1 β γ
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Fig. 3. Immunoblotting of mouse casein components secreted from cultured primary mouse mammary epithelial cells. The cells (1.5106/well) were cultured for 72 h with hormones, and culture media were subjected to Western blotting as described in Materials and Methods. The final concentrations of hormones were 5 g/ml for insulin and PRL, 310 6 for cortisol and 110 11–110 7 for 1,25-DHCC. Lane 1, 0.5 g mouse whole casein; Lanes 2–7, media from mammary cell cultures in the presence of insulin+PRL+cortisol. Concentrations of 1,25-DHCC were Lane 2, 0; Lane 3, 10 11 ; Lane 4, 10 10 ; Lane 5, 10 9 ; Lane 6, 10 8 ; Lane 7, 10 7 .
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Fig. 5. Dose dependency of immunoblotting for mouse casein components. The immuno-stained bands were scanned on a densitometer. The maximal value of each component was made as 100. Data are expressed as mean.. from three independent experiments. ( ), ; ( ); ; (), .
casein for s1- and -casein, and 0.04 and 1 for -casein. *
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Fig. 4. Dose-dependent effect of 1,25-DHCC on casein secretion by primary mouse mammary epithelial cells. Immunostained casein bands after Immunoblotting were densitometrically analysed as described in Materials and Methods. Data are expressed as mean.. from three independent experiments. Means with asterisks are significantly different from each control by Dunnet’s multiple range test (P<0.05). ( ), ; ( ), ; (), .
1,25-DHCC during 3-day culture (data not shown). Dose dependency of immunoblotting for casein components is shown in Figure 5. A correlation between the amount of whole casein and density of bands was observed between 0.1 and 1.4 g whole
The present results obtained by using a serum-free culture system clearly show that 1,25-DHCC stimulates growth and casein synthesis of mammary epithelial cells at low concentrations (10 12– 10 10 ) and inhibits them at high concentrations (over 10 8 ). FBS masks the stimulatory effect but not the inhibitory effect of 1,25-DHCC on cell growth. Most reports on several other types of cells agree well with this general feature of the regulation of cell growth by 1,25-DHCC. Inhibition of proliferation by 1,25-DHCC, mostly at concentrations higher than 10 9 , was reported for various cell lines cultured in the presence of FBS or calf serum (Abe et al., 1981; Miyaura et al., 1981; Hosomi et al., 1983; Kuribayashi et al., 1983; Dokoh et al., 1984; Chouvet et al., 1986). In contrast, stimulation of growth by 1,25-DHCC was not observed in serum-containing cultures, as observed in the present study. These observations suggest the possibility that serum contains some factors which may mask the stimulatory effect by 1,25-DHCC. In fact, serum contains steroids, including glucocorticoids (Frampton et al., 1983), and forms of vitamin D3 derivatives (Hosomi et al., 1983) and their binding proteins. It is highly probable that these factors interfere with or diminish the effects of added steroids. Indeed, treatment of serum with charcoal removes interfering factors,
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allowed the stimulatory effect of 1,25-DHCC to be revealed (Freak et al., 1981). The inhibition of cell growth by higher concentration of 1,25-DHCC may be considered as a pharmacological effect in terms of serum concentrations and dissociation constants (Kd) of these steroids. In normal mouse mammary glands, binding of 1,25DHCC has a Kd of 210 9 (Colston et al., 1980), and glucocorticoids a Kd of 3.910 9 (Chomczynski and Zwierzchowski, 1976), respectively. The minimum concentration of cortisol required for stimulating casein synthesis by mammary epithelial cells is 110 9 (T. Kanazawa, unpublished data), which is in good agreement with the Kd value. In the present study, the inhibitory effect of 1,25-DHCC was observed at concentrations over 210 8 , substantially higher than the Kd value, while the stimulatory effect was observed at concentrations around the Kd value. The concentration of 1,25-DHCC in rats during pregnancy and lactation is reported to range from 25–158 pg/ml (6.510 11–4.110 10 ) (Halloran et al., 1979). From these facts, it is likely that the inhibitory effect of 1,25-DHCC at high concentrations on growth of normal mammary epithelial cells is not physiological, but pharmacological. Scatchard analysis of 1,25-DHCC binding to its receptor from various cells showed high binding affinity Kd =1– 2010 11 (Colston et al., 1980; Eisman et al., 1980; Fry et al., 1980; Reinhardt and Conrad, 1980; Freak et al., 1981; Hosomi et al., 1983), whereas most of the inhibitory effects of 1,25-DHCC have been observed at higher concentrations (Abe et al., 1981; Miyaura et al., 1981; Hosomi et al., 1983; Kuribayashi et al., 1983; Dokoh et al., 1984; Chouvet et al., 1986). Taking together with the generally accepted relationship between physiologically effective dose and Kd values of steroids, 1,25DHCC may normally exert its physiological effect within the concentration range of the Kd value. 1,25-DHCC has been shown to act more effectively than glucocorticoids in inducing differentiation of myeloid leukemia cells (Abe et al., 1981) and normal myelocytes (McCarthy et al., 1983). In the present study, however, 1,25-DHCC did not substitute for cortisol in the differentiation of mammary epithelial cells. Glucocorticoids induce a number of cellular changes, leading directly to lactogenesis. Effects of glucocorticoids include increase in ribosomal binding to the endoplasmic reticulum (Oka and Topper, 1971), increase in the number of prolactin binding sites in mammary glands of mice ovariectomized and adrenalectomized during pregnancy (Harigaya et al., 1982), and increased synthesis of casein messenger RNA
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(Ganguly et al., 1980). However, glucocorticoids cannot stimulate synthesis of casein without prolactin and insulin. These findings suggest that glucocorticoids are essential for induction of differentiation in mammary epithelial cells. On the other hand, 1,25-DHCC does not seem to be essential for differentiation of mammary epithelial cells, since physiological and superphysiological concentrations (10 11–10 7 ) of 1,25-DHCC did not induce casein synthesis in the absence of cortisol. Physiological low concentration of 1,25-DHCC, however, did not inhibit the differentiation of primary mammary epithelial cells induced by the presence of cortisol. Rather casein secretion, a marker of differentiated mammary epithelial cells, was enhanced by 1,25-DHCC at lower than 110 8 , in the presence of insulin, prolactin and cortisol, in the present study. Although the present immunoblotting is not sufficiently quantitative and the observed augmentation in casein secretion is small, this result suggests that 1,25-DHCC synergizes with these hormones which are essential for differentiation of normal mouse mammary epithelial cells. This finding does not contradict the earlier report that PRL increases specific binding of 1,25-DHCC in cultured mammary glands while 1,25-DHCC augments uptake of calcium by cultured mammary glands in the presence of insulin, cortisol and PRL (Mezzetti et al., 1988). It is not clear how 1,25DHCC synergizes in the synthesis of casein. However, it is noteworthy that 1,25-DHCC enhances elongation of chromogranin-A peptide chain by increasing recruitment of its mRNA into the polyribosomes in bovine parathyroid cells (Mouland and Hendy, 1992). This mode of action of 1,25-DHCC may account for the increase in casein synthesis in the present study. From these experiments, we conclude that: (1) 1,25-DHCC and cortisol both affect the growth and differentiation of mammary epithelial cells of the mouse in dose-dependent ways though 1,25DHCC does not substitute for cortisol in inducing differentiation of the cells; (2) 1,25-DHCC synergizes with cortisol for the differentiation; and (3) inhibition of cell growth by 1,25-DHCC is not directly associated with induction of differentiation in the cells.
ACKNOWLEDGEMENT This work was supported in part by a Grant-inAid for Cancer Research from the Ministry of Education, Science and Culture of Japan.
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