0013-7227/91/1283-1574$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 3 Printed in U.S.A.
Annexin-I Regulation in Response to Suckling and Rat Mammary Cell Differentiation* KENNETH R. HORLICK, MEHRDAD GANJIANPOURf, SUSAN C. FROST, AND HARRY S. NICK* Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610
removal of the suckling stimulus caused a rapid increase in mRNA expression, but that translation of message was delayed, possibly until the gland was irreversibly committed to involution. Since high levels of annexin-I are associated with the quiescent epithelial cell, annexin-I may play an important role in blocking differentiation of the mammary gland. (Endocrinology 128: 1574-1579,1991)
ABSTRACT. Expression of annexin-I was investigated in the rat mammary gland during pregnancy, lactation, and involution. Both the mRNA and protein were very abundant in the mature virgin gland, but declined significantly by midpregnancy. In the lactating gland, little or no annexin-I was detected. After weaning, mRNA and protein levels increased dramatically, corresponding to glandular involution. We also show that premature
A
tein synthesis (Wong, W., S. C. Frost, and H. S. Nick, unpublished observations). Another hormone-dependent effect related to annexin-I has been revealed with the identification of a PRL-inducible gene isolated from the pigeon crop-sac, which shows a high level of similarity to the mammalian protein (25). The crop-sac, like the more complex mammary gland, contains epithelial cells that differentiate into nutrient-secreting cells during pregnancy (26). The connection between PRL and the pigeon annexin-I-like gene together with the importance of PRL in mammary gland function (27) prompted us to study annexin-I expression in the rat mammary gland during pregnancy, lactation, and involution to determine whether PRL might be involved in its regulation. Here we show that mammary annexin-I mRNA and protein levels exhibit striking changes during these periods, and that removal of the nerve stimuli associated with suckling, through either normal or premature weaning, dramatically induces annexin-I synthesis.
NNEXIN-I, formerly called lipocortin-I (1), is one of the best characterized members of the annexins, a group of calcium-dependent phospholipid-binding proteins that share about 50% sequence homology. Proteins that are part of this family include lipocortins (2), calpactins (3), calelectrins (4-6), calcimedins (7), anticoagulant protein (8), endonexins (4, 5, 9, 10), and chromobindins (11). Various annexins have been implicated in cellular processes, including modulation of phospholipase-A2 activity and inflammation (12), exocytosis (13, 14), differentiation (15), blood coagulation (16), the immune response (17), membrane-cytoskeletal linkage (18), and intracellular signal transduction (19, 20). Most recently, lipocortin-III has been identified as inositol 1,2cyclic phosphate 2-phosphohydrolase, an enzyme important in metabolizing phosphoinositol intermediates (21). With the large number of implied cellular roles, a definitive and widely accepted function for annexin-I or the other members of the family has not been determined. Studies regarding the regulation of annexin-I expression have focused on glucocorticoids, because annexin-I was initially defined as a glucocorticoid-induced antiinflammatory protein (22-24). We have shown that annexin-I mRNA and protein are induced by dexamethasone in fibroblasts and epithelial cells by a process that is transcriptionally dependent and requires de novo pro-
Materials and Methods Animal usage
Received October 9, 1990. * This work was supported by the NIH. t Supported by a NIH Medical Student Training Grant. X To whom all correspondence and requests for reprints should be addressed.
All animal experimentation was conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Timed pregnant Sprague-Dawley rats (225-250 g) were obtained from Charles River Laboratories (Wilmington, MA). For each time point, a single animal was killed by cervical dislocation under anesthesia. The two lateral abdominal glands were immediately dissected; one was used for RNA isolation, and the other for protein analysis. Each tissue was weighed and added immediately to the appropriate homogeni-
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND zation buffer as described below. All experiments were repeated with at least two separate groups of pregnant rats. RNA isolation Total RNA was isolated by homogenizing tissues in a guanidinium thiocyanate-sarcosyl solution, followed by acid phenolchloroform extraction and ethanol precipitation, as described previously (28). Typically, about 1 g tissue was lysed in 5 ml thiocyanate solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 100 mM 2mercaptoethanol. To this solution, 300 n\ 2 M sodium acetate (pH 4.0), 3 ml H2O-saturated phenol, and 1 ml chloroformisoamyl alcohol (24:1) were added in stepwise fashion. The mixture was centrifuged at 10,000 x g for 10 min to separate phases. The aqueous phase was mixed with 3 ml isopropanol, and total RNA was precipitated by incubating the mixture at -20 C for at least 1 h, followed by centrifugation at 10,000 X g for 30 min. The RNA pellet was then resuspended in 600 /A guanidine thiocyanate solution and further purified by isopropanol and ethanol precipitation.
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Western analysis Mammary tissue was weighed and homogenized in 10 ml 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 255 mM sucrose supplemented with 0.1% aprotinin. A Polytron (Brinkmann, Westbury, NY) was used for homogenization (two times at halfmaximal speed for 10 sec each). After the suspension was spun at 212,000 x g for 70 min at 4 C, a Markwell protein assay was performed on the supernatant. One hundred micrograms of total protein were run on a 10% polyacrylamide reducing gel and transferred to nitrocellulose. The membrane was blocked for 3 h using 20 mM Tris-HCl (pH 7.4)-0.5 M NaCl (TBS) supplemented with 3% BSA and 0.05% Tween and incubated for 16 h at 4 C with an antiannexin-I antibody at a 1:1000 dilution in 3% BSA-TBS. The preparation and specificity of this antibody have been described previously (2). The membrane was washed for 1 h with 1% BSA-TBS and then incubated with 1 x 107 cpm 125I-labeled protein-A for 1 h. After three 20-min washes in 25 ml TBS, the membrane was air dried and exposed for 3-5 days to Kodak X-Omat film. Nonimmune serum showed no cross-reactivity.
Northern analysis An aliquot of 20 ng total RNA was loaded on a 1% MOPS [3-(N-morpholino)propanesulfonic acid]-formaldehyde agarose gel as described previously (29). The gel was run for 12-16 h at 40 V, with constant buffer recirculation. The gel was then subjected to three 30-min washes with water, one 45-min wash in 0.05 N NaOH and 0.01 M NaCl, one 45-min wash in 0.1 M Tris-HCl (pH 8.0), and a 30-min wash in a solution containing 0.089 M Tris-borate, 0.089 M boric acid, and 0.002 M EDTA, pH 8.0. The RNA was electrophoretically transferred from the gel to a noncharged nylon membrane (GeneScreen, New England Nuclear, Boston, MA) for 1 h. The RNA was immobilized on the membrane by cross-linking with UV light (30) and analyzed with a specific radiolabeled probe.
Results and Discussion
To investigate annexin-I mRNA expression during rat mammary gland differentiation and involution, we carried out Northern analysis using a mouse annexin-I cDNA probe. mRNA levels in mature virgin animals were compared with those in animals that were pregnant, lactating, or weaned (Fig. 1). The mRNA was very abundant in the nonpregnant state. At midpregnancy, corresponding to the time when epithelial cells begin differentiation (27, 32), a large decline was observed. By day 22 of pregnancy (1-2 days before birth), mRNA levels were decreased by 50-fold compared to those in the virgin animals when analyzed by densitometry. At birth a small Probe synthesis and hybridization but reproducible increase occurred. With the onset of A mouse annexin-I cDNA was provided by William Wong lactation, annexin-I mRNA was undetectable and refrom this laboratory. It was composed of a 900-basepair insert mained so for the entire suckling period. After weaning that contained only coding region. The coding region included (30 days after birth), the mRNA rose to levels comall but 10 N-terminal amino acids. Close homology exists parable to those in virgin animals. To illustrate the between mouse and rat annexin-I mRNA at the nucleotide proliferative nature of the mammary gland at selected (88%) and protein (93%) levels. A rat Cu/Zn superoxide distimes during pregnancy, we determined levels of histone mutase cDNA was provided by Jan-Ling Hsu from this laboH4 mRNA, which is known to increase during DNA ratory. The mouse histone H4 cDNA was from Dr. Gary Stein. synthesis in association with cellular proliferation (33). Probes were synthesized using [a-32P]dATP (New England Nuclear; 800 fiCi/mmol) by either random primer extension As shown in Fig. 1, a rise in histone H4 mRNA was (31) or an M13 single stranded probe synthesis method, using apparent on day 11 of pregnancy, when mammary epian M13mpl8 clone containing the mouse annexin-I cDNA thelial cells undergo a secondary phase of proliferation insert in the appropriate orientation to produce a probe com(32). Low levels of histone H4 expression were observed plementary to mRNA (30). Blots were prehybridized for 15-30 throughout the later stages of pregnancy and lactation, min in a buffer containing 1% BSA (Sigma), 1 mM EDTA, 0.5 corresponding to the slower growth and differentiation M NaHPO4 (pH 7.2), and 7% sodium dodecyl sulfate at 60 C. of the gland (32). Most striking was the increase in After addition of the labeled probe, the blots were hybridized histone message at weaning, a time when the gland at 60 C for 12-16 h. The blots were then subjected to three 10undergoes involution and new epithelial cells replace min washes in 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), and 1% secretory cells. Copper/zinc superoxide dismutase (SOD) sodium dodecyl sulfate at 65 C and exposed to Kodak X-Omat mRNA was chosen as an internal control because it AR film (Eastman Kodak, Rochester, NY) for various periods shows little regulation under many metabolic conditions of time.
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND PREGNANT
Endo• 1991 Voll28-No3
LACTATING
NP FlG. 1. Regulation of mammary annexin-I mRNA during pregnancy, lactation, and weaning. Total RNA was isolated from mammary tissue of female rats that were mature virgins (NP), pregnant for various lengths of time (days), had just given birth (B), or had suckled pups for various lengths of time (days) or after weaning (W; 30 days after giving birth). mRNA levels for histone H4 and Cu/Zn superoxide dismutase are shown below lipocortin-I.
Annexin I
Htstone
Cu/Zn SOD
(Visner, G., and H. S. Nick, unpublished observations). Although SOD mRNA levels did not change during pregnancy, expression was decreased abruptly at the onset of lactation, but then remained constant until weaning. To examine whether the annexin-I protein followed a similar expression pattern, extracted mammary tissue proteins were analyzed by immunoblotting (Fig. 2A). Also shown are mammary annexin-I mRNA levels from
mRNA
the same animals. The annexin-I-specific antibody revealed abundant levels of annexin-I protein in the virgin rat gland. Like mRNA, the protein declined during pregnancy, was nearly undetectable during lactation, and increased at weaning. Densitometric analysis of protein and mRNA levels illustrates the close correspondence between the two (Fig. 2B). Given that annexin-I mRNA and protein levels increased upon weaning, the role of the suckling stimulus in annexin-I regulation was investigated. mRNA levels were determined in mammary tissue of dams that had pups withdrawn at birth and at specific times during lactation (Fig. 3). If pups were removed at birth, completely preventing the suckling stimulus, mammary annexin-I mRNA levels increased significantly in the dams as early as 6 h after removal, reaching a plateau by 12 h. If pups were withdrawn after 7 days of suckling, the increase in annexin-I mRNA was delayed by several hours and peaked after 24 h. Expression of SOD mRNA did not change during withdrawal (data not shown). Interestingly, annexin-I protein was nearly undetectable for more than 24 h after pups were removed either at pups removed a t time after removal:
7doyt
Birth 0
6
12 24 5d 0
6
12 24 5d
mRNA 1L
7L
15L
W
FlG. 2. Regulation of annexin-I mRNA and protein in rat mammary tissue during gland development and involution. A, Total RNA and protein were extracted from mammary tissue of rats at specific stages of pregnancy (P) and lactation (L), as described in Fig. 1. Northern and Western analyses were performed as described in Materials and Methods. B, Quantitation of the results in A by video densitometry (Bio Image Visage 60).
protein FlG. 3. Increase in annexin-I mRNA and protein levels after removal of the suckling stimulus. Pups were withdrawn from dams at birth or after 7 days of suckling. Total RNA was isolated from mammary tissue of dams 0, 6,12, 24, and 120 h (5 days) after pup withdrawal.
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND
birth or after 7 days of suckling. However, protein levels were restored by 5 days after removal. To examine the specificity of annexin-I regulation after pup withdrawal, annexin-I mRNA levels were measured in the mammary gland and several other tissues from animals whose pups were withdrawn prematurely. These experiments demonstrated that annexin-I mRNA levels were constant in the heart, kidney, and hindleg muscle of dams compared to the significant rise in mammary tissue expression over the 5-day withdrawal period (Fig. 4). Thus, the regulation of annexin-I mRNA in the mammary gland after pup removal was a tissue specific phenomenon. It is likely that hormonal changes during pregnancy and lactation influence the pattern of mammary annexin-I mRNA and protein expression. Based on the knowledge of a PRL-inducible annexin-I-like gene in the pigeon crop-sac, we have explored the effects of PRL on mammalian annexin-I gene expression. These experiments involved the use of the midpregnancy-derived mammary epithelial cell line HCll, which had been clonally selected for its responsiveness to PRL (34). Treatment of these cells with PRL, however, caused no change in annexin-I mRNA expression, which was also the case when mammary explants were treated with PRL in culture (data not shown). However, circulating levels of PRL in the rat are low until 1 day before birth, when they rise dramatically (35). Together, these data suggest that the down-regulation of annexin-I gene expression is not mediated by PRL action. However, two placental lactogens (I and II) have been identified in the rat which appear on day 8 of pregnancy, show peak activity on day 12 (32), and, thus, inversely correlate with annexin-I mRNA and protein between days 11-15. These lactogens are thought to be of primary importance in mammary gland development by providing a mammotropic stimulus. We are further investigating mammary epithelial cell lines for hormonal effects on annexin-I expression in order to better understand the regulation we observed in the mammary gland during differentiation and involution. Although PRL did not affect expression of annexin-I mRNA in the mouse mammary epithelial cell line HCll, we did observe high levels of the message (data not shown). Thus, it is likely that the resting ductal epithelial Mammary tissue
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cells, which give rise to secretory cells during pregnancy, are responsible for the high annexin-I expression we observed in the virgin gland. Immunolocalization of two other annexins, annexin-II and calelectrin, in virgin mammary epithelial cells by Rocha and co-workers (3638) supports this hypothesis. In contrast, secretory cells of the lactating gland were essentially devoid of these proteins (36). The ductal cells, which are dormant in the mature virgin animal and reported to be arrested in the Gi phase of the cell cycle, begin proliferation and differentiation into secretory cells around midpregnancy (27, 32). The striking decline in annexin-I mRNA and protein levels that we observed in the rat mammary gland during pregnancy corresponds to the onset of these changes in epithelial cell morphology. We propose that the high level of annexin-I in the virgin mammary gland may be involved in maintaining the undifferentiated state. This leads us to consider what regulates annexin-I. Although the lactogens may play some role, it is well known that epidermal growth factor is important in signaling mammary gland growth during pregnancy (39). The epidermal growth factor receptor, which is known to phosphorylate annexin-I (40), reaches its highest levels in the mammary gland around midpregnancy (41). Perhaps phosphorylation of annexin-I during pregnancy alters its activity, which is followed by a subsequent down-regulation of annexin-I expression. Although it has been demonstrated that serine/threonine phosphorylation of annexin-I blocks its phospholipase-inhibitory activity and alters its Ca2+/phospholipid-binding properties (42-45), it is yet to be investigated whether tyrosine phosphorylation, too, is inhibitory. Nevertheless, this event may be necessary to initiate epithelial cell differentiation. Soon after natural weaning or premature termination of the suckling stimulus by pup removal, a subpopulation of the mammary secretory epithelial cells reverts back to the resting state, while the remainder of the cells die (46, 47). No irreversible changes in the gland occur immediately after cessation of suckling, because lactation can recommence several days after weaning (48). The subset of epithelial cells that revert to the resting state may be responsible for the rapid increase in mammary annexinI mRNA levels after removal of the suckling stimulus. These cells may receive a signal from the sympathetic nervous system when suckling ceases which causes a KMney
Muscle
time after removal:
FiG. 4. Specificity of the mammary annexin-I mRNA regulation after pup withdrawal. Pups were removed from dams after 15 days of suckling, and total RNA was isolated from mammary and other tissues of dams 0, 6,12, 24, and 120 h (5 days) after removal.
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND
rapid transcriptional activation of annexin-I in preparation for return to the resting state. As shown in Fig. 3, translation of annexin-I mRNA appears to be delayed and requires more than 24 h after cessation of suckling. This delay may correspond to the time when glandular involution becomes committed, resulting in secretory cell death. This appears to occur approximately 5 days after weaning (48) and, therefore, correlates with the time at which maximal levels of annexin-I are reached. We believe that the high annexin-I levels observed in the involuted gland after natural weaning (Fig. 2) indicate the presence of new resting epithelial cells, which, like those in the mature virgin gland, may undergo proliferation and differentiation with the onset of a new pregnancy.
Acknowledgment We would like to thank Dr. Blake Pepinsky (Biogen) for helpful discussion and providing his antiannexin-I antibody («646).
References 1. Crumpton MJ, Dedman JR 1990 Protein terminology tangle. Nature 345:12 2. Huang K-S, Wallner BP, Mattaliano RJ, Tizard R, Burne C, Frey A, Hession C, McGray P, Sinclair LK, Chow EP, Browning JL, Ramachandran KL, Tang J, Smart JE, Pepinsky RB 1986 Two human 35 kd inhibitors of phospholipase A2 are related to substrates of pp60V8rc and the epidermal growth factor receptor/kinase. Cell 46:191-199 3. Glenney Jr JR 1986 Two related but distinct forms of the Mr 36,000 tyrosine kinase substrate (calpactin) that interacts with phospholipids and actin in a Ca2+-dependent manner. Proc Natl Acad Sci USA 83:4258-4262 4. Geisow MJ, Fritsche U, Hexham JM, Dash B, Johnson T 1986 A consensus amino-acid sequence repeat in torpedo and mammalian Ca2+-dependent membrane-binding proteins. Nature 320:636-638 5. Geisow MJ, Walker JH 1986 New proteins involved in cell regulation by Ca2+ and phospholipids. Trends Biochem Sci 11:420-423 6. Sudhof TC, Slaughter CA, Leznicki I, Barjon P, Reynolds GA 1988 Human 67-kDa calelectrin contains a duplication of four repeats found in 35-kDa lipocortins. Proc Natl Acad Sci USA 85:664-668 7. Smith VL, Dedman JR 1986 An immunological comparison of several novel calcium-binding proteins. J Biol Chem 261:1581515818 8. Funakoshi T, Hendrickson LE, McMullen BA, Fujikawa K 1987 Primary structure of human placental anticoagulant protein. Biochemistry 26:8087-8092 9. Weber K, Johnsson N, Plesmann U, Van PN, Soling H, Ampe C, Vandekerckhove J 1987 The amino acid sequence of protein II and its phosphorylation site for protein kinase C; the domain structure Ca2+-modulated lipid binding proteins. EMB0 J 6:1599-1604 10. Schlaepfer DD, Mehlman T, Burgess WH, Haigler HT 1987 Structural and functional characterizaton of endonexin II, a calciumand phospholipid-binding protein. Proc Natl Acad Sci USA 84:6078-6082 11. Creutz CE, Zakas WJ, Hamman HC, Crane S, Martin WH, Gould KL, Oddie KM, Parsons SJ 1987 Identification of chromaffin granule-binding proteins. J Biol Chem 262:1860-1868 12. Flower RJ 1988 Lipocortin and the mechanism of action of the glucocorticoids. Br J Pharmacol 94:987-1001 13. Drust D, Creutz C 1988 Aggregation of chromaffin granules by calpactin at micromolar levels of calcium. Nature 331:88-91
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14. Burgonyne RD 1988 Calpactin in exocytosis? Nature 331:20 15. Violette SM, King I, Browning JL, Pepinsky RB, Wallner BP, Sartorelli AC 1990 Role of lipocortin I in the glucocorticoid induction of the terminal differentiation of a human squamous carcinoma. J Cell Physiol 142:70-77 16. Tait JF, Sakata M, McMullen BA, Miao CH, Funakoshi T, Hendrickson L, Fujikawa K 1988 Placental anticoagulant proteins: isolation and comparative characterization of four members of the lipocortin family. Biochemistry 27:6268-6276 17. Hirata F, Iwata M 1983 Role of lipomodulin, a phospholipase inhibitory protein, in immunoregulation by thymocytes. J Immunol 130:1930-1936 18. Roy-Choudhury S, Mishra VS, Low MG, Das M 1988 A phospholipid is the membrane anchoring domain of a protein growth factor of molecular mass 34 kDa in placental trophoblasts. Proc Natl Acad Sci USA 85:2014-2018 19. Ullrich A, Schlessinger J 1990 Signal transduction by receptors with tyrosine kinase activity. Cell 61:203-212 20. Cooper JA, Hunter T 1983 Identification and characterization of cellular targets for tyrosine protein kinases. J Biol Chem 258:11081115 21. Ross TS, Tait JF, Majerus PW 1990 Identity of inositol 1,2-cyclic
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phosphate 2-phosphohydrolase with lipocortin III. Science 248:605-607 Flower RJ, Blackwell GJ 1979 Anti-inflammatory steroids induce biosynthesis of a phospholipase A2 inhibitor which prevents prostaglandin generation. Nature 278:456-459 Blackwell GJ, Carnuccio R, Di Rosa M, Flower RJ, Parente L, Persico P 1980 Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids. Nature 287:147-149 Hirata F, Schiffman D, Venkatasubramanian K, Salomon D, Axelrod J 1980 A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids. Proc Natl Acad Sci USA 77:2533-2536 Horseman ND 1989 A prolactin-inducible gene product which is a member of the calpactin/lipocortin family. Mol Endocrinol 3:773779 Pukac LA, Horseman ND 1986 Regulation of cloned prolactininducible genes in pigeon crop. Mol Endocrinol 1:188-194 Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049-1106 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 Sambrook J, Fritsch EF, Maniatis T 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Church GM, Gilbert W 1984 Genomic sequencing. Proc Natl Acad Sci USA 81:1991-1995 Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Thordarson G, Talamantes F 1987 Role of the placenta in mammary gland development and function. In: Neville MC, Daniel CW (eds) The Mammary Gland: Development, Regulation, and Function. Plenum Press, New York, pp 459-498 Seiler-Tuyns A, Birnstiel ML 1981 Structure and expression in Lcells of a cloned H4 histone gene of the mouse. J Mol Biol 151:607625 Ball RK, Friis RR, Schoenenberger CA, Doppoler W, Groner B 1988 Prolactin regulation of /3-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J 7:2089-2095 Rosenblatt JS, Siegel HI, Mayer AD 1979 Progress in the study of maternal behavior in the rat: hormonal, nonhormonal, sensory, and developmental aspects. In: Rosenblatt RA, Hinde CB, Busnel M-C (eds) Advances in the Study of Behavior. Academic Press, New York, pp 225-311 Lozano JJ, Silberstein GB, Hwang S-I, Haindl AH, Rocha V 1989a Developmental regulation of calcium-binding proteins (calelectrins and calpactin I) in mammary glands. J Cell Physiol 138:503-510
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND 37. Horn YK, Sudhof TC, Lozano JL, Haindl AH, Rocha V 1988 Mammary gland Ca2+-binding (-dependent) proteins: identification as calelectrins and calpactin I/p36. J Cell Physiol 135:435-442 38. Lozano JL, Haindl AH, Rocha V 1989b Purification, characterization, and localization of 70 kDa calcium-sensitive protein (calelectrin) from mammary glands. J Cell Physiol 141:318-324 39. Taketani Y, Oka T 1983 Possible physiological role of epidermal growth factor in the development of the mouse mammary gland during pregnancy. FEBS Lett 152:256-260 40. Sawyer ST, Cohen S 1985 Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells. J Biol Chem 260:8233-8236 41. Edery M, Pang K, Larson L, Colosi T, Nandi S 1985 Epidermal growth factor receptor levels in mouse mammary glands in various physiological states. Endocrinology 117:405-411 42. Ando Y, Imamura S, Hong Y-M, Owada MK, Kakunaga T, Kannagi R 1989 Enhancement of calcium sensitivity of lipocortin I in phospholipid binding induced by limited proteolysis and phosphorylation at the amino terminus as analyzed by phospholipid affinity
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column chromatography. J Biol Chem 264:6948-6955 43. Touqui L, Rothhut B, Shaw AM, Fradin A, Vargaftig BB, RussoMarie F 1986 Platelet activation-a role for a 40K anti-phospholipase A2 protein indistinguishable from lipocortin. Nature 321:177180 44. Hirata F, Matsuda K, Notsu Y, Hattori T, Del Carmine R 1984 Phosphorylation at a tyrosine residue of lipomodulin in mitogenstimulated murine thymocytes. Proc Natl Acad Sci USA 81:47174721 45. Hirata F 1981 The regulation of lipomodulin, a phospholipase inhibitory protein, in rabbit neutrophils by phosphorylation. J Biol Chem 256:7730-7733 46. Hollmann KM 1974 Cytology and fine structure of the mammary gland. In: Larson BL, Smith VR (eds) Lactation-A Comprehensive Treatise. Academic Press, New York, pp 3-95 47. Borellini F, Oka T 1989 Growth control and differentiation in mammary epithelial cells. Env Health Perspect 80:85-99 48. Silver IA 1956 Vascular changes in the mammary gland during engorgement with milk. J Physiol 133:65P
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Vol. 128, No. 3 Printed in U.S.A.
Annexin-I Regulation in Response to Suckling and Rat Mammary Cell Differentiation* KENNETH R. HORLICK, MEHRDAD GANJIANPOURf, SUSAN C. FROST, AND HARRY S. NICK* Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610
removal of the suckling stimulus caused a rapid increase in mRNA expression, but that translation of message was delayed, possibly until the gland was irreversibly committed to involution. Since high levels of annexin-I are associated with the quiescent epithelial cell, annexin-I may play an important role in blocking differentiation of the mammary gland. (Endocrinology 128: 1574-1579,1991)
ABSTRACT. Expression of annexin-I was investigated in the rat mammary gland during pregnancy, lactation, and involution. Both the mRNA and protein were very abundant in the mature virgin gland, but declined significantly by midpregnancy. In the lactating gland, little or no annexin-I was detected. After weaning, mRNA and protein levels increased dramatically, corresponding to glandular involution. We also show that premature
A
tein synthesis (Wong, W., S. C. Frost, and H. S. Nick, unpublished observations). Another hormone-dependent effect related to annexin-I has been revealed with the identification of a PRL-inducible gene isolated from the pigeon crop-sac, which shows a high level of similarity to the mammalian protein (25). The crop-sac, like the more complex mammary gland, contains epithelial cells that differentiate into nutrient-secreting cells during pregnancy (26). The connection between PRL and the pigeon annexin-I-like gene together with the importance of PRL in mammary gland function (27) prompted us to study annexin-I expression in the rat mammary gland during pregnancy, lactation, and involution to determine whether PRL might be involved in its regulation. Here we show that mammary annexin-I mRNA and protein levels exhibit striking changes during these periods, and that removal of the nerve stimuli associated with suckling, through either normal or premature weaning, dramatically induces annexin-I synthesis.
NNEXIN-I, formerly called lipocortin-I (1), is one of the best characterized members of the annexins, a group of calcium-dependent phospholipid-binding proteins that share about 50% sequence homology. Proteins that are part of this family include lipocortins (2), calpactins (3), calelectrins (4-6), calcimedins (7), anticoagulant protein (8), endonexins (4, 5, 9, 10), and chromobindins (11). Various annexins have been implicated in cellular processes, including modulation of phospholipase-A2 activity and inflammation (12), exocytosis (13, 14), differentiation (15), blood coagulation (16), the immune response (17), membrane-cytoskeletal linkage (18), and intracellular signal transduction (19, 20). Most recently, lipocortin-III has been identified as inositol 1,2cyclic phosphate 2-phosphohydrolase, an enzyme important in metabolizing phosphoinositol intermediates (21). With the large number of implied cellular roles, a definitive and widely accepted function for annexin-I or the other members of the family has not been determined. Studies regarding the regulation of annexin-I expression have focused on glucocorticoids, because annexin-I was initially defined as a glucocorticoid-induced antiinflammatory protein (22-24). We have shown that annexin-I mRNA and protein are induced by dexamethasone in fibroblasts and epithelial cells by a process that is transcriptionally dependent and requires de novo pro-
Materials and Methods Animal usage
Received October 9, 1990. * This work was supported by the NIH. t Supported by a NIH Medical Student Training Grant. X To whom all correspondence and requests for reprints should be addressed.
All animal experimentation was conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Timed pregnant Sprague-Dawley rats (225-250 g) were obtained from Charles River Laboratories (Wilmington, MA). For each time point, a single animal was killed by cervical dislocation under anesthesia. The two lateral abdominal glands were immediately dissected; one was used for RNA isolation, and the other for protein analysis. Each tissue was weighed and added immediately to the appropriate homogeni-
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND zation buffer as described below. All experiments were repeated with at least two separate groups of pregnant rats. RNA isolation Total RNA was isolated by homogenizing tissues in a guanidinium thiocyanate-sarcosyl solution, followed by acid phenolchloroform extraction and ethanol precipitation, as described previously (28). Typically, about 1 g tissue was lysed in 5 ml thiocyanate solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 100 mM 2mercaptoethanol. To this solution, 300 n\ 2 M sodium acetate (pH 4.0), 3 ml H2O-saturated phenol, and 1 ml chloroformisoamyl alcohol (24:1) were added in stepwise fashion. The mixture was centrifuged at 10,000 x g for 10 min to separate phases. The aqueous phase was mixed with 3 ml isopropanol, and total RNA was precipitated by incubating the mixture at -20 C for at least 1 h, followed by centrifugation at 10,000 X g for 30 min. The RNA pellet was then resuspended in 600 /A guanidine thiocyanate solution and further purified by isopropanol and ethanol precipitation.
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Western analysis Mammary tissue was weighed and homogenized in 10 ml 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 255 mM sucrose supplemented with 0.1% aprotinin. A Polytron (Brinkmann, Westbury, NY) was used for homogenization (two times at halfmaximal speed for 10 sec each). After the suspension was spun at 212,000 x g for 70 min at 4 C, a Markwell protein assay was performed on the supernatant. One hundred micrograms of total protein were run on a 10% polyacrylamide reducing gel and transferred to nitrocellulose. The membrane was blocked for 3 h using 20 mM Tris-HCl (pH 7.4)-0.5 M NaCl (TBS) supplemented with 3% BSA and 0.05% Tween and incubated for 16 h at 4 C with an antiannexin-I antibody at a 1:1000 dilution in 3% BSA-TBS. The preparation and specificity of this antibody have been described previously (2). The membrane was washed for 1 h with 1% BSA-TBS and then incubated with 1 x 107 cpm 125I-labeled protein-A for 1 h. After three 20-min washes in 25 ml TBS, the membrane was air dried and exposed for 3-5 days to Kodak X-Omat film. Nonimmune serum showed no cross-reactivity.
Northern analysis An aliquot of 20 ng total RNA was loaded on a 1% MOPS [3-(N-morpholino)propanesulfonic acid]-formaldehyde agarose gel as described previously (29). The gel was run for 12-16 h at 40 V, with constant buffer recirculation. The gel was then subjected to three 30-min washes with water, one 45-min wash in 0.05 N NaOH and 0.01 M NaCl, one 45-min wash in 0.1 M Tris-HCl (pH 8.0), and a 30-min wash in a solution containing 0.089 M Tris-borate, 0.089 M boric acid, and 0.002 M EDTA, pH 8.0. The RNA was electrophoretically transferred from the gel to a noncharged nylon membrane (GeneScreen, New England Nuclear, Boston, MA) for 1 h. The RNA was immobilized on the membrane by cross-linking with UV light (30) and analyzed with a specific radiolabeled probe.
Results and Discussion
To investigate annexin-I mRNA expression during rat mammary gland differentiation and involution, we carried out Northern analysis using a mouse annexin-I cDNA probe. mRNA levels in mature virgin animals were compared with those in animals that were pregnant, lactating, or weaned (Fig. 1). The mRNA was very abundant in the nonpregnant state. At midpregnancy, corresponding to the time when epithelial cells begin differentiation (27, 32), a large decline was observed. By day 22 of pregnancy (1-2 days before birth), mRNA levels were decreased by 50-fold compared to those in the virgin animals when analyzed by densitometry. At birth a small Probe synthesis and hybridization but reproducible increase occurred. With the onset of A mouse annexin-I cDNA was provided by William Wong lactation, annexin-I mRNA was undetectable and refrom this laboratory. It was composed of a 900-basepair insert mained so for the entire suckling period. After weaning that contained only coding region. The coding region included (30 days after birth), the mRNA rose to levels comall but 10 N-terminal amino acids. Close homology exists parable to those in virgin animals. To illustrate the between mouse and rat annexin-I mRNA at the nucleotide proliferative nature of the mammary gland at selected (88%) and protein (93%) levels. A rat Cu/Zn superoxide distimes during pregnancy, we determined levels of histone mutase cDNA was provided by Jan-Ling Hsu from this laboH4 mRNA, which is known to increase during DNA ratory. The mouse histone H4 cDNA was from Dr. Gary Stein. synthesis in association with cellular proliferation (33). Probes were synthesized using [a-32P]dATP (New England Nuclear; 800 fiCi/mmol) by either random primer extension As shown in Fig. 1, a rise in histone H4 mRNA was (31) or an M13 single stranded probe synthesis method, using apparent on day 11 of pregnancy, when mammary epian M13mpl8 clone containing the mouse annexin-I cDNA thelial cells undergo a secondary phase of proliferation insert in the appropriate orientation to produce a probe com(32). Low levels of histone H4 expression were observed plementary to mRNA (30). Blots were prehybridized for 15-30 throughout the later stages of pregnancy and lactation, min in a buffer containing 1% BSA (Sigma), 1 mM EDTA, 0.5 corresponding to the slower growth and differentiation M NaHPO4 (pH 7.2), and 7% sodium dodecyl sulfate at 60 C. of the gland (32). Most striking was the increase in After addition of the labeled probe, the blots were hybridized histone message at weaning, a time when the gland at 60 C for 12-16 h. The blots were then subjected to three 10undergoes involution and new epithelial cells replace min washes in 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), and 1% secretory cells. Copper/zinc superoxide dismutase (SOD) sodium dodecyl sulfate at 65 C and exposed to Kodak X-Omat mRNA was chosen as an internal control because it AR film (Eastman Kodak, Rochester, NY) for various periods shows little regulation under many metabolic conditions of time.
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND PREGNANT
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LACTATING
NP FlG. 1. Regulation of mammary annexin-I mRNA during pregnancy, lactation, and weaning. Total RNA was isolated from mammary tissue of female rats that were mature virgins (NP), pregnant for various lengths of time (days), had just given birth (B), or had suckled pups for various lengths of time (days) or after weaning (W; 30 days after giving birth). mRNA levels for histone H4 and Cu/Zn superoxide dismutase are shown below lipocortin-I.
Annexin I
Htstone
Cu/Zn SOD
(Visner, G., and H. S. Nick, unpublished observations). Although SOD mRNA levels did not change during pregnancy, expression was decreased abruptly at the onset of lactation, but then remained constant until weaning. To examine whether the annexin-I protein followed a similar expression pattern, extracted mammary tissue proteins were analyzed by immunoblotting (Fig. 2A). Also shown are mammary annexin-I mRNA levels from
mRNA
the same animals. The annexin-I-specific antibody revealed abundant levels of annexin-I protein in the virgin rat gland. Like mRNA, the protein declined during pregnancy, was nearly undetectable during lactation, and increased at weaning. Densitometric analysis of protein and mRNA levels illustrates the close correspondence between the two (Fig. 2B). Given that annexin-I mRNA and protein levels increased upon weaning, the role of the suckling stimulus in annexin-I regulation was investigated. mRNA levels were determined in mammary tissue of dams that had pups withdrawn at birth and at specific times during lactation (Fig. 3). If pups were removed at birth, completely preventing the suckling stimulus, mammary annexin-I mRNA levels increased significantly in the dams as early as 6 h after removal, reaching a plateau by 12 h. If pups were withdrawn after 7 days of suckling, the increase in annexin-I mRNA was delayed by several hours and peaked after 24 h. Expression of SOD mRNA did not change during withdrawal (data not shown). Interestingly, annexin-I protein was nearly undetectable for more than 24 h after pups were removed either at pups removed a t time after removal:
7doyt
Birth 0
6
12 24 5d 0
6
12 24 5d
mRNA 1L
7L
15L
W
FlG. 2. Regulation of annexin-I mRNA and protein in rat mammary tissue during gland development and involution. A, Total RNA and protein were extracted from mammary tissue of rats at specific stages of pregnancy (P) and lactation (L), as described in Fig. 1. Northern and Western analyses were performed as described in Materials and Methods. B, Quantitation of the results in A by video densitometry (Bio Image Visage 60).
protein FlG. 3. Increase in annexin-I mRNA and protein levels after removal of the suckling stimulus. Pups were withdrawn from dams at birth or after 7 days of suckling. Total RNA was isolated from mammary tissue of dams 0, 6,12, 24, and 120 h (5 days) after pup withdrawal.
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND
birth or after 7 days of suckling. However, protein levels were restored by 5 days after removal. To examine the specificity of annexin-I regulation after pup withdrawal, annexin-I mRNA levels were measured in the mammary gland and several other tissues from animals whose pups were withdrawn prematurely. These experiments demonstrated that annexin-I mRNA levels were constant in the heart, kidney, and hindleg muscle of dams compared to the significant rise in mammary tissue expression over the 5-day withdrawal period (Fig. 4). Thus, the regulation of annexin-I mRNA in the mammary gland after pup removal was a tissue specific phenomenon. It is likely that hormonal changes during pregnancy and lactation influence the pattern of mammary annexin-I mRNA and protein expression. Based on the knowledge of a PRL-inducible annexin-I-like gene in the pigeon crop-sac, we have explored the effects of PRL on mammalian annexin-I gene expression. These experiments involved the use of the midpregnancy-derived mammary epithelial cell line HCll, which had been clonally selected for its responsiveness to PRL (34). Treatment of these cells with PRL, however, caused no change in annexin-I mRNA expression, which was also the case when mammary explants were treated with PRL in culture (data not shown). However, circulating levels of PRL in the rat are low until 1 day before birth, when they rise dramatically (35). Together, these data suggest that the down-regulation of annexin-I gene expression is not mediated by PRL action. However, two placental lactogens (I and II) have been identified in the rat which appear on day 8 of pregnancy, show peak activity on day 12 (32), and, thus, inversely correlate with annexin-I mRNA and protein between days 11-15. These lactogens are thought to be of primary importance in mammary gland development by providing a mammotropic stimulus. We are further investigating mammary epithelial cell lines for hormonal effects on annexin-I expression in order to better understand the regulation we observed in the mammary gland during differentiation and involution. Although PRL did not affect expression of annexin-I mRNA in the mouse mammary epithelial cell line HCll, we did observe high levels of the message (data not shown). Thus, it is likely that the resting ductal epithelial Mammary tissue
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cells, which give rise to secretory cells during pregnancy, are responsible for the high annexin-I expression we observed in the virgin gland. Immunolocalization of two other annexins, annexin-II and calelectrin, in virgin mammary epithelial cells by Rocha and co-workers (3638) supports this hypothesis. In contrast, secretory cells of the lactating gland were essentially devoid of these proteins (36). The ductal cells, which are dormant in the mature virgin animal and reported to be arrested in the Gi phase of the cell cycle, begin proliferation and differentiation into secretory cells around midpregnancy (27, 32). The striking decline in annexin-I mRNA and protein levels that we observed in the rat mammary gland during pregnancy corresponds to the onset of these changes in epithelial cell morphology. We propose that the high level of annexin-I in the virgin mammary gland may be involved in maintaining the undifferentiated state. This leads us to consider what regulates annexin-I. Although the lactogens may play some role, it is well known that epidermal growth factor is important in signaling mammary gland growth during pregnancy (39). The epidermal growth factor receptor, which is known to phosphorylate annexin-I (40), reaches its highest levels in the mammary gland around midpregnancy (41). Perhaps phosphorylation of annexin-I during pregnancy alters its activity, which is followed by a subsequent down-regulation of annexin-I expression. Although it has been demonstrated that serine/threonine phosphorylation of annexin-I blocks its phospholipase-inhibitory activity and alters its Ca2+/phospholipid-binding properties (42-45), it is yet to be investigated whether tyrosine phosphorylation, too, is inhibitory. Nevertheless, this event may be necessary to initiate epithelial cell differentiation. Soon after natural weaning or premature termination of the suckling stimulus by pup removal, a subpopulation of the mammary secretory epithelial cells reverts back to the resting state, while the remainder of the cells die (46, 47). No irreversible changes in the gland occur immediately after cessation of suckling, because lactation can recommence several days after weaning (48). The subset of epithelial cells that revert to the resting state may be responsible for the rapid increase in mammary annexinI mRNA levels after removal of the suckling stimulus. These cells may receive a signal from the sympathetic nervous system when suckling ceases which causes a KMney
Muscle
time after removal:
FiG. 4. Specificity of the mammary annexin-I mRNA regulation after pup withdrawal. Pups were removed from dams after 15 days of suckling, and total RNA was isolated from mammary and other tissues of dams 0, 6,12, 24, and 120 h (5 days) after removal.
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ANNEXIN-I REGULATION IN THE RAT MAMMARY GLAND
rapid transcriptional activation of annexin-I in preparation for return to the resting state. As shown in Fig. 3, translation of annexin-I mRNA appears to be delayed and requires more than 24 h after cessation of suckling. This delay may correspond to the time when glandular involution becomes committed, resulting in secretory cell death. This appears to occur approximately 5 days after weaning (48) and, therefore, correlates with the time at which maximal levels of annexin-I are reached. We believe that the high annexin-I levels observed in the involuted gland after natural weaning (Fig. 2) indicate the presence of new resting epithelial cells, which, like those in the mature virgin gland, may undergo proliferation and differentiation with the onset of a new pregnancy.
Acknowledgment We would like to thank Dr. Blake Pepinsky (Biogen) for helpful discussion and providing his antiannexin-I antibody («646).
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