The Putative Tumor Suppressor Deleted in Malignant Brain Tumors 1 Is an Estrogen-Regulated Gene in Rodent and Primate Endometrial Epithelium
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内分泌学杂志 2005年第3期
Reproductive Therapeutics (S.T., E.P., D.H.-J., J.-Z.G., S.L., G.A.), Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Raritan, New Jersey 08869; and Target Validation Team (D.L., M.R.D.), Johnson & Johnson Pharmaceutical Research and Development, Spring House, Pennsylvania 19477
Address all correspondence and requests for reprints to: George Allan, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Room B-115, 1000 US Route 202 South, P.O. Box 300, Raritan, New Jersey 08869. E-mail: gallan@prdus.jnj.com.
Abstract
Deleted in malignant brain tumors 1 (DMBT1) is a candidate suppressor of malignancies of the brain, lung, gut, and breast. We have been studying gene expression in the uterus in the presence of estrogens and their antagonists. Here, we show that DMBT1 RNA levels are robustly increased by estrogen treatment in the uteri of ovariectomized monkeys and rats. In monkeys, the progestin antagonist mifepristone inhibits estrogen-dependent uterine proliferation. As determined by a microarray experiment and quantitative analysis of RNA levels, mifepristone inhibited estrogenic induction of DMBT1. DMBT1 was not expressed in intact monkeys that were treated with a gonadotropin agonist to suppress steroidogenesis. An in vitro transfection study with human DMBT1 promoter constructs showed that an Alu site approximately 3000 nucleotides upstream of the gene mediates estrogenic regulation. Surprisingly, the estrogen antagonists tamoxifen, raloxifene, and ICI 182,780 also induced gene expression via this Alu site. Rodents represent a more convenient model system for studying uterine biology than monkeys. In rats, uterine DMBT1 RNA levels were dramatically up-regulated by estrogen. Consistent with the transfection study, tamoxifen and raloxifene increased DMBT1 RNA levels in vivo, but ICI 182,780 inhibited an estrogen-induced increase. Immunohistochemical studies showed that DMBT1 is specifically induced in glandular and luminal epithelia of the rat endometrium. Our experiments establish that DMBT1 is an estrogen-responsive gene with a possible role in endometrial proliferation or differentiation, and they have implications for the putative tumor suppressive and mucosal protective functions of DMBT1 in the uterus.
Introduction
THE UTERINE ENDOMETRIUM is subject to dynamic alterations in structural morphology in response to a changing hormonal milieu during the menstrual cycle. Structural changes in the endometrium occur to prepare for implantation and are accompanied by equally dramatic changes in gene expression (1, 2). Identification of the genes involved in endometrial turnover and implantation will have a range of applications, including the development of new diagnostic tools and therapies for endometriosis, endometrial cancer, and female infertility. Likewise, it will enable the identification of new drug discovery targets for these and other gynecological disorders, as well as for contraception and for hormone replacement.
The steroid hormones estrogen and progesterone are directly responsible for the gene expression changes that underlie endometrial reorganization. Disruption of estrogen or progestin action in the uterus is the mode of action of current treatments for endometriosis through regulation of the hypothalamic-pituitary-gonadal axis. This is also the basis for combined estrogen/progestin oral contraceptives (3, 4). Several steroidal and nonsteroidal estrogens and progestins are used clinically. Raloxifene is a selective estrogen receptor modulator (SERM) that is an estrogen antagonist in uterus and breast and an estrogen agonist in bone and is used for the prevention and treatment of osteoporosis. Tamoxifen is a SERM with estrogen antagonist activity in breast, but agonist activity in bone and uterus, that is used for breast cancer treatment. ICI 182,780 (Fulvestrant) is a pure in vivo estrogen antagonist that has been approved for treating tamoxifen-resistant breast cancer (5). Mifepristone is a progestin receptor antagonist that has proven effective at ameliorating the symptoms of endometriosis (6). Mifepristone has the interesting property of inhibiting estrogen activity in the primate uterus; thus, it blocks the proliferative activity of estrogen in the endometrium (7, 8, 9). This may explain the ability of mifepristone to reduce the sizes of endometriotic lesions. In addition, including mifepristone or other progestin antagonists in hormone replacement or contraceptive regimens may prevent unwanted stimulation of the endometrium by the estrogen component.
The deleted in malignant brain tumors 1 (DMBT1) gene was shown to be deleted or underexpressed in malignant gliomas, lung, gut, and breast cancers (10, 11, 12, 13, 14), although its designation as a tumor suppressor is controversial (15, 16). DMBT1 has respiratory tract and salivary variants also known as glycoprotein-340 (GP340) and salivary agglutinin (SAG) in humans. DMBT1 is known as hensin, ebnerin, or CRP-ductin in other mammalian species. It encodes a large secreted glycoprotein with 13 scavenger receptor cysteine-rich domains separated by serine- and threonine-rich interspersed domains, plus a pair of carboxyl-terminal C1r/C1s-Uegf-Bmp1 domains and a zona pellucida domain (10). A protein with this domain composition is likely to have extensive protein-protein interactions. DMBT1 has at least two functions that have been identified to date: 1) mucosal protection and immunity and 2) epithelial differentiation. Evidence for the former includes the fact that DMBT1 can bind to bacteria and interacts with the opsonin lung surfactant protein D, a mucosal lectin (17, 18, 19). DMBT1GP340 is secreted to the respiratory lumen (17) and DMBT1SAG is a component of saliva (20). Both of these proteins also bind to surfactant protein D. In addition, DMBT1 is expressed throughout the immune system and localizes to tumor-associated macrophages (21). On the other hand, DMBT1 is overexpressed in regenerating liver cells (22), whereas hensin can switch the polarity of collecting duct epithelial cells and induce terminal differentiation in the kidney (23). DMBT1, CRP-ductin, and hensin can also be secreted to the extracellular matrices of intestinal cells (21, 24), a location that is less consistent with immune protection than it is with epithelial differentiation and cell-extracellular matrix interactions. The functional duality of DMBT1 is reminiscent of the related Mac-2 binding protein and the unrelated but similar mucins (25).
Here, we show for the first time that DMBT1 is an estrogen-regulated gene. It is strongly estrogen-induced in both primate and rodent uterus, and the induced protein is localized to the epithelial layer of the endometrium. Our data reveal a new aspect to the biology of DMBT1, and they suggest that it may be involved in endometrial growth and/or differentiation.
Materials and Methods
Animal studies and RNA preparations
All animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult female cynomolgus monkeys (Macaca fascicularis) (Covance Research Products, Princeton, NJ) were ovariectomized (OVX) by a midventral laparotomy in the follicular phase of the menstrual cycle. After the 4th wk after ovariectomy, groups of three OVX monkeys were given an estrogen or placebo implant (vehicle control) for a 19-d treatment cycle. One group was also administered mifepristone (Sigma, St. Louis, MO) by oral gavage, at a daily dose of 10 mg/kg in 0.5% methylcellulose. An additional group of intact monkeys were given a gonadotropin agonist (Depo-Lupron, TAP Pharmaceuticals, Lake Forest, IL) on d 2 and 28 of the menstrual cycle and were killed on d 41. After treatment, the OVX monkeys were killed, and tissue biopsies were taken from the endometria. Total RNA was prepared from the biopsies using RNAzol (Tel-Test, Friendswood, TX), and an RNeasy Mini Kit (QIAGEN, Valencia, CA).
Approximately 22-d-old female Sprague Dawley rats (Charles River, Wilmington, MA) were treated for 3 d with vehicle, estrogen, and/or SERMs or an estrogen antagonist at various doses. Alternatively, 6-wk-old OVX Wistar rats (Charles River) were dosed for either 1 d or 6 wk with vehicle or test compounds. All treatments were delivered orally, once daily, in 0.5% methylcellulose or sesame oil. After treatment, the uteri were removed for immunohistochemistry. In addition, total RNA was purified from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) as directed by the manufacturer.
Microarray analysis
Chips were generated and hybridized by the Microarray Group, Johnson & Johnson Pharmaceutical Research and Development, L.L.C. (La Jolla, CA). Approximately 5000 cDNAs printed on the microarrays were from the Integrated Molecular Analysis of Genomes and their Expression (IMAGE) consortium and Incyte libraries. All clones were sequence-verified before PCR amplification. The IMAGE clones were purchased from the Human UniGene Library (Research Genetics, Huntsville, AL). To make the probe from the sample RNA, one round of T7 polymerase-based linear RNA amplification was performed by RT of RNA with a T7 promoter oligo(dT) primer, and Cy3-dCTP-labeled fluorescent cDNA probes were synthesized from the amplified RNA as described (26). The probes were heated to 95 C for 2 min, cooled, and applied to the slides. The slides were covered with glass coverslips, sealed, and hybridized at 42 C overnight. Microarrays were scanned with an Agilent G2565AA Microarray Scanner (Agilent Technologies, Palo Alto, CA). Fluorescence intensity for each feature of the array was obtained using Imagene 4.2 software (BioDiscovery, Los Angeles, CA). Each gene identifier was spotted twice on each chip, and duplicate chips were hybridized to each labeled sample. Thus, quadruplicate data points were generated for each gene identifier (n = 4). Normalized microarray spot intensities were analyzed to generate in silico electronic Northern comparisons using Excel for Windows (Microsoft, Seattle, WA) and GraphPad Prism (GraphPad Software, San Diego, CA).
Northern analysis
Two to five micrograms of total RNA was added to formaldehyde loading dye and separated on 1x 3[N-morpholino]propanesulfonic acid and 1.25% SeaKem Gold Agarose Reliant gels (BioWhittaker, Rockland, ME). The RNA was transferred to a Hybond-N membrane (Amersham, Piscataway, NJ), after which the membranes were stained with ethidium bromide and photographed for lane loading control. A partial rat DMBT1 clone (Incyte Genomics, Palo Alto, CA) was digested with EcoRI and NotI (Promega, Madison, WI) to release a 1.2-kbp DNA fragment. A random-primed probe was made from the purified DNA fragment. The blot was prehybridized in 10 ml RapidHyb (Amersham) at 65 C, then hybridized in 10 ml RapidHyb + 25 μl probe for 10 h at 65 C. After hybridization, the membrane was washed at 65 C with in 0.5x saline sodium phosphate EDTA buffer + 0.5% sodium dodecyl sulfate + 0.5% pyrophosphate. After the last wash, the membrane was exposed to a phosphor imaging screen. The phosphor screen was scanned with a STORM 840 (Amersham) using IQ Tools 2.2 software (Amersham). The hybridized probe was visualized and quantified with ImageQuant 5.2 (Amersham).
Quantitative PCR
Quantitative RT-PCR of DMBT1 from monkey, and rat RNA samples were carried out using a One-Step RT-PCR Master Mix Kit (Applied Biosystems, Foster City, CA) and an ABI Prism 7000 Sequence Detection System (Applied Biosystems), as described by the manufacturer. Commercially available primers and FAM-labeled MGB probes for human and rat DMBT1 were obtained from Applied Biosystems. mRNA levels were normalized against 18S ribosomal RNA levels determined using primers and a VIC- and TAMRA-labeled probe from the same vendor.
Luciferase reporter assays
HEK293 human embryonic kidney cells were seeded in assay medium (phenol red-free DMEM/F12, 10% charcoal/dextran-treated fetal bovine serum, penicillin/streptomycin) in 96-well culture dishes at 10,000 cells/well. One day later, cells were transfected with 10 ng/well reporter plasmid and 55 ng/well receptor plasmid (pcDNA3.1/hER or pcDNA3.1/hER?) using 1 μl/well Lipofectamine 2000 (Invitrogen). Transfection proceeded for approximately 16 h, after which the transfection mixture was replaced with assay medium containing vehicle or test compound at the appropriate concentration. After a 24-h incubation, Steady-Glo luciferase reagent (Promega) was added and luciferase activity was determined on an MLX Microtiter Plate Luminometer (Dynex Technologies, Chantilly, VA).
Plasmid constructs
DMBT#1.
An upstream region of the human DMBT1 gene from –1349 to +42 was cloned into the pGL3basic plasmid (Promega). This region of the DMBT1 promoter was amplified by touchdown PCR (27) from human genomic DNA (BD Biosciences, Palo Alto, CA). To facilitate molecular cloning, the restriction sites MluI and HindIII were incorporated into the primer sequences. The resulting PCR product was ligated to pGEM-T Easy (Promega), after which it was subcloned into pGL3basic.
DMBT#2.
A region spanning nucleotides –2923 to –926 of the DMBT1 promoter was amplified as above. The 2-kbp PCR product was ligated to pGEM-T Easy. The PCR product was removed from the pGEM-T vector by digestion with KpnI and subcloned into DMBT#1. The resulting subclones were screened for the correct orientation of the 2-kbp fragment by digestion with NdeI. The final DMBT#2 construct has a regulatory region consisting of nucleotides –2923 through +42 of the DMBT1 gene.
DMBT#3.
The pGEM-T Easy plasmid containing the DMBT1 PCR product spanning –2923 to –974 was digested with KpnI, and the resulting fragment was cloned into pTA-Luc (BD Biosciences).
Oligo.
An upstream region of the human DMBT1 gene from –2765 to –2733 was cloned into the pTA-Luc plasmid. Annealed complementary oligonucleotides were cloned into SmaI-digested pGL3promoter plasmid (Promega), after which the insert was removed by digestion with BglII and MluI and cloned into pTA-Luc.
All clones were verified by sequencing of PCR-generated regions. The construction of ER/pcDNA3.1, ER?/pcDNA3.1, and the vitellogenin ERE pTA-Luc reporter is described in Ref. 28 .
Immunohistochemistry
Tissues were trimmed and processed for paraffin embedding according to conventional methods. Five-micron sections were cut, mounted onto SuperFrost Plus+ (Fisher Scientific, Pittsburgh, PA) microscopic slides, and dried overnight. The protocols for routine single immunohistochemistry have been described previously (29). Briefly, tissue sections on microscopic slides were dewaxed and rehydrated. Slides were microwaved for 5 min in Target buffer (Dako, Carpenteria, CA), cooled, placed in PBS (pH 7.4) and treated with 3% (vol/vol) H2O2 for 10 min at room temperature. All incubations (30 min each) and washes were performed at room temperature. Normal rabbit blocking serum (Vector Laboratories, Burlingame, CA) was placed on all slides for 10 min. After a brief rinse in PBS, sections were treated with goat polyclonal DMBT1 primary antibody (1:25, Hypromatrix, Worcester, MA). Slides were then washed in PBS and treated with rabbit antigoat biotinylated secondary antibodies (Vector Laboratories). After washing in PBS, the avidin-biotin-horseradish peroxidase complex reagent (Vector Laboratories) was added. All slides were washed and treated with 3,3'-diaminobenzidine (Biomeda, Foster City, CA) two times for 5 min each, rinsed in distilled water, and counterstained with hematoxylin.
Results
DMBT1 RNA levels in monkey endometrium
A monkey study was performed to assess the effect of progesterone antagonists on endometrial proliferation in the presence of estrogen. Monkeys (three per group) were OVX and given subcutaneous implants of an estrogen or placebo capsule, then dosed for 19 d (two thirds of a regular menstrual cycle) with vehicle or mifepristone at 10 mg/kg. This dose has previously been shown to inhibit endometrial proliferation (7, 8, 9). An additional group was left intact but was given gonadotropin injections at the beginning and end of one menstrual cycle.
We were interested in examining changes in RNA levels in the uteri of these animals in response to estrogen and mifepristone. Microarrays containing approximately 5000 cDNAs were used to analyze RNA levels. Samples from three animals from each treatment group were hybridized to the microarrays. Using normalized hybridization intensities, data from estrogen-treated samples were compared with controls. The most highly up-regulated gene in all three animals was DMBT1, with an average increase in RNA level of 37-fold (data not shown). The background tends to be high in microarray experiments, so that the real message level of a gene is often underestimated (28). To verify these results, quantitative RT-PCR was performed on some of the RNA samples (Fig. 1). DMBT1 RNA levels were found to be increased several thousand-fold after estrogen treatment. Individual animals had DMBT1 RNA levels 2800-, 3200-, and 8800-fold higher than the vehicle control. Mifepristone prevented the increase in DMBT1 RNA levels in all three monkeys, as assessed by microarray analysis and PCR (Fig. 1). No DMBT1 RNA was detected in gonadotropin-treated monkey endometria.
FIG. 1. Induction of DMBT1 by estrogen in monkey endometrium as assessed by quantitative RT-PCR. Mean ± SD, n = 3 for OVX estrogen, n = 1 for OVX vehicle, and n = 2 for OVX E + mif 10 mg/kg·d and intact GnRH. Statistics: the OVX estrogen and E + mif groups were compared with the intact GnRH group using unpaired two-tailed Student’s t tests. **, P < 0.0001. E, Estrogen implant; mif, mifepristone; GnRH, gonadotropin.
Regulation of the DMBT1 gene by an Alu ERE
To identify an estrogen-responsive sequence in the human DMBT1 promoter, we cloned fragments of the promoter into a luciferase expression vector (Fig. 2A). DMBT#1, which includes the sequence from nucleotides –1349 through +42, did not show any estrogen responsiveness when transfected into human embryonic kidney (HEK293) cells in the presence of a human estrogen receptor expression vector (data not shown). An additional fragment was added to DMBT#1 to make DMBT#2, covering nucleotides –2923 through +42. Although DMBT#2 did not respond to estrogen, when we tested the partial sequence –2923 through –974 (DMBT#3), there was a 3-fold induction of luciferase expression by the steroid (Fig. 2B).
FIG. 2. Human DMBT1 is regulated by an Alu ERE. A, Luciferase reporter constructs of the DMBT1 promoter. The scale (in bases) is at the bottom, with the location of the Alu repeat represented by the black bar. B, Luciferase reporter assay of DMBT1 promoter fragments in HEK293 cells cotransfected with an estrogen receptor expression vector. All samples were normalized to DMBT#1/vehicle, except for the ERE reporter, which was normalized only to vehicle. Mean ± SD, n = 4. The data shown are representative of two experiments. Statistics: for each reporter, each group was compared with the vehicle control (top) or to the estradiol-treated group (bottom) using one-way ANOVA with Dunnett’s post test. #, P < 0.01. The ERE reporter has two copies of the vitellogenin gene ERE (28 ) in the plasmid pTA-Luc. C, Alignment of the DMBT1 Alu ERE (Oligo) with the consensus Alu ERE identified by Norris et al. (30 ) and the consensus classic ERE (45 ). Conserved half sites are in bold and underlined, with nonconserved nucleotides in lowercase. D, Luciferase assay of the oligo reporter (oligo) or the empty pTA-Luc reporter (pTA) in HEK293 cells cotransfected with an estrogen receptor () or estrogen receptor ? (?) expression vector. Concentrations of estradiol are in nanomolar. ERE, ERE pTA-Luc reporter. Mean ± SD, n = 4. The data shown are representative of two experiments. Statistics: the ERE reporter or the oligo reporter was compared with the empty pTA reporter at 100 nM estradiol for each receptor, using one-way ANOVA with Dunnett’s post test. #, P < 0.01; *, P < 0.05.
Sequence analysis of DMBT#3 indicated that the best ERE sequence was within an Alu repeat that is encompassed by this clone. Alu repeats containing EREs have been previously described, with a consensus sequence of GGTCAxxxxxxxxxGACCAxxxTGTCC (30). A similar sequence was found within this Alu repeat. An alignment of the consensus Alu ERE with that in DMBT#3, and the consensus ERE derived from the chicken vitellogenin gene is shown in Fig. 2C. The DMBT#3 Alu ERE sequence was cloned into a luciferase reporter vector to make the oligo construct (Fig. 2A), which was induced 8-fold by estrogen in the presence of cotransfected receptor (Fig. 2B). Neither DMBT#3 nor the oligo reporter was induced by estrogen in HEK293 cells in the absence of cotransfected receptor (data not shown).
The SERMs raloxifene and the estrogen antagonist ICI 182,780 antagonized estrogen induction via dual vitellogenin EREs, whereas tamoxifen acted as an agonist on this reporter (Fig. 2B). In contrast, all three of these compounds acted as agonists on DMBT#3 and the oligo reporter and had little activity on the DMBT#2 reporter.
Estradiol induced expression from the oligo reporter dose-dependently in the presence of estrogen receptor , but estrogen receptor ? lacked activity on this reporter (Fig. 2D). The empty pTA-Luc reporter was not induced by estrogen in the presence of either receptor.
DMBT1 RNA levels in rat uterus
The rat uterus is a convenient model system for analyzing estrogen-dependent gene expression changes. We used Northern blotting to determine whether DMBT1 is regulated in rat uteri as it is in the primate. RNA was prepared from uteri of immature rats that had been treated with 70 or 140 μg/kg·d estrone or vehicle for 3 d. Northern blot analysis using a rat DMBT1 cDNA probe showed that a high steady-state level of DMBT1 mRNA was induced in the rat uterus (Fig. 3A). Induction was maximal at the lowest dose tested, 70 μg/kg·d. In a separate experiment using OVX mature rats, ethinyl estradiol treatment for 1 d (RNA preparation approximately 24 h after a single dose) resulted in a significant increase in DMBT1 mRNA levels, as assessed by quantitative PCR (Fig. 3B). Induction was maximal (6-fold) at a dose of 0.3 mg/kg estradiol. Cotreatment with a progestin (medroxyprogesterone acetate) blocked the increase in mRNA levels in a dose-dependent manner, an effect that was inhibited in turn by 10 mg/kg of the progestin antagonist, mifepristone.
FIG. 3. DMBT1 is up-regulated in rat uterus. A, Northern blotting of RNA prepared from uteri of immature rats treated with estrone for 3 d was hybridized with a rat DMBT1 cDNA fragment. Each lane contains RNA from individual rats. The major DMBT1 RNA is indicated at the left; standards are indicated at the right. The lower panel shows ethidium bromide staining of the filter (28S) as a loading control. B, DMBT1 expression in rat uteri as assessed by quantitative RT-PCR. OVX mature rats were dosed for 1 d with the indicated compounds. The doses shown are in mg/kg·d. Mean ± SD, n = 3. Statistics: one-way ANOVA with Dunnett’s post test. #, P < 0.01, comparing the EE groups with vehicle. ##, P < 0.01, comparing the MPA groups to the 0.3 mg/kg·d EE group. EE, ethinyl estradiol; MPA, medroxyprogesterone acetate; Mif, mifepristone.
To determine the effects of SERMs on DMBT1 expression, groups of immature rats were treated with the SERMs tamoxifen and raloxifene or the ER antagonist ICI 182,780 (AstraZeneca, Waltham, MA), for 3 d. The resulting uterine weights at necropsy are shown in Fig. 4A. As expected, raloxifene and ICI 182,780 did not stimulate uterine weights, whereas tamoxifen tended to weakly stimulate it. In the presence of estrogen, all three compounds antagonized estrogen-dependent increases in tissue weight (data not shown). A Northern blot of DMBT1 with the RNA from these uteri (Fig. 4B) showed that tamoxifen and raloxifene both increased DMBT1 RNA levels to an extent similar to that of estrone, whereas ICI 182,780 did not increase them. Tamoxifen and raloxifene did not antagonize the increased RNA level induced by estrone, whereas the ICI compound did (data not shown). Thus, tamoxifen and raloxifene, but not ICI 182,780, are agonists of DMBT1 RNA levels in rat uterus.
FIG. 4. DMBT1 RNA level distinguishes between SERMs and complete antagonists. A, Uterine weights. Data shown are the means from three immature animals, with error bars representing SD values. Statistics: each group was compared with the vehicle group, using one-way ANOVA with Dunnett’s post test. #, P < 0.01; *, P < 0.05. Estrone was administered at 70 μg/kg·d, and tamoxifen, raloxifene, and ICI 182,780 were dosed at 1 mg/kg·d. B, Northern blot of RNA prepared from the uteri reported in A. The lower panel shows ethidium bromide staining of the filter (28S) as a loading control.
Immunohistochemical analysis of uterine expression
Mature, OVX, or sham-operated rats were dosed with ethinyl estradiol, tamoxifen, or raloxifene for 6 wk. Uteri were removed, sectioned, and stained for DMBT1 expression using a polyclonal goat antibody against the human protein (Fig. 5). Compared with the sham (Fig. 5A) and the OVX control (Fig. 5B), estrogen treatment produced the expected thickening of the luminal epithelium (Fig. 5C), which was even more pronounced after tamoxifen treatment (Fig. 5D) but not with raloxifene treatment (Fig. 5E). This effect on the epithelium correlated with gross effects on uterine weight (Fig. 4A). DMBT1 staining was restricted to the luminal epithelium, with little or no staining in glandular epithelia or in stromal cells. However, staining in the luminal epithelium was variable, with some cells intensely stained (arrows), others weakly stained, and others not stained at all. Raloxifene and especially tamoxifen appeared to produce higher cell levels of DMBT1 than estrogen. Staining was extra-nuclear in all cases. In sham-operated and OVX, estrogen-treated uterine epithelium, staining was predominantly at the luminal aspect of the cell (Fig. 5, A and C). In contrast, staining was uniformly distributed throughout positive cells in epithelium from SERM-treated uteri (Fig. 5, D and E).
FIG. 5. DMBT1 expression in rat uterine epithelium. Immunohistochemical analysis using a DMBT1 antibody. A, Sham-operated. B, OVX, vehicle control. C, OVX, 0.1 mg/kg ethinyl estradiol-treated. D, OVX, 1 mg/kg tamoxifen-treated. E, OVX, 1 mg/kg raloxifene-treated. The arrows show the presence of intense cytoplasmic DMBT1 labeling in several epithelial cells. Bar, 25 μm. All panels are at the same magnification.
Discussion
DMBT1 is a complex protein structurally and functionally. Here, we show that DMBT1 is expressed under the control of estrogen in uterine epithelium, a site with ongoing cycles of proliferation and differentiation and an important role in immune protection for its mucosal secretions.
DMBT1 is strongly up regulated in monkey endometrium and in rat uterine epithelium by estrogen (Figs. 1 and 3). DMBT1 has been shown previously to be expressed at a higher level in endometrium during the estrogen-dominant proliferative phase relative to the progesterone-dominant secretory phase in intact monkeys (31). Gene induction occurs rapidly in vivo, after 1 d of estrogen treatment in rats (Fig. 3B), and is inhibited dose-dependently by a progestin. The antiestrogenic effect of the progestin is inhibited by cotreatment of the rats with a progestin antagonist. These properties are seen with other estrogen-dependent marker genes, such as that for complement component 3 (32), and they suggest that DMBT1 expression tracks with proliferation of the uterine epithelium. Consistent with this, the antiproliferative compounds mifepristone and ICI 182,780 also inhibited estrogenic induction of DMBT1 in monkeys and rats, respectively (Fig. 1; data not shown). In addition, tamoxifen, a SERM that increases epithelial thickness in the human and rat uterus (33) (Fig. 5D) and is considered an estrogen agonist in that tissue, strongly stimulated DMBT1 expression (Fig. 5D). Raloxifene has no stimulatory activity in the human uterus, but does in rodents, where it moderately increases uterine weight (34) and epithelial cell thickness (Ref. 35 ; see also Fig. 5, compare B and E). Thus, induction of DMBT1 may be a marker of uterine epithelial stimulation by estrogen agonists and partial agonists.
The data reported suggest a few possible roles for the protein in the uterus. First, DMBT1 may play more of a role in supporting epithelial cell proliferation than has been postulated for other tissues. Alternatively, DMBT1 expression may be a preparatory signal for subsequent differentiation of the endometrium in the presence of progesterone. Others have shown that, in the gut, DMBT1 is localized in intracellular vesicles of less differentiated crypt cells and is found exclusively in the extracellular matrix of terminally differentiated villus cells (23). It would be worth determining the cellular location of DMBT1 in estrogen-treated epithelium; following the logic that estrogen-treated epithelium is less differentiated than progesterone-treated epithelium, the protein should be predominantly intracellular. It was not possible to determine this from our immunohistochemistry data, beyond noting that the protein appeared to be diffused throughout SERM-exposed cells (Fig. 5, D and E), and expressed at the luminal aspect of estrogen-treated cells (Fig. 5C).
Another possibility is that DMBT1 has a protective role in the uterus. Cervical mucus is important to the health of the reproductive tract, where it serves both to protect against infection and to facilitate the movement of sperm into the uterus (36). The components of cervical mucus are influenced by the hormonal milieu (37). It is not known whether DMBT1 is a component of uterine mucus, but it is present in lung secretions (17) and functional parallels between DMBT1 and mucins have been noted (25, 38). In lung, DMBT1 is known to have antibacterial properties (17, 18, 19, 20). Finally, it has been suggested that DMBT1 may be a local regulator of homeostasis in epithelial cells, linking mucosal inflammation to epithelial regeneration (25, 39). The functions proposed for DMBT1 in uterine mucosal immunity and epithelial turnover may not be mutually exclusive. Further work with knockout mice is being pursued to elucidate the function(s) of this protein in the reproductive tract.
An estrogen-regulated promoter element was identified upstream of the human DMBT1 gene (Fig. 2). The element was an Alu site, some of which have previously been shown to carry estrogen response elements (30). For example, an Alu sequence may mediate estrogenic regulation of the BRCA1 gene (30, 40). The DMBT1 promoter Alu ERE that we identified is close to the consensus Alu ERE identified by Norris et al. (30) (Fig. 2C). There are several additional Alu sites between 3,000 and 40,000 bases upstream of the DMBT1 transcription initiation site, some of which are related to the consensus Alu ERE. It is likely that other, yet-to-be-identified elements in the DMBT1 gene upstream region also play a role in estrogen regulation. In addition, a reporter (DMBT#3; Fig. 2A) that contains nearly 2,000 nucleotides, including the entire Alu sequence, was up-regulated 5-fold, whereas a reporter (DMBT#2) that contains the entire promoter region from nucleotide +42 through –2923 did not respond to estrogen. This suggests that, in addition to estrogen-responsive sequences, there are other regulatory elements that act to repress DMBT1 induction. This is consistent with a previous report of a silencing element between –1057 and –2109 (41). Finally, Alu sites are present in primates, but not in rodents (42). Therefore, estrogenic regulation of the rat DMBT1 gene must proceed by a different mechanism from the human gene. This is also true for the human BRCA1 gene, whose promoter contains Alu regulatory elements and is differently regulated in humans and rodents (30, 40).
Both tamoxifen and raloxifene acted as agonists on DMBT1 promoter reporters in transfected cells (Fig. 2B). This was consistent with their ability to increase uterine DMBT1 RNA levels and protein in rats (Figs. 3A and 5). In contrast, ICI 182,780 acted as an agonist in vitro, whereas it antagonized estrogen-dependent increases in DMBT1 RNA in rats. ICI 182,780 is a pure antagonist of estrogenic induction of ERE-based reporters in transfected cells but can induce expression from AP1- and cyclic AMP response element-based reporters (43). ICI 182,780 can act as an agonist on the glyceraldehyde-3-phosphate dehydrogenase gene in sheep endometrium (44). It is possible that the DMBT1 Alu sequence reveals a new aspect to regulation by this antagonist. Finally, the fact that the ICI compound is an antagonist of DMBT1 induction in vivo indicates that the transfection system does not replicate the full physiological activity of this compound, probably because we are using only a fragment of the gene’s regulatory sequences.
In conclusion, this work reveals a new aspect to the biology of DMBT1. Expression of the protein is strongly estrogen-dependent in uterine epithelia. Future work will address the roles of DMBT1 in endometrial proliferation and differentiation, as well as the implications of hormone regulation for tumor suppression in uterus.
Acknowledgments
We thank Gary Hodgen and Bob Williams (Eastern Virginia Medical School, Norfolk, VA) and Ralph Richart (Columbia University, New York, NY) for the monkey study, the La Jolla microarray group for the chip experiment, and Jan Mollenhauer (Deutsches Krebsforschungszentrum, Heidelburg, Germany) for helpful discussions and critical review of the manuscript. We also acknowledge the contributions of Drs. Joanna Clancy, Stephen Palmer, and James Hutchison to this work.
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Address all correspondence and requests for reprints to: George Allan, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Room B-115, 1000 US Route 202 South, P.O. Box 300, Raritan, New Jersey 08869. E-mail: gallan@prdus.jnj.com.
Abstract
Deleted in malignant brain tumors 1 (DMBT1) is a candidate suppressor of malignancies of the brain, lung, gut, and breast. We have been studying gene expression in the uterus in the presence of estrogens and their antagonists. Here, we show that DMBT1 RNA levels are robustly increased by estrogen treatment in the uteri of ovariectomized monkeys and rats. In monkeys, the progestin antagonist mifepristone inhibits estrogen-dependent uterine proliferation. As determined by a microarray experiment and quantitative analysis of RNA levels, mifepristone inhibited estrogenic induction of DMBT1. DMBT1 was not expressed in intact monkeys that were treated with a gonadotropin agonist to suppress steroidogenesis. An in vitro transfection study with human DMBT1 promoter constructs showed that an Alu site approximately 3000 nucleotides upstream of the gene mediates estrogenic regulation. Surprisingly, the estrogen antagonists tamoxifen, raloxifene, and ICI 182,780 also induced gene expression via this Alu site. Rodents represent a more convenient model system for studying uterine biology than monkeys. In rats, uterine DMBT1 RNA levels were dramatically up-regulated by estrogen. Consistent with the transfection study, tamoxifen and raloxifene increased DMBT1 RNA levels in vivo, but ICI 182,780 inhibited an estrogen-induced increase. Immunohistochemical studies showed that DMBT1 is specifically induced in glandular and luminal epithelia of the rat endometrium. Our experiments establish that DMBT1 is an estrogen-responsive gene with a possible role in endometrial proliferation or differentiation, and they have implications for the putative tumor suppressive and mucosal protective functions of DMBT1 in the uterus.
Introduction
THE UTERINE ENDOMETRIUM is subject to dynamic alterations in structural morphology in response to a changing hormonal milieu during the menstrual cycle. Structural changes in the endometrium occur to prepare for implantation and are accompanied by equally dramatic changes in gene expression (1, 2). Identification of the genes involved in endometrial turnover and implantation will have a range of applications, including the development of new diagnostic tools and therapies for endometriosis, endometrial cancer, and female infertility. Likewise, it will enable the identification of new drug discovery targets for these and other gynecological disorders, as well as for contraception and for hormone replacement.
The steroid hormones estrogen and progesterone are directly responsible for the gene expression changes that underlie endometrial reorganization. Disruption of estrogen or progestin action in the uterus is the mode of action of current treatments for endometriosis through regulation of the hypothalamic-pituitary-gonadal axis. This is also the basis for combined estrogen/progestin oral contraceptives (3, 4). Several steroidal and nonsteroidal estrogens and progestins are used clinically. Raloxifene is a selective estrogen receptor modulator (SERM) that is an estrogen antagonist in uterus and breast and an estrogen agonist in bone and is used for the prevention and treatment of osteoporosis. Tamoxifen is a SERM with estrogen antagonist activity in breast, but agonist activity in bone and uterus, that is used for breast cancer treatment. ICI 182,780 (Fulvestrant) is a pure in vivo estrogen antagonist that has been approved for treating tamoxifen-resistant breast cancer (5). Mifepristone is a progestin receptor antagonist that has proven effective at ameliorating the symptoms of endometriosis (6). Mifepristone has the interesting property of inhibiting estrogen activity in the primate uterus; thus, it blocks the proliferative activity of estrogen in the endometrium (7, 8, 9). This may explain the ability of mifepristone to reduce the sizes of endometriotic lesions. In addition, including mifepristone or other progestin antagonists in hormone replacement or contraceptive regimens may prevent unwanted stimulation of the endometrium by the estrogen component.
The deleted in malignant brain tumors 1 (DMBT1) gene was shown to be deleted or underexpressed in malignant gliomas, lung, gut, and breast cancers (10, 11, 12, 13, 14), although its designation as a tumor suppressor is controversial (15, 16). DMBT1 has respiratory tract and salivary variants also known as glycoprotein-340 (GP340) and salivary agglutinin (SAG) in humans. DMBT1 is known as hensin, ebnerin, or CRP-ductin in other mammalian species. It encodes a large secreted glycoprotein with 13 scavenger receptor cysteine-rich domains separated by serine- and threonine-rich interspersed domains, plus a pair of carboxyl-terminal C1r/C1s-Uegf-Bmp1 domains and a zona pellucida domain (10). A protein with this domain composition is likely to have extensive protein-protein interactions. DMBT1 has at least two functions that have been identified to date: 1) mucosal protection and immunity and 2) epithelial differentiation. Evidence for the former includes the fact that DMBT1 can bind to bacteria and interacts with the opsonin lung surfactant protein D, a mucosal lectin (17, 18, 19). DMBT1GP340 is secreted to the respiratory lumen (17) and DMBT1SAG is a component of saliva (20). Both of these proteins also bind to surfactant protein D. In addition, DMBT1 is expressed throughout the immune system and localizes to tumor-associated macrophages (21). On the other hand, DMBT1 is overexpressed in regenerating liver cells (22), whereas hensin can switch the polarity of collecting duct epithelial cells and induce terminal differentiation in the kidney (23). DMBT1, CRP-ductin, and hensin can also be secreted to the extracellular matrices of intestinal cells (21, 24), a location that is less consistent with immune protection than it is with epithelial differentiation and cell-extracellular matrix interactions. The functional duality of DMBT1 is reminiscent of the related Mac-2 binding protein and the unrelated but similar mucins (25).
Here, we show for the first time that DMBT1 is an estrogen-regulated gene. It is strongly estrogen-induced in both primate and rodent uterus, and the induced protein is localized to the epithelial layer of the endometrium. Our data reveal a new aspect to the biology of DMBT1, and they suggest that it may be involved in endometrial growth and/or differentiation.
Materials and Methods
Animal studies and RNA preparations
All animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult female cynomolgus monkeys (Macaca fascicularis) (Covance Research Products, Princeton, NJ) were ovariectomized (OVX) by a midventral laparotomy in the follicular phase of the menstrual cycle. After the 4th wk after ovariectomy, groups of three OVX monkeys were given an estrogen or placebo implant (vehicle control) for a 19-d treatment cycle. One group was also administered mifepristone (Sigma, St. Louis, MO) by oral gavage, at a daily dose of 10 mg/kg in 0.5% methylcellulose. An additional group of intact monkeys were given a gonadotropin agonist (Depo-Lupron, TAP Pharmaceuticals, Lake Forest, IL) on d 2 and 28 of the menstrual cycle and were killed on d 41. After treatment, the OVX monkeys were killed, and tissue biopsies were taken from the endometria. Total RNA was prepared from the biopsies using RNAzol (Tel-Test, Friendswood, TX), and an RNeasy Mini Kit (QIAGEN, Valencia, CA).
Approximately 22-d-old female Sprague Dawley rats (Charles River, Wilmington, MA) were treated for 3 d with vehicle, estrogen, and/or SERMs or an estrogen antagonist at various doses. Alternatively, 6-wk-old OVX Wistar rats (Charles River) were dosed for either 1 d or 6 wk with vehicle or test compounds. All treatments were delivered orally, once daily, in 0.5% methylcellulose or sesame oil. After treatment, the uteri were removed for immunohistochemistry. In addition, total RNA was purified from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) as directed by the manufacturer.
Microarray analysis
Chips were generated and hybridized by the Microarray Group, Johnson & Johnson Pharmaceutical Research and Development, L.L.C. (La Jolla, CA). Approximately 5000 cDNAs printed on the microarrays were from the Integrated Molecular Analysis of Genomes and their Expression (IMAGE) consortium and Incyte libraries. All clones were sequence-verified before PCR amplification. The IMAGE clones were purchased from the Human UniGene Library (Research Genetics, Huntsville, AL). To make the probe from the sample RNA, one round of T7 polymerase-based linear RNA amplification was performed by RT of RNA with a T7 promoter oligo(dT) primer, and Cy3-dCTP-labeled fluorescent cDNA probes were synthesized from the amplified RNA as described (26). The probes were heated to 95 C for 2 min, cooled, and applied to the slides. The slides were covered with glass coverslips, sealed, and hybridized at 42 C overnight. Microarrays were scanned with an Agilent G2565AA Microarray Scanner (Agilent Technologies, Palo Alto, CA). Fluorescence intensity for each feature of the array was obtained using Imagene 4.2 software (BioDiscovery, Los Angeles, CA). Each gene identifier was spotted twice on each chip, and duplicate chips were hybridized to each labeled sample. Thus, quadruplicate data points were generated for each gene identifier (n = 4). Normalized microarray spot intensities were analyzed to generate in silico electronic Northern comparisons using Excel for Windows (Microsoft, Seattle, WA) and GraphPad Prism (GraphPad Software, San Diego, CA).
Northern analysis
Two to five micrograms of total RNA was added to formaldehyde loading dye and separated on 1x 3[N-morpholino]propanesulfonic acid and 1.25% SeaKem Gold Agarose Reliant gels (BioWhittaker, Rockland, ME). The RNA was transferred to a Hybond-N membrane (Amersham, Piscataway, NJ), after which the membranes were stained with ethidium bromide and photographed for lane loading control. A partial rat DMBT1 clone (Incyte Genomics, Palo Alto, CA) was digested with EcoRI and NotI (Promega, Madison, WI) to release a 1.2-kbp DNA fragment. A random-primed probe was made from the purified DNA fragment. The blot was prehybridized in 10 ml RapidHyb (Amersham) at 65 C, then hybridized in 10 ml RapidHyb + 25 μl probe for 10 h at 65 C. After hybridization, the membrane was washed at 65 C with in 0.5x saline sodium phosphate EDTA buffer + 0.5% sodium dodecyl sulfate + 0.5% pyrophosphate. After the last wash, the membrane was exposed to a phosphor imaging screen. The phosphor screen was scanned with a STORM 840 (Amersham) using IQ Tools 2.2 software (Amersham). The hybridized probe was visualized and quantified with ImageQuant 5.2 (Amersham).
Quantitative PCR
Quantitative RT-PCR of DMBT1 from monkey, and rat RNA samples were carried out using a One-Step RT-PCR Master Mix Kit (Applied Biosystems, Foster City, CA) and an ABI Prism 7000 Sequence Detection System (Applied Biosystems), as described by the manufacturer. Commercially available primers and FAM-labeled MGB probes for human and rat DMBT1 were obtained from Applied Biosystems. mRNA levels were normalized against 18S ribosomal RNA levels determined using primers and a VIC- and TAMRA-labeled probe from the same vendor.
Luciferase reporter assays
HEK293 human embryonic kidney cells were seeded in assay medium (phenol red-free DMEM/F12, 10% charcoal/dextran-treated fetal bovine serum, penicillin/streptomycin) in 96-well culture dishes at 10,000 cells/well. One day later, cells were transfected with 10 ng/well reporter plasmid and 55 ng/well receptor plasmid (pcDNA3.1/hER or pcDNA3.1/hER?) using 1 μl/well Lipofectamine 2000 (Invitrogen). Transfection proceeded for approximately 16 h, after which the transfection mixture was replaced with assay medium containing vehicle or test compound at the appropriate concentration. After a 24-h incubation, Steady-Glo luciferase reagent (Promega) was added and luciferase activity was determined on an MLX Microtiter Plate Luminometer (Dynex Technologies, Chantilly, VA).
Plasmid constructs
DMBT#1.
An upstream region of the human DMBT1 gene from –1349 to +42 was cloned into the pGL3basic plasmid (Promega). This region of the DMBT1 promoter was amplified by touchdown PCR (27) from human genomic DNA (BD Biosciences, Palo Alto, CA). To facilitate molecular cloning, the restriction sites MluI and HindIII were incorporated into the primer sequences. The resulting PCR product was ligated to pGEM-T Easy (Promega), after which it was subcloned into pGL3basic.
DMBT#2.
A region spanning nucleotides –2923 to –926 of the DMBT1 promoter was amplified as above. The 2-kbp PCR product was ligated to pGEM-T Easy. The PCR product was removed from the pGEM-T vector by digestion with KpnI and subcloned into DMBT#1. The resulting subclones were screened for the correct orientation of the 2-kbp fragment by digestion with NdeI. The final DMBT#2 construct has a regulatory region consisting of nucleotides –2923 through +42 of the DMBT1 gene.
DMBT#3.
The pGEM-T Easy plasmid containing the DMBT1 PCR product spanning –2923 to –974 was digested with KpnI, and the resulting fragment was cloned into pTA-Luc (BD Biosciences).
Oligo.
An upstream region of the human DMBT1 gene from –2765 to –2733 was cloned into the pTA-Luc plasmid. Annealed complementary oligonucleotides were cloned into SmaI-digested pGL3promoter plasmid (Promega), after which the insert was removed by digestion with BglII and MluI and cloned into pTA-Luc.
All clones were verified by sequencing of PCR-generated regions. The construction of ER/pcDNA3.1, ER?/pcDNA3.1, and the vitellogenin ERE pTA-Luc reporter is described in Ref. 28 .
Immunohistochemistry
Tissues were trimmed and processed for paraffin embedding according to conventional methods. Five-micron sections were cut, mounted onto SuperFrost Plus+ (Fisher Scientific, Pittsburgh, PA) microscopic slides, and dried overnight. The protocols for routine single immunohistochemistry have been described previously (29). Briefly, tissue sections on microscopic slides were dewaxed and rehydrated. Slides were microwaved for 5 min in Target buffer (Dako, Carpenteria, CA), cooled, placed in PBS (pH 7.4) and treated with 3% (vol/vol) H2O2 for 10 min at room temperature. All incubations (30 min each) and washes were performed at room temperature. Normal rabbit blocking serum (Vector Laboratories, Burlingame, CA) was placed on all slides for 10 min. After a brief rinse in PBS, sections were treated with goat polyclonal DMBT1 primary antibody (1:25, Hypromatrix, Worcester, MA). Slides were then washed in PBS and treated with rabbit antigoat biotinylated secondary antibodies (Vector Laboratories). After washing in PBS, the avidin-biotin-horseradish peroxidase complex reagent (Vector Laboratories) was added. All slides were washed and treated with 3,3'-diaminobenzidine (Biomeda, Foster City, CA) two times for 5 min each, rinsed in distilled water, and counterstained with hematoxylin.
Results
DMBT1 RNA levels in monkey endometrium
A monkey study was performed to assess the effect of progesterone antagonists on endometrial proliferation in the presence of estrogen. Monkeys (three per group) were OVX and given subcutaneous implants of an estrogen or placebo capsule, then dosed for 19 d (two thirds of a regular menstrual cycle) with vehicle or mifepristone at 10 mg/kg. This dose has previously been shown to inhibit endometrial proliferation (7, 8, 9). An additional group was left intact but was given gonadotropin injections at the beginning and end of one menstrual cycle.
We were interested in examining changes in RNA levels in the uteri of these animals in response to estrogen and mifepristone. Microarrays containing approximately 5000 cDNAs were used to analyze RNA levels. Samples from three animals from each treatment group were hybridized to the microarrays. Using normalized hybridization intensities, data from estrogen-treated samples were compared with controls. The most highly up-regulated gene in all three animals was DMBT1, with an average increase in RNA level of 37-fold (data not shown). The background tends to be high in microarray experiments, so that the real message level of a gene is often underestimated (28). To verify these results, quantitative RT-PCR was performed on some of the RNA samples (Fig. 1). DMBT1 RNA levels were found to be increased several thousand-fold after estrogen treatment. Individual animals had DMBT1 RNA levels 2800-, 3200-, and 8800-fold higher than the vehicle control. Mifepristone prevented the increase in DMBT1 RNA levels in all three monkeys, as assessed by microarray analysis and PCR (Fig. 1). No DMBT1 RNA was detected in gonadotropin-treated monkey endometria.
FIG. 1. Induction of DMBT1 by estrogen in monkey endometrium as assessed by quantitative RT-PCR. Mean ± SD, n = 3 for OVX estrogen, n = 1 for OVX vehicle, and n = 2 for OVX E + mif 10 mg/kg·d and intact GnRH. Statistics: the OVX estrogen and E + mif groups were compared with the intact GnRH group using unpaired two-tailed Student’s t tests. **, P < 0.0001. E, Estrogen implant; mif, mifepristone; GnRH, gonadotropin.
Regulation of the DMBT1 gene by an Alu ERE
To identify an estrogen-responsive sequence in the human DMBT1 promoter, we cloned fragments of the promoter into a luciferase expression vector (Fig. 2A). DMBT#1, which includes the sequence from nucleotides –1349 through +42, did not show any estrogen responsiveness when transfected into human embryonic kidney (HEK293) cells in the presence of a human estrogen receptor expression vector (data not shown). An additional fragment was added to DMBT#1 to make DMBT#2, covering nucleotides –2923 through +42. Although DMBT#2 did not respond to estrogen, when we tested the partial sequence –2923 through –974 (DMBT#3), there was a 3-fold induction of luciferase expression by the steroid (Fig. 2B).
FIG. 2. Human DMBT1 is regulated by an Alu ERE. A, Luciferase reporter constructs of the DMBT1 promoter. The scale (in bases) is at the bottom, with the location of the Alu repeat represented by the black bar. B, Luciferase reporter assay of DMBT1 promoter fragments in HEK293 cells cotransfected with an estrogen receptor expression vector. All samples were normalized to DMBT#1/vehicle, except for the ERE reporter, which was normalized only to vehicle. Mean ± SD, n = 4. The data shown are representative of two experiments. Statistics: for each reporter, each group was compared with the vehicle control (top) or to the estradiol-treated group (bottom) using one-way ANOVA with Dunnett’s post test. #, P < 0.01. The ERE reporter has two copies of the vitellogenin gene ERE (28 ) in the plasmid pTA-Luc. C, Alignment of the DMBT1 Alu ERE (Oligo) with the consensus Alu ERE identified by Norris et al. (30 ) and the consensus classic ERE (45 ). Conserved half sites are in bold and underlined, with nonconserved nucleotides in lowercase. D, Luciferase assay of the oligo reporter (oligo) or the empty pTA-Luc reporter (pTA) in HEK293 cells cotransfected with an estrogen receptor () or estrogen receptor ? (?) expression vector. Concentrations of estradiol are in nanomolar. ERE, ERE pTA-Luc reporter. Mean ± SD, n = 4. The data shown are representative of two experiments. Statistics: the ERE reporter or the oligo reporter was compared with the empty pTA reporter at 100 nM estradiol for each receptor, using one-way ANOVA with Dunnett’s post test. #, P < 0.01; *, P < 0.05.
Sequence analysis of DMBT#3 indicated that the best ERE sequence was within an Alu repeat that is encompassed by this clone. Alu repeats containing EREs have been previously described, with a consensus sequence of GGTCAxxxxxxxxxGACCAxxxTGTCC (30). A similar sequence was found within this Alu repeat. An alignment of the consensus Alu ERE with that in DMBT#3, and the consensus ERE derived from the chicken vitellogenin gene is shown in Fig. 2C. The DMBT#3 Alu ERE sequence was cloned into a luciferase reporter vector to make the oligo construct (Fig. 2A), which was induced 8-fold by estrogen in the presence of cotransfected receptor (Fig. 2B). Neither DMBT#3 nor the oligo reporter was induced by estrogen in HEK293 cells in the absence of cotransfected receptor (data not shown).
The SERMs raloxifene and the estrogen antagonist ICI 182,780 antagonized estrogen induction via dual vitellogenin EREs, whereas tamoxifen acted as an agonist on this reporter (Fig. 2B). In contrast, all three of these compounds acted as agonists on DMBT#3 and the oligo reporter and had little activity on the DMBT#2 reporter.
Estradiol induced expression from the oligo reporter dose-dependently in the presence of estrogen receptor , but estrogen receptor ? lacked activity on this reporter (Fig. 2D). The empty pTA-Luc reporter was not induced by estrogen in the presence of either receptor.
DMBT1 RNA levels in rat uterus
The rat uterus is a convenient model system for analyzing estrogen-dependent gene expression changes. We used Northern blotting to determine whether DMBT1 is regulated in rat uteri as it is in the primate. RNA was prepared from uteri of immature rats that had been treated with 70 or 140 μg/kg·d estrone or vehicle for 3 d. Northern blot analysis using a rat DMBT1 cDNA probe showed that a high steady-state level of DMBT1 mRNA was induced in the rat uterus (Fig. 3A). Induction was maximal at the lowest dose tested, 70 μg/kg·d. In a separate experiment using OVX mature rats, ethinyl estradiol treatment for 1 d (RNA preparation approximately 24 h after a single dose) resulted in a significant increase in DMBT1 mRNA levels, as assessed by quantitative PCR (Fig. 3B). Induction was maximal (6-fold) at a dose of 0.3 mg/kg estradiol. Cotreatment with a progestin (medroxyprogesterone acetate) blocked the increase in mRNA levels in a dose-dependent manner, an effect that was inhibited in turn by 10 mg/kg of the progestin antagonist, mifepristone.
FIG. 3. DMBT1 is up-regulated in rat uterus. A, Northern blotting of RNA prepared from uteri of immature rats treated with estrone for 3 d was hybridized with a rat DMBT1 cDNA fragment. Each lane contains RNA from individual rats. The major DMBT1 RNA is indicated at the left; standards are indicated at the right. The lower panel shows ethidium bromide staining of the filter (28S) as a loading control. B, DMBT1 expression in rat uteri as assessed by quantitative RT-PCR. OVX mature rats were dosed for 1 d with the indicated compounds. The doses shown are in mg/kg·d. Mean ± SD, n = 3. Statistics: one-way ANOVA with Dunnett’s post test. #, P < 0.01, comparing the EE groups with vehicle. ##, P < 0.01, comparing the MPA groups to the 0.3 mg/kg·d EE group. EE, ethinyl estradiol; MPA, medroxyprogesterone acetate; Mif, mifepristone.
To determine the effects of SERMs on DMBT1 expression, groups of immature rats were treated with the SERMs tamoxifen and raloxifene or the ER antagonist ICI 182,780 (AstraZeneca, Waltham, MA), for 3 d. The resulting uterine weights at necropsy are shown in Fig. 4A. As expected, raloxifene and ICI 182,780 did not stimulate uterine weights, whereas tamoxifen tended to weakly stimulate it. In the presence of estrogen, all three compounds antagonized estrogen-dependent increases in tissue weight (data not shown). A Northern blot of DMBT1 with the RNA from these uteri (Fig. 4B) showed that tamoxifen and raloxifene both increased DMBT1 RNA levels to an extent similar to that of estrone, whereas ICI 182,780 did not increase them. Tamoxifen and raloxifene did not antagonize the increased RNA level induced by estrone, whereas the ICI compound did (data not shown). Thus, tamoxifen and raloxifene, but not ICI 182,780, are agonists of DMBT1 RNA levels in rat uterus.
FIG. 4. DMBT1 RNA level distinguishes between SERMs and complete antagonists. A, Uterine weights. Data shown are the means from three immature animals, with error bars representing SD values. Statistics: each group was compared with the vehicle group, using one-way ANOVA with Dunnett’s post test. #, P < 0.01; *, P < 0.05. Estrone was administered at 70 μg/kg·d, and tamoxifen, raloxifene, and ICI 182,780 were dosed at 1 mg/kg·d. B, Northern blot of RNA prepared from the uteri reported in A. The lower panel shows ethidium bromide staining of the filter (28S) as a loading control.
Immunohistochemical analysis of uterine expression
Mature, OVX, or sham-operated rats were dosed with ethinyl estradiol, tamoxifen, or raloxifene for 6 wk. Uteri were removed, sectioned, and stained for DMBT1 expression using a polyclonal goat antibody against the human protein (Fig. 5). Compared with the sham (Fig. 5A) and the OVX control (Fig. 5B), estrogen treatment produced the expected thickening of the luminal epithelium (Fig. 5C), which was even more pronounced after tamoxifen treatment (Fig. 5D) but not with raloxifene treatment (Fig. 5E). This effect on the epithelium correlated with gross effects on uterine weight (Fig. 4A). DMBT1 staining was restricted to the luminal epithelium, with little or no staining in glandular epithelia or in stromal cells. However, staining in the luminal epithelium was variable, with some cells intensely stained (arrows), others weakly stained, and others not stained at all. Raloxifene and especially tamoxifen appeared to produce higher cell levels of DMBT1 than estrogen. Staining was extra-nuclear in all cases. In sham-operated and OVX, estrogen-treated uterine epithelium, staining was predominantly at the luminal aspect of the cell (Fig. 5, A and C). In contrast, staining was uniformly distributed throughout positive cells in epithelium from SERM-treated uteri (Fig. 5, D and E).
FIG. 5. DMBT1 expression in rat uterine epithelium. Immunohistochemical analysis using a DMBT1 antibody. A, Sham-operated. B, OVX, vehicle control. C, OVX, 0.1 mg/kg ethinyl estradiol-treated. D, OVX, 1 mg/kg tamoxifen-treated. E, OVX, 1 mg/kg raloxifene-treated. The arrows show the presence of intense cytoplasmic DMBT1 labeling in several epithelial cells. Bar, 25 μm. All panels are at the same magnification.
Discussion
DMBT1 is a complex protein structurally and functionally. Here, we show that DMBT1 is expressed under the control of estrogen in uterine epithelium, a site with ongoing cycles of proliferation and differentiation and an important role in immune protection for its mucosal secretions.
DMBT1 is strongly up regulated in monkey endometrium and in rat uterine epithelium by estrogen (Figs. 1 and 3). DMBT1 has been shown previously to be expressed at a higher level in endometrium during the estrogen-dominant proliferative phase relative to the progesterone-dominant secretory phase in intact monkeys (31). Gene induction occurs rapidly in vivo, after 1 d of estrogen treatment in rats (Fig. 3B), and is inhibited dose-dependently by a progestin. The antiestrogenic effect of the progestin is inhibited by cotreatment of the rats with a progestin antagonist. These properties are seen with other estrogen-dependent marker genes, such as that for complement component 3 (32), and they suggest that DMBT1 expression tracks with proliferation of the uterine epithelium. Consistent with this, the antiproliferative compounds mifepristone and ICI 182,780 also inhibited estrogenic induction of DMBT1 in monkeys and rats, respectively (Fig. 1; data not shown). In addition, tamoxifen, a SERM that increases epithelial thickness in the human and rat uterus (33) (Fig. 5D) and is considered an estrogen agonist in that tissue, strongly stimulated DMBT1 expression (Fig. 5D). Raloxifene has no stimulatory activity in the human uterus, but does in rodents, where it moderately increases uterine weight (34) and epithelial cell thickness (Ref. 35 ; see also Fig. 5, compare B and E). Thus, induction of DMBT1 may be a marker of uterine epithelial stimulation by estrogen agonists and partial agonists.
The data reported suggest a few possible roles for the protein in the uterus. First, DMBT1 may play more of a role in supporting epithelial cell proliferation than has been postulated for other tissues. Alternatively, DMBT1 expression may be a preparatory signal for subsequent differentiation of the endometrium in the presence of progesterone. Others have shown that, in the gut, DMBT1 is localized in intracellular vesicles of less differentiated crypt cells and is found exclusively in the extracellular matrix of terminally differentiated villus cells (23). It would be worth determining the cellular location of DMBT1 in estrogen-treated epithelium; following the logic that estrogen-treated epithelium is less differentiated than progesterone-treated epithelium, the protein should be predominantly intracellular. It was not possible to determine this from our immunohistochemistry data, beyond noting that the protein appeared to be diffused throughout SERM-exposed cells (Fig. 5, D and E), and expressed at the luminal aspect of estrogen-treated cells (Fig. 5C).
Another possibility is that DMBT1 has a protective role in the uterus. Cervical mucus is important to the health of the reproductive tract, where it serves both to protect against infection and to facilitate the movement of sperm into the uterus (36). The components of cervical mucus are influenced by the hormonal milieu (37). It is not known whether DMBT1 is a component of uterine mucus, but it is present in lung secretions (17) and functional parallels between DMBT1 and mucins have been noted (25, 38). In lung, DMBT1 is known to have antibacterial properties (17, 18, 19, 20). Finally, it has been suggested that DMBT1 may be a local regulator of homeostasis in epithelial cells, linking mucosal inflammation to epithelial regeneration (25, 39). The functions proposed for DMBT1 in uterine mucosal immunity and epithelial turnover may not be mutually exclusive. Further work with knockout mice is being pursued to elucidate the function(s) of this protein in the reproductive tract.
An estrogen-regulated promoter element was identified upstream of the human DMBT1 gene (Fig. 2). The element was an Alu site, some of which have previously been shown to carry estrogen response elements (30). For example, an Alu sequence may mediate estrogenic regulation of the BRCA1 gene (30, 40). The DMBT1 promoter Alu ERE that we identified is close to the consensus Alu ERE identified by Norris et al. (30) (Fig. 2C). There are several additional Alu sites between 3,000 and 40,000 bases upstream of the DMBT1 transcription initiation site, some of which are related to the consensus Alu ERE. It is likely that other, yet-to-be-identified elements in the DMBT1 gene upstream region also play a role in estrogen regulation. In addition, a reporter (DMBT#3; Fig. 2A) that contains nearly 2,000 nucleotides, including the entire Alu sequence, was up-regulated 5-fold, whereas a reporter (DMBT#2) that contains the entire promoter region from nucleotide +42 through –2923 did not respond to estrogen. This suggests that, in addition to estrogen-responsive sequences, there are other regulatory elements that act to repress DMBT1 induction. This is consistent with a previous report of a silencing element between –1057 and –2109 (41). Finally, Alu sites are present in primates, but not in rodents (42). Therefore, estrogenic regulation of the rat DMBT1 gene must proceed by a different mechanism from the human gene. This is also true for the human BRCA1 gene, whose promoter contains Alu regulatory elements and is differently regulated in humans and rodents (30, 40).
Both tamoxifen and raloxifene acted as agonists on DMBT1 promoter reporters in transfected cells (Fig. 2B). This was consistent with their ability to increase uterine DMBT1 RNA levels and protein in rats (Figs. 3A and 5). In contrast, ICI 182,780 acted as an agonist in vitro, whereas it antagonized estrogen-dependent increases in DMBT1 RNA in rats. ICI 182,780 is a pure antagonist of estrogenic induction of ERE-based reporters in transfected cells but can induce expression from AP1- and cyclic AMP response element-based reporters (43). ICI 182,780 can act as an agonist on the glyceraldehyde-3-phosphate dehydrogenase gene in sheep endometrium (44). It is possible that the DMBT1 Alu sequence reveals a new aspect to regulation by this antagonist. Finally, the fact that the ICI compound is an antagonist of DMBT1 induction in vivo indicates that the transfection system does not replicate the full physiological activity of this compound, probably because we are using only a fragment of the gene’s regulatory sequences.
In conclusion, this work reveals a new aspect to the biology of DMBT1. Expression of the protein is strongly estrogen-dependent in uterine epithelia. Future work will address the roles of DMBT1 in endometrial proliferation and differentiation, as well as the implications of hormone regulation for tumor suppression in uterus.
Acknowledgments
We thank Gary Hodgen and Bob Williams (Eastern Virginia Medical School, Norfolk, VA) and Ralph Richart (Columbia University, New York, NY) for the monkey study, the La Jolla microarray group for the chip experiment, and Jan Mollenhauer (Deutsches Krebsforschungszentrum, Heidelburg, Germany) for helpful discussions and critical review of the manuscript. We also acknowledge the contributions of Drs. Joanna Clancy, Stephen Palmer, and James Hutchison to this work.
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