Life Sciences, Vol. 50, pp. 791-798 Printed in the U S A
Pergamon Press
F U N C T I O N A L EXPRESSION OF VIP RECEPTORS IN NORMAL, I M M O R T A L I Z E D AND T R A N S F O R M E D M A M M A R Y E P I T H E L I A L CELLS Philippe Berthon, Ladislav Mirossay1, Susumo Ito2, Fabien Calvo3 and Christian Gespach. INSERM U.55, Unit~ de recherches sur les Neuropeptides Digestifs et le Diab~te, H6pital Saint- Antoine, 75571 PARIS Cedex 12, France. 1. Department of Pharmacology, Medical Faculty, P.J. Safarik University, Tr. SNP 1, 04001 KOSICE, Czechoslovakia. 2. Harvard Medical School, Department of Anatomy and Cellular Biology, Boston, MA 02115, USA. 3. Laboratoire de Pharmacologie, Institut de G~n6tique Mol~culaire, H6pital Saint- Louis, 75475 PARIS Cedex 10, France. (Received in final form January 14, 1992)
Summary The effect of VIP and its related peptides on cAMP production has been characterized: 1) in long term culture of normal human mammary epithelial cells (HMEC); 2) in immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40; 3) in the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1 D, described to exhibit normal morphogenesis in vivo and functional differentiation in vitro. Basal cAMP levels were increased 1.5- to 8.7- fold in mammary epithelial cells (p < 0.001 - 0.05), with a potency EC50 = 0.02 - 0.6 nM VlP. The pharmacological specificity of the VIP receptors coupled to cAMP generation was established according to the following potency sequence: VIP > PACAP-38 > helodermin > PHM, PHV > helospectin 1 >> hpGRF, secrefin in HMEC, VlP > PACAP-38 > helodermin > helospectin 1, PHM, PHV > hpGRF > secretin in $1T3 cells, and VlP, PHI, helodermin > PHV > rhGRF > secretin in HC-11 cells. Our data demonstrate the presence of functional, highly sensitive and specific VIP receptors in normal, immortalized and transformed mammary epithelial cells, suggesting a regulatory role for this neuropeptide on the growth, differentiation and function in normal and neoplastic breast tissue. Vasoactive intestinal peptide (VlP) is widely distributed in the brain and peripheral nervous systems, including the gastrointestinal tract and pancreas (1). Its biological effects are mediated by specific binding sites coupled with the adenylate cyclase system (2, 3). Covalent labeling of these binding sites in cellular membranes, using 1251-VIP as a ligand and chemical cross- linkers such as dithiobis (succinimidyl propionate) and disuccinimidyl suberate, revealed that the VIP receptor has an apparent molecular weight of Mr 30,000 to 80,000 depending on the tissues investigated, which include liver, stomach, intestine and lung (4-11). Activation of VIP receptors and increased intracellular cyclic AMP levels (cAMP) are associated with cAMP- dependent protein kinase activation and phosphorylation of specific target proteins. Accordingly, the human VlP receptor gene encodes a 362- amino acid polypeptide with structural homologies with other G protein- coupled receptors, including a seven transmembrane structure (12). A wide range of biological functions has been described for VlP, including the relaxation of the smooth muscle and the stimulation of enzyme, water and electrolyte secretion in the gastroenteropancreatic, cardiovascular and respiratory systems, and of hormonal secretion by the pancreas, adrenal and pituitary glands (13-16).
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.
792
VIP Receptors in Mammary Epithelial Cells
Vol. 50, No. ii, 1992
The high levels of VIP in human milk, the increased VIP gene expression and VIP plasma levels in response to suckling in lactating animals and nursing women (17-21) imply that VlP has a physiological role in regulating milk secretion and ejection from mammary gland ducts. The aim of the present study was to investigate the presence of VlP receptors in human and mouse mammary epithelial cells derived from normal breast or established at various stages of neoplastic transformation. The effects of VIP and its related peptides on cAMP production were therefore characterized pharmacologically in 1) long- term cultures of normal human mammary epithelial cells (HMEC), 2) immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40 (22, 23), and 3) the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1D, reported to exhibit normal morphogenesis in vivo and functional differentiation in vitro (24, 25).
Methods Cell culture and transfection: Primary cultures of normal HMEC from mammoplasty patients were plated in medium A, consisting of calcium- free DMEM/F12 media ( 1 v / 1 v , IJB - France) containing 2 mM glutamine, 10 p.g/ml insulin (Sigma), 5 x 10-6 M cortisol (Sigma), 50 IU/ml penicillin, 50 lig/ml streptomycin, 100 ng/ml cholera toxin (Sigma), 2 ng/ml EGF (Paesel, West Germany) and 1.05 mM CaCI2, supplemented with 5 % Ca2+- chelated horse serum (Gibco). Between days 7 and 10 after plating, medium A was replaced by the same medium except that the calcium concentration was 60 p~M instead of 1.05 mM (medium B). Normal HMEC were treated with dispase (grade II, Boehringer) and passaged by transfer of either freefloating or adherent cells. For transfection, about 20,000 HMEC from 2 mammoplasty specimens were seeded for 24 hours in 24- well plates (Falcon) in 0.5 ml of medium B. HMEC were then transfected by calcium- phosphate precipitation with 2 p.g of the pMK16- SV40 (ori-) plasmid in 50 Ill of medium B (24, 25). Next, HMEC were incubated for 5 hours at 37°C, washed twice, exposed for 1 h at 37°C to 1 ml medium B, and fed every 3 days with 0.5 ml of the same medium. The SV40 large T- transfected cells were designated $1T3 and $3T3 for the immortalized cell lines, and $2T2 and NS2T2 for the tumorigenic and transformed cell lines. These ST human mammary epithelial cell lines were used for long term cultures in medium B without cholera toxin and passaged at confluency with 0.05% trypsin and 0.02% EDTA. Transfected HMEC were fixed in 2 % formaldehyde, 4 % glutaraldehyde and 0.06 % picric acid, post- fixed in osmium tetroxide, and processed for electron microscopy. In the mouse mammary epithelial cell line HC-11, a clone isolated from the COMMA-1D cell line, production of the milk protein ~-casein is regulated by lactogenic hormones (24). This HC-11 clone was generously given by Dr. Roland Ball (Ludwig Institute for Cancer Research, Bern, Switzerland). HC-11 cells were cultured in RPMI 1640 medium (Gibco) with 10 % heat- inactivated fetal calf serum (Biological Industries, Israel), 10 ng/ml epidermal growth factor (EGF), 5 lig/ml insulin, 50 IU/ml penicillin and 50 p.g/ml streptomycin.
VIP receptor binding and cAMP generation assay: Binding assays were performed on HC-11 cultures by incubation of 200,000 cells for 30 min at 15°C in 500 p.I of the binding assay buffer containing 35 mM Tris- HCI (pH 7.5), 2 % BSA, 50 mM NaCI, 0.6 mg/ml bacitracin, 5 x 10-11M 1251- VIP and concentrations of unlabeled VIP and related peptides ranging from 10 "11 to 10-6 M Cells were then washed and centrifuged, and the pellet was counted for cell- associated 1251-VIP in a Packard autogamma counter. To assess the effect of VIP on cAMP generation in mouse and human mammary epithelial cells, normal HMEC and mouse HC-11 cells were removed from culture flasks by exposure to 0.02 % EDTA in Ca2+-, Mg 2+- free phosphate buffer saline. Next, 200,000 cells per tube were preincubated for 10 min at 20°C in 400 p.I of the same 35 mM Tris- HCI buffer containing 50 mM NaCI and 1 mM IBMX as phosphodiesterase inhibitor. The incubation lasted 60 rain at the same temperature in the presence or absence of various concentrations of VIP or its structurally related peptides listed below. Cyclic AMP production in ST cells was measured when the adherent cell layers reached the density of 3 x 105 cells/well (24 wells, Corning). Cultured ST cells were incubated as described above, and the reaction was stopped by adding 50 ILl of 11 N HCIO4. Intracellular and extracellular cAMP released during incubation was determined using a radioimmunoassay (26). VIP- related peptides and chemicals: Porcine VIP was purchased from Pr. V. Mutt (Department of Biochemistry II, Karolinska Institutet, Stockholm, Sweden). This VIP was used because its aminoacid sequence is identical to that of human VIP. Porcine VIP was iodinated by the chloramine T method and purified by HPLC. The VIP- related peptides were human secretin, human peptide with N-terminal histidine and C-terminal methionine (PHM), human peptide with N-terminal histidine and C-terminal valine (PHV), rat peptide with N-terminal histidine and C-terminal isoleucine (PHI), the truncated form of glucagon- like peptide- 1 (TGLP- 1) and helospectin 1. These peptides were purchased from Peninsula Laboratories (St.
Vol. 50, No. 11, 1992
rIP Receptors in Mammary Epithelial Cells
793
Helens, England). Crystallized highly purified pomine glucagon (batch 42306) was from Novo Research Institute (Bagsvaerd, Denmark), rat hypothalamic growth hormone- releasing factor (rhGRF) and human pancreatic growth hormone- releasing factor (hpGRF) were kindly given by Dr. J. Rivier and Dr. W. Vale (Salk Institute, San Diego, CA, USA), and helodermin was purchased from Novabiochem (Switzerland). Ovine hypothalamic PACAP-38 (27) synthetized by solid- phase techniques was generously donated by Dr. A. Arimura (Tulane University, Belle Chasse, CA, USA). 1251- labeled Na was from the Radiochemical Centre (Amersham, UK). All other chemicals were of analar grade.
Results Normal HMEC: Cyclic AMP levels in normal HMEC rose 3.2- fold, from 37 + 8.3 to 119 -+ 32.6 pmol/106 cells, after the addition of 10-7 M VIP (p < 0.02, n=6). As shown in Figure 1, half- maximal stimulation was observed at the concentration of EC50 = 1.7 x 10 -10 M VIP. The VIP analogues stimulated cAMP generation in the following order of potency: VIP > PACAP- 38 > helodermin > PHM, PHV • helospectin 1 >> hpGRF, secretin. Therefore, VIP was respectively 6, 24 and 290 times more potent than PACAP- 38, helodermin and PHM or PHV. The VIP- related peptides exhibited parallel dose- response curves and similar extent of maximal stimulation to those observed for VIP, PACAP- 36, helodermin, PHV and PHM.
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Pergamon Press
F U N C T I O N A L EXPRESSION OF VIP RECEPTORS IN NORMAL, I M M O R T A L I Z E D AND T R A N S F O R M E D M A M M A R Y E P I T H E L I A L CELLS Philippe Berthon, Ladislav Mirossay1, Susumo Ito2, Fabien Calvo3 and Christian Gespach. INSERM U.55, Unit~ de recherches sur les Neuropeptides Digestifs et le Diab~te, H6pital Saint- Antoine, 75571 PARIS Cedex 12, France. 1. Department of Pharmacology, Medical Faculty, P.J. Safarik University, Tr. SNP 1, 04001 KOSICE, Czechoslovakia. 2. Harvard Medical School, Department of Anatomy and Cellular Biology, Boston, MA 02115, USA. 3. Laboratoire de Pharmacologie, Institut de G~n6tique Mol~culaire, H6pital Saint- Louis, 75475 PARIS Cedex 10, France. (Received in final form January 14, 1992)
Summary The effect of VIP and its related peptides on cAMP production has been characterized: 1) in long term culture of normal human mammary epithelial cells (HMEC); 2) in immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40; 3) in the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1 D, described to exhibit normal morphogenesis in vivo and functional differentiation in vitro. Basal cAMP levels were increased 1.5- to 8.7- fold in mammary epithelial cells (p < 0.001 - 0.05), with a potency EC50 = 0.02 - 0.6 nM VlP. The pharmacological specificity of the VIP receptors coupled to cAMP generation was established according to the following potency sequence: VIP > PACAP-38 > helodermin > PHM, PHV > helospectin 1 >> hpGRF, secrefin in HMEC, VlP > PACAP-38 > helodermin > helospectin 1, PHM, PHV > hpGRF > secretin in $1T3 cells, and VlP, PHI, helodermin > PHV > rhGRF > secretin in HC-11 cells. Our data demonstrate the presence of functional, highly sensitive and specific VIP receptors in normal, immortalized and transformed mammary epithelial cells, suggesting a regulatory role for this neuropeptide on the growth, differentiation and function in normal and neoplastic breast tissue. Vasoactive intestinal peptide (VlP) is widely distributed in the brain and peripheral nervous systems, including the gastrointestinal tract and pancreas (1). Its biological effects are mediated by specific binding sites coupled with the adenylate cyclase system (2, 3). Covalent labeling of these binding sites in cellular membranes, using 1251-VIP as a ligand and chemical cross- linkers such as dithiobis (succinimidyl propionate) and disuccinimidyl suberate, revealed that the VIP receptor has an apparent molecular weight of Mr 30,000 to 80,000 depending on the tissues investigated, which include liver, stomach, intestine and lung (4-11). Activation of VIP receptors and increased intracellular cyclic AMP levels (cAMP) are associated with cAMP- dependent protein kinase activation and phosphorylation of specific target proteins. Accordingly, the human VlP receptor gene encodes a 362- amino acid polypeptide with structural homologies with other G protein- coupled receptors, including a seven transmembrane structure (12). A wide range of biological functions has been described for VlP, including the relaxation of the smooth muscle and the stimulation of enzyme, water and electrolyte secretion in the gastroenteropancreatic, cardiovascular and respiratory systems, and of hormonal secretion by the pancreas, adrenal and pituitary glands (13-16).
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.
792
VIP Receptors in Mammary Epithelial Cells
Vol. 50, No. ii, 1992
The high levels of VIP in human milk, the increased VIP gene expression and VIP plasma levels in response to suckling in lactating animals and nursing women (17-21) imply that VlP has a physiological role in regulating milk secretion and ejection from mammary gland ducts. The aim of the present study was to investigate the presence of VlP receptors in human and mouse mammary epithelial cells derived from normal breast or established at various stages of neoplastic transformation. The effects of VIP and its related peptides on cAMP production were therefore characterized pharmacologically in 1) long- term cultures of normal human mammary epithelial cells (HMEC), 2) immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40 (22, 23), and 3) the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1D, reported to exhibit normal morphogenesis in vivo and functional differentiation in vitro (24, 25).
Methods Cell culture and transfection: Primary cultures of normal HMEC from mammoplasty patients were plated in medium A, consisting of calcium- free DMEM/F12 media ( 1 v / 1 v , IJB - France) containing 2 mM glutamine, 10 p.g/ml insulin (Sigma), 5 x 10-6 M cortisol (Sigma), 50 IU/ml penicillin, 50 lig/ml streptomycin, 100 ng/ml cholera toxin (Sigma), 2 ng/ml EGF (Paesel, West Germany) and 1.05 mM CaCI2, supplemented with 5 % Ca2+- chelated horse serum (Gibco). Between days 7 and 10 after plating, medium A was replaced by the same medium except that the calcium concentration was 60 p~M instead of 1.05 mM (medium B). Normal HMEC were treated with dispase (grade II, Boehringer) and passaged by transfer of either freefloating or adherent cells. For transfection, about 20,000 HMEC from 2 mammoplasty specimens were seeded for 24 hours in 24- well plates (Falcon) in 0.5 ml of medium B. HMEC were then transfected by calcium- phosphate precipitation with 2 p.g of the pMK16- SV40 (ori-) plasmid in 50 Ill of medium B (24, 25). Next, HMEC were incubated for 5 hours at 37°C, washed twice, exposed for 1 h at 37°C to 1 ml medium B, and fed every 3 days with 0.5 ml of the same medium. The SV40 large T- transfected cells were designated $1T3 and $3T3 for the immortalized cell lines, and $2T2 and NS2T2 for the tumorigenic and transformed cell lines. These ST human mammary epithelial cell lines were used for long term cultures in medium B without cholera toxin and passaged at confluency with 0.05% trypsin and 0.02% EDTA. Transfected HMEC were fixed in 2 % formaldehyde, 4 % glutaraldehyde and 0.06 % picric acid, post- fixed in osmium tetroxide, and processed for electron microscopy. In the mouse mammary epithelial cell line HC-11, a clone isolated from the COMMA-1D cell line, production of the milk protein ~-casein is regulated by lactogenic hormones (24). This HC-11 clone was generously given by Dr. Roland Ball (Ludwig Institute for Cancer Research, Bern, Switzerland). HC-11 cells were cultured in RPMI 1640 medium (Gibco) with 10 % heat- inactivated fetal calf serum (Biological Industries, Israel), 10 ng/ml epidermal growth factor (EGF), 5 lig/ml insulin, 50 IU/ml penicillin and 50 p.g/ml streptomycin.
VIP receptor binding and cAMP generation assay: Binding assays were performed on HC-11 cultures by incubation of 200,000 cells for 30 min at 15°C in 500 p.I of the binding assay buffer containing 35 mM Tris- HCI (pH 7.5), 2 % BSA, 50 mM NaCI, 0.6 mg/ml bacitracin, 5 x 10-11M 1251- VIP and concentrations of unlabeled VIP and related peptides ranging from 10 "11 to 10-6 M Cells were then washed and centrifuged, and the pellet was counted for cell- associated 1251-VIP in a Packard autogamma counter. To assess the effect of VIP on cAMP generation in mouse and human mammary epithelial cells, normal HMEC and mouse HC-11 cells were removed from culture flasks by exposure to 0.02 % EDTA in Ca2+-, Mg 2+- free phosphate buffer saline. Next, 200,000 cells per tube were preincubated for 10 min at 20°C in 400 p.I of the same 35 mM Tris- HCI buffer containing 50 mM NaCI and 1 mM IBMX as phosphodiesterase inhibitor. The incubation lasted 60 rain at the same temperature in the presence or absence of various concentrations of VIP or its structurally related peptides listed below. Cyclic AMP production in ST cells was measured when the adherent cell layers reached the density of 3 x 105 cells/well (24 wells, Corning). Cultured ST cells were incubated as described above, and the reaction was stopped by adding 50 ILl of 11 N HCIO4. Intracellular and extracellular cAMP released during incubation was determined using a radioimmunoassay (26). VIP- related peptides and chemicals: Porcine VIP was purchased from Pr. V. Mutt (Department of Biochemistry II, Karolinska Institutet, Stockholm, Sweden). This VIP was used because its aminoacid sequence is identical to that of human VIP. Porcine VIP was iodinated by the chloramine T method and purified by HPLC. The VIP- related peptides were human secretin, human peptide with N-terminal histidine and C-terminal methionine (PHM), human peptide with N-terminal histidine and C-terminal valine (PHV), rat peptide with N-terminal histidine and C-terminal isoleucine (PHI), the truncated form of glucagon- like peptide- 1 (TGLP- 1) and helospectin 1. These peptides were purchased from Peninsula Laboratories (St.
Vol. 50, No. 11, 1992
rIP Receptors in Mammary Epithelial Cells
793
Helens, England). Crystallized highly purified pomine glucagon (batch 42306) was from Novo Research Institute (Bagsvaerd, Denmark), rat hypothalamic growth hormone- releasing factor (rhGRF) and human pancreatic growth hormone- releasing factor (hpGRF) were kindly given by Dr. J. Rivier and Dr. W. Vale (Salk Institute, San Diego, CA, USA), and helodermin was purchased from Novabiochem (Switzerland). Ovine hypothalamic PACAP-38 (27) synthetized by solid- phase techniques was generously donated by Dr. A. Arimura (Tulane University, Belle Chasse, CA, USA). 1251- labeled Na was from the Radiochemical Centre (Amersham, UK). All other chemicals were of analar grade.
Results Normal HMEC: Cyclic AMP levels in normal HMEC rose 3.2- fold, from 37 + 8.3 to 119 -+ 32.6 pmol/106 cells, after the addition of 10-7 M VIP (p < 0.02, n=6). As shown in Figure 1, half- maximal stimulation was observed at the concentration of EC50 = 1.7 x 10 -10 M VIP. The VIP analogues stimulated cAMP generation in the following order of potency: VIP > PACAP- 38 > helodermin > PHM, PHV • helospectin 1 >> hpGRF, secretin. Therefore, VIP was respectively 6, 24 and 290 times more potent than PACAP- 38, helodermin and PHM or PHV. The VIP- related peptides exhibited parallel dose- response curves and similar extent of maximal stimulation to those observed for VIP, PACAP- 36, helodermin, PHV and PHM.
,oo
7'/ J/F L.s, {
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.~
I
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\ \T,Helodermin PACAP-38
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