Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Developmental Biology 274 (2004) 31 – 44 www.elsevier.com/locate/ydbio

Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro Naoko Nukumi, Kayoko Ikeda, Megumi Osawa, Tokuko Iwamori, Kunihiko Naito, Hideaki Tojo* Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Received for publication 14 October 2003, revised 30 March 2004, accepted 28 April 2004 Available online 10 August 2004

Abstract

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo. D 2004 Elsevier Inc. All rights reserved. Keywords: Cell cycle; Cell proliferation; FACScan; HC11 cells; Mammary; Epithelial cells; Transgenic mice; WAP

Introduction Whey acidic protein (WAP) has been found in the milk of a limited number of species of rodents, including mice (Hennighausen and Sippel, 1982a) and rats (Campbell et al., 1984). In addition, WAP has been identified in camels (Beg et al., 1984) and rabbits (Devinoy et al., 1988), and more recently in the milk of pigs (Rival et al., 2001; Simpson et al., 1998), wallabies (Simpson et al., 2000), red kangaroos (Nicholas et al., 2001), and brush tail possums (Demmer et * Corresponding author. Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Fax: +81 3 5841 8191. E-mail address: [email protected] (H. Tojo). 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.04.040

al., 2001). WAP gene expression occurs in mammary epithelial cells during late pregnancy and throughout lactation, and is regulated by a variety of lactogenic hormones, including insulin, glucocorticoid, and prolactin (Burdon et al., 1991a; Doppler et al., 1991; Vonderhaar and Ziska, 1989). WAP proteins have a signal peptide (Dandekar et al., 1982) and two domain structures that can be identified at the four-disulfide core (4-DSC) domain, which is composed of eight cysteine residues in a conserved arrangement (Hennighausen and Sippel, 1982b). The 4-DSC domain arrangement is not exclusive to the WAP family; several other proteins containing 4-DSC domains have been identified (Dandekar et al., 1982; Garczynski and Goetz, 1997; Heinzel et al., 1986; Kirchhoff et al., 1991; Wideow et al., 1990), which contain either single or multiple copies of

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the 4-DSC domain. Generally, these proteins are secreted and may function as protease inhibitors (Larsen et al., 1998; Ranganathan et al., 2000). Thus, based on the limited sequence identity with known protease inhibitors, it has been postulated that WAP may also be a secreting protease inhibitor (Nicholas et al., 1997; Ranganathan et al., 2000). However, previous studies have demonstrated that rat WAP does not exhibit any protease inhibitory activity in elastase I and II and chymotrypsin assays (Burdon et al., 1991b). Transgenic mothers in which a mouse genomic WAP transgene is expressed precociously have significantly impaired development of the mammary gland and have failed to lactate (Burdon et al., 1991b; Shamay et al., 1992). It has also been reported that high expression of WAP in MMTV-LTR/WAP transgenic mice results in hyperplasia of coagulation glands and impaired mammary development (Hennighausen et al., 1994). On the other hand, Guntzberg et al. (1991) and Wen et al. (1995) have reported, based on their RT-PCR analyses, that WAP is expressed in various exocrine tissues of lactating mice. Such findings suggest that WAP is important as a nutrient in milk for growth, but that it also may exert some other biological function not only in the mammary gland but also in other tissues. To date, few studies have investigated the true function of WAP. To determine whether WAP functions in any tissue types other than the mammary glands, we generated transgenic mice ubiquitously expressing WAP and investigated their phenotypes. WAP function was also investigated by an in vitro system using mammary epithelial cells (HC11 cells). Here, we report that the ubiquitous and forced production of WAP not only inhibits lobulo-alveolar development in vivo but also the cell cycle progression of HC11 cells in vitro.

A 452-bp segment of the WAP cDNA was inserted into a pGEM-T Easy vector (Promega) and amplified. We determined the DNA sequences of the PCR products and judged the results to indicate a complete cDNA encoding of the full open reading frame of mouse WAP. EGFP in pCX-EGFP plasmid (kindly provided by Dr. M. Okabe, Osaka University, Japan) was excised, and in turn replaced by the EcoRI/EcoRI WAP cDNA fragment (452 bp). The pCX-WAP plasmid contained an enhancer of cytomegalo virus, a chicken h-actin promoter with splicing sequences, WAP cDNA, and the rabbit h-globin 3V flanking sequences. For the production of transgenic mice, the SspI/HindIII DNA fragment (2.98 kb) was excised from a pCXN-WAP and separated by electrophoresis using a 1% agarose gel, and then purified by CsCl ultracentrifugation according to the method of Hogan et al. (1994). For the transfection of plasmids into cultured cells, pCXN-WAP plasmid, pCXN-EGFP plasmid as a marker gene, and pCXN plasmid without the coding region as an empty plasmid (mock) were used, respectively. CAG/WAP transgenic mice The purified DNA fragment (approximately 2000 copies) was microinjected using standard methodology into the pronucleus of BDF1 (C57BL/6XDBA/2) eggs (Hogan et al., 1994). The pups were screened for the transgene by PCR and Southern blot analysis of genomic DNA obtained from the tip of the tail. The primers 5V-ATG-CGT-TGC-CTCATC-AGC-C-3V (forward) and 5V-GAC-AGG-CAG-GGATGG-C-3V (reverse) were used for the detection of WAP cDNA by PCR, and WAP cDNA labeled with 32P a-dCTP was used as a probe for Southern blot analysis. Whole mounts of mammary glands

Materials and methods Animals Mice were purchased from a laboratory animal dealer (SLC, Shizuoka, Japan) and were housed under regulated temperature (22–258C), humidity (40–60%), and illumination cycles (14-h light, 10-h dark); food and water were supplied ad libitum. The experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals from the University of Tokyo College of Agriculture. Gene construction Mouse WAP cDNA was made by reverse transcription using total RNAs that were extracted from the mammary glands of C57BL/6J female mice in late pregnancy using Trizol Reagent (Gibson BRL) according to the manufacturer’s instructions. PCR was carried out using the following primers: 5V-ATG-CGT-TGC-CTC-ATC-AGC-C-3V (forward) and 5V-GAC-AGG-CAG-GGA-TGG-C-3V (reverse).

The third thoracic mammary glands were removed from the transgenic and control mice. Each mammary gland was dissected from the inner surface of the skin, retaining some of the peripheral connective tissues, spread on a cork board, and fixed in freshly prepared Bouin’s solution. The glands were then defatted overnight in acetone. The whole mounts were stained with Mayer’s hematoxylin, embedded in Canadian balsam, and photographed. Histological examination of mammary glands Tissues were embedded in optimal cutting temperature compound (Sakura Finetechnical) and frozen in liquid nitrogen. Frozen sections, 5-Am thick, were mounted on vectabond-coated slides and fixed in 3% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. The sections were then blocked with 0.3% H2O2 in methanol for 30 min at room temperature and then 8% skim milk at 48C overnight. After washing with PBS (pH 7.4), the sections were incubated with primary antibodies, anti-WAP rabbit serum in a 1/100 dilution with 1% BSA in PBS for 1 h. The

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sections were rinsed in PBS (pH 7.4) and then incubated for 40 min with the biotin-labeled mouse anti-rabbit secondary antibody (B5283) (Sigma) in a 1/400 dilution. Signals were detected by the horseradish-peroxidase-labeled avidin–biotin complex (ABC)-mediated method (Vector Laboratories) using diaminobenzidine (DAB) as a substrate. To avoid background staining, slides were preincubated with 10% rabbit serum for 30 min before incubation for 18 h at 48C with the specific antibody directed against mouse WAP.

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Cell culture

RNA was extracted from the tissues using Trizol Reagent (Gibco BRL) according to the manufacturer’s instructions. Total RNA (10 Ag) was fractionated through a 2% agarose formaldehyde gel, transferred to a nylon membrane (Pall Biosupport), and then cross-linked by exposure to ultraviolet irradiation. The membrane was prehybridized, hybridized with the mouse WAP cDNA labeled with 32P a-dCTP, and then washed twice in 2 SSC/0.1% SDS at room temperature, followed by a wash in 0.2 SSC/0.1% SDS at 658C. The signals were visualized by exposure of the membrane to X-ray film (Fuji Film).

Two independent mammary epithelial cell lines derived from mouse pregnant mammary glands were used: HC11 cells (kindly provided by Dr. R.K. Ball, Fredrich Miescher Institute, Switzerland) (Ball et al., 1998) and EpH4K6 cells (kindly provided by Dr. W. Birchmeier, Max-DelbritckCenter, Berlin, Germany) (Niemann et al., 1998). NIH3T3 cells (American Type Culture Collection) from mouse fetal cells were used as a control for comparison with the mammary epithelial cells. The HC11 cells were grown in a complete medium, RPMI1640 (Gibco BRL), containing 10% (v/v) heat-inactivated FBS, 5 Ag/ml of insulin, 10 ng/ ml of epidermal growth factor, and 50 Ag/ml of gentamicin. The EpH4/K6 cells were grown in a complete medium, Dulbecco’s Modified Eagle Medium (DMEM) (Gibco BRL), containing 10% (v/v) heat inactivated FBS, 3.7 g/l of NaHCO3, and 105 U/l of penicillin and gentamicin. The NIH3T3 cells were grown in a complete medium, i.e., Dulbecco’s Modified Eagle Medium containing 10% (v/v) heat-inactivated FBS, 100,000 U/l of penicillin, and 10 mg/l of streptomycin. All cells were cultured in a humidified atmosphere of 5% CO2 in air at 378C.

Antibodies

Transfection of plasmids

For the immunoblotting experiments, goat antibody against mouse WAP (Jackson Immune Research Lab), mouse antibodies against human cyclin D1 and Cdk4 (Santa Cruz Biotechnology), and rabbit antibody against human cyclin D3 and cyclin E (Santa Cruz Biotechnology) were used as the primary antibodies. Anti-mouse, antirabbit, and anti-goat IgG conjugated with horseradish peroxidase were used as the secondary antibodies.

The plasmids were transfected by the use of the Profection Mammalian Transfection System Calcium Phosphate (Promega) according to the manufacturer’s instructions. For the establishment of stable plasmid expression, linearized plasmids were transfected into the cells and, after culturing the cells in complete medium for 2 days, cells demonstrating stable expression of plasmids were selected by passing of the culture for 10 days in a complete medium containing G418 (700 Ag). The efficiency of plasmid transfection was confirmed by observing GFP expression levels in the EGFP-transfected cells.

Northern blot analysis

Western blot analysis Total protein was extracted from the cells by homogenizing them in CytoBustor Protein Extraction Reagent (Novagen) including 1 mM DTT, 0.5 mM PMSF, 2 Ag/ml of aprotinin, 2 Ag/ml pepstatin, and 2 Ag/ml leupeptin; the proteins were then collected by centrifugation. The protein concentration of the extracts was determined using the Bradford method (Bradford, 1976) and the samples were used for Western blot analysis. The proteins were separated on SDS-PAGE (10% or 15%) gel and transferred to a membrane by semidry blotting. The membrane was blocked for 1 h in phosphate-buffered saline (PBS) containing 3% skim milk. After washing the membrane with PBS containing 0.1% Tween 20 (PBST), it was incubated with primary antibody overnight at 48C. After washing with PBST, the membrane was incubated with peroxidase-conjugated secondary antibody for 1.5 h at room temperature. After the membrane had been washed with PBST, the specific antibody signals were detected with ECL (Amersham Pharmacia Biotech), followed by exposure to X-ray film.

RT-PCR One microgram of total RNA was denatured and reverse-transcribed into cDNA using SuperScript II (Promega) in a reaction volume of 10 IU, according to the manufacturer’s protocol. PCR was carried out, and the products were then sequenced and confirmed to be the appropriate transcript. We used the following primer sequences for the amplification of target genes: 5V-ATGCGT-TGC-CTC-ATC-AGC-C-3V (forward) and 5V-GACAGG-CAG-GGA-TGG-C-3V(reverse) for WAP cDNA; 5VCCT-GCA-TGT-TCG-TGG-CCT-CT-3Vand 5V-GAA-AGTGCG-TTG-TGC-GGT-AGC-3Vfor cyclin D1;5V-AGACCT-TCA-TCG-CTC-TGT-GC-3Vand 5V-TAG-CAG-ATGACG-AAC-ACG-CC-3V for cyclin D2; 5V-CCG-TGATTG-CGC-ACG-ACT-TC-3V and 5V-TCT-GTG-GGAGTG-CTG-GTC-TG-3V for cyclin D3;5V-AGC-GAG-GATAGC-AGT-CAG-CC-3V and 5V-TTT-CAT-CCC-CGG-

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AGC-AAG-CG-3V for cyclin E; 5V-TGA-AGG-TCG-GTGCAA-CGG-ATT-TGG-C-3V and 5V-CAT-GTA-GGC-CATGAG-GTC-CAC-CAC-3V for G3PDH (an endogenous control). The quantitative conditions of the PCR reaction were 28 or 31 cycles of 1 min at 948C, 1 min at 588C, and 1 min at 728C. The size of each PCR product was confirmed by electrophoresis using 1% agarose gels in TAE buffer. The RT-PCR products were semiquantified by NIH image software (NIH, Bethesda, MD, USA, Version 1.60) and were expressed as relative amounts in relation to the PCR product of G3PDH mRNA. Cell proliferation assay Cell-Counting Kit-8 solution (Wako Pure Chemical) (Ishiyama et al., 1996) was added to a medium containing the cells in a 96-well dish reacted at 378C for 1 h, according to the manufacturer’s instructions. The number of viable cells was measured at OD 450 nm using a Microplate Reader (Bio-Rad). Assay of apoptosis in vivo and in vitro Apoptotic cells in the mammary glands of transgenic mice were examined by the In Situ Cell Death Detection Kit (Boehringer Mannheim) together with the TUNEL method. The extent of apoptosis observed is indicated here as the number of apoptotic nuclei counted as a percentage of the total cells visualized. For the in vitro assay of apoptosis, WAP-clonal cells were analyzed according to a previously reported method (Li et al., 1999). Equal numbers of clones of WAP-clonal HC11 cells and nontransfected (control) cells were cultured for 2 days in a 12-well dish. The cells were rinsed once with PBS and fixed with 3% paraformaldehyde at 48C overnight, washed three times with PBS, and then treated with 0.1% Triton X-100 at room temperature for 15 min. After being rinsed with PBS three times, the cells were stained with 1 Ag/ml of Hoechst 33342 (Sigma) solution on PBS–glycerol (vol/vol 1:1) at room temperature for 30 min. The number of condensed or fragmented nuclei among the cells in a dish was calculated under a fluorescence microscope at an excitation wavelength of 365 nm. Cell cycle analysis Incorporation of bromodeoxyuridine (BrdU) into the cultured cells was determined by the recommended protocol of the Cell Proliferation ELISA, BrdU (Roche). For the FACScan analysis, adherent cells were trypsinized and fixed in cold 70% ethanol for 4 h. Fixed cells were washed with PBS and incubated with 5 Ag/ml of RNase A (Sigma), and then the cells were stained with 25 Ag/ml of propidium iodine (Aldrich Chemicals) for 15 min at 48C. The stained cells were analyzed on a FACScan cytofluorimeter equipped with the ModeFit LT Software program (Verity).

Examination of the paracrine action of WAP The paracrine action of WAP produced in WAP-clonal cells upon cell proliferation was investigated by the following two methods. First, WAP-clonal HC11 cells (#24) were cultured for 48 h after confluence was reached. The culture medium was recovered and then exchanged for medium in which control HC11 or NIH3T3 cells had been cultured. After the medium exchange, cells were cultured for 24, 48, 72, and 96 h, respectively, and assayed for potential for cell proliferation according to the method described in the bCell proliferation assayQ section. Secondly, WAP activity was also investigated using transwell cell culture chamber inserts (Corning Costar). WAP-clonal cells (104/chamber) were seeded on an upper microporous membrane (pore size, 0.4 Am) in a transwell filled with an appropriate medium for HC11 or NIH3T3 cells; the control HC11 or NIH3T3 cells (3  104/chamber) were seeded on the glass cover of a lower plate. After 96 h of co-culture, the cells on the glass cover were recovered and immunochemically stained with WAP antibody. Evidence for the absence of endogenous WAP expression in the cells on the lower plate was confirmed by RT-PCR using the total RNA extracted from those cells after co-culture. Immunocytochemistry The cultured cells were fixed in 3% paraformaldehyde in PBS for 20 min, then in PBS or PBS containing 0.5% Triton X-100 for 15 min at 48C. After being washed in PBS, the cells were blocked for 30 min in 8% skim milk in PBS, and then were incubated with the rabbit anti-mouse WAP polyclonal antibody in a 1/100 dilution overnight at 48C, followed by washing three times in PBS. The porcine anti-rabbit rhodamine-conjugated secondary antibody (DAKO Immunoglobulins) were applied at a 1/50 dilution with 8% skim milk in PBS, and the cells were incubated for 1 h. Images were observed by confocal microscopy (Carl Zeiss). Detection of WAP in the cultured medium The WAP-clonal cells were cultured in complete medium without FBS for 2 days after 80% confluence was reached in medium containing 10% fetal bovine serum. The medium in the dish was collected by centrifugation (10 min, 3000 rpm), condensed in a Centricon YH-10 (Milipore), and stored at 808C until use. WAP protein was detected by Western blotting, as described in the bMaterials and methodsQ section. Statistical analysis Data were analyzed by Student’s t test. Differences between the mean values were considered statistically significant at P b 0.05.

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Results Phenotype of transgenic mice First, to confirm the expression of WAP in various tissues other than in the mammary glands of normal lactating mice, we analyzed RNAs from various tissues by RT-PCR. WAP mRNA was detected in muscle, liver, lung, and vagina although the expression levels were very low compared with those in the mammary glands (Fig. 1A). To determine whether WAP plays a role in various tissues other than the mammary gland, CAG/WAP transgenic mice were produced that ubiquitously expressed WAP. Ten pups (six females and four males) were found to be transgenic by Southern blot analysis (Fig. 1B) and by PCR analyses of tail DNAs. The pups that were born from two transgenic lines showed poor growth or death during nursing. For example, all of the pups from the first parturition of the #7 founder mother died while nursing within 3 days after birth. In the third parturition of the #7 founder, we exchanged her pups for those born from a normal female at 3 days after birth. All of the seven pups born from the normal mother showed significantly retarded growth under the nursing of the transgenic mother and died 3 days after the exchange, whereas the pups from the transgenic mother grew normally under the nursing of a normal mother. We sacrificed pregnant and lactating transgenic females and collected their tissues. Northern blot analyses showed that the female #7 transgenic founder expressed the exogenous WAP gene at a high level in various tissues,

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including the mammary gland (Fig. 1C). Whole-mount species of the mammary glands of this transgenic female showed unique development (Fig. 2), including poor development of lobulo-alveolar structures and acinous formation considering the dam’s state of midpregnancy (Fig. 2A) and lactation (Fig. 2B), compared with normal mice (C and D in Fig. 2). The results from the examination of mammary glands of transgenic females suggested that the synthesis of milk proteins was inadequate, causing retarded growth or death of the pups. Despite the high expression of the WAP transgene in various tissues, the #7 transgenic mothers grew normally and showed normal parturition and nursing behavior. One other transgenic founder showed slight impairment of the development of the mammary gland, but her pups nonetheless grew normally. The transgenic female offspring of eight other transgenic founders showed no abnormalities of the mammary glands. The hematoxylin and eosin stained transverse sections showed that the development of the lobulo-alveolar structures was remarkably impaired in the transgenic tissues (A and C in Fig. 3) compared with that of normal mammary tissue (B and D in Fig. 3). Exogenous WAP was expressed extensively in the mammary glands of pregnant and lactating transgenic females. This expression was observed not only in the alveolar gland, but also in other connective tissues (E and G in Fig. 3). WAP was not yet expressed in the mammary glands of normal 10-day-pregnant females (F in Fig. 3). To determine the cause of impaired lobuloalveolar development in transgenic mice, we used the TUNEL method to investigate the rate of extent of apoptosis in the lobulo-alveolar structures. No significant apoptosis was observed in the lobulo-alveolar cells of transgenic mice (data not shown). Effects of forced WAP expression on proliferation of mammary epithelial cells

Fig. 1. Representative WAP expression in normal lactating mice and analyses of transgenic mice. (A) RT-PCR of WAP expression in various tissues on day 14 of lactation. An aliquot of 1/100 of entire RT products from RNA of the mammary glands was used for PCR. G3PDH mRNA was used as an internal control. Ma, mammary gland. WAP was expressed in several tissues as well as in the mammary gland; Mu, muscle; Li, liver; Su, submandibular gland; Lu, lung; Ut, uterus; Va, vagina; Cr, cerebrum; Cl, cerebellum; P, positive control (pCX/WAP); N, negative control. (B) Southern blot analysis of four of the resultant transgenic founders. Genomic DNA from the tail was digested with EcoRI and hybridized with a WAP cDNA probe. The 452-bp WAP transgene was excised by EcoRI; (C) Northern blot analysis of a transgenic female of #7 founder (U7). S, spleen; H, heart; Lu, lung; Li, liver; K, kidney; M, mammary gland; T, testis.

We first investigated the effects of the plasmid transfection on the proliferation of HC11 cells by transient expression of exogenous WAP. The transfection of empty plasmids (mock) or EGFP plasmids did not affect the relative numbers of live cells in either the HC11 or NIH3T3 cells (Fig. 4). The number of live cells among HC11 cells transfected with WAP plasmids was significantly (P b 0.05) lower at 2 days of culture after plasmid transfection, compared with the number of live cells in the mock or EGFP plasmid groups. This difference was not observed in the case of NIH3T3 cells. Next, we used the clonal HC11 (#24), EpH4K6 (#19), and NIH3T3 cells (#13), which showed the highest levels of stable WAP expression among the established clones. For HC11 cells, we used other WAP-clonal cells (#59) that had an expression level approximately one third of that of the clone #24 cells as an additional clone of HC11 cells. The production of WAP in WAP-clonal cells and its secretion into the cultured medium were confirmed. We detected

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Fig. 2. Whole-mount observation of mammary glands from normal (non-Tg) and transgenic mice (Tg) on day 10 of pregnancy (P10) and on day 7 of lactation (L7). Lobulo-alveolar structures were significantly impaired in the transgenic mice (A, B) compared to the normal mice (C, D). Note that the sizes of the main ducts in the transgenic mammary glands do not differ significantly from those of the normal mammary glands.

mRNA by RT-PCR in the WAP-clonal cell lines (HC11#24 and NIH3T3#13) (a in Fig. 5A). Immunoblot analysis showed that WAP-clonal cells expressed WAP and secreted it into the cultured medium: A 14-kDa WAP protein was detected in cell extracts prepared from the WAP-clonal cells (b in Fig. 5A), and it was also detected in the cultured medium (c in Fig. 5A). We investigated the effects of WAP expression on the proliferation of WAP-clonal cells and found a significant inhibition of cell proliferation in WAP-clonal HC11 cells (#24) at 48 h after of culturing, compared with that of nontransfected cells (Fig. 5B); moreover, the inhibitory effect became even more significant at 96 h. Significant inhibition of cell proliferation in the WAP-clonal HC11#59 cells was not observed until 96 h of culturing. Inhibitory effects of WAP expression on cell proliferation were also shown in WAP-clonal EpH4/K6 cells (Fig. 5B). There was no difference in the proliferation of NIH3T3 cells between the nontransfected and WAP-clonal cells throughout the culturing period, although the expression of WAP transgene was rather higher than that of WAP-clonal cells (#24) (data not shown). Paracrine action of WAP In the subsequent experiments, HC11#24 cells that showed the highest expression of WAP transgene among the clones were used as the representative mammary epithelial cells. To investigate the function of extracellular WAP secreted from WAP-clonal cells, the nontransfected cells were cultured in the cultured medium of WAP-clonal cells. The experiments involving the exchange of cultured medium showed that the cultured medium of WAP-clonal cells significantly inhibited the proliferation of control

HC11 cells at 48 h of culture after medium exchange, but not that of NIH3T3 cells (Fig. 6A). This effect increased with the passage of time during the culture period. To test for the paracrine action of WAP, we used a co-culture system with a transwell insert. WAP-clonal cells were cultured on transwell inserts and placed over control HC11 or NIH3T3 cells that had been seeded without WAP expression. As shown in Fig. 6B, the WAP secreted from the WAP-clonal HC11 cells was vectorially transported and was able to bind to control HC11 cells without WAP expression. In contrast, NIH3T3 cells were not stained with the antibody against WAP, thereby indicating that transported WAP does not bind to NIH3T3 cells. Effects of WAP on cell cycle and cyclins To determine how the proliferation of HC11 cells was inhibited by forced WAP expression, we first examined apoptosis in the WAP-clonal HC11 cells. There was no difference in the number of condensed or fragmented nuclei between WAP-clonal cells and nontransfected cells; the rate of apoptosis in nontransfected cells was 1.72% (total cell number of control HC11 cells investigated = 407 cells), and rates among the four clones ranged from 1.62% to 1.91% (total cell number of four WAP-clonal cell lines = 1894 cells). Next, we examined the BrdU incorporation and the proportion of cell cycle progression in the WAP-clonal cells. The percentage of BrdU-positive cells was significantly lower in the WAP-clonal cells than in the nontransfected HC11 cells or the WAP-clonal NIH3T3 cells (Fig. 7A). We further investigated the effects of WAP on cell cycle progression by FACScan. The proportion of WAP-clonal HC11 cells in the G0/G1 phase showed higher than the

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Fig. 3. Immunohistochemical examination of mammary tissues from normal and transgenic mice at 10 days of pregnancy (P10) and 7 days of lactation (L7). Pups born from the transgenic female were exchanged by those from a normal female at 3 days after parturition. The glands from pregnant and lactating transgenic mice (Tg) indicate very impaired development of lobulo-alveolar cells compared with those from normal mice (non Tg). WAP was not yet expressed in mammary tissue of normal mice at 10 days of pregnancy (F), whereas WAP was expressed over an extensive area of mammary tissue of transgenic mice (E, G). HE, hematoxylin and eosin staining (A, B, C, and D); WAP, immunostained with an antibody to WAP(E,F, G, and H); Arrows indicate mammary glands. *, muscle; +, fat tissue; Original magnification, 100.

Fig. 4. Effects of transfection and transient WAP expression on the proliferation of growing HC11 and NIH3T3 cells. The expression of EGFP and the transfection of mock plasmids did not affect proliferation of any of the cells used in this study. The WAP forced expression significantly (*P b 0.05) inhibited the proliferation of HC11 cells but not that of HIH3T3 cells. The transgene expression was normalized by G3PDH expression as an internal control. The results represent the mean of triplicate experiments and are expressed as number of cells. The values indicate the means F SEM of three experiments.

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Fig. 5. Effects of WAP expression on the proliferation of WAP-clonal cells. (A) Determination of WAP production in WAP-clonal cells and its secretion into medium. (a) mRNA, RT-PCR of RNAs from cells; (b) Cell lysate, Western blot of cell lysate from cells; (c) Conditioned medium, Western blot of serum-free medium in which cells were cultured for 48 h. (B) Time course of proliferation of WAP-clonal HC11, EpH4/K6, and NIH3T3 cells after the start of culturing. The proliferation of WAP-clonal mammary epithelial cells was significantly depressed at 48 h in HC11#24, at 96 h in HC11#59, or at 72 h in EpH4/K6#13 of culturing. The expression level of WAP in HC11#59 was approximately one third of that of HC11#24 cells. Proliferation was not affected in NIH3T3 cells by WAP expression throughout the culture period. The results represent the means F SEM of three experiments. WT, nontransfected; #13, #19, #24, and #59, clone number; *P b 0.05; **P b 0.01.

proportion of nontransfected HC11 cells in the G0/G1 phase (Fig. 7B). In contrast, there was no difference in cell cycle proportions between nontransfected cells and WAP-clonal cells among the NIH3T3 cells. The G0/G1 phase was longer in the WAP-clonal HC11 cells than in the nontransfected HC11 cells. The cells were cultured in conditioned medium after being synchronized at the G0 phase, and they were subsequently analyzed by FACScan; it was found that the proportion of WAP-clonal HC11 cells that remained in the G0/G1 phase was significantly higher (81.1%) than that of the nontransfected HC11 cells (61.5%) at 12 h of culturing. The FACScan analysis also demonstrated that forced WAP expression did not enhance apoptosis in HC11 cells. Finally, we investigated the effects of WAP on the expression of the G1-phase cyclins D1, D2, D3, and E using

WAP-clonal HC11and NIH3T3 cells. The summarized results of these RT-PCR analyses are shown in Fig. 8A. The expression of both cyclin D1 and D3 was significantly lower in WAP-clonal cells than in the control cells: The expression of these cyclins was low in HC11 cells in the presence of WAP expression. There was no difference in the expression of the G1 phase cyclins between WAP-clonal cells and control NIH3T3 cells. No overt differences in either cyclin D2 or cyclin E expression were observed among WAP-clonal HC11 cells or control cells. Western blot analyses confirmed the depression of cyclin D1 and D3 expression in WAP-clonal HC11 cells (Fig. 8B). To further confirm the inhibitory effects of WAP against cyclin D1 expression, we cultured WAP-clonal HC11 and control HC11 cells that were synchronized to the G0 phase in

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Fig. 6. Evidence for the paracrine activity of WAP secreted from WAP-clonal cells. (A) Inhibitory effect of medium in which WAP-clonal cells were cultured on the proliferation of nontransfected HC11 or NIH3T3 cells. WT, nontransfected HC11 cells; #13 and #24, clone number. Each data point indicates the means F SEM of three experiments. *P b 0.05; **P b 0.025. (B) Immunocytochemical observation of vectorially transported WAP. WAP-clonal HC11 cells were cocultured with nontransfected HC11 and NIH3T3 cells for 96 h in a transwell insert system. WAP bound to nontransfected HC11 cells but not to NIH3T3 cells. Binding of WAP to HC11 cells was dissolved by Triton treatment (+Triton).

conditioned medium containing WAP and then the cells were examined for the expression of cyclin D1 with a timelapse culture. The elevation of cyclin D1 expression was significantly depressed in WAP-clonal HC11 cells at 10 h of culture after the cells were synchronized in the G0 phase, compared to that in the control HC11 cells (C and D in Fig. 8). In particular, we noted that the mean amount of cyclin D1 produced in the WAP-clonal HC11 cells was about half of that in the control HC11 cells at 12 h of culturing (P b 0.05) (Fig. 8D).

Discussion In vivo experiments To date, some insights regarding the possible functions of WAP have been proposed. For example, Robinson et al. (1995) studied WAP/WAP transgenic mice in which WAP is synthesized precociously, and they proposed a model for the regulation of mammary alveolar cell differentiation whereby the precocious expression of WAP results in the terminal differentiation of alveolar cells already at midpregnancy, which in turn prevents the alveolar structures from

proliferating. However, only a few attempts at determining the function of WAP have previously been carried out (Burdon et al., 1991a,b; Robinson et al., 1995; Shamay et al., 1992); this lack of investigative reports is likely to the fact that WAP had previously been identified in the milk of only a limited number of mammalian species (Beg et al., 1984; Campbell et al., 1984; Devinoy et al., 1988; Hennighausen and Sippel, 1982a). In recent years, WAP has also been identified in the milk of pigs (Rival et al., 2001; Simpson et al., 1998), wallabies (Simpson et al., 2000), red kangaroos (Nicholas et al., 2001), and brushtail possums (Demmer et al., 2001). Our results indicate that precocious and ubiquitous expression of WAP impairs only the development of mammary glands; it does not appear to inhibit other physiological functions in transgenic females. On the other hand, it has been reported that MMTV-LTR/WAP transgenic mice show hyperplasia of the coagulation gland epithelium in addition to impaired mammary development (Hennighausen et al., 1994). The discrepancy in phenotypes between CAG/WAP and MMTV-LTR/WAP transgenic mice may be attributable to the difference in expression level of WAP transgene in the coagulation glands owing to the different transgene. The lobulo-alveolar

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Fig. 7. Effects of forced WAP expression on cell cycle progression of HC11 cells. (A) Comparison of BrdU incorporation in WAP-clonal HC11 and NIH3T3 cells. The values indicate the percentages of BrdU incorporated into cells (BrdU/cell) at 4 h after BrdU was added. The incorporation of BrdU in HC11 cells was significantly lower (*P b 0.05) in WAP-clonal cells (WAP clone) than in nontransfected cells (WT). The values indicate the means F SEM of three experiments. (B) FACScan analysis of WAP-clonal HC11 and NIH3T3 cells. Cells were cultured for 96 h, and the adherent cells were trypsinized, fixed in 70% ethanol, stained with propidium iodide, and analyzed on a FACScan cytofluorimeter (see "bMaterials and methodsQ section for details). HC11 (synchronized), HC11 cells were synchronized. Numbers indicate the percentages of cells in different phases of the cell cycle.

structures in the mammary glands of postpartum transgenic mice were found to resemble those of midpregnant, nontransgenic mice. The marked epithelial cell proliferation that usually occurs during days 15–19 of pregnancy was absent, indicating that partial alveolar cells failed to develop functionally. Poor lobulo-alveolar development caused agalactia and resulted in the death of pups born nursing with an affected mother. These findings clearly

demonstrate that WAP regulates the development of the mammary glands of females throughout late pregnancy and lactation. Because it has been postulated that the mammary glands of these transgenic mice are under the normal control of various lactogenic hormones, as are those of normal mice during pregnancy and lactation (Lyons, 1958), it is thought that precocious expression and overexpression of WAP inhibit alveolar development, working against the

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Fig. 8. Effects of forced WAP expression on synthesis of cyclin proteins. (A) RT-PCR analysis of the expression of G1-phase cyclins (cyclin D1, D2, and D3) and cyclin E in WAP-clonal HC11 and NIH3T3 cells that were cultured for 96 h. RT-PCR products were semiquantified by NIH image software and were expressed as relative amounts in relation to the PCR product of G3PDH mRNA. The values indicate the means F SEM of three experiments. WT, nontransfected cells; #13 and #24, WAP-clone number. (B) Western blot analysis of cyclin D1, D3, E, and CDK4 in cultured cells at 96 h of culturing. (C) Western blotting of cyclin D1 and CDK4 in WAP-clonal HC11 cells that were cultured in conditioned medium for 0, 10, 12, and 14 h after the cells were synchronized in the G0 phase. The elevation of cyclin D1 expression was significantly depressed in WAP-clonal HC11 cells at 10 h of culture. (D) Relative amount of cyclin D1 protein expressed in WAP-clonal and nontransfected HC11 cells. The amount of cyclin D1 protein was semiquantified by NIH image software and is indicated as the relative amounts in relation to the amount of CDK4 protein. The values indicate the means F SEM of three experiments. *P b 0.05; **P b 0.025.

influence of those hormones on mammary glands. The impaired lobulo-alveolar development of our transgenic mice very much resembled that of Id2 (the helix-loop-helix inhibitor) null mice with a lactation defect (Mori et al., 2000). Id2 null mammary glands exhibit a defect in cell proliferation, significant BrdU incorporation, and enhanced apoptosis. In our experiments, apoptosis was not enhanced in the mammary glands of transgenic females. Therefore, we believe that the mechanism underlying WAP function in the epithelial proliferation of mammary glands differs from that underlying Id2 function.

It is noteworthy that ubiquitous expression and overexpression of WAP did not appear to affect any overt physiological function other than to induce impaired development of the lobulo-alveolar cells of the mammary glands in our transgenic mice. The present results may also help to explain our previous findings of no histological or physiological abnormalities in transgenic mice overexpressing the WAP gene under the control of the metallothionein-I gene promoter (Hase et al., 1996). Impairment of mammary gland development in our transgenic females was not as severe as that in WAP/WAP transgenic animals (Burdon et al., 1991b;

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Shamay et al., 1992), possibly because the activity of the WAP promoter overcomes that of the CAG promoter in the mammary glands, resulting in tissue-specific expression. In vitro experiments The forced and high expression of exogenous WAP significantly inhibited the proliferation of HC11 and EpH4/ K6 cells established from mammary glands, whereas it did not affect that of NIH3T3 cells established from mouse fibroblasts. The results of the experiments involving medium exchange and a co-culture system with a transwell insert demonstrated that WAP acts on HC11 cells through a paracrine mechanism. The finding that vectorially transported WAP binds to HC11 cells but not to NIH3T3 cells clearly indicates that the inhibitory action of WAP on cell proliferation is specific to mammary epithelial cells. Our BrdU incorporation and FACScan analyses indicated that the cell cycle progression from the G0/G1 phase to the S phase was inhibited in WAP-clonal HC11 cells and also apoptosis was not enhanced in those cells. RT-PCR and Western blot analyses of the expression of G1-phase cyclins demonstrated that forced WAP expression in HC11 cells significantly depresses the expression of cyclins D1 and D3, but does not affect the expression of other cyclins such as cyclins D2 and E. D-cyclins are core components of the cell cycle (Sherr and Roberts, 1999), and they show significant amino acid similarity (Inaba et al., 1992; Xiong et al., 1992). Cyclin D1 is believed to play a critical role in the progression from the G1 to the S phase of the cell cycle. Overexpression of cyclin D1 is seen in a large number of human cancers, including mammary and liver cancer (Bartkova et al., 1995; Dickson et al., 1995; Weinstat-Saslow et al., 1995), and transgenic mice overexpressing cyclin D1 under the control of the mouse mammary tumor virus (MMTV) promoter develop mammary carcinomas (Wang et al., 1994). In contrast, adult female mice lacking cyclin D1 fail to undergo the massive epithelial proliferation of mammary glands during pregnancy, despite normal levels of ovarian steroid hormones (Sicinski et al., 1995). Thus, cyclin D1 has been found to regulate the epithelial proliferation of mammary glands. The results of our in vivo and in vitro experiments, when taken together with those of previous studies of WAP/WAP transgenic animals (Burdon et al., 1991b; Robinson et al., 1995; Shamay et al., 1992), strongly indicate that WAP may play a role in the cell cycle progression of mammary epithelial cells during pregnancy and lactation, in particular at the point in the cycle from G1 to S-phase entry, via the regulation of cyclin D1 expression. Cyclin-D3-deficient mice have also been found to be viable, but they display lymphoid abnormalities (Ciemerych et al., 2002). A previous study of mice expressing a single D-type cyclin showed that cyclin-D3-only mice have abnormal cerebra (Ciemerych et al., 2002). In our study, the expression of cyclin D3, as well as that of cyclin D1, significantly

decreased in WAP-clonal cells in comparison to control cells; however, the mechanism responsible for the decrease in cyclin D3 expression and WAP function in WAP-clonal HC11 cells remains obscure. The WAP proteins contain four-disulfide (4-DSC) domains, and each disulfide domain is composed of approximately 50 amino acids, including eight cysteine residues in a conserved arrangement. The WAP motif, consisting of two 4-DSC domains, is found in several other proteins with membrane-adhesion properties or with inhibition properties, suggesting that its motif is important with regard to the biological role of these proteins (Ranganathan et al., 2000; Simpson et al., 2000). A variety of mucosal proteins that are composed of one or multiple copies of a 4DSC domain have shown protease-inhibitor functions. Based on the limited sequence identity of WAP with known protease inhibitors, it has been suggested that WAP may be a protease inhibitor. If WAP does indeed posses protease inhibitor activity, it may be directed against specific protease(s). This putative WAP function is supported by evidence showing no abnormalities in the physiology of the present transgenic mice that express WAP ubiquitously. Proteases and protease inhibitors are thought to regulate tissue formation and remodeling during the development of the mammary glands (Fata et al., 1999; Kramps et al., 1988; Lee et al., 2000; Lyons et al., 1988). Therefore, it will be necessary in future studies to consider WAP function in terms of both protease–protease inhibitor interactions and cell–cell interactions in the mammary gland.

Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 14360173, 1465101 to K.N. and Nos. 10356010, 13876061, and 14360174 to H.T.) from the Ministry of Education, Science, Sports, and Culture of Japan. We thank Dr. Yamanouchi K for helpful suggestions on this work.

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