Molecular and Cellular Endocrinology, 69 (1990) 217-226 Elsevier Scientific Ireland, Ltd.

MOLCEL

02248

Proliferation of bovine undifferentiated mammary epithelial cells in vitro is modulated by G-proteins Avi Shamay

I, Mark Pines 2, Michal Waksman



and Arieh Gertler

1

’ Department of Biochemistry and Human Nutrition, Faculty of Agriculture, The Hebrew Universrty of Jerusalem, Rehovot 76100, Israel, and ’ Institute of Animal Science, Agricultural Research Organization. The Volcani Center, Rehovot 76100, Israel (Received

Key words; Proliferation; Cholera gland, bovine

toxin; Pertussis

14 August

1989; accepted

toxin; Insulin-like

growth

12 December

1989)

factor I; Epidermal

growth

factor;

G-protein;

Mammary

Summary Several CAMP-elevating agents such as cholera toxin (CT), forskolin and 3-isobutyl-1-methylxanthine (IBMX) exhibited weak mitogenic activity on bovine undifferentiated mammary epithelial cells in three-dimensional collagen culture. CT and IBMX strongly synergized with epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) or both, but not with 10% fetal calf serum (FCS). Permeable CAMP analogs also synergized with IGF-I. Other hormones such as ovine prolactin, bovine growth hormone, estrogen or progesterone were not mitogenic and not synergistic with EGF, IGF-I, CT and FCS. Pertussis toxin (PT) reduced the DNA synthesis in cells cultured in the basal medium and attenuated 40-90% of the mitogenic activity stimulated by 10% FCS. PT inhibition of DNA synthesis was accompanied by ADP-ribosylation of 40 kDa and 41 kDa membrane proteins. The 41 kDa protein cross-reacted with antibodies that recognize the Gi-protein of the adenylate cyclase system, indicating the involvement of the latter in the mitogenic process. The nature of the second protein remains unknown. The present results suggest that the mitogenesis of normal mammary epithelial cells which is stimulated by IGF-I, EGF and other factors found in FCS is mediated through both CAMP-dependent and independent pathways. These pathways include PT-sensitive GTP-binding proteins.

Introduction The complexity of various factors involved in the control of the growth of bovine mammary gland in vivo prompted us to establish, as a tool for study, an in vitro system of bovine undifferentiated mammary epithelial cells cultured in the

Address for correspondence: Prof. A. Gertler, Department of Biochemistry and Human Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel. 0303-7207/90/$03.50

0 1990 Elsevier Scientific

Publishers

Ireland,

three-dimensional collagen gel (Shamay and Gertler, 1986), according to Richards et al. (1983). Using this system, we found that insulin at pharmacological rather than physiological and insulin-like growth factor I (IGF-I) in nM concentration stimulated DNA synthesis and growth (Shamay et al., 1988). The maximal activity of both hormones was, however, only 2540% of that of medium supplemented with 10% fetal calf serum (FCS), indicating involvement of additional factors. Epidermal growth factor (EGF) was a weak mitogen but a mixture of EGF and IGF-I strongly synergized with FCS resulting in a 100% enhanceLtd.

mcnt of DNA synthesis as compared to the medium supplemented with 10% FCS only. These results raised a question about the nature of additional growth factors found in FCS and their interaction with each other. Several lines of investigation lead to the possibility that these factors may activate the cAMP pathway. Sheffield et al. (1985) have shown that daily injections of cholera toxin (CT) stimulated the in vivo development of mammary gland in female mice. This effect was strongly dependent on 17~-estradi~~l and progesterone and abolished in ovariectomized mice. The effect of CT or other cAMP elevating agents was also observed in vitro in rat mammary epithelial cells cultured on the surface of collagen gels (Ethier et al.. 19X7) or in mice mammary epithelial cells cultured in or on the surface of collagen gels (Imagawn et al.. 198X). In both reports pharmacological (S-10 pg/ml) concentrations of insulin were used. Thus, it was possible that the mitogenic effect of CT or other cAMP elevating factors was dependent on a sitnultaneous presence of high ~OIl~entrations of insulin. Clear conclusion could not be drawn since both effects were not dissociated. As shown in our previous study (Shamay et al., 19X8) and by others (Decks et al., 198X), insulin at those concentrations acts through an IGF-I growth promoting mechanism. The present study was aimed at investigating the interaction between the cAMP modulating agents such as cholera and pertussis toxins. forskolin and 3-isobutyl-l-methylxanthine (IBMX) and growth factors that stimulate the proliferation of bovine mammary epitheli~~l cells. Since both cholera and pertussis toxins act through ADPribosylation of specific GTP-binding proteins. an attempt was made to identify these proteins in bovine mammary gland and to establish their possible involvement in the growth of bovine mammary tissue. A preliminary report of the present work was presented at the Annual Meeting of the Endocrine Society (Shamay et al., 1989). Materials and methods

M-199 medium powder (Gibco Co.. Grand Island. NY, U.S.A.) was dissolved in glass-distilled

water ~(~IltaiI~il~g It) mM Hepes. pH 7.4. sterilized by vacuum filtration through 0.2 pm filters and kept at 4°C up to 3 weeks. An antibiotic-antimycotic mixture consisting of S.OOO,OOOli pencillin, 50,000 1-16 streptomycin and 125 pg fungizone/ml (Biolab, Jerusalem. Israel) was stored frozen and added to the medium (2 ml/l) before use. Collagenase (type II, 135 U/mg) was obtained from Worthington Biochemical Corp. (Freehold, NY. U.S.A.). Anti-CAMP antibody and lyophilized crude Protein A were from Bio-Yeda (Rehovot, Israel). Purified transduciI1 from bovine rod outer segment and anti-serum AS-6 that was prepared against the carboxyf terminal decapeptide (KENLKDCGLS) of transducin cY-subunit, which interacts with Gi proteins in various tissues (Cerione et al., 1988). were a gift from Dr. A. Spiegel, NIH (Bethesda, MD. U.S.A.). Deoxyribonuclease type 1, hyaluronidase (type 1-S. 300 U/mg), bovine insulin, 3-isobutyl-l-methylxanthine (IBMX), pertussis toxin (PT), cholera toxin, 8-bromo-CAMP. dibutyryl CAMP. forskolin, CAMP. tissue culture grade transferrin. bovine serum albumin. soybean trypsin inhibitor, reduced glutathione and selenic acid were purchased from Sigma Co. (St. Louis. MO. U.S.A.). Nitex filters of various sizes were obtained from Tetko Co. (Elmsford, NY. U.S.A.). fetal calf serum (FCS) from Sera-Lab (Sussex, U.K.), epidermal growth factor (EGF) receptor grade was purchased from Collaborative Research (Waltham. MA. U.S.A.). and human insulin-like growth factor I (IGF-I), prepared by recombinant DNA methodology (Saito et al.. 1986) was obtained from Fujisawa Pharnla~ellti~al Co. (Osaka. Japan). Ovine prolactin ~NIADDK-oPR~-1~) and bovine growth hormone (NIH-GH-BIX) were a gift of the National Hormone Pituitary Program (University of Maryland School of Medicine). Basal medium was prepared from M-199 medium by addition of 0.25% bovine serum albumin. bovine insulin (10 ng/ml), transferrin (5 fig/ml), selenic acid (1 ng/ml). reduced glutathione (1 pgg/ml) and soybean trypsin inhibitor (100 U/ml). Collagen gels were prepared according to Richards et al. (1983). [LY-PI&$ ~~H]thymidine (70-85 Ci,/mmol). ni~otinamjde dinucleotide. [“‘PINAD (2.5 Ci/mmol) and [i2SI]Protein A were purchased from Amersham (Buckinghamshire, U.K.).

219

Cell culture Mammary epithelial cells were prepared from healthy Israeli-Friesian 4- to 6-month-old female calves as described previously (Shamay et al., 1988). Briefly, mammary tissue was cut into small pieces, finely chopped and placed in a 500 ml Erlenmeyer flask containing M-199 medium supplemented with collagenase (1 mg/ml), hyaluronidase (1 mg/ml) and bovine insulin (1 pg/ml), in a ratio of 10 ml medium per 1 g tissue. The flask was swirled on a gyratory water bath at 100 rpm at 37’C for 3-4 h. At the end of the incubation, the suspension was passed through a Nitex filter (200 pm) and the cell clumps were collected by centrifugation (4 min at 300 x g). Then the clumps were washed 3-5 times with M-199 medium and kept on ice until embedded in collagen. Clumps consisting of 50-200 epithelial cells; each was suspended in minimal volume of M-199 medium and added to the cold collagen solution, yielding a final concentration of 4-8 x 10’ cells/ml. Cell number was determined by counting the nuclei after treatment with 0.2% crystal violet in 0.1 M citric acid. Cell viability as determined with trypan blue was over 90%. The collagen-cell suspension (0.5 ml) was overlaid on 0.3 ml pre-gelled collagen in each well of 24 multiwell plates and allowed to gel at room temperature. As soon as the layer gelled, 1 ml of M-199 medium containing the appropriate additives was added. The cells were cultured at 37 o C in air/CO, (95 : 5) atmosphere and the medium was changed every 48 h. Thymidine incorporation Cells were pulsed with 1 PCi [3H]thymidine for 24 h. The medium was aspirated from the wells. Gels were lifted carefully with broad-tipped forceps and placed in 1.5 ml Eppendorf tubes. 50 ~1 of 4.3 M acetic acid were added to each tube and the tubes were incubated for 20 min at 37 o C to dissolve the gels. The cells were recovered by centrifugation (5 min at 500 x g) and washed consecutively with 4% perchloric acid (PCA), 80% EtOH, 100% EtOH and 4% PCA. After hydrolysis in 0.5 ml 6% PCA (80 o C, 1 h), the suspension was mixed with 10 ml of scintillation liquid and counted in a Beckman /3-scintillation counter. Since the results were calculated as cpm incorpo-

rated per well they represent a combined function of rate of DNA synthesis and number of cells and thus measure the overall effect of growth factors (GFs) on cell proliferation.

Membrane preparation and ADP-ribosylation Collagen gels containing clumps of cells, cultured previously in either absence or presence of PT were solubilized with acetic acid as described above. Then the cells were pelleted (5 min, 500 x g), resuspended in homogenizing buffer (10 mM Tris, pH 7.5, 2 mM magnesium chloride, 2 mM dithiothreitol (DTT) and 0.1 mM EDTA), and homogenized with a motor-driven Teflon pestle. The homogenate was centrifuged at low speed (2000 rpm in a Sorvall SS-34 rotor) for 5 min and the pellet discarded. The supernatant was centrifuged at 15,000 rpm for 20 min and the pellet collected, resuspended in buffer, and frozen at - 20 o C until use. Pertussis toxin was preactivated in 250 mM Tris buffer (pH 8.0) containing 50 mM DTT for 1 h at 37°C. Membranes (200 pg protein) were incubated with 10 pg of the toxin, 30 PCi of [j2 PINAD and with 2.5 mM ATP for 1 h. Purified bovine transducin was ADP-ribosylated as previously described (Pines et al., 1985). The reaction was terminated by centrifugation. The supernatant was discharged and the pellet was dissolved in sample buffer containing sodium dodecyl sulfate (SDS) and /?-mercaptoethanol, boiled for 5 min and subjected to SDS/polyacrylamide gel electrophoresis (PAGE) (12.5%) followed by autoradiography.

CAMP assay Aliquots of 0.8 ml of cell suspension were withdrawn, pelleted in Eppendorf tubes (3 min, 500 X g), resuspended in 0.1 ml of 0.1 M KCl, and boiled for 3 min. Then 0.5 ml of double-distilled H,O was added and after centrifugation 0.5 ml samples were acetylated. 50 ~1 aliquots were removed for CAMP determination. The radioimmunoassay (RIA) was performed according to Harper and Brooker (1975) as modified by Pines et al. (1988). The bound ligand was separated from the free ligand by binding the antibody to protein A. The sensitivity of the assay ranged from 1 to 4 fmol. The cross-reactivity of ATP

220

(fmol required for 50% inhibition serum) was > lo5 fmol.

with CAMP anti-

Statistical analysis Statistical analyses were performed by Student’s two-tailed t-test for unpaired comparison or by analysis of variance followed by Duncan’s multiple range test. All parametric data are expressed as the mean rf SE. Each experimental treatment was repeated at least 2-3 times, using tissue derived from different animals each time. Since variations in the absolute values were observed, in spite of the fact that the relative values were similar, one or two experiments were presented in each case. Results Effect of various factors on CAMP production The CAMP level was determined in cell clumps prior to the seeding in the collagen gels. Cells were suspended in the basal medium and exposed to various factors for 90 min in the presence of 1 mM IBMX. The cells were collected and the intracellular level of CAMP was determined. In absence of any factors the level of CAMP (mean + SE) was 2.9 f 0.5 pmol/lO’ cells. Neither FCS (lo%), prostaglandin E, (PGE,) (100 PM), IGF-I (50 ng/ml), EGF (50 ng/ml) nor PT (100 ng/ml) significantly (P < 0.05) changed the level of CAMP. Cholera toxin (100 ng/ml) and forskolin (100 FM) augmented respectively the CAMP levels to 45.2 + 1.2 and 34.0 & 1.7 pmol/lO’ cells (P < 0.001). Combined effects of EGF, IGF-I and insulin on 3[HJthymidine incorporation Addition of as little as 2 ng/ml EGF to the basal medium significantly increased the incorporation of [3H]thymidine. Maximal increase was achieved at the range of 20-200 ng/ml. The overall maxima1 increases observed in several experiments were 1.6 to 4-fold over the cells cultured in the basal medium, as compared to lo- to 30-fold increases in cultures supplemented with 10% FCS (data not shown). This obviously reflects both the increase in the total number of cells that occurred through the 5 days of the experimental treatment as well as the enhanced rate of DNA synthesis. As presented in Fig. 1, the mitogenic effects of EGF

h

9 g

da

P sT

Yt 0

r

z

$0

0

10

103

1000

INSULIN (ng/

10,000

50,000

KS

ml)

Fig. 1. Effect of EGF on DNA synthesis in bovine mammary epithelial cells cultured in the presence of increasing concentrations of insulin. Cells were cultured for 6 days in the presence of increasing concentrations of insulin. added to basal insulin devoid serum-free medium, with (striped bars) or without (open bars) EGF (100 ngfml). A pulse of ~3H~thy~dine (1 pCi/weIl) was given for the last 24 h. The results are presented asmean+SE(n=3).

was strongly stimulated by insulin. insulin was a potent mitogen in supraphysiological concentrations and EGF enhanced its effect at all concentrations tested. At 10 and 50 pg/ml of insulin, addition of EGF resulted in 70% of the mitogenic effect achieved by 10% FCS. As shown in Table 1, IGF-I and EGF exhibited a strong synergistic effect. Addition of both factors to medium supplemented with 10% FCS resulted in a further 212% (expt. 1) or 71% (expt. 2) enhancement of DNA synthesis. Similar enhancement (50-155%) was also observed in three additional experiments (not shown) carried out with tissue derived from different animals. Effect of cholera toxin with or without other growth factors on r3~Jthymidine incorporation As shown in Fig. 2. cholera toxin potentiated the IGF-I- and EGF-stimulated [ ‘HJthy~dine incorporation in a dose-dependent manner. A strong synergism between CT and EGF or IGF-I also was found when all three factors were added together. The combination of EGF (20 ng/ml), IGF-I (50 ng/ml) and CT (0.1 ng/ml or more) resulted in DNA synthesis that equalled the value obtained in the presence of 10% FCS. However,

221 TABLE 1 COMBINED EFFECTS OF IGF-I, EGF AND FCS ON THE DNA SYNTHESIS IN BOVINE MAMMARY EPITHELIAL CELLS IN BASAL SERUM-FREE MEDIA Factor added



[ 3H]Thymidine incorporation (dpm/well

None IGF-I EGF FCS (10%) IGF-I + FCS EGF + FCS IGF-I + EGF IGF-I + EGF+ FCS

x lo-‘)

*

Expt. 1

Expt. 2

12.0* 0.5 a 52.0* 11.01 ’ 35.9* 2.6 b 121.0* 7.1 e 301.0*15.6 = 242.8 f 14.2 b 112.2f 6.3 d 379.8 f 74.0 d

12.7f 1.1 48.1* 5.9 29.lf 3.0 206.9 f 18.5 437.5 f 38.7 487.9 f 63.0 172.1 f 22.1 751.0*51.3

a = ’ d = ’ d d

The concentration of IGF-I and EGF were 50 ng/ml and 20 ng/ml, respectively. The concentration of FCS was 10%. Mean f SE, n = 3, 24 h pulse after 6 days in culture; means with different letters within the same column are significantly (P -z 0.05) different by Duncan’s multiple comparison test.

manner. As shown in Fig. 3, up to a 5-fold increase over the value obtained in the basal medium was achieved with 250 PM of forskolin and a 3-fold increase with 1 mM of IBMX. It should be noted that the maximal effect of forskolin in this experiment was even higher than that of CT (100 ng/ml). The effect of IBMX was further tested in media supplemented with CT, IGF-I, EGF (Table 2). It was found to be additive to the effect of CT, synergistic to the effects of IGF-I, EGF or both, while addition to 10% FCS-supplemented medium

r 24

$

BASAL MEDIUM

.? I P 20

the addition of CT to 10% FCS-supplemented medium did not further elevate the DNA synthesis (not shown). Cholera toxin alone was also mitogenic and a statistically significant increase in DNA synthesis was observed at 0.01 ng/ml. The maximal effect of CT was achieved at lo-100 ng/ml and consisted of lo-158 of that observed in 10% FCS-supplemented medium. This maximal effect of CT was obtained in most experiments, but values resulting in up to 25% of 10% FCS-supplemented medium were observed occasionally. Other hormones, such as estrogen (10 ng/ml), progesterone (100 ng/ml), bovine growth hormone (bGH) (1 pg/ml) or bovine prolactin (bPRL) (1 pg/ml) had no significant effect on DNA synthesis when added to the basal medium or to the basal medium supplemented with IGF-I (100 ng/ml), EGF (100 ng/ml), cholera toxin (100 ng/ml) or EGF and cholera toxin (results not shown). Effects of forskolin, IBMX and 8-bromo-CAMP on r3H]thymidine incorporation Two additional CAMP elevating agents, forskolin and IBMX increased the rate of [3H]thymidine incorporation in a dose-dependent

l-+

IGF +_EGF

BASAL + IGF

MEDIUM:

_

12 -

8-

6-

0

00010.r

o1 IO100 CHOLERA lOXIN (nglml)

Fig. 2. Effect of CT on DNA synthesis in bovine mammary epithelial cells cultured in the absence or presence of IGF-I or EGF. Cells were cultured for 6 days in basal serum-free medium, containing increasing concentrations of CT (open bars, upper left), supplemented with IGF-I (50 ng/ml, crosshatched bars, lower left), EGF (20 ng/ml. hatched bars, upper right) (cross-striped bars, lower right). A pulse of [ 3H]thymidine (1 uCi/well) was given for the last 24 h. The results are presented as mean f SE (n = 3). The respective value obtained in cells cultured in 10% FCS-supplemented medium was 129,000 f 2500.

_ +

t +t

1 .T

Fig. 3. Effect of IBMX and forskolin on DNA synthesis in bovine mammary epithelial cells. Cells were cultured for 6 days in the presence of increasing concentrations of IBMX (A). forskolin (0). 10% FCS or 100 ng/ml of CT (bars) in basal serum-free medium. A pulse of [3H]thymidine (1 pCi/well) was given for the last 24 h. The results are presented as mean+SE(n = 3).

had no effect at all. The effect of the CAMP analog, S-bromo-CAMP, was tested in two separate experiments using 0.01, 0.1 and 1.0 mM concentrations. When added to the basal medium

TABLE

2

EFFECT OF CHOLERA TOXIN, IGF-I, EGF AND FCS ON THE DNA SYNTHESIS IN BOVINE MAMMARY EPITHELIAL CELLS IN THE ABSENCE OR PRESENCE OF IBMX (0.1 mM) IN BASAL SERUM-FREE MEDIA Factor

added



[ 3H]Thymidine incorporation (dpm/well X lo- a) * Without

None CT ’ IGF-I3 EGF ’ IGF-I + EGF 3 FCS (10%)

4.8 f 15.7 i 28.6* 23.8 f 88.6 f 102.7 +

IBMX 0.5 0.1 1.1 1.4 0.7 3.2

’ h ’ ’ d e

MEDIUM

BASAL

MEDIUM

:

T +

g4 2 a0 5 20

FCS

+ EGF

0 0.01 0.1 1 10100 PERTUSSIS

0 0.01 0.1 1 10100 TOXIN (nglml)

Fig. 4. Effect of PT on DNA synthesis in bovine mammary epithelial cells cultured in the absence or presence of IGF-I. EGF, IGF-I + EGF or FCS. Cells were cultured for 6 days in basal serum-free medium, containing increasing concentrations of PT (open bars, upper left), supplemented with IGF-I (50 “g/ml. cross-hatched bars, middle left), EGF (20 “g/ml. hatched bars, lower left). IGF-I + EGF (cross-striped bars, upper right) or 10% FCS (striped bars. lower right). A pulse of [“Hjthymidine (1 pCi/welI) was given for the last 24 h. The results are presented as mean + SE (n = 3).

P

value ’

With IBMX 9.9* 0.3 a 24.0+ 1.7 ’ 73.6 * 10.7 c 53.5+ 9.6 = 118.2* 5.9 d 98.5rt 4.2 d

BASAL

IGF + EGF

0.05 0.01 0.01 0.01 0.01 NS

’ The concentrations of CT, IGF-I and EGF were 100 ng/mI, 50 ng/ml and 20 “g/ml, respectively. * Meanf SE, n = 3, 24 h pulse after 6 days in culture; means with different letters within the same column are significantly (P < 0.05) different by Duncan’s multiple comparison test. ’ Difference between the treatments with and without IBMX, compared by Student’s two-tailed t-test; NS, non-significant.

it had no effect in one experiment and slight numerical increase in the second. However, it enhanced by 50-100’56 the rnitogenic effect of IGF-I. The maximal effect was at 0.1 mM in the first and at 1 mM in the second experiment. Another permeable CAMP analog (dibutyryl CAMP) was also tested. It had no mitogenic activity but significantly enhanced the effect of IGF-I. Effect of pertussis toxin on [‘Hjthymidine incorporation Addition of PT to basal medium decreased the [3H]thymidine incorporation (Fig. 4). PT also exhibited remarkable inhibitory effects on FCSstimulated DNA synthesis and at 10 or 100 ng/ml

223 TABLE

3

EFFECT OF PERTUSSIS TOXIN ON THE CHOLERA TOXIN-, IGF-I-, EGF- AND FCS-STIMULATED DNA SYNTHESIS IN BOVINE MAMMARY EPITHELIAL CELLS Cholera toxin

Pertussis toxin

[ 3H]Thymidine (dpm/welI

X

incorporation

10m3)'

(ng/m1)bbdml) Factoraddd None 0

0

0

10

6.8fO.l 4.4+0.3

100 100 100 100

0 1 10 100

16.9 50.9 11.1+1.7 7.9kO.l 3.7 *0.5

IGF-I + EGF * FCS (10%) cd 157.5*10.1 b de 94.3+ 0.6 b a b c =

’ Mean+ SE, n = 3, 24 h pulse with different letters within cantly (P < 0.05) different by test. * The concentrations of IGF-I 20 ng/ml, respectively.

345.1 f 55.3 242.4k39.7 105.5* 4.8 140.1529.7

375.Ok49.0 = 69.0+ 9.9 ’

a 303.2 f 35.1 ab 123.6k20.9 b 77.9* 1.6 b 110.3* 4.0

a b b b

after 6 days in culture; means the same column are signifiDuncan’s multiple comparison and EGF were 50 ng/ml

and

caused over 80% inhibition. The inhibitory effect of PT on the EGF- or IGF-I-stimulated DNA synthesis was less pronounced and did not exceed ca. 50% even at 100 ng/ml. The ability of PT to inhibit the DNA synthesis stimulated by IGF-I, EGF and FCS in the presence of 10 ng/ml of CT was also tested (Table 3). As shown the effect of CT (100 ng/ml) was abrogated progressively by l-100 ng/ml of PT. Cholera toxin strongly synergized with IGF-I -t EGF. Addition of PT attenuated this effect but the decrease was partial and gradual. PT inhibited also the FCS-stimulated DNA synthesis in a pattern similar to that exhibited in the absence of CT (Fig. 4). Similar results were also obtained with 10 ng/ml CT and l-100 ng/ml PT (not shown). ADP-ribosylation of G-proteins ADP-ribosylation of membranes, prepared from cells cultured in 10% FCS by PT revealed a broad band around 40 kDa (Fig. 5A, lane 1). In a subsequent experiment, and after culturing the cells without or with increasing concentrations of PT for 6 days, it was found that this band consist of two major PT substrates with M, of 40 and 41 kDa (Fig. SB, lanes 3 and 4). The ADP-ribosyla-

tion of these proteins was unaltered when the cells were precultured with CT (Fig. 5A, lane 2) but abrogated when the cells were precultured with PT, even that the protein concentration loaded on the gel was doubled (Fig. 5A, lane 3). Although PT-dependent ribosylation of other minor proteins cannot be excluded, no such proteins could be identified under our experimental conditions. Culturing of the cells with different PT concentrations caused subsequent decrease in the radiolabelling of these proteins, suggesting previous ADP-ribosylation by the toxin in culture (Fig. 5B, lanes 2-5). Culturing the cells with (Fig. SB, lane 5) or without (Fig. 5B, lane 6) 10% FCS did not change the ADP-ribosylation. In order to identify the PT-sensitive proteins, specific anti-Gi antibodies were used (Fig. 6A, lane 2). These antibodies, prepared against the a-subunit of transducin, the GTP-binding protein of the vision system (Cerione et al., 1988) recognize the inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. Only one of the PT substrates in the mammary gland cell membranes (the 41 kDa protein), which

II

A 1234

5

123456 -

180

-

-

116 84

-

58 48.5

-

36.5

-

26.5

Fig. 5. ADP ribosylation of membranes from pertussis or cholera toxin-treated cells by pertussis toxin. Membranes were prepared from cells cultured for 6 days in medium supplemented with (A) lane 1: 10% FCS; lane 2: CT (10 ng/ml); lane 3: PT (10 ng/ml); (E) lanes 2-5: 10% FCS, containing respectively (in ng/ml) 100, 10, 1 and 0 PT; lane 6: basal medium. Lane 4 in A represents PT-independent ADP-ribosylation of membranes from cells cultured in medium supplemented with 10% FCS only; lane 5 in A and lane 1 in B show the ADP-ribosylation of purified transducin by PT. At the end of incubation the membranes were subjected to SDS/PAGE on 12.5% gels, dried and autoradiographed.

224

12 A

12 f B '&

116

- 04 - 58 - 48.5

LaMi

- 36.5

626.5

c

Fig. 6. Immunoblot of mammary epithelial cell membranes with anti-bovine rod outer segment transducin. Membranes were prepared from untreated epithelial cells as described in Materials and Methods and subjected to SDS/PAGE on 12.5% gels. The proteins from half of the gel (A) were transblotted into nitrocellulose and the other half of the gel (B) was stained with Coomassie blue B. The blot was incubated for 1 h with anti-a-subunit of transducin, washed, incubated for 4 h with [“‘I]Protein A, dried and autoradiographed. Lanes 1 in A and B: transducin; lanes 2 in A and B: mammary cell membranes.

probably the a-subunit by these antibodies.

is

of Gi, was recognized

Discussion Both our present and previous (Shamay et al., 1988) results indicate that EGF alone exhibits a mitogenic effect on bovine mammary epithelial cells. Its activity is, however, substantially elevated in the presence of insulin. In doses up to 100 ng/ml the effect is mainly additive, while at l-50 pg/rnl concentration of insulin, the effect is synergistic and finally accounts for up to 70% of the effect achieved in the presence of 10% FCS. We have shown previously (Shamay et al., 1988) that

the mitogenic effect of insulin is most likely mediated through IGF-I receptors. Thus, it is reasonable to assume that synergism of EGF and insulin results from the same type of interaction. This suggestion is substantiated by the finding that the effects of EGF and IGF-I are additive or even synergistic (see Table 1 and Figs. 2 and 4). The extent of this additive effect is variable and may range from 10% (see Table 1 in Shamay et al., 1988) to a 100% or even more. It is also evident that the effects of IGF-I and EGF are additive or even synergistic to those of factors present in FCS. Six experiments with tissue obtained from different calves have been carried out until the present time. Results of these experiments (Table 1, and similar unpublished data from three additional experiments) show that addition of IGF-I + EGF to 10% FCS-supplemented medium enhanced the mitogenic effect of FCS by 50-200%. The reason for this variability is not clear and could result from the different state of differentiation of the mammary tissue in vivo prior to the slaughter. It is now evident that the profound stimulatory effects of EGF and IGF-I on thymidine incorporation are not limited to in vitro systems, but were also demonstrated by elevating the amount of mammary DNA in in vivo infusion experiments in ruminants (Collier and McGrath, 1988; McGrath and Collier, 1988). It should be noted that existence of specific receptors for IGF-I (Dehoff et al., 1988) EGF (Spitzer and Grosse, 1987) in bovine mammary gland was reported recently. We have previously shown that our culture system consisted of a small amount of fibroblasts, though their relative number did not increase through 2-8 days of culture (Shamay et al., 1988). The increase in thymidine incorporation was also always accompanied by a visible increase in the size of the epithelial clumps (Shamay and Gertler, 1986). However, the existence of other cell types such as myoepithelial cells cannot be excluded. thus leading to a possibility that the GFs and toxin effects may result not only from a direct effect on the epithelial cells but also from paracrine interactions. The present results are insufficient to distinguish between these possibilities. The main part of the present work was devoted to study the effects of IBMX and cholera and pertussis toxins and their interaction with IGF-I,

225 TABLE

4

SEMI-QUANTITIVE SUMMARY OF THE DIRECT EFFECTS AND INTERACTIONS OF MAJOR FACTORS ING DNA SYNTHESIS IN UNDIFFERENTIATED BOVINE MAMMARY EPITHELIAL CELLS IN VITRO Factor assayed None IBMX Cholera toxin Pertussis toxin

Factor

compared



None

Cholera

0 + + -

+ ++ 0

STIMULAT-

toxin

IGF-I

EGF

IGF-I + EGF

FCS 2

++ ++++ ++++ +/+ +

+ +++ +++

+++ ++++ +++++ ++

+++++ +++++ +++++ +

o/+

’ 0 = basal medium; - = less than basal medium; + to + + + + + = increasingly stimulatory effect on DNA synthesis. * Addition of IGF-I + EGF to 10% FCS-supplemented medium resulted in an further 1.5 to 3-fold enhancement of the DNA synthesis.

EGF and unidentified GFs present in FCS. Semiquantitative results of all experiments were summarized in Table 4. It seems that the mitogenic process is mediated in part through a CAMP-dependent event. This is documented by the following findings: (1) CT exhibits a mitogenic activity which is potentiated by IBMX; (2) forskolin is also mitogenic; (3) the mitogenic activities of EGF and IGF-I are potentiated by IBMX; and (4) 8-bromo-CAMP and dibutyryl-CAMP enhanced the effect of IGF-I. The mitogenic events also include a CAMP-independent mitogenic pathway. This is documented by the effects of IGF-I, EGF and their mutual synergism. Both types of mitogenic pathways strongly synergize. Neither CT nor IBMX synergized, however, with the effect of FCS. This indicates that factors stimulating the CAMP-mediated effect on DNA synthesis by either indirect stimulation of CAMP formation (10% FCS-supplemented medium did not increase the intracellular level of CAMP in freshly prepared cells), or by inhibition of its degradation may already be present in FCS at optimal concentrations. Additional factors must, however, be present there as well, since the overall effect of the FCS is much higher than that of CT, IBMX or both. It seems that these factors are distinct from IGF-I and EGF. This conclusion is based on the finding that while the combination of IGF-I, EGF and CT resulted in DNA synthesis which was equal to that achieved in the medium supplemented with 10% FCS (Fig. 2) combination of IGF-I, EGF and 10% FCS resulted in a further (up to 3-fold) increase in DNA synthesis.

Pertussis toxin attenuated the effect of CT. Most likely this effect was not connected to the ribosylation of Gi protein that affects adenylate cyclase directly or indirectly (Helmreich and Pfeuffer, 1985). Such a modification would release tonic inhibition of the adenylate cyclase and led to enhanced proliferation, in contrast to the experimental results. The ability of PT to inhibit drastically (80-908) the effect of FCS and, partially, the effect of IGF-I, EGF or both, hints at the possibility that an additional PT-sensitive mitogenie pathway(s) may exist as well. This result also suggests that other GFs existing in FCS stimulate the cell proliferation through a PT-sensitive protein. Our finding that the mammary cell membranes have an additional G-protein that is ribosylated by PT, but distinct from Gi, supports this notion. The nature of this PT-sensitive pathway is at present unknown but it may be a reflection of a second PT substrate interacting with phosphatidylinositol-specific phospholipase C. It should be noted that two independent hormone-stimulated DNA synthesis pathways have been demonstrated in fibroblasts (Chambard et al., 1987). The first was activated by IGF-I or fibroblast growth factor and not inhibited by PT. The other, stimulated by thrombin and mediated through activation of phosphatidylinositol 4,5-b& phosphate-specific phospholipase C, was strongly inhibited by PT with I&, (amount required for 50% inhibition) = 0.1 ng/ml. A 41 kDa G-protein was identified as a mediator of this pathway. Mitogenic activity of bombesin-like growth factors in Swiss 3T3 fibroblasts was also mediated through

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a PT-sensitive G-protein (Letterio et al., 1986). On the other hand, in the EGF-dependent human breast cancer cell line MDA-468 SA, PT almost completely attenuated the EGF-stimulated cell growth both in anchorage-dependent and anchorage-independent conditions (Church and Buick, 1988). A 41 kDa G-protein was identified as a target of PT action leading to the possibility that. like in fibroblasts, the effect of EGF may be mediated through a G-protein. It has been recently demonstrated that the action of an unidentified growth factor present in FCS is mediated through a PT-sensitive Gi,-protein in rat glioma C6BUI cells (Milligan, 1989). Until now, PT action in normal mammary gland was not studied and no information on PT- or CT-sensitive G-proteins is available. Our present and previous results suggest that DNA synthesis and cell proliferation in normal bovine undifferentiated mammary gland may be a product of several synergic stimulants: IGF-I, EGF and other unidentified GFs existing in the FCS. These factors may activate CAMP-dependent and -independent pathways and be regulated through a PT-sensitive GTP-binding protein. Acknowledgements We thank Dr. M. Niwa of the Fujisawa Pharmaceutical Co. (Osaka, Japan) for supplying us with IGF-I, Drs. G. Brenner and D. Hochman of the Marbek Co., Israel, for their help at the slaughterhouse, Dr. R.J. Collier for reading the manuscript, Mr. S. Salame for the mammary glands and Mrs. M. Rockitter for her secretarial assistance. We also thank the National Hormone Pituitary Program (University of Maryland School of Medicine) for their generous gift of ovine prolactin and bovine growth hormone. This investigation was supported by a grant from the National Council for Research and Development (Israel), grant No. 2662-l-87. One of us (A.S.) is a recipient of a stipendium from the Baron Hirsch Fund.

References Chamhard, J.C.. Paw S.. L’AlIemain. G. and Pouyssegur. J. (19X7) Nature 326. 800-X03. Church. J.G. and Buick. R.N. (1988) J. Biol. Chem. 263, 4242-4246. Cerione. R.A.. Kroll. S., RaJaram. R.. Unson, C., Goldamidth. P. and Spregel, A.M. (19X8) J. Biol. Chem. 263, 9345-9352. Collier. R.J. and McGrath. M.F. (198X) J. Dairy Sci. 71 (Suppl.). 22X (abstract). Deeks. S., Richards, J. and Nandi, S. (1988) Exp. Cell Res. 174. 44X-460. Dehoff. M.H., Elgin. R.G.. Collier, R.J. and Clemmons, D.R. (I 988) Endocrinology 122, 2412-2417. Ethier. S.P.. Kudla. A. and Cundlff. K.C. (19X7) J. Cell. Physiol. 132. 161-167. Harper, J.F. and Brooker. G. (1975) J. Cyclic Nucleotide Res. 1, 207%?18. Helmreich. E.J.M. and Pfeuffer. T. (1985) Trends Pharmacol. SCI. 6. 43X-443. Imagawa, W., Bandyopadhyay. G.K., Wallace. D. and Nandi, S. (1988) J. Cell. Physiol. 135. 509-515. Letterio, J.J., Coughlin. S.R. and Williams, L.T. (1986) Science 234. 1137-1119. McGrath, M.F. and Collier, R.J. (19X8) J. Dairy Sci. 71 (Suppl.), 229 (abstract). Milligan. G. (1989) Cell. Signal. 1. 65-72. Pines. M., Gierschik, P., Milligan, G., Klee, W. and Spiegel, A.M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4095-4099. Pines, M., Ashkenazi, A., Cohen-Chapnik. N.. Binder. L. and Gertler. A. (1988) J. Cell. B&hem. 37, 119-129. Richards, J., Larson, L., Guzman. R.. Tomooka, Y.. Osborn, R.. Imagawa. W. and Nandi, S. (1983) J. Tissue Cult. Methods 8. 31-36. Saito. Y., Yamada, H.. Niwa. M. and Ueda. 1. (1986) J. Biochem. (Tokyo) 101, 123- 134. Shamay. A. and Gertler, A. (1986) Cell Biol. Int. Rep. 10. 923-930. Shamay. A.. Cohen. N., Niwa, M. and Gertler, A. (1988a) Endocrinology 123, 804-809. Shamay, A., Pines, M.. Waxman, M. and Gertler. A. (1988h) in Proceedings 71st Meeting Endocrinology Society, Seattle, WA, Abstract No. 1313. Sheffield, L.G., Sinha, H.A. and Welsch. C.W. (1985) Endocrinology 117, 1864-1869. Spitzer. E. and Grosse, R. (1987) B&hem. Int. 14, 5X1-588.

Proliferation of bovine undifferentiated mammary epithelial cells in vitro is modulated by G-proteins.

Several cAMP-elevating agents such as cholera toxin (CT), forskolin and 3-isobutyl-1-methylxanthine (IBMX) exhibited weak mitogenic activity on bovine...
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