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

Regulatory function of whey acidic 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 al., 2001).WAPgeneexpressionoccursinmammary epithelial cells during late pregnancy and throughout Whey acidic protein (WAP) has been found in the milk of lactation, and is regulated by a variety of lactogenic a limited number of species of rodents, including mice hormones, including insulin, glucocorticoid, and prolactin (Hennighausen and Sippel, 1982a) and rats (Campbell et al., (Burdon et al., 1991a; Doppler et al., 1991; Vonderhaar and 1984). In addition, WAP has been identified in camels (Beg Ziska, 1989). WAP have a signal peptide (Dandekar et al., 1984) and rabbits (Devinoy et al., 1988), and more et al., 1982) and two domain structures that can be identified recently in the milk of pigs (Rival et al., 2001; Simpson et al., at the four-disulfide core (4-DSC) domain, which is 1998), wallabies (Simpson et al., 2000), red kangaroos composed of eight cysteine residues in a conserved arrange- (Nicholas et al., 2001), and brush tail possums (Demmer et ment (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 * Corresponding author. Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 identified (Dandekar et al., 1982; Garczynski and Goetz, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Fax: +81 3 5841 8191. 1997; Heinzel et al., 1986; Kirchhoff et al., 1991; Wideow et E-mail address: [email protected] (H. Tojo). al., 1990), which contain either single or multiple copies of

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.04.040 32 N. Nukumi et al. / Developmental Biology 274 (2004) 31–44 the 4-DSC domain. Generally, these proteins are secreted A 452-bp segment of the WAP cDNA was inserted into a and may function as inhibitors (Larsen et al., 1998; pGEM-T Easy vector (Promega) and amplified. We deter- Ranganathan et al., 2000). Thus, based on the limited mined the DNA sequences of the PCR products and judged sequence identity with known protease inhibitors, it has been the results to indicate a complete cDNA encoding of the full postulated that WAP may also be a secreting protease open reading frame of mouse WAP. EGFP in pCX-EGFP inhibitor (Nicholas et al., 1997; Ranganathan et al., 2000). plasmid (kindly provided by Dr. M. Okabe, Osaka University, However, previous studies have demonstrated that rat WAP Japan) was excised, and in turn replaced by the EcoRI/EcoRI does not exhibit any protease inhibitory activity in elastase I WAP cDNA fragment (452 bp). The pCX-WAP plasmid and II and chymotrypsin assays (Burdon et al., 1991b). contained an enhancer of cytomegalo virus, a chicken h-actin Transgenic mothers in which a mouse genomic WAP promoter with splicing sequences, WAP cDNA, and the transgene is expressed precociously have significantly rabbit h-globin 3V flanking sequences. For the production of impaired development of the mammary gland and have transgenic mice, the SspI/HindIII DNA fragment (2.98 kb) failed to lactate (Burdon et al., 1991b; Shamay et al., 1992). was excised from a pCXN-WAP and separated by electro- It has also been reported that high expression of WAP in phoresis using a 1% agarose gel, and then purified by CsCl MMTV-LTR/WAP transgenic mice results in hyperplasia of ultracentrifugation according to the method of Hogan et al. coagulation glands and impaired mammary development (1994). For the transfection of plasmids into cultured cells, (Hennighausen et al., 1994). On the other hand, Guntzberg et pCXN-WAP plasmid, pCXN-EGFP plasmid as a marker al. (1991) and Wen et al. (1995) have reported, based on their gene, and pCXN plasmid without the coding region as an RT-PCR analyses, that WAP is expressed in various exocrine empty plasmid (mock) were used, respectively. tissues of lactating mice. Such findings suggest that WAP is important as a nutrient in milk for growth, but that it also may CAG/WAP transgenic mice exert some other biological function not only in the mammary gland but also in other tissues. To date, few The purified DNA fragment (approximately 2000 copies) studies have investigated the true function of WAP. To was microinjected using standard methodology into the determine whether WAP functions in any tissue types other pronucleus of BDF1 (C57BL/6XDBA/2) eggs (Hogan et al., than the mammary glands, we generated transgenic mice 1994). The pups were screened for the transgene by PCR ubiquitously expressing WAP and investigated their pheno- and Southern blot analysis of genomic DNA obtained from types. WAP function was also investigated by an in vitro the tip of the tail. The primers 5V-ATG-CGT-TGC-CTC- system using mammary epithelial cells (HC11 cells). Here, ATC-AGC-C-3V (forward) and 5V-GAC-AGG-CAG-GGA- we report that the ubiquitous and forced production of WAP TGG-C-3V (reverse) were used for the detection of WAP not only inhibits lobulo-alveolar development in vivo but cDNA by PCR, and WAP cDNA labeled with 32P a-dCTP also the cell cycle progression of HC11 cells in vitro. was used as a probe for Southern blot analysis.

Whole mounts of mammary glands Materials and methods The third thoracic mammary glands were removed from Animals the transgenic and control mice. Each mammary gland was dissected from the inner surface of the skin, retaining some Mice were purchased from a laboratory animal dealer of the peripheral connective tissues, spread on a cork board, (SLC, Shizuoka, Japan) and were housed under regulated and fixed in freshly prepared Bouin’s solution. The glands temperature (22–258C), humidity (40–60%), and illumina- were then defatted overnight in acetone. The whole mounts tion cycles (14-h light, 10-h dark); food and water were were stained with Mayer’s hematoxylin, embedded in supplied ad libitum. The experiments were conducted Canadian balsam, and photographed. according to the Guidelines for the Care and Use of Laboratory Animals from the University of Tokyo College Histological examination of mammary glands of Agriculture. Tissues were embedded in optimal cutting temperature Gene construction compound (Sakura Finetechnical) and frozen in liquid nitrogen. Frozen sections, 5-Am thick, were mounted on Mouse WAP cDNA was made by reverse transcription vectabond-coated slides and fixed in 3% paraformaldehyde using total RNAs that were extracted from the mammary in PBS (pH 7.4) for 15 min at room temperature. The glands of C57BL/6J female mice in late pregnancy using sections were then blocked with 0.3% H2O2 in methanol for Trizol Reagent (Gibson BRL) according to the manufactur- 30 min at room temperature and then 8% skim milk at 48C er’s instructions. PCR was carried out using the following overnight. After washing with PBS (pH 7.4), the sections primers: 5V-ATG-CGT-TGC-CTC-ATC-AGC-C-3V (for- were incubated with primary antibodies, anti-WAP rabbit ward) and 5V-GAC-AGG-CAG-GGA-TGG-C-3V (reverse). serum in a 1/100 dilution with 1% BSA in PBS for 1 h. The N. Nukumi et al. / Developmental Biology 274 (2004) 31–44 33 sections were rinsed in PBS (pH 7.4) and then incubated for Cell culture 40 min with the biotin-labeled mouse anti-rabbit secondary antibody (B5283) (Sigma) in a 1/400 dilution. Signals were Two independent mammary epithelial cell lines derived detected by the horseradish-peroxidase-labeled avidin–bio- from mouse pregnant mammary glands were used: HC11 tin complex (ABC)-mediated method (Vector Laboratories) cells (kindly provided by Dr. R.K. Ball, Fredrich Miescher using diaminobenzidine (DAB) as a substrate. To avoid Institute, Switzerland) (Ball et al., 1998) and EpH4K6 cells background staining, slides were preincubated with 10% (kindly provided by Dr. W. Birchmeier, Max-Delbritck- rabbit serum for 30 min before incubation for 18 h at 48C Center, Berlin, Germany) (Niemann et al., 1998). NIH3T3 with the specific antibody directed against mouse WAP. cells (American Type Culture Collection) from mouse fetal cells were used as a control for comparison with the Northern blot analysis mammary epithelial cells. The HC11 cells were grown in a complete medium, RPMI1640 (Gibco BRL), containing RNA was extracted from the tissues using Trizol Reagent 10% (v/v) heat-inactivated FBS, 5 Ag/ml of insulin, 10 ng/ (Gibco BRL) according to the manufacturer’s instructions. ml of epidermal growth factor, and 50 Ag/ml of gentamicin. Total RNA (10 Ag) was fractionated through a 2% agarose The EpH4/K6 cells were grown in a complete medium, formaldehyde gel, transferred to a nylon membrane (Pall Dulbecco’s Modified Eagle Medium (DMEM) (Gibco Biosupport), and then cross-linked by exposure to ultra- BRL), containing 10% (v/v) heat inactivated FBS, 3.7 g/l violet irradiation. The membrane was prehybridized, hybri- of NaHCO3, and 105 U/l of penicillin and gentamicin. The dized with the mouse WAP cDNA labeled with 32P a-dCTP, NIH3T3 cells were grown in a complete medium, i.e., and then washed twice in 2Â SSC/0.1% SDS at room Dulbecco’s Modified Eagle Medium containing 10% (v/v) temperature, followed by a wash in 0.2Â SSC/0.1% SDS at heat-inactivated FBS, 100,000 U/l of penicillin, and 10 mg/l 658C. The signals were visualized by exposure of the of streptomycin. All cells were cultured in a humidified membrane to X-ray film (Fuji Film). atmosphere of 5% CO2 in air at 378C.

Antibodies Transfection of plasmids

For the immunoblotting experiments, goat antibody The plasmids were transfected by the use of the against mouse WAP (Jackson Immune Research Lab), Profection Mammalian Transfection System Calcium Phos- mouse antibodies against human cyclin D1 and Cdk4 phate (Promega) according to the manufacturer’s instruc- (Santa Cruz Biotechnology), and rabbit antibody against tions. For the establishment of stable plasmid expression, human cyclin D3 and cyclin E (Santa Cruz Biotechnology) linearized plasmids were transfected into the cells and, after were used as the primary antibodies. Anti-mouse, anti- culturing the cells in complete medium for 2 days, cells rabbit, and anti-goat IgG conjugated with horseradish demonstrating stable expression of plasmids were selected peroxidase were used as the secondary antibodies. by passing of the culture for 10 days in a complete medium containing G418 (700 Ag). The efficiency of plasmid Western blot analysis transfection was confirmed by observing GFP expression levels in the EGFP-transfected cells. Total protein was extracted from the cells by homoge- nizing them in CytoBustor Protein Extraction Reagent RT-PCR (Novagen) including 1 mM DTT, 0.5 mM PMSF, 2 Ag/ml of aprotinin, 2 Ag/ml pepstatin, and 2 Ag/ml leupeptin; the One microgram of total RNA was denatured and proteins were then collected by centrifugation. The protein reverse-transcribed into cDNA using SuperScript II (Prom- concentration of the extracts was determined using the ega) in a reaction volume of 10 IU, according to the Bradford method (Bradford, 1976) and the samples were manufacturer’s protocol. PCR was carried out, and the used for Western blot analysis. The proteins were separated products were then sequenced and confirmed to be the on SDS-PAGE (10% or 15%) gel and transferred to a appropriate transcript. We used the following primer membrane by semidry blotting. The membrane was blocked sequences for the amplification of target genes: 5V-ATG- for 1 h in phosphate-buffered saline (PBS) containing 3% CGT-TGC-CTC-ATC-AGC-C-3V (forward) and 5V-GAC- skim milk. After washing the membrane with PBS contain- AGG-CAG-GGA-TGG-C-3V(reverse) for WAP cDNA; 5V- ing 0.1% Tween 20 (PBST), it was incubated with primary CCT-GCA-TGT-TCG-TGG-CCT-CT-3Vand 5V-GAA-AGT- antibody overnight at 48C. After washing with PBST, the GCG-TTG-TGC-GGT-AGC-3Vfor cyclin D1;5V-AGA- membrane was incubated with peroxidase-conjugated sec- CCT-TCA-TCG-CTC-TGT-GC-3Vand 5V-TAG-CAG-ATG- ondary antibody for 1.5 h at room temperature. After the ACG-AAC-ACG-CC-3V for cyclin D2; 5V-CCG-TGA- membrane had been washed with PBST, the specific TTG-CGC-ACG-ACT-TC-3V and 5V-TCT-GTG-GGA- antibody signals were detected with ECL (Amersham GTG-CTG-GTC-TG-3V for cyclin D3;5V-AGC-GAG-GAT- Pharmacia Biotech), followed by exposure to X-ray film. AGC-AGT-CAG-CC-3V and 5V-TTT-CAT-CCC-CGG- 34 N. Nukumi et al. / Developmental Biology 274 (2004) 31–44

AGC-AAG-CG-3V for cyclin E; 5V-TGA-AGG-TCG-GTG- Examination of the paracrine action of WAP CAA-CGG-ATT-TGG-C-3V and 5V-CAT-GTA-GGC-CAT- GAG-GTC-CAC-CAC-3V for G3PDH (an endogenous The paracrine action of WAP produced in WAP-clonal control). The quantitative conditions of the PCR reaction cells upon cell proliferation was investigated by the were 28 or 31 cycles of 1 min at 948C, 1 min at 588C, and following two methods. First, WAP-clonal HC11 cells 1 min at 728C. The size of each PCR product was (#24) were cultured for 48 h after confluence was reached. confirmed by electrophoresis using 1% agarose gels in The culture medium was recovered and then exchanged for TAE buffer. The RT-PCR products were semiquantified by medium in which control HC11 or NIH3T3 cells had been NIH image software (NIH, Bethesda, MD, USA, Version cultured. After the medium exchange, cells were cultured 1.60) and were expressed as relative amounts in relation to for 24, 48, 72, and 96 h, respectively, and assayed for the PCR product of G3PDH mRNA. potential for cell proliferation according to the method described in the bCell proliferation assayQ section. Secondly, Cell proliferation assay WAP activity was also investigated using transwell cell culture chamber inserts (Corning Costar). WAP-clonal cells Cell-Counting Kit-8 solution (Wako Pure Chemical) (104/chamber) were seeded on an upper microporous (Ishiyama et al., 1996) was added to a medium containing membrane (pore size, 0.4 Am) in a transwell filled with an the cells in a 96-well dish reacted at 378C for 1 h, according appropriate medium for HC11 or NIH3T3 cells; the control to the manufacturer’s instructions. The number of viable HC11 or NIH3T3 cells (3 Â 104/chamber) were seeded on cells was measured at OD 450 nm using a Microplate the glass cover of a lower plate. After 96 h of co-culture, the Reader (Bio-Rad). cells on the glass cover were recovered and immunochemi- cally stained with WAP antibody. Evidence for the absence Assay of apoptosis in vivo and in vitro of endogenous WAP expression in the cells on the lower plate was confirmed by RT-PCR using the total RNA Apoptotic cells in the mammary glands of transgenic mice extracted from those cells after co-culture. were examined by the In Situ Cell Death Detection Kit (Boehringer Mannheim) together with the TUNEL method. Immunocytochemistry The extent of apoptosis observed is indicated here as the number of apoptotic nuclei counted as a percentage of the The cultured cells were fixed in 3% paraformaldehyde total cells visualized. For the in vitro assay of apoptosis, in PBS for 20 min, then in PBS or PBS containing 0.5% WAP-clonal cells were analyzed according to a previously Triton X-100 for 15 min at 48C. After being washed in reported method (Li et al., 1999). Equal numbers of clones of PBS, the cells were blocked for 30 min in 8% skim milk in WAP-clonal HC11 cells and nontransfected (control) cells PBS, and then were incubated with the rabbit anti-mouse were cultured for 2 days in a 12-well dish. The cells were WAP polyclonal antibody in a 1/100 dilution overnight at rinsed once with PBS and fixed with 3% paraformaldehyde at 48C, followed by washing three times in PBS. The porcine 48C overnight, washed three times with PBS, and then treated anti-rabbit rhodamine-conjugated secondary antibody with 0.1% Triton X-100 at room temperature for 15 min. (DAKO Immunoglobulins) were applied at a 1/50 dilution After being rinsed with PBS three times, the cells were with 8% skim milk in PBS, and the cells were incubated stained with 1 Ag/ml of Hoechst 33342 (Sigma) solution on for 1 h. Images were observed by confocal microscopy PBS–glycerol (vol/vol 1:1) at room temperature for 30 min. (Carl Zeiss). The number of condensed or fragmented nuclei among the cells in a dish was calculated under a fluorescence micro- Detection of WAP in the cultured medium scope at an excitation wavelength of 365 nm. The WAP-clonal cells were cultured in complete medium Cell cycle analysis without FBS for 2 days after 80% confluence was reached in medium containing 10% fetal bovine serum. The medium in Incorporation of bromodeoxyuridine (BrdU) into the the dish was collected by centrifugation (10 min, 3000 rpm), cultured cells was determined by the recommended condensed in a Centricon YH-10 (Milipore), and stored at protocol of the Cell Proliferation ELISA, BrdU (Roche). À808C until use. WAP protein was detected by Western For the FACScan analysis, adherent cells were trypsinized blotting, as described in the bMaterials and methodsQ and fixed in cold 70% ethanol for 4 h. Fixed cells were section. washed with PBS and incubated with 5 Ag/ml of RNase A (Sigma), and then the cells were stained with 25 Ag/ml of Statistical analysis propidium iodine (Aldrich Chemicals) for 15 min at 48C. The stained cells were analyzed on a FACScan cyto- Data were analyzed by Student’s t test. Differences fluorimeter equipped with the ModeFit LT Software between the mean values were considered statistically program (Verity). significant at P b 0.05. N. Nukumi et al. / Developmental Biology 274 (2004) 31–44 35

Results including the mammary gland (Fig. 1C). Whole-mount species of the mammary glands of this transgenic female Phenotype of transgenic mice showed unique development (Fig. 2), including poor development of lobulo-alveolar structures and acinous First, to confirm the expression of WAP in various tissues formation considering the dam’s state of midpregnancy other than in the mammary glands of normal lactating mice, (Fig. 2A) and lactation (Fig. 2B), compared with normal we analyzed RNAs from various tissues by RT-PCR. WAP mice (C and D in Fig. 2). The results from the examination mRNA was detected in muscle, liver, lung, and vagina of mammary glands of transgenic females suggested that the although the expression levels were very low compared with synthesis of milk proteins was inadequate, causing retarded those in the mammary glands (Fig. 1A). growth or death of the pups. Despite the high expression of To determine whether WAP plays a role in various the WAP transgene in various tissues, the #7 transgenic tissues other than the mammary gland, CAG/WAP trans- mothers grew normally and showed normal parturition and genic mice were produced that ubiquitously expressed WAP. nursing behavior. One other transgenic founder showed Ten pups (six females and four males) were found to be slight impairment of the development of the mammary transgenic by Southern blot analysis (Fig. 1B) and by PCR gland, but her pups nonetheless grew normally. analyses of tail DNAs. The pups that were born from two The transgenic female offspring of eight other transgenic transgenic lines showed poor growth or death during founders showed no abnormalities of the mammary glands. nursing. For example, all of the pups from the first The hematoxylin and eosin stained transverse sections parturition of the #7 founder mother died while nursing showed that the development of the lobulo-alveolar struc- within 3 days after birth. In the third parturition of the #7 tures was remarkably impaired in the transgenic tissues (A founder, we exchanged her pups for those born from a and C in Fig. 3) compared with that of normal mammary normal female at 3 days after birth. All of the seven pups tissue (B and D in Fig. 3). Exogenous WAP was expressed born from the normal mother showed significantly retarded extensively in the mammary glands of pregnant and growth under the nursing of the transgenic mother and died lactating transgenic females. This expression was observed 3 days after the exchange, whereas the pups from the not only in the alveolar gland, but also in other connective transgenic mother grew normally under the nursing of a tissues (E and G in Fig. 3). WAP was not yet expressed in normal mother. the mammary glands of normal 10-day-pregnant females (F We sacrificed pregnant and lactating transgenic females in Fig. 3). To determine the cause of impaired lobulo- and collected their tissues. Northern blot analyses showed alveolar development in transgenic mice, we used the that the female #7 transgenic founder expressed the TUNEL method to investigate the rate of extent of apoptosis exogenous WAP gene at a high level in various tissues, 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

We first investigated the effects of the plasmid trans- fection 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, Fig. 1. Representative WAP expression in normal lactating mice and compared with the number of live cells in the mock or analyses of transgenic mice. (A) RT-PCR of WAP expression in various EGFP plasmid groups. This difference was not observed in tissues on day 14 of lactation. An aliquot of 1/100 of entire RT products the case of NIH3T3 cells. 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 Next, we used the clonal HC11 (#24), EpH4K6 (#19), several tissues as well as in the mammary gland; Mu, muscle; Li, liver; Su, and NIH3T3 cells (#13), which showed the highest levels of submandibular gland; Lu, lung; Ut, uterus; Va, vagina; Cr, cerebrum; Cl, stable WAP expression among the established clones. For cerebellum; P, positive control (pCX/WAP); N, negative control. (B) HC11 cells, we used other WAP-clonal cells (#59) that had Southern blot analysis of four of the resultant transgenic founders. Genomic an expression level approximately one third of that of the 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) clone #24 cells as an additional clone of HC11 cells. The Northern blot analysis of a transgenic female of #7 founder (U7). S, spleen; production of WAP in WAP-clonal cells and its secretion H, heart; Lu, lung; Li, liver; K, kidney; M, mammary gland; T, testis. into the cultured medium were confirmed. We detected 36 N. Nukumi et al. / Developmental Biology 274 (2004) 31–44

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 HC11 cells at 48 h of culture after medium exchange, but and NIH3T3#13) (a in Fig. 5A). Immunoblot analysis not that of NIH3T3 cells (Fig. 6A). This effect increased showed that WAP-clonal cells expressed WAP and secreted with the passage of time during the culture period. To test it into the cultured medium: A 14-kDa WAP protein was for the paracrine action of WAP, we used a co-culture detected in cell extracts prepared from the WAP-clonal cells system with a transwell insert. WAP-clonal cells were (b in Fig. 5A), and it was also detected in the cultured cultured on transwell inserts and placed over control HC11 medium (c in Fig. 5A). or NIH3T3 cells that had been seeded without WAP We investigated the effects of WAP expression on the expression. As shown in Fig. 6B, the WAP secreted from proliferation of WAP-clonal cells and found a significant the WAP-clonal HC11 cells was vectorially transported and inhibition of cell proliferation in WAP-clonal HC11 cells was able to bind to control HC11 cells without WAP (#24) at 48 h after of culturing, compared with that of expression. In contrast, NIH3T3 cells were not stained with nontransfected cells (Fig. 5B); moreover, the inhibitory the antibody against WAP, thereby indicating that trans- effect became even more significant at 96 h. Significant ported WAP does not bind to NIH3T3 cells. inhibition of cell proliferation in the WAP-clonal HC11#59 cells was not observed until 96 h of culturing. Inhibitory Effects of WAP on cell cycle and cyclins effects of WAP expression on cell proliferation were also shown in WAP-clonal EpH4/K6 cells (Fig. 5B). There was To determine how the proliferation of HC11 cells was no difference in the proliferation of NIH3T3 cells between inhibited by forced WAP expression, we first examined the nontransfected and WAP-clonal cells throughout the apoptosis in the WAP-clonal HC11 cells. There was no culturing period, although the expression of WAP transgene difference in the number of condensed or fragmented nuclei was rather higher than that of WAP-clonal cells (#24) (data between WAP-clonal cells and nontransfected cells; the rate not shown). of apoptosis in nontransfected cells was 1.72% (total cell number of control HC11 cells investigated = 407 cells), and Paracrine action of WAP rates among the four clones ranged from 1.62% to 1.91% (total cell number of four WAP-clonal cell lines = 1894 In the subsequent experiments, HC11#24 cells that cells). showed the highest expression of WAP transgene among Next, we examined the BrdU incorporation and the the clones were used as the representative mammary proportion of cell cycle progression in the WAP-clonal cells. epithelial cells. To investigate the function of extracellular The percentage of BrdU-positive cells was significantly WAP secreted from WAP-clonal cells, the nontransfected lower in the WAP-clonal cells than in the nontransfected cells were cultured in the cultured medium of WAP-clonal HC11 cells or the WAP-clonal NIH3T3 cells (Fig. 7A). We cells. The experiments involving the exchange of cultured further investigated the effects of WAP on cell cycle medium showed that the cultured medium of WAP-clonal progression by FACScan. The proportion of WAP-clonal cells significantly inhibited the proliferation of control HC11 cells in the G0/G1 phase showed higher than the N. Nukumi et al. / Developmental Biology 274 (2004) 31–44 37

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, 100Â.

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. 38 N. Nukumi et al. / Developmental Biology 274 (2004) 31–44

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 WAP-clonal HC11and NIH3T3 cells. The summarized (Fig. 7B). In contrast, there was no difference in cell cycle results of these RT-PCR analyses are shown in Fig. 8A. proportions between nontransfected cells and WAP-clonal The expression of both cyclin D1 and D3 was significantly cells among the NIH3T3 cells. The G0/G1 phase was longer lower in WAP-clonal cells than in the control cells: The in the WAP-clonal HC11 cells than in the nontransfected expression of these cyclins was low in HC11 cells in the HC11 cells. The cells were cultured in conditioned medium presence of WAP expression. There was no difference in the after being synchronized at the G0 phase, and they were expression of the G1 phase cyclins between WAP-clonal subsequently analyzed by FACScan; it was found that the cells and control NIH3T3 cells. No overt differences in proportion of WAP-clonal HC11 cells that remained in the either cyclin D2 or cyclin E expression were observed G0/G1 phase was significantly higher (81.1%) than that of among WAP-clonal HC11 cells or control cells. Western the nontransfected HC11 cells (61.5%) at 12 h of culturing. blot analyses confirmed the depression of cyclin D1 and D3 The FACScan analysis also demonstrated that forced WAP expression in WAP-clonal HC11 cells (Fig. 8B). To further expression did not enhance apoptosis in HC11 cells. confirm the inhibitory effects of WAP against cyclin D1 Finally, we investigated the effects of WAP on the expression, we cultured WAP-clonal HC11 and control expression of the G1-phase cyclins D1, D2, D3, and E using HC11 cells that were synchronized to the G0 phase in N. Nukumi et al. / Developmental Biology 274 (2004) 31–44 39

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 co- cultured 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 proliferating. However, only a few attempts at determining were examined for the expression of cyclin D1 with a time- the function of WAP have previously been carried out lapse culture. The elevation of cyclin D1 expression was (Burdon et al., 1991a,b; Robinson et al., 1995; Shamay et significantly depressed in WAP-clonal HC11 cells at 10 h of al., 1992); this lack of investigative reports is likely to the culture after the cells were synchronized in the G0 phase, fact that WAP had previously been identified in the milk of compared to that in the control HC11 cells (C and D in Fig. only a limited number of mammalian species (Beg et al., 8). In particular, we noted that the mean amount of cyclin 1984; Campbell et al., 1984; Devinoy et al., 1988; D1 produced in the WAP-clonal HC11 cells was about half Hennighausen and Sippel, 1982a). In recent years, WAP of that in the control HC11 cells at 12 h of culturing (P b has also been identified in the milk of pigs (Rival et al., 0.05) (Fig. 8D). 2001; Simpson et al., 1998), wallabies (Simpson et al., 2000), red kangaroos (Nicholas et al., 2001), and brushtail possums (Demmer et al., 2001). Discussion Our results indicate that precocious and ubiquitous expression of WAP impairs only the development of In vivo experiments mammary glands; it does not appear to inhibit other physiological functions in transgenic females. On the other To date, some insights regarding the possible functions of hand, it has been reported that MMTV-LTR/WAP trans- WAP have been proposed. For example, Robinson et al. genic mice show hyperplasia of the coagulation gland (1995) studied WAP/WAP transgenic mice in which WAP is epithelium in addition to impaired mammary development synthesized precociously, and they proposed a model for the (Hennighausen et al., 1994). The discrepancy in pheno- regulation of mammary alveolar cell differentiation whereby types between CAG/WAP and MMTV-LTR/WAP trans- the precocious expression of WAP results in the terminal genic mice may be attributable to the difference in differentiation of alveolar cells already at midpregnancy, expression level of WAP transgene in the coagulation which in turn prevents the alveolar structures from glands owing to the different transgene. The lobulo-alveolar 40 N. Nukumi et al. / Developmental Biology 274 (2004) 31–44

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 demonstrate that WAP regulates the development of the mice were found to resemble those of midpregnant, mammary glands of females throughout late pregnancy and nontransgenic mice. The marked epithelial cell proliferation lactation. Because it has been postulated that the mammary that usually occurs during days 15–19 of pregnancy was glands of these transgenic mice are under the normal absent, indicating that partial alveolar cells failed to control of various lactogenic hormones, as are those of develop functionally. Poor lobulo-alveolar development normal mice during pregnancy and lactation (Lyons, 1958), caused agalactia and resulted in the death of pups born it is thought that precocious expression and overexpression nursing with an affected mother. These findings clearly of WAP inhibit alveolar development, working against the N. Nukumi et al. / Developmental Biology 274 (2004) 31–44 41

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 It is noteworthy that ubiquitous expression and over- impaired lobulo-alveolar development of our transgenic expression of WAP did not appear to affect any overt mice very much resembled that of Id2 (the helix-loop-helix physiological function other than to induce impaired devel- inhibitor) null mice with a lactation defect (Mori et al., opment of the lobulo-alveolar cells of the mammary glands 2000). Id2 null mammary glands exhibit a defect in cell in our transgenic mice. The present results may also help to proliferation, significant BrdU incorporation, and enhanced explain our previous findings of no histological or physio- apoptosis. In our experiments, apoptosis was not enhanced logical abnormalities in transgenic mice overexpressing the in the mammary glands of transgenic females. Therefore, WAP gene under the control of the metallothionein-I gene we believe that the mechanism underlying WAP function in promoter (Hase et al., 1996). Impairment of mammary gland the epithelial proliferation of mammary glands differs from development in our transgenic females was not as severe as that underlying Id2 function. that in WAP/WAP transgenic animals (Burdon et al., 1991b; 42 N. Nukumi et al. / Developmental Biology 274 (2004) 31–44

Shamay et al., 1992), possibly because the activity of the decreased in WAP-clonal cells in comparison to control WAP promoter overcomes that of the CAG promoter in the cells; however, the mechanism responsible for the decrease mammary glands, resulting in tissue-specific expression. in cyclin D3 expression and WAP function in WAP-clonal HC11 cells remains obscure. In vitro experiments The WAP proteins contain four-disulfide (4-DSC) domains, and each disulfide domain is composed of The forced and high expression of exogenous WAP approximately 50 amino acids, including eight cysteine significantly inhibited the proliferation of HC11 and EpH4/ residues in a conserved arrangement. The WAP motif, K6 cells established from mammary glands, whereas it did consisting of two 4-DSC domains, is found in several other not affect that of NIH3T3 cells established from mouse proteins with membrane-adhesion properties or with inhib- fibroblasts. The results of the experiments involving ition properties, suggesting that its motif is important with medium exchange and a co-culture system with a transwell regard to the biological role of these proteins (Ranganathan insert demonstrated that WAP acts on HC11 cells through a et al., 2000; Simpson et al., 2000). A variety of mucosal paracrine mechanism. The finding that vectorially trans- proteins that are composed of one or multiple copies of a 4- ported WAP binds to HC11 cells but not to NIH3T3 cells DSC domain have shown protease-inhibitor functions. clearly indicates that the inhibitory action of WAP on cell Based on the limited sequence identity of WAP with known proliferation is specific to mammary epithelial cells. Our protease inhibitors, it has been suggested that WAP may be BrdU incorporation and FACScan analyses indicated that a protease inhibitor. If WAP does indeed posses protease the cell cycle progression from the G0/G1 phase to the S inhibitor activity, it may be directed against specific phase was inhibited in WAP-clonal HC11 cells and also protease(s). This putative WAP function is supported by apoptosis was not enhanced in those cells. RT-PCR and evidence showing no abnormalities in the physiology of the Western blot analyses of the expression of G1-phase cyclins present transgenic mice that express WAP ubiquitously. demonstrated that forced WAP expression in HC11 cells and protease inhibitors are thought to regulate significantly depresses the expression of cyclins D1 and D3, tissue formation and remodeling during the development of but does not affect the expression of other cyclins such as the mammary glands (Fata et al., 1999; Kramps et al., 1988; cyclins D2 and E. Lee et al., 2000; Lyons et al., 1988). Therefore, it will be D-cyclins are core components of the cell cycle (Sherr necessary in future studies to consider WAP function in and Roberts, 1999), and they show significant amino acid terms of both protease–protease inhibitor interactions and similarity (Inaba et al., 1992; Xiong et al., 1992). Cyclin D1 cell–cell interactions in the mammary gland. 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 Acknowledgments mammary and liver cancer (Bartkova et al., 1995; Dickson et al., 1995; Weinstat-Saslow et al., 1995), and transgenic This work was supported in part by Grants-in-Aid for mice overexpressing cyclin D1 under the control of the Scientific Research (Nos. 14360173, 1465101 to K.N. and mouse mammary tumor virus (MMTV) promoter develop Nos. 10356010, 13876061, and 14360174 to H.T.) from the mammary carcinomas (Wang et al., 1994). In contrast, adult Ministry of Education, Science, Sports, and Culture of female mice lacking cyclin D1 fail to undergo the massive Japan. We thank Dr. Yamanouchi K for helpful suggestions epithelial proliferation of mammary glands during preg- on this work. nancy, 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. References The results of our in vivo and in vitro experiments, when taken together with those of previous studies of WAP/WAP Ball, R.K., Friis, R.R., Schoenenberger, C.A., Doppler, W., Groner, B., transgenic animals (Burdon et al., 1991b; Robinson et al., 1998. Prolactin regulation of beta-casein gene expression and of a 1995; Shamay et al., 1992), strongly indicate that WAP may cytosolic 120-kD protein in a cloned mouse mammary epithelial cell play a role in the cell cycle progression of mammary line. 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