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PIP gene during pre- and early post-natal development
Molecular and Cellular Endocrinology 205 (2003) 33 /41 www.elsevier.com/locate/mce
Expression of the mouse homologue for the human GCDFP-15/PIP gene during pre- and early post-natal development Beverley Lee a, Geetanjalee Modha a, Peter H. Watson a, Janice Dodd b, Sandy Troup a, Anne Blanchard a, Yvonne Myal a,* a
Department of Pathology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Man., Canada R3E 0W3 b Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Man., Canada R3E 0W3 Received 21 December 2002; accepted 15 May 2003
Abstract The function of the mouse submaxillary gland/prolactin inducible protein (mSMGP/mPIP), the homologue of the human gross cystic disease fluid protein 15 (GCDFP-15)/prolactin inducible protein (hPIP) remains unknown. The human gene, normally expressed in apocrine glands of healthy individuals, is aberrantly expressed in human breast cancers where it is regulated by hormones including androgens, and in prostate cancers. We have previously reported that in the adult mouse and rat, gene expression is tissue-specific for the salivary and lacrimal glands, and is hormonally regulated. In this study, we examine the endogenous pattern of mouse SMGP/PIP (mSMGP/mPIP) gene expression in mid- and late-embryonic, and in early postnatal development. Gene expression was analyzed by RT-PCR followed by Southern blot analysis, and by in situ hybridization. Gene expression was detected in the submandibular gland as early as embryonic day 14 (E14), a period that coincides with the initiation of submandibular gland development in the embryo, suggesting that mSMGP/mPIP may have a functional role in the developing gland. Nearing the end of gestation, E18, mSMGP/mPIP transcripts were localized in the proacinar cells of the gland, and gene expression continued to be maintained following birth. In addition, during early postnatal development, mSMGP/mPIP gene expression was detected in the other two major salivary glands, the sublingual and parotid, as well as in the lacrimal gland and in reproductive tissues. In the prostate, gene expression was turned off by 10 weeks of age. The spatial and temporal pattern of the mSMGP/mPIP gene expression, in addition to our recent demonstration that mSMGP/mPIP is found in mouse saliva and can bind bacteria, suggest that this protein may have a protective role in the mouse. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Submandibular gland; Prostate; Gene expression; Development; mSMGP/mPIP; GCDFP-15/hPIP
1. Introduction The mouse submaxillary gland protein (mSMGP) was initially identified as a highly expressed gene in the submandibular (submaxillary) gland of the adult mouse (Windass et al., 1984; Myal et al., 1994). The 576 bp mSMGP cDNA (GenBank Accession # NM_008843) encodes a secreted polypeptide sequence of 146 amino acids, containing a 25 amino acid signal peptide (Myal et al., 1994) and has a calculated Mr of 14 kDa. In the adult rodent, mSMGP transcripts were localized to the
* Corresponding author. Tel.: /1-204-789-3874; fax: /1-204-7893931. E-mail address: [email protected] (Y. Myal).
acinar cells (Myal et al., 1994; Mirels et al., 1998), the major secretory cells of the gland. The levels of mSMGP gene expression in the submandibular gland of the male or female mouse were found to be similar, suggesting that in this tissue, gene expression is not differentially regulated by androgen or estrogen. mSMGP was also found to be expressed in the parotid and sublingual salivary glands of the rat, as well as in the lacrimal gland. In the lacrimal gland, androgen was found to be a potent inhibitor of mSMGP gene expression (Myal et al., 1994). At the amino acid level, the mSMGP exhibits a 51% identity with the human gross cystic disease fluid protein-15 (GCDFP-15; Haagensen and Mazoujian, 1986), which is also known as the prolactin inducible
0303-7207/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0303-7207(03)00210-7
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protein (hPIP; Shiu and Iwasiow, 1985; Murphy et al., 1987a), extraparotid glycoprotein (EP-GP; Schenkels et al., 1993), secretory actin binding protein (SABP; Akiyama and Kimura, 1990) and glycoprotein 17 (gp17; Autiero et al., 1991). In healthy humans, the secreted protein has been identified in saliva, tears, sweat, seminal plasma, submucosal glands of the lung and in amniotic fluid (Murphy et al., 1987b) and is thought to play a role in host immunity. The GCDFP-15/hPIP has been shown to, (a) bind CD4 and inhibit CD4-mediated T cell programmed cell death (Autiero et al., 1995) and, (b) interfere with the HIV envelope protein gp-120 interaction with CD4 (Zhang et al., 1997). The GCDFP-15/hPIP gene appears to be aberrantly expressed in breast tumor biopsies and human breast cancer cell lines, where, in the latter, it is regulated by several hormones, most potently by the interleukins, gluccocorticoids and androgen (Murphy et al., 1987a; Blais et al., 1994, 1995, 1996; Clark et al., 1999; Carsol et al., 2002). Interestingly, a recent report suggests that GCDFP-15/hPIP, isolated from apocrine epithelia and breast tumors, is a novel aspartyl proteinase (Caputo et al., 2000). However, the GCDFP-15/hPIP gene is not expressed in healthy mammary glands (Haagensen and Mazoujian, 1986). Neither is the mSMGP (also referred to as mouse PIP) gene expressed in normal rodent mammary glands, nor in rodent mammary tumors (Myal et al., 1994). Whether the mSMGP/mPIP binds to CD4 and is an aspartyl proteinase is not known. The rat homologue (rPIP) was recently isolated and found to share a high degree of similarity (80%) with the mSMGP/mPIP at the amino acid level (Mirels et al., 1998). With regards to tissue specificity, the expression of this gene in both the salivary and lacrimal glands appears to be conserved across species (Myal et al., 1994; Mirels et al., 1998; Myal and Shiu, 2000). Studies by us and by others have pointed to an association between this gene and reproductive organs, and/or steroid hormone-dependent tissues in humans and rodents. For example, Clark et al. (1999) observed a low level of expression of GCDFP-15/hPIP gene expression in human ovaries, whereas, Akiyama and Kimura (1990) and Autiero et al. (1991) have isolated GCDFP15/hPIP from human seminal plasma. Also, Autiero et al. (1999) and Myal et al. (1989) have identified restriction fragment length polymorphisms in the GCDFP-15/hPIP gene in human prostate and breast cancers. Furthermore, we have previously demonstrated that androgen was a potent inhibitor of gene expression in the rat lacrimal gland (Myal et al., 1994). Analysis of the mSMGP/mPIP pattern of gene expression at different developmental stages will provide insight into the many possible roles for mSMGP/mPIP in vivo. In the present study we examined the expression of the mSMGP/mPIP gene during embryonic and early postnatal development in the mouse.
2. Material and methods 2.1. Screening of poly A RNA blot for the developmental and tissue specific pattern of mSMGP/ mPIP gene expression A commercially available RNA Master Blot (Clontech, Palo Alto, CA, USA) containing poly A RNAs extracted from several adult mouse tissues and whole mouse embryos, was hybridized with the mSMGP/mPIP cDNA probe (570 bp; Myal et al., 1994) according to the manufacturer’s instructions. Briefly, the RNA Master Blot was pre-hybridized at 65 8C for 1 h followed by overnight hybridization at 65 8C with a random primed 32 P-labelled full-length mSMGP/mPIP cDNA (Myal et al., 1994). The membrane was washed twice with 200 ml wash solution 1 (2 / SSC, 1% SDS) at 65 8C for 20 min followed by a final wash with prewarmed wash solution 2 (0.1 / SSC, 0.5% SDS) at 65 8C for 20 min. The blot was then exposed to Kodak X-OMAT AR film (InterScience, Markham, ON, Canada) at /70 8C with an intensifying screen for 3 h. 2.2. Animals Four to 6 week old CD1 mice were obtained from and housed at the facilities of the Central Animal Care Services, Faculty of Medicine, University of Manitoba (Winnipeg, MB Canada). Mice were mated overnight and the females were checked for vaginal plugs the next morning. The first appearance of the vaginal plug was counted as day 1 of gestation. 2.3. Tissue collection Pregnant females were sacrificed at days 14, 15, 16, 17, 18, 19, 20 and in some cases day 21 of gestation and whole fetuses were removed from the dams and immediately euthanized. Depending on the ages, fetuses were divided either into two (upper [U] and lower [L]) or three parts (head, neck and forelimbs [U], abdominal or mid region [M] and the pelvic region including the tail and hind limbs [L]), prior to freezing or fixing. E14 to E17 embryos were divided into two parts whereas the larger embryos, E18 to E21, were sectioned into three parts. Various other tissues were further dissected from an E18 embryo for analysis of gene expression. Young pups, 1, 3 or 5 days old were also collected, tissues immediately dissected and either frozen at /70 8C or fixed in 4% paraformaldehyde overnight. The paraformaldehyde was poured off the following day and replaced by 70% ethanol for short-term storage. As well, tissues were also collected from 4, 6, 8 and 10 week old males and from lactating females. In specific cases, (embryonic or early postnatal prostates and ovaries; days 1, 3 and 5), samples were pooled in order to collect
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sufficient amount of tissue for total RNA extraction. Following the castration of 6-week old male mice, prostates were collected at 2 and 4 week post surgery and at 2 week post-castration followed by 3 or 6 days hormone replacement with dihydrotestosterone (DHT; 3 mg/kg per day) subcutaneously. 2.4. Isolation of total RNA Total RNA was extracted from frozen tissues using TRIzol Reagent (Invitrogen Corporation, Burlington, ON, Canada) according to the protocol outlined by the manufacturer. The pellet was washed with 75% cold ethanol, air dried, dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at /70 8C. The purity and the yield of the RNA samples were determined spectrophotometrically. Total RNA (1 mg) was subjected to agarose gel electrophoresis and stained with ethidium bromide to ascertain the integrity of the 18S and 28S ribosomal RNA.
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GAC TCG AGT CGA CAT CGA TT 3? (backward). For amplification of the control gene, mGAPDH, the following primers were used: 5? TCA TCA TCT CCG CCC CTT CTG C 3? (forward) and 5? GTC CAC CAC CCT GTT GCT GTA G 3? (backward). PCR amplification of the mSMGP/mPIP cDNA was carried out using the following profile: an initial step at 94 8C for 5 min, then 35 cycles of 94 8C for 1 min (denaturation), 50 8C for 2 min (annealing) and 72 8C for 1 min (elongation). PCR amplification of the mGAPDH cDNA proceeded using the following profile: an initial step at 94 8C for 2 min, then 24 cycles of 94 8C for 30 s, 50 8C for 30 s and 72 8C for 30 s. All samples were finally incubated at 72 8C for 10 min. The products were electrophoresed in a 1% agarose gel stained with ethidium bromide and visualized with long wave ultraviolet light. A 100 base pair DNA ladder (Invitrogen Corporation) was used as a molecular standard. Three independent PCRs were performed for each sample. 2.6. Southern blot analysis
2.5. Reverse-transcription polymerase chain reaction To remove any contaminating DNA, total RNA used for RT-PCR was pretreated with one unit of Amplification Grade DNase I (Invitrogen Corporation). A mixture of 1 mg RNA, 1 ml DNase I (1.0 U), 1 ml 10 / DNase I reaction buffer (200 mM Tris /HCl pH 8.4, 20 mM MgCl2, 500 mM KCl), and 7 ml DEPC-treated water was prepared on ice, then incubated for 15 min at 22 8C. The DNase I was inactivated at 65 8C for 10 min in the presence of 2.5 mM EDTA (pH 8.0). Following DNase I treatment, 1 mg of total RNA was reverse transcribed using RT buffer (Invitrogen Corporation; 50 mM Tris /HCl, 75 mM KCl, 3 mM MgCl2), 200 nM of each dNTP (Amersham Pharmacia, Baie d’Urfe´, PQ, Canada), 1 mg BSA (New England Biolabs, Mississauga, ON, Canada), 250 ng Oligo dT RT primer with a restriction enzyme cassette (GACTCGAGTCGACATCGAT18), 0.01 M DTT (Invitrogen Corporation), 19 units of RNase inhibitor (RNA guard; Amersham Pharmacia) and 200 units of Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada) in a final volume of 50 ml. The reaction mixture was incubated at 37 8C for 2 h using a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA, USA). One tenth (5 ml) of each RT reaction was used for PCR in a final volume of 50 ml containing PCR buffer (Amersham Pharmacia; 10 mM Tris /HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl), 200 nM of each dNTP, 2.5 units of Taq DNA Polymerase (Amersham Pharmacia) and the appropriate primers. For amplification of the mSMGP/mPIP gene, the following primers were used: 5? TGG TGT TCT GAC TTC TCC AC 3? (forward) and
Following PCR amplification, 20 ml aliquots of the PCR products were electrophoresed in a 1% agarose gel. The gel was then agitated in a denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 1 h at room temperature. The solution was poured off and replaced by neutralization solution (1 M Tris /HCl, pH 8.0, 1.5 M NaCl) and agitated for 1 h overnight. The DNA was transferred onto a nitrocellulose membrane (0.45 mm pore size; Micron Separations, Westborough, MA, USA), which was then baked for 2 h at 80 8C. The blot was prehybridized at 42 8C for 2 h in 50% (v/v) formamide, 1 / SSPE buffer (0.15 M NaCl, 0.01 M NaH2PO4, 1 mM EDTA, pH 7.7), 1/ Denhardt’s Solution (0.02% [w/v] each of Ficoll 400, polyvinylpyrrolidone and BSA), 0.1% sodium dodecyl sulfate (SDS) and 250 mg/ml denatured salmon sperm DNA. For hybridization, a fresh 10 ml of the same solution was used with the addition of 30 ng of a random primed 32P-labelled mSMGP/mPIP cDNA probe and hybridized overnight at 42 8C. Nitrocellulose membranes were washed twice with 2/ SSC (0.3 M NaCl, 0.03 M sodium citrate)/0.1% SDS at room temperature for 10 min followed by prewarmed 0.1 / SSC/0.1% SDS at 65 8C for 5 min. All membranes were exposed to X-ray film at /70 8C with an intensifying screen. 2.7. In situ hybridization Submandibular glands were dissected from an E18 mouse embryo and placed in 4% paraformaldehyde for 24 h, then washed with 70% ethanol and processed for paraffin sectioning. In situ hybridization was performed using three 5 mm paraffin sections as previously described by Al-Haddad et al. (1999). A linearized pVZ I
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plasmid containing the mSMGP/mPIP cDNA insert was used to generate 35S-UTP-labelled sense and antisense RNA probes using the Riboprobe Gemini II Core System kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The riboprobes were purified using a Quick spin G-50 Sephadex column (Roche, Indianapolis, IN). Tissue sections were hybridized to either sense or antisense probes (at a concentration of 1 /106 cpm/ml) at 42 8C overnight. For autoradiographic detection, the slides were dipped in Kodak NTB-2 emulsion (InterScience) at 40 8C, dried for 1 h in a humidified chamber and placed in black light tight slide boxes at 4 8C for a 4 week exposure. Slides were developed, counterstained with Lee’s methylene blue and basic fuchsin and photographed (Leica, Wetzlar, Germany).
3. Results 3.1. mSMGP/mPIP gene expression in whole mouse embryos and adult prostate As a first step in examining gene expression in embryonic tissues, poly A RNAs derived from whole mouse embryos and adult tissues were screened with a 32 P-labeled mSMGP/mPIP cDNA probe. Included on the blot was RNA from the adult mouse submandibular gland, which was used as a positive control. mSMGP/ mPIP mRNA transcripts were not detected at E7, E11, E15 or at E17 (Fig. 1, position E1, E2, E3, E4). However, a low level of the mSMGP/mPIP gene expression was detected in the prostate (Fig. 1, position 3D). All other tissues examined were negative for mSMGP/mPIP gene expression.
3.2. mSMGP/mPIP gene expression during embryonic development Whole mouse embryos were sectioned into two or three parts depending on age as described in Section 2 in order to minimize as much as possible, the dilution of any detectable signal obtained by RT-PCR or by Southern blot analysis. The results showed that the upper body region (U) of the embryos, which contained the head and neck, was positive for mSMGP/mPIP gene expression, and this expression was apparent as early as embryonic day 14 (E14) (Fig. 2B). Both the middle and lower sections of the embryos were negative for mSMGP/mPIP gene expression in all ages (E14 /21) analyzed (data not shown). We further analyzed the upper body with and without the submandibular gland in order to establish the origin of the observed mSMGP/mPIP gene expression. Using RT-PCR and Southern blot analysis, we demonstrated that mSMGP/mPIP was highly expressed in the upper body region plus submandibular gland, but not expressed in the upper body region minus the submandibular gland (Fig. 2D). When analyzed alone, the submandibular gland was shown to highly express the mSMGP/mPIP gene (Fig. 2B). The expression of the mSMGP/mPIP mRNA in the E18 submandibular gland was further demonstrated by in situ hybridization analysis. The results showed that the mSMGP/mPIP gene expression was specific for the proacinar cells (Fig. 3C). No hybridization signals were observed with the control 35S-labelled mSMGP/mPIP sense probe (Fig. 3B). Several other tissues were also dissected from the E18 embryo and found negative for mSMGP/mPIP gene expression by RT-PCR and Southern blot analysis (Fig. 4A, B).
Fig. 1. Analysis of mSMGP/mPIP gene expression in mouse embryo and adult tissues. A commercially available RNA Master Blot (Clontech) containing poly A RNAs from different mouse tissues (2 mg each) was screened with 32P-labelled mSMGP/mPIP cDNA. The mSMGP/mPIP gene was expressed in the submandibular (C4) and in the prostate (D3) glands.
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Fig. 2. mSMGP/mPIP gene expression in the submandibular glands of mouse embryos. RNA was extracted from either whole upper body segments (U) containing intact submandibular glands (SMGs) of day 14 /16 embryos (E14 to E16), or from the SMGs alone from embryos ages E17 to postnatal day 1 (1d), and analyzed by RT-PCR. PCR products were electrophoresed on a 1% agarose gel (A). mSMGP/ mPIP gene expression was detected in E14 to E16 embryos by Southern blot analysis following a 3-day exposure to X-ray film (B), whereas in E17 to 1 day old mice gene expression was detected only after 2 h. In panel C, the results of RT-PCR amplification of the housekeeping gene mouse GAPDH are compared with RT-PCR of mSMGP/mPIP. In panel D, Southern blot analysis of the RT-PCR products from an E17 mouse embryo [upper body sections (U)] with (/) and without (/) the submandibular gland, is shown.
3.3. mSMGP/mPIP expression during early postnatal development Tissue specificity was further assessed in pups ranging in ages from day 1 to 4 week old. In each specific age group mSMGP/mPIP gene was consistently expressed in the submandibular gland (Figs. 5 and 6). The mSMGP/ mPIP gene expression was also detected in the other two major salivary glands, the sublingual and the parotid and in the lacrimal gland of 5 day old mice (Fig. 5). mSMGP/mPIP gene expression was also assessed in the reproductive tissues during early postnatal development. Gene expression was not detected in the prostate, uterus or ovary at day 1 (Fig. 6A, B) or day 3
Fig. 3. Localization of mSMGP/mPIP transcripts in the mouse embryonic (E18) submandibular gland. E18 submandibular gland (SMG) tissue sections were stained with Hematoxylin and Eosin (A). In situ hybridization was carried out using specific 35S-labelled mSMGP/mPIP riboprobes. Hybridization of the SMG with sense (B) and antisense mSMGP/mPIP probes (C), is shown. mSMGP/mPIP transcripts were found to be abundantly expressed in the proacinar cells (pac) but not in the ductal cells. Magnification /400.
postpartum. However, by day 5 mSMGP/mPIP gene expression was apparent in all three tissues (Fig. 6A, B). Further analysis of prostate tissue showed that gene expression persisted up to 4 weeks of age and was still evident in some animals up to 8 weeks of age, although at this time, gene expression was substantially reduced.
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Fig. 4. Tissue specific mSMGP/mPIP gene expression in the E18 mouse embryo. Total RNAs from various E18 embryo tissues (1 mg each) analyzed by RT-PCR using mSMGP/mPIP specific primers PCR products, were electrophoresed on a 1% agarose gel (A) and DNA transferred to nitrocellulose. Southern blot analysis of PCR products using 32P-labelled mSMGP/mPIP cDNA probe (B). RT minus (/) reaction and the amplification of the housekeeping gene, mouse GAPDH (C), were used as controls.
Fig. 6. Expression of the mSMGP/mPIP gene in reproductive tissues. Total RNAs (1 mg) from the prostate, uterus and ovary derived from E18, postnatal day 1 (1d) and day 5 (5d) mice, adult 4 week old males and lactating females were analyzed by RT-PCR using mSMGP/mPIP specific primers. PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide (A), followed by Southern blot analysis (B). Four week old male SMG total RNA was used as a positive control and RT reaction minus reverse transcriptase enzyme (RT-), as a negative control. The mGAPDH amplification products are shown in (C). Gene expression in the prostate of mice ages E18 to 10 weeks old is shown (D). Temporal expression of the mSMGP/mPIP gene was detected between the ages of 5 days to 4 weeks. Submaxillary gland was used as a positive control.
4. Discussion
Fig. 5. Early postnatal expression of the mSMGP/mPIP gene in other major salivary glands and in the lacrimal gland. Total RNAs (1 mg) derived from the sublingual (SLG), parotid (PG) and lacrimal (LG) glands of day 5 (5d) and 4 week old mice were analyzed by RT-PCR using mSMGP/mPIP specific primers. PCR products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining (A), followed by Southern blot analysis (B). Four week SMG total RNA was used as a positive control and RT reaction minus reverse transcriptase enzyme (RT-), as a negative control. The amplification of the mGAPDH gene was used to evaluate the amount of RNA template (C).
No gene expression was detected in prostates from 10 week old mice (Fig. 6D). Moreover, mSMGP/mPIP gene expression was not induced by castration and the re-administration of DHT up to 6 days post-castration further failed to induce gene expression (data not shown).
Homologues of the human GCDFP-15/hPIP gene have been identified in the mouse (Myal et al., 1994) and more recently in the rat (Mirels et al., 1998). In both human and rodents, the gene is highly expressed in the salivary glands. The identification of a mouse homologue presents an excellent opportunity to study gene function through the use of homologous recombination (gene knockout) experiments. Very little, however, is known about the pattern of gene expression in the developing mouse. Our data clearly demonstrates that the mSMGP/ mPIP gene is expressed early in the development of the mouse. In the head and neck region, gene expression was detected as early as embryonic day 14 (E14), a time which coincides with the first appearance of the primordial cells of the developing submandibular gland (Kaufman and Bard, 1999). Furthermore, using E17 embryos, we have demonstrated that once the submandibular gland was removed, the mSMGP/mPIP gene expression was no longer detectable. However, we cannot completely rule out the possibility that contaminating sublingual gland tissue may have also contrib-
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uted to the overall signal observed because, (a) of the close proximity of the sublingual to the submandibular gland, (b) the initiation of differentiation is also thought to occur at the same time (Kaufman and Bard, 1999) and (c) gene expression of rPIP, the rat homologue, has recently been documented in the sublingual gland of the newborn rat (Mirels et al., 1998). Nevertheless, it is unlikely that any of the observed gene expression could be attributed to either the parotid gland (the other major salivary gland) or to the lacrimal gland, since, in the parotid gland, secretory cell differentiation only occurs 1 day following birth (Redman and Sweeny, 1971), and in the lacrimal gland, such differentiation occurs even slightly later (Kaufman and Bard, 1999). Additional evidence comes from the observation that 1 day old parotid glands were found to be negative for mSMGP/ mPIP gene expression. In situ hybridization analysis was also carried out on embryonic (E18) submandibular glands. At this time the gland is more developed and the cell types are clearly discernible. The submandibular gland now comprises of two cell types; the terminal cells and the proacinar cells, the latter being the precursors of the acinar cells in the adult mouse/rat (Cutler and Chaudhry, 1974). Consistent with results observed in the adult rat (Mirels et al., 1998; Myal et al., 1994), the abundantly expressed mSMGP/mPIP transcripts were found localized to the proacinar cells of the embryonic submandibular gland (Fig. 3). Several other E18 tissues, including tails and skin (which contain sweat glands) and reproductive organs, were also negative for mSMGP/mPIP gene expression. Altogether, these data seem to indicate that the early expression of mSMGP/mPIP gene in the embryo is closely associated with the initiation of the submandibular, and possibly the sublingual gland development. In view of the fact that multiple wellformed Golgi units and strands of dilated rough endoplasmic reticulum are already present in the E15 submandibular gland (Cutler and Chaudhry, 1974), and the observation that the mSMGP/mPIP gene is highly expressed in the proacinar cells of the embryonic submandibular gland, we suggest that the mSMGP/ mPIP has a function related to the development of the gland and/or one related to a regulatory or protective role in the developing embryo. We further examined mSMGP/mPIP gene expression during early mouse postnatal development. Through RT-PCR and Southern blot analysis, the mSMGP/mPIP gene expression was found in the submandibular, sublingual and in the parotid glands by d5 following parity. The mSMGP/mPIP is, therefore, one of the few salivary proteins expressed in all three major salivary glands. The discovery of a weak positive signal identified in prostate tissue on a commercial poly A RNA blot prompted us to further analyze mSMGP/mPIP gene
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expression in the reproductive tissues. Since the specific ages of the mice from which these tissues were derived were not available for the commercial blot, we then analyzed tissues from early developing prostate to adult. mSMGP/mPIP gene expression was detected in the prostate at day 5 and at 4 and 8 week but not at E18, day 1, day 3 or at 10 week of age (Fig. 6D). Interestingly, this period (day 5 to 4 week), during which the mSMGP/mPIP gene expression is turned on in the prostate coincides with a period of rapid ductal branching (day 5 to 4 week) in response to androgens (Cunha et al., 1987). The absence of mSMGP/mPIP gene expression in the adult mouse prostate is consistent with previous observations made by us (Myal et al., 1994) in the rat. In that study, we also demonstrated that androgen regulated gene expression in the lacrimal gland (Myal et al., 1994) but surprisingly, not in the rodent submandibular gland, a known androgen responsive tissue. Since mSMGP/mPIP appeared to be ‘‘turned off’’ in the adult prostate, we examined whether castration induced gene expression. However, castration and the further re-administration of DHT, failed to induce mSMGP/mPIP gene expression. Indeed, the possibility exists that co-factor(s) important for mSMGP/mPIP gene expression, are present during early postnatal development but not in the adult mouse. Taken together, these data suggest that in the mouse, the regulation of the mSMGP/mPIP gene by androgens, occur in a time-dependent and tissue-specific manner. The mSMGP/mPIP gene expression was also demonstrated in the mouse uterus and ovary at day 5 following parity. It is interesting to note that weak expression of the GCDFP-15/hPIP gene has also been observed in the human ovary (Clark et al., 1999). There are some key species similarities that exists in the pattern of gene and protein expression that allow us to speculate on some possible roles for PIP. The human and rodent PIP genes are expressed in exocrine organs and the PIP protein is found in the fluid secretions from these organs. In addition, both the human GCDFP-15/ hPIP (Schenkels et al., 1997) and more recently the mSMGP/mPIP (Lee et al., 2002) have been identified in saliva and shown to bind several species of oral and nonoral bacteria. Also, there are other known proteins such as salivary peroxidases (Yamashina and Barka, 1974; Kruse et al., 1998) which, like PIP, are expressed in both salivary and reproductive tissues and which have been shown to play a role in mucosal defense (Carlsson, 1987). These studies suggest that the mSMGP/mPIP may also have a similar function. In summary, we have shown that the mSMGP/mPIP gene is expressed in the submandibular gland in the developing embryo and in the major salivary glands and the reproductive organs during early postnatal development. The developmental and tissue specific pattern of the mSMGP/mPIP gene expression pattern, together
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with the identification of the protein in saliva and its ability to bind to bacteria (Lee et al., 2002) lend support to our hypothesis that mSMGP/mPIP may play a role in non-immune host defense. Further studies to determine which specific cell types express the mSMGP/mPIP gene in the reproductive tissues should give more insight into the different biological roles of the mSMGP/mPIP gene in different organs.
Acknowledgements The authors would like to thank Ms Barbara Iwasiow and Ms Pat Sheppard for technical assistance. This work was funded by NSERC (Y.M.).
References Akiyama, K., Kimura, H., 1990. Isolation of a new actin-binding protein from human seminal plasma. Biochim. Biophys. Acta 1040, 206 /210. Al-Haddad, S., Zhang, Z., Leygue, E., Snell, L., Huang, A., Niu, Y., Hiller-Hitchcock, T., Hole, K., Murphy, L.C., Watson, P.H., 1999. Psoriasin (S100A7) expression and invasive breast cancer. Am. J. Pathol. 155, 2057 /2066. Autiero, M., Abrescia, P., Guardiola, J., 1991. Interaction of seminal plasma proteins with cell surface antigens, presence of a CD4binding glycoprotein in human seminal plasma. Exp. Cell Res. 197, 268 /271. Autiero, M., Cammarota, G., Friedlein, A., Zulauf, M., Chiappetta, G., Dragone, V., Gaurdiola, J., 1995. A 17-kDa CD4-binding glycoprotein present in human seminal plasma and in breast tumor cells. Eur. J. Immunol. 25, 1461 /1464. Autiero, M., Culerrier, R., Bouchier, C., Basmaciogullari, S., Gaubin, M., El Marhomy, S., Blanchet, P., Paradis, V., Jardin, A., Guardiola, J., Piatier-Tonneau, D., 1999. Abnormal restriction pattern of PIP gene associated with human primary prostate cancers. DNA Cell Biol. 18, 481 /487. Blais, Y., Sugimoto, K., Carriere, M.C., Haagensen, D.E., Labrie, F., Simard, J., 1994. Potent stimulatory effect of interleukin-1 alpha on apolipoprotein D and gross cystic disease fluid protein-15 expression in human breast-cancer cells. Int. J. Cancer 59, 400 /407. Blais, Y., Sugimoto, K., Carriere, M.C., Haagensen, D.E., Labrie, F., Simard, J., 1995. Interleukin-6 inhibits the potent stimulatory action of androgens, glucocorticoids and interleukin-1 alpha on apolipoprotein D and GCDFP-15 expression in human breast cancer cells. Int. J. Cancer 62, 732 /737. Blais, Y., Gingras, S., Haagensen, D.E., Labrie, F., Simard, J., 1996. Interleukin-4 and interleukin-13 inhibit estrogen-induced breast cancer cell proliferation and stimulate GCDFP-15 expression in human breast cancer cells. Mol. Cell. Endocrinol. 121, 11 /18. Caputo, E., Manco, G., Mandrich, L., Guardiola, J., 2000. A novel aspartyl proteinase from apocrine epithelia and breast tumors. J. Biol. Chem. 275, 7935 /7941. Carlsson, J., 1987. Salivary peroxidase, an important part of our defense against oxygen toxicity. J. Oral Pathol. 16, 412 /416. Carsol, J.L., Gingras, S., Simard, J., 2002. Synergistic action of prolactin (PRL) and androgen on PRL-inducible protein gene expression in human breast cancer cells: a unique model for functional cooperation between signal transducer and activator of transcription-5 and androgen receptor. Mol. Endocrinol. 16, 1696 /1710.
Clark, J.W., Snell, L., Shiu, R.P.C., Orr, F.W., Maitre, N., Vary, C.P.H., Cole, D.J., Watson, P.H., 1999. The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis. Br. J. Cancer 81, 1002 / 1008. Cunha, G.R., Donjacour, A.A., Cooke, P.S., Mee, S., Bigsby, R.M., Higgins, S.J., Sugimura, Y., 1987. The endocrinology and developmental biology of the prostate. Endocr. Rev. 8, 338 / 362. Cutler, L.S., Chaudhry, A.P., 1974. Cytodifferentiation of the acinar cells of the rat submandibular gland. Dev. Biol. 41, 31 /41. Haagensen, D.E., Jr, Mazoujian, G., 1986. Biochemistry and immunohistochemistry of fluid proteins of the breast in gross cystic disease. In: Haagensen, C.D. (Ed.), Diseases of the Breast. Saunders, Philadelphia, pp. 474 /500. Kaufman, M.H., Bard, J.B.L., 1999. The Anatomical Basis of Mouse Development. Academic Press, San Diego. Kruse, D.H., Redman, R.S., Mansson-Rahemtulla, B., 1998. Salivary peroxidase immunohistochemistry in the developing rat submandibular gland. J. Dent. Res. 77A, 232. Lee, B., Bowden, G.H.W., Myal, Y., 2002. Identification of mouse submaxillary gland protein in mouse saliva and its binding to mouse oral bacteria. Arch. Oral Biol. 47, 327 /332. Mirels, L., Hand, A.R., Branin, H.J., 1998. Expression of gross cystic disease fluid protein-15/prolactin-inducible protein in rat salivary glands. J. Histochem. Cytochem. 46, 1061 /1071. Murphy, L.C., Tsuyuki, D., Myal, Y., Shiu, R.P.C., 1987a. Isolation and sequencing of a cDNA clone for a prolactin-inducible protein (PIP). Regulation of PIP gene expression in the human breast cancer cell line, T-47D. J. Biol. Chem. 262, 15236 / 15241. Murphy, L.C., Lee-Wing, M., Goldenberg, G.J., Shiu, R.P.C., 1987b. Expression of the gene encoding a prolactin-inducible protein by human breast cancers in vivo, correlation with steroid receptor status. Cancer Res. 47, 4160 /4164. Myal, Y., Shiu, R.P.C., 2000. The physiology and pathology of an apocrine protein, the prolactin-inducible protein (PIP)/gross cystic disease fluid protein (GCDFP-15). In: Pandalai, S.G. (Ed.), Recent Research Developments in Endocrinology, vol. I. Transworld Research Network, pp. 321 /335. Myal, Y., Gregory, C.A., Karpan, C., Hamerton, J.L., Shiu, R.P.C., 1989. Taq1 and RsaI RFLP’s at the prolactin inducible protein (PIP) locus on chromosome 7. Nucleic Acids Res. 17, 5879. Myal, Y., Iwasiow, B., Yarmill, A., Harrison, E., Paterson, J.A., Shiu, R.P.C., 1994. Tissue-specific androgen-inhibited gene expression of a submaxillary gland protein, a rodent homolog of the human prolactin-inducible protein/GCDFP-15 gene. Endocrinology 135, 1605 /1610. Redman, R.S., Sweeny, L.M., 1971. Morphologic and biochemical observations on the development of the rat parotid gland. Dev. Biol. 25, 248 /279. Shiu, R.P.C., Iwasiow, B., 1985. Prolactin-inducible proteins in human breast cancer cells. J. Biol. Chem. 260, 11307 /11313. Schenkels, L.C., Lautenberg, A.J., Vermin, E.C., New Amidogen, A.V., 1993. Interaction of the salivary glycoprotein EP-GP with the bacterium Streptococcus salivarius HB. J. Dent. Res. 72, 1559 / 1565. Schenkels, L.C., Walgreen-Weterings, E., Oomen, L.C., Bolscher, J.G., Veerman, E.C., Nieuw Amerongen, A.V., 1997. In vivo binding of the salivary glycoprotein EP-GP (identical to GCDFP-15) to oral and non-oral bacteria detection and identification of EP-GP binding species. Biol. Chem. 378, 83 / 88.
B. Lee et al. / Molecular and Cellular Endocrinology 205 (2003) 33 /41 Windass, J.D., Mullins, J.J., Beecroft, L.J., George, H., Meacock, P.A., Williams, B.R., Brammer, W.J., 1984. Molecular cloning of cDNAs from androgen-independent mRNA species of DBA/2 mouse sub-maxillary glands. Nucleic Acids Res. 12, 1361 /1376. Yamashina, S., Barka, T., 1974. Peroxidase activity in the developing rat submandibular gland. Lab. Invest. 31, 82 /89.
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Zhang, X., Gaubin, M., Briant, L., Srikantan, V., Murali, R., Saragovi, U., Weiner, D., Devaux, C., Autiero, M., PiatierTonneau, D., Greene, M.I., 1997. Synthetic CD4 exocyclics inhibit binding of human immunodeficiency virus type 1 envelope to CD4 and virus replication in T lymphocytes. Nat. Biotechnol. 15, 150 / 154.
Expression of the mouse homologue for the human GCDFP-15/PIP gene during pre- and early post-natal development Beverley Lee a, Geetanjalee Modha a, Peter H. Watson a, Janice Dodd b, Sandy Troup a, Anne Blanchard a, Yvonne Myal a,* a
Department of Pathology, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Man., Canada R3E 0W3 b Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Man., Canada R3E 0W3 Received 21 December 2002; accepted 15 May 2003
Abstract The function of the mouse submaxillary gland/prolactin inducible protein (mSMGP/mPIP), the homologue of the human gross cystic disease fluid protein 15 (GCDFP-15)/prolactin inducible protein (hPIP) remains unknown. The human gene, normally expressed in apocrine glands of healthy individuals, is aberrantly expressed in human breast cancers where it is regulated by hormones including androgens, and in prostate cancers. We have previously reported that in the adult mouse and rat, gene expression is tissue-specific for the salivary and lacrimal glands, and is hormonally regulated. In this study, we examine the endogenous pattern of mouse SMGP/PIP (mSMGP/mPIP) gene expression in mid- and late-embryonic, and in early postnatal development. Gene expression was analyzed by RT-PCR followed by Southern blot analysis, and by in situ hybridization. Gene expression was detected in the submandibular gland as early as embryonic day 14 (E14), a period that coincides with the initiation of submandibular gland development in the embryo, suggesting that mSMGP/mPIP may have a functional role in the developing gland. Nearing the end of gestation, E18, mSMGP/mPIP transcripts were localized in the proacinar cells of the gland, and gene expression continued to be maintained following birth. In addition, during early postnatal development, mSMGP/mPIP gene expression was detected in the other two major salivary glands, the sublingual and parotid, as well as in the lacrimal gland and in reproductive tissues. In the prostate, gene expression was turned off by 10 weeks of age. The spatial and temporal pattern of the mSMGP/mPIP gene expression, in addition to our recent demonstration that mSMGP/mPIP is found in mouse saliva and can bind bacteria, suggest that this protein may have a protective role in the mouse. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Submandibular gland; Prostate; Gene expression; Development; mSMGP/mPIP; GCDFP-15/hPIP
1. Introduction The mouse submaxillary gland protein (mSMGP) was initially identified as a highly expressed gene in the submandibular (submaxillary) gland of the adult mouse (Windass et al., 1984; Myal et al., 1994). The 576 bp mSMGP cDNA (GenBank Accession # NM_008843) encodes a secreted polypeptide sequence of 146 amino acids, containing a 25 amino acid signal peptide (Myal et al., 1994) and has a calculated Mr of 14 kDa. In the adult rodent, mSMGP transcripts were localized to the
* Corresponding author. Tel.: /1-204-789-3874; fax: /1-204-7893931. E-mail address: [email protected] (Y. Myal).
acinar cells (Myal et al., 1994; Mirels et al., 1998), the major secretory cells of the gland. The levels of mSMGP gene expression in the submandibular gland of the male or female mouse were found to be similar, suggesting that in this tissue, gene expression is not differentially regulated by androgen or estrogen. mSMGP was also found to be expressed in the parotid and sublingual salivary glands of the rat, as well as in the lacrimal gland. In the lacrimal gland, androgen was found to be a potent inhibitor of mSMGP gene expression (Myal et al., 1994). At the amino acid level, the mSMGP exhibits a 51% identity with the human gross cystic disease fluid protein-15 (GCDFP-15; Haagensen and Mazoujian, 1986), which is also known as the prolactin inducible
0303-7207/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0303-7207(03)00210-7
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protein (hPIP; Shiu and Iwasiow, 1985; Murphy et al., 1987a), extraparotid glycoprotein (EP-GP; Schenkels et al., 1993), secretory actin binding protein (SABP; Akiyama and Kimura, 1990) and glycoprotein 17 (gp17; Autiero et al., 1991). In healthy humans, the secreted protein has been identified in saliva, tears, sweat, seminal plasma, submucosal glands of the lung and in amniotic fluid (Murphy et al., 1987b) and is thought to play a role in host immunity. The GCDFP-15/hPIP has been shown to, (a) bind CD4 and inhibit CD4-mediated T cell programmed cell death (Autiero et al., 1995) and, (b) interfere with the HIV envelope protein gp-120 interaction with CD4 (Zhang et al., 1997). The GCDFP-15/hPIP gene appears to be aberrantly expressed in breast tumor biopsies and human breast cancer cell lines, where, in the latter, it is regulated by several hormones, most potently by the interleukins, gluccocorticoids and androgen (Murphy et al., 1987a; Blais et al., 1994, 1995, 1996; Clark et al., 1999; Carsol et al., 2002). Interestingly, a recent report suggests that GCDFP-15/hPIP, isolated from apocrine epithelia and breast tumors, is a novel aspartyl proteinase (Caputo et al., 2000). However, the GCDFP-15/hPIP gene is not expressed in healthy mammary glands (Haagensen and Mazoujian, 1986). Neither is the mSMGP (also referred to as mouse PIP) gene expressed in normal rodent mammary glands, nor in rodent mammary tumors (Myal et al., 1994). Whether the mSMGP/mPIP binds to CD4 and is an aspartyl proteinase is not known. The rat homologue (rPIP) was recently isolated and found to share a high degree of similarity (80%) with the mSMGP/mPIP at the amino acid level (Mirels et al., 1998). With regards to tissue specificity, the expression of this gene in both the salivary and lacrimal glands appears to be conserved across species (Myal et al., 1994; Mirels et al., 1998; Myal and Shiu, 2000). Studies by us and by others have pointed to an association between this gene and reproductive organs, and/or steroid hormone-dependent tissues in humans and rodents. For example, Clark et al. (1999) observed a low level of expression of GCDFP-15/hPIP gene expression in human ovaries, whereas, Akiyama and Kimura (1990) and Autiero et al. (1991) have isolated GCDFP15/hPIP from human seminal plasma. Also, Autiero et al. (1999) and Myal et al. (1989) have identified restriction fragment length polymorphisms in the GCDFP-15/hPIP gene in human prostate and breast cancers. Furthermore, we have previously demonstrated that androgen was a potent inhibitor of gene expression in the rat lacrimal gland (Myal et al., 1994). Analysis of the mSMGP/mPIP pattern of gene expression at different developmental stages will provide insight into the many possible roles for mSMGP/mPIP in vivo. In the present study we examined the expression of the mSMGP/mPIP gene during embryonic and early postnatal development in the mouse.
2. Material and methods 2.1. Screening of poly A RNA blot for the developmental and tissue specific pattern of mSMGP/ mPIP gene expression A commercially available RNA Master Blot (Clontech, Palo Alto, CA, USA) containing poly A RNAs extracted from several adult mouse tissues and whole mouse embryos, was hybridized with the mSMGP/mPIP cDNA probe (570 bp; Myal et al., 1994) according to the manufacturer’s instructions. Briefly, the RNA Master Blot was pre-hybridized at 65 8C for 1 h followed by overnight hybridization at 65 8C with a random primed 32 P-labelled full-length mSMGP/mPIP cDNA (Myal et al., 1994). The membrane was washed twice with 200 ml wash solution 1 (2 / SSC, 1% SDS) at 65 8C for 20 min followed by a final wash with prewarmed wash solution 2 (0.1 / SSC, 0.5% SDS) at 65 8C for 20 min. The blot was then exposed to Kodak X-OMAT AR film (InterScience, Markham, ON, Canada) at /70 8C with an intensifying screen for 3 h. 2.2. Animals Four to 6 week old CD1 mice were obtained from and housed at the facilities of the Central Animal Care Services, Faculty of Medicine, University of Manitoba (Winnipeg, MB Canada). Mice were mated overnight and the females were checked for vaginal plugs the next morning. The first appearance of the vaginal plug was counted as day 1 of gestation. 2.3. Tissue collection Pregnant females were sacrificed at days 14, 15, 16, 17, 18, 19, 20 and in some cases day 21 of gestation and whole fetuses were removed from the dams and immediately euthanized. Depending on the ages, fetuses were divided either into two (upper [U] and lower [L]) or three parts (head, neck and forelimbs [U], abdominal or mid region [M] and the pelvic region including the tail and hind limbs [L]), prior to freezing or fixing. E14 to E17 embryos were divided into two parts whereas the larger embryos, E18 to E21, were sectioned into three parts. Various other tissues were further dissected from an E18 embryo for analysis of gene expression. Young pups, 1, 3 or 5 days old were also collected, tissues immediately dissected and either frozen at /70 8C or fixed in 4% paraformaldehyde overnight. The paraformaldehyde was poured off the following day and replaced by 70% ethanol for short-term storage. As well, tissues were also collected from 4, 6, 8 and 10 week old males and from lactating females. In specific cases, (embryonic or early postnatal prostates and ovaries; days 1, 3 and 5), samples were pooled in order to collect
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sufficient amount of tissue for total RNA extraction. Following the castration of 6-week old male mice, prostates were collected at 2 and 4 week post surgery and at 2 week post-castration followed by 3 or 6 days hormone replacement with dihydrotestosterone (DHT; 3 mg/kg per day) subcutaneously. 2.4. Isolation of total RNA Total RNA was extracted from frozen tissues using TRIzol Reagent (Invitrogen Corporation, Burlington, ON, Canada) according to the protocol outlined by the manufacturer. The pellet was washed with 75% cold ethanol, air dried, dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at /70 8C. The purity and the yield of the RNA samples were determined spectrophotometrically. Total RNA (1 mg) was subjected to agarose gel electrophoresis and stained with ethidium bromide to ascertain the integrity of the 18S and 28S ribosomal RNA.
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GAC TCG AGT CGA CAT CGA TT 3? (backward). For amplification of the control gene, mGAPDH, the following primers were used: 5? TCA TCA TCT CCG CCC CTT CTG C 3? (forward) and 5? GTC CAC CAC CCT GTT GCT GTA G 3? (backward). PCR amplification of the mSMGP/mPIP cDNA was carried out using the following profile: an initial step at 94 8C for 5 min, then 35 cycles of 94 8C for 1 min (denaturation), 50 8C for 2 min (annealing) and 72 8C for 1 min (elongation). PCR amplification of the mGAPDH cDNA proceeded using the following profile: an initial step at 94 8C for 2 min, then 24 cycles of 94 8C for 30 s, 50 8C for 30 s and 72 8C for 30 s. All samples were finally incubated at 72 8C for 10 min. The products were electrophoresed in a 1% agarose gel stained with ethidium bromide and visualized with long wave ultraviolet light. A 100 base pair DNA ladder (Invitrogen Corporation) was used as a molecular standard. Three independent PCRs were performed for each sample. 2.6. Southern blot analysis
2.5. Reverse-transcription polymerase chain reaction To remove any contaminating DNA, total RNA used for RT-PCR was pretreated with one unit of Amplification Grade DNase I (Invitrogen Corporation). A mixture of 1 mg RNA, 1 ml DNase I (1.0 U), 1 ml 10 / DNase I reaction buffer (200 mM Tris /HCl pH 8.4, 20 mM MgCl2, 500 mM KCl), and 7 ml DEPC-treated water was prepared on ice, then incubated for 15 min at 22 8C. The DNase I was inactivated at 65 8C for 10 min in the presence of 2.5 mM EDTA (pH 8.0). Following DNase I treatment, 1 mg of total RNA was reverse transcribed using RT buffer (Invitrogen Corporation; 50 mM Tris /HCl, 75 mM KCl, 3 mM MgCl2), 200 nM of each dNTP (Amersham Pharmacia, Baie d’Urfe´, PQ, Canada), 1 mg BSA (New England Biolabs, Mississauga, ON, Canada), 250 ng Oligo dT RT primer with a restriction enzyme cassette (GACTCGAGTCGACATCGAT18), 0.01 M DTT (Invitrogen Corporation), 19 units of RNase inhibitor (RNA guard; Amersham Pharmacia) and 200 units of Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada) in a final volume of 50 ml. The reaction mixture was incubated at 37 8C for 2 h using a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA, USA). One tenth (5 ml) of each RT reaction was used for PCR in a final volume of 50 ml containing PCR buffer (Amersham Pharmacia; 10 mM Tris /HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl), 200 nM of each dNTP, 2.5 units of Taq DNA Polymerase (Amersham Pharmacia) and the appropriate primers. For amplification of the mSMGP/mPIP gene, the following primers were used: 5? TGG TGT TCT GAC TTC TCC AC 3? (forward) and
Following PCR amplification, 20 ml aliquots of the PCR products were electrophoresed in a 1% agarose gel. The gel was then agitated in a denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 1 h at room temperature. The solution was poured off and replaced by neutralization solution (1 M Tris /HCl, pH 8.0, 1.5 M NaCl) and agitated for 1 h overnight. The DNA was transferred onto a nitrocellulose membrane (0.45 mm pore size; Micron Separations, Westborough, MA, USA), which was then baked for 2 h at 80 8C. The blot was prehybridized at 42 8C for 2 h in 50% (v/v) formamide, 1 / SSPE buffer (0.15 M NaCl, 0.01 M NaH2PO4, 1 mM EDTA, pH 7.7), 1/ Denhardt’s Solution (0.02% [w/v] each of Ficoll 400, polyvinylpyrrolidone and BSA), 0.1% sodium dodecyl sulfate (SDS) and 250 mg/ml denatured salmon sperm DNA. For hybridization, a fresh 10 ml of the same solution was used with the addition of 30 ng of a random primed 32P-labelled mSMGP/mPIP cDNA probe and hybridized overnight at 42 8C. Nitrocellulose membranes were washed twice with 2/ SSC (0.3 M NaCl, 0.03 M sodium citrate)/0.1% SDS at room temperature for 10 min followed by prewarmed 0.1 / SSC/0.1% SDS at 65 8C for 5 min. All membranes were exposed to X-ray film at /70 8C with an intensifying screen. 2.7. In situ hybridization Submandibular glands were dissected from an E18 mouse embryo and placed in 4% paraformaldehyde for 24 h, then washed with 70% ethanol and processed for paraffin sectioning. In situ hybridization was performed using three 5 mm paraffin sections as previously described by Al-Haddad et al. (1999). A linearized pVZ I
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plasmid containing the mSMGP/mPIP cDNA insert was used to generate 35S-UTP-labelled sense and antisense RNA probes using the Riboprobe Gemini II Core System kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The riboprobes were purified using a Quick spin G-50 Sephadex column (Roche, Indianapolis, IN). Tissue sections were hybridized to either sense or antisense probes (at a concentration of 1 /106 cpm/ml) at 42 8C overnight. For autoradiographic detection, the slides were dipped in Kodak NTB-2 emulsion (InterScience) at 40 8C, dried for 1 h in a humidified chamber and placed in black light tight slide boxes at 4 8C for a 4 week exposure. Slides were developed, counterstained with Lee’s methylene blue and basic fuchsin and photographed (Leica, Wetzlar, Germany).
3. Results 3.1. mSMGP/mPIP gene expression in whole mouse embryos and adult prostate As a first step in examining gene expression in embryonic tissues, poly A RNAs derived from whole mouse embryos and adult tissues were screened with a 32 P-labeled mSMGP/mPIP cDNA probe. Included on the blot was RNA from the adult mouse submandibular gland, which was used as a positive control. mSMGP/ mPIP mRNA transcripts were not detected at E7, E11, E15 or at E17 (Fig. 1, position E1, E2, E3, E4). However, a low level of the mSMGP/mPIP gene expression was detected in the prostate (Fig. 1, position 3D). All other tissues examined were negative for mSMGP/mPIP gene expression.
3.2. mSMGP/mPIP gene expression during embryonic development Whole mouse embryos were sectioned into two or three parts depending on age as described in Section 2 in order to minimize as much as possible, the dilution of any detectable signal obtained by RT-PCR or by Southern blot analysis. The results showed that the upper body region (U) of the embryos, which contained the head and neck, was positive for mSMGP/mPIP gene expression, and this expression was apparent as early as embryonic day 14 (E14) (Fig. 2B). Both the middle and lower sections of the embryos were negative for mSMGP/mPIP gene expression in all ages (E14 /21) analyzed (data not shown). We further analyzed the upper body with and without the submandibular gland in order to establish the origin of the observed mSMGP/mPIP gene expression. Using RT-PCR and Southern blot analysis, we demonstrated that mSMGP/mPIP was highly expressed in the upper body region plus submandibular gland, but not expressed in the upper body region minus the submandibular gland (Fig. 2D). When analyzed alone, the submandibular gland was shown to highly express the mSMGP/mPIP gene (Fig. 2B). The expression of the mSMGP/mPIP mRNA in the E18 submandibular gland was further demonstrated by in situ hybridization analysis. The results showed that the mSMGP/mPIP gene expression was specific for the proacinar cells (Fig. 3C). No hybridization signals were observed with the control 35S-labelled mSMGP/mPIP sense probe (Fig. 3B). Several other tissues were also dissected from the E18 embryo and found negative for mSMGP/mPIP gene expression by RT-PCR and Southern blot analysis (Fig. 4A, B).
Fig. 1. Analysis of mSMGP/mPIP gene expression in mouse embryo and adult tissues. A commercially available RNA Master Blot (Clontech) containing poly A RNAs from different mouse tissues (2 mg each) was screened with 32P-labelled mSMGP/mPIP cDNA. The mSMGP/mPIP gene was expressed in the submandibular (C4) and in the prostate (D3) glands.
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Fig. 2. mSMGP/mPIP gene expression in the submandibular glands of mouse embryos. RNA was extracted from either whole upper body segments (U) containing intact submandibular glands (SMGs) of day 14 /16 embryos (E14 to E16), or from the SMGs alone from embryos ages E17 to postnatal day 1 (1d), and analyzed by RT-PCR. PCR products were electrophoresed on a 1% agarose gel (A). mSMGP/ mPIP gene expression was detected in E14 to E16 embryos by Southern blot analysis following a 3-day exposure to X-ray film (B), whereas in E17 to 1 day old mice gene expression was detected only after 2 h. In panel C, the results of RT-PCR amplification of the housekeeping gene mouse GAPDH are compared with RT-PCR of mSMGP/mPIP. In panel D, Southern blot analysis of the RT-PCR products from an E17 mouse embryo [upper body sections (U)] with (/) and without (/) the submandibular gland, is shown.
3.3. mSMGP/mPIP expression during early postnatal development Tissue specificity was further assessed in pups ranging in ages from day 1 to 4 week old. In each specific age group mSMGP/mPIP gene was consistently expressed in the submandibular gland (Figs. 5 and 6). The mSMGP/ mPIP gene expression was also detected in the other two major salivary glands, the sublingual and the parotid and in the lacrimal gland of 5 day old mice (Fig. 5). mSMGP/mPIP gene expression was also assessed in the reproductive tissues during early postnatal development. Gene expression was not detected in the prostate, uterus or ovary at day 1 (Fig. 6A, B) or day 3
Fig. 3. Localization of mSMGP/mPIP transcripts in the mouse embryonic (E18) submandibular gland. E18 submandibular gland (SMG) tissue sections were stained with Hematoxylin and Eosin (A). In situ hybridization was carried out using specific 35S-labelled mSMGP/mPIP riboprobes. Hybridization of the SMG with sense (B) and antisense mSMGP/mPIP probes (C), is shown. mSMGP/mPIP transcripts were found to be abundantly expressed in the proacinar cells (pac) but not in the ductal cells. Magnification /400.
postpartum. However, by day 5 mSMGP/mPIP gene expression was apparent in all three tissues (Fig. 6A, B). Further analysis of prostate tissue showed that gene expression persisted up to 4 weeks of age and was still evident in some animals up to 8 weeks of age, although at this time, gene expression was substantially reduced.
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Fig. 4. Tissue specific mSMGP/mPIP gene expression in the E18 mouse embryo. Total RNAs from various E18 embryo tissues (1 mg each) analyzed by RT-PCR using mSMGP/mPIP specific primers PCR products, were electrophoresed on a 1% agarose gel (A) and DNA transferred to nitrocellulose. Southern blot analysis of PCR products using 32P-labelled mSMGP/mPIP cDNA probe (B). RT minus (/) reaction and the amplification of the housekeeping gene, mouse GAPDH (C), were used as controls.
Fig. 6. Expression of the mSMGP/mPIP gene in reproductive tissues. Total RNAs (1 mg) from the prostate, uterus and ovary derived from E18, postnatal day 1 (1d) and day 5 (5d) mice, adult 4 week old males and lactating females were analyzed by RT-PCR using mSMGP/mPIP specific primers. PCR products were electrophoresed on a 1% agarose gel and stained with ethidium bromide (A), followed by Southern blot analysis (B). Four week old male SMG total RNA was used as a positive control and RT reaction minus reverse transcriptase enzyme (RT-), as a negative control. The mGAPDH amplification products are shown in (C). Gene expression in the prostate of mice ages E18 to 10 weeks old is shown (D). Temporal expression of the mSMGP/mPIP gene was detected between the ages of 5 days to 4 weeks. Submaxillary gland was used as a positive control.
4. Discussion
Fig. 5. Early postnatal expression of the mSMGP/mPIP gene in other major salivary glands and in the lacrimal gland. Total RNAs (1 mg) derived from the sublingual (SLG), parotid (PG) and lacrimal (LG) glands of day 5 (5d) and 4 week old mice were analyzed by RT-PCR using mSMGP/mPIP specific primers. PCR products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining (A), followed by Southern blot analysis (B). Four week SMG total RNA was used as a positive control and RT reaction minus reverse transcriptase enzyme (RT-), as a negative control. The amplification of the mGAPDH gene was used to evaluate the amount of RNA template (C).
No gene expression was detected in prostates from 10 week old mice (Fig. 6D). Moreover, mSMGP/mPIP gene expression was not induced by castration and the re-administration of DHT up to 6 days post-castration further failed to induce gene expression (data not shown).
Homologues of the human GCDFP-15/hPIP gene have been identified in the mouse (Myal et al., 1994) and more recently in the rat (Mirels et al., 1998). In both human and rodents, the gene is highly expressed in the salivary glands. The identification of a mouse homologue presents an excellent opportunity to study gene function through the use of homologous recombination (gene knockout) experiments. Very little, however, is known about the pattern of gene expression in the developing mouse. Our data clearly demonstrates that the mSMGP/ mPIP gene is expressed early in the development of the mouse. In the head and neck region, gene expression was detected as early as embryonic day 14 (E14), a time which coincides with the first appearance of the primordial cells of the developing submandibular gland (Kaufman and Bard, 1999). Furthermore, using E17 embryos, we have demonstrated that once the submandibular gland was removed, the mSMGP/mPIP gene expression was no longer detectable. However, we cannot completely rule out the possibility that contaminating sublingual gland tissue may have also contrib-
B. Lee et al. / Molecular and Cellular Endocrinology 205 (2003) 33 /41
uted to the overall signal observed because, (a) of the close proximity of the sublingual to the submandibular gland, (b) the initiation of differentiation is also thought to occur at the same time (Kaufman and Bard, 1999) and (c) gene expression of rPIP, the rat homologue, has recently been documented in the sublingual gland of the newborn rat (Mirels et al., 1998). Nevertheless, it is unlikely that any of the observed gene expression could be attributed to either the parotid gland (the other major salivary gland) or to the lacrimal gland, since, in the parotid gland, secretory cell differentiation only occurs 1 day following birth (Redman and Sweeny, 1971), and in the lacrimal gland, such differentiation occurs even slightly later (Kaufman and Bard, 1999). Additional evidence comes from the observation that 1 day old parotid glands were found to be negative for mSMGP/ mPIP gene expression. In situ hybridization analysis was also carried out on embryonic (E18) submandibular glands. At this time the gland is more developed and the cell types are clearly discernible. The submandibular gland now comprises of two cell types; the terminal cells and the proacinar cells, the latter being the precursors of the acinar cells in the adult mouse/rat (Cutler and Chaudhry, 1974). Consistent with results observed in the adult rat (Mirels et al., 1998; Myal et al., 1994), the abundantly expressed mSMGP/mPIP transcripts were found localized to the proacinar cells of the embryonic submandibular gland (Fig. 3). Several other E18 tissues, including tails and skin (which contain sweat glands) and reproductive organs, were also negative for mSMGP/mPIP gene expression. Altogether, these data seem to indicate that the early expression of mSMGP/mPIP gene in the embryo is closely associated with the initiation of the submandibular, and possibly the sublingual gland development. In view of the fact that multiple wellformed Golgi units and strands of dilated rough endoplasmic reticulum are already present in the E15 submandibular gland (Cutler and Chaudhry, 1974), and the observation that the mSMGP/mPIP gene is highly expressed in the proacinar cells of the embryonic submandibular gland, we suggest that the mSMGP/ mPIP has a function related to the development of the gland and/or one related to a regulatory or protective role in the developing embryo. We further examined mSMGP/mPIP gene expression during early mouse postnatal development. Through RT-PCR and Southern blot analysis, the mSMGP/mPIP gene expression was found in the submandibular, sublingual and in the parotid glands by d5 following parity. The mSMGP/mPIP is, therefore, one of the few salivary proteins expressed in all three major salivary glands. The discovery of a weak positive signal identified in prostate tissue on a commercial poly A RNA blot prompted us to further analyze mSMGP/mPIP gene
39
expression in the reproductive tissues. Since the specific ages of the mice from which these tissues were derived were not available for the commercial blot, we then analyzed tissues from early developing prostate to adult. mSMGP/mPIP gene expression was detected in the prostate at day 5 and at 4 and 8 week but not at E18, day 1, day 3 or at 10 week of age (Fig. 6D). Interestingly, this period (day 5 to 4 week), during which the mSMGP/mPIP gene expression is turned on in the prostate coincides with a period of rapid ductal branching (day 5 to 4 week) in response to androgens (Cunha et al., 1987). The absence of mSMGP/mPIP gene expression in the adult mouse prostate is consistent with previous observations made by us (Myal et al., 1994) in the rat. In that study, we also demonstrated that androgen regulated gene expression in the lacrimal gland (Myal et al., 1994) but surprisingly, not in the rodent submandibular gland, a known androgen responsive tissue. Since mSMGP/mPIP appeared to be ‘‘turned off’’ in the adult prostate, we examined whether castration induced gene expression. However, castration and the further re-administration of DHT, failed to induce mSMGP/mPIP gene expression. Indeed, the possibility exists that co-factor(s) important for mSMGP/mPIP gene expression, are present during early postnatal development but not in the adult mouse. Taken together, these data suggest that in the mouse, the regulation of the mSMGP/mPIP gene by androgens, occur in a time-dependent and tissue-specific manner. The mSMGP/mPIP gene expression was also demonstrated in the mouse uterus and ovary at day 5 following parity. It is interesting to note that weak expression of the GCDFP-15/hPIP gene has also been observed in the human ovary (Clark et al., 1999). There are some key species similarities that exists in the pattern of gene and protein expression that allow us to speculate on some possible roles for PIP. The human and rodent PIP genes are expressed in exocrine organs and the PIP protein is found in the fluid secretions from these organs. In addition, both the human GCDFP-15/ hPIP (Schenkels et al., 1997) and more recently the mSMGP/mPIP (Lee et al., 2002) have been identified in saliva and shown to bind several species of oral and nonoral bacteria. Also, there are other known proteins such as salivary peroxidases (Yamashina and Barka, 1974; Kruse et al., 1998) which, like PIP, are expressed in both salivary and reproductive tissues and which have been shown to play a role in mucosal defense (Carlsson, 1987). These studies suggest that the mSMGP/mPIP may also have a similar function. In summary, we have shown that the mSMGP/mPIP gene is expressed in the submandibular gland in the developing embryo and in the major salivary glands and the reproductive organs during early postnatal development. The developmental and tissue specific pattern of the mSMGP/mPIP gene expression pattern, together
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with the identification of the protein in saliva and its ability to bind to bacteria (Lee et al., 2002) lend support to our hypothesis that mSMGP/mPIP may play a role in non-immune host defense. Further studies to determine which specific cell types express the mSMGP/mPIP gene in the reproductive tissues should give more insight into the different biological roles of the mSMGP/mPIP gene in different organs.
Acknowledgements The authors would like to thank Ms Barbara Iwasiow and Ms Pat Sheppard for technical assistance. This work was funded by NSERC (Y.M.).
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