Lactoferrin gene expression is estrogen ... - Semantic Scholar

Molecular Human Reproduction Vol.8, No.1 pp. 58–67, 2002

Lactoferrin gene expression is estrogen responsive in human and rhesus monkey endometrium Christina T.Teng1,5, Wesley Gladwell1, Clara Beard1, David Walmer1,2, Ching S.Teng3 and Robert Brenner4 1Gene

Regulation Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, 2Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Duke University Medical Center, Durham, NC 27710, 3Department of APR, School of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina and 4Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, OR 97006, USA 5To

whom correspondence should be addressed at: P.O.Box 12233, MD E2-01, RTP, NC 27709, USA. E-mail: [email protected]

We have previously shown that the estrogen responsiveness of the human lactoferrin gene in a transient transfection system is mediated through an imperfect estrogen response element (ERE) and a steroidogenic factor 1 binding element (SFRE) 26 bp upstream from ERE. Reporter constructs containing SFRE and ERE respond to estrogen stimulation in a dose-dependent manner, whereas mutations at either one of the response elements severely impaired the estrogen responsiveness. In this study, we demonstrated that estrogen receptor (ERα) binds to the human lactoferrin gene ERE and forms two complexes in an electrophoresis mobility shift assay (EMSA). These complexes could be supershifted by an antibody to ERα. We also showed that in normal cycling women, lactoferrin gene expression in the endometrium increases during the proliferative phase and diminishes during the luteal phase. This in-vivo study thus supported the finding from transient transfection experiments that the human lactoferrin gene expression is elevated in an environment with a high level of estrogen. The estrogen effect on lactoferrin gene expression in the rhesus monkey endometrium was studied by Western blotting and immunohistochemistry. The immunohistochemistry results showed that immunoreactive lactoferrin protein was not detectable in the untreated ovariectomized monkey endometrium, was elevated by estrogen treatment, and was suppressed by sequential, combined estrogen plus progesterone treatment. In conclusion, this study has shown that lactoferrin gene expression is responsive to estrogen in primate endometrium. Key words: endometrium/estrogen response element/human and monkey/lactoferrin/lactoferrin gene promoter

Introduction Lactoferrin, a non-haem iron-binding glycoprotein, was first discovered in milk and later found in the wet surface mucosa epithelium. It is a major protein in the secondary granules of neutrophils and is present in many biological secretions including the saliva, tears and semen (Sanchez et al., 1992; Levay and Viljoen, 1995; Lonnerdal and Iyer, 1995; Nuijens et al., 1996). The protein has been shown to kill bacteria, play an immunomodulatory role and participate in inflammatory response (Sanchez et al., 1992; Levay and Viljoen, 1995; Lonnerdal and Iyer, 1995; Nuijens et al., 1996). However, its mechanism of action is presently unknown. In the mouse uterus, lactoferrin expression is up-regulated by both estrogens and epidermal growth factor (EGF) (Teng, 1999). The mRNA and protein expression of lactoferrin can be induced several hundred-fold in the uterus of immature mice by estrogen treatment (Teng et al., 1986; Pentecost and Teng, 1987). DNA 58

elements of the mouse lactoferrin gene responsible for estrogen and EGF stimulation have been identified and characterized in a transiently transfected cell culture system (Liu and Teng, 1991, 1992; Liu et al., 1993; Shi and Teng, 1994, 1996). The estrogen response element (ERE) of the mouse lactoferrin gene consists of an imperfect palindromic AGGTCA motif, which overlaps with a chicken ovalbumin upstream promoter (COUP)-transcription factor (TF) binding site (Liu and Teng, 1992). Estrogen receptor alpha (ERα) binds COUP/ERE and forms stable complexes with a half-life of 30 min, whereas an interaction between COUP-TF and the DNA elements lasts only 5 min (Liu et al., 1993). Thus, estrogen-bound ERα activates the mouse lactoferrin gene readily in the transfection system and in the mouse uterus. During the mouse estrus cycle, lactoferrin content in the uterine epithelium fluctuates with the level of circulating estrogen (Newbold et al., 1992; Walmer et al., 1992). For example, the expression of the © European Society of Human Reproduction and Embryology

Lactoferrin gene is regulated by estrogen in primate endometrium

uterine lactoferrin gene increases during proestrus, becomes highest at estrus, declines during metestrus, and is undetectable at diestrus. These changes suggest that the lactoferrin protein plays an important role in the physiology of the mouse uterus. As with the mouse lactoferrin gene, an imperfect ERE, located at similar position in the human lactoferrin gene, can be activated by estrogen in transiently transfected cells (Teng et al., 1992). However, in the human lactoferrin gene, an additional element, Steroid Factor 1 Response Element (SFRE) (Yang and Teng, 1994), located 26 bp upstream from the ERE, is involved in estrogen responsiveness (Yang et al., 1996). Mutations at SFRE reduce the activity of ERα to 50% although the ERE remains intact. Also, an estrogen receptor-related receptor (ERRα1) binds to SFRE and is responsible for modulating the transcriptional activity mediated by ERα (Yang et al., 1996; Zhang and Teng, 2000). Therefore, estrogen regulation of the human lactoferrin gene appears to be subject to an additional level of fine-tuning as compared to the mouse (Teng et al., 1992; Yang et al., 1996). Lactoferrin was observed in the human endometrium at various stages of menstrual cycle more than 30 years ago (Masson et al., 1968; Tourville et al., 1970). However, examination of lactoferrin expression in the human endometrium during the menstrual cycle by immunohistochemistry has yielded inconsistent results. It has been found to be highly expressed in the endometrium of both proliferative (Kelver et al., 1996) or secretory phase (Tourville et al., 1970; Walmer et al., 1995). However, most of the studies were conducted with polyclonal rabbit anti-lactoferrin serum that may also react with other members of the transferrin gene family since they share 60– 100% sequence identity at certain regions of the protein (Pentecost and Teng, 1987). The human studies have also been constrained by limited samples and variations among the human subjects. It has also been reported that lactoferrin gene expression in normal and pathological human endometrium is up-regulated by estrogen (Walmer et al., 1995; Kelver et al., 1996). The goal of the present study was to investigate human endometrium lactoferrin expression at various stages of the normal cycle in carefully staged tissue samples with a wellcharacterized antiserum to lactoferrin. In addition, we examined lactoferrin expression in the endometrium of rhesus monkey under experimentally controlled hormonal conditions.

Materials and methods Patient population and sample preparation The inclusion criteria of women in the study and preparation of the endometrium samples have been described previously (Bush et al., 1998). The samples were collected in accordance with the guidelines of the Internal Review Board committee at Duke University Medical Center and informed consent was obtained from the patients. Briefly, two sets of endometrium samples (100297 as Set 1 and 102897 as Set 2) were collected from women of reproductive age (28–48 years old and with spontaneous menstrual cycles occurring every 26–35 days), whose uterus was removed during hysterectomy. All uteri with severe pathology, excessive bleeding, fibroids and endometrosis were excluded from the study. Screening for uterine pathology included a clinical history, physical examination, documenting regular monthly menses at a normal interval and three independent blinded readings

by a single board-certified gynaecological pathologist to rule out unsuspected histological abnormalities. The endometrium was scraped from the fundus in a uniform fashion with a scalpel and was immediately placed into solubilization buffer [1% Triton X-100, 2 mmol/l EDTA, 2 mmol/l EGTA, 1 mmol/l Na3VO4, 20 mmol/l NaF, 50 µmol/l Na2Mo4O4, aprotinin and leupeptin (20 µg/ml each), and 15 µmol/l (4-amidinophenyl)-methansulphonyl fluoride in 20 mmol/l HEPES, pH 7.4] and homogenized with a Virtishear (Virtis Corp., Gardiner, NY, USA) in the cold. After centrifugation at 21 000 g for 1 min, the supernatant was divided into small aliquots and stored at –70°C until use. The protein concentration was determined with the Pierce BCA Protein Assay (Pierce, Rockford, IL, USA). Human milk was obtained from a lactating mother with a milk pump. Human prostate tissue (from a normal 67 year old white male) was obtained from the Sourthern Division of Cooperative Human Tissue Network (Birmingham, AL, USA). Rhesus monkey sample preparation Animal treatments and endometrial collection have been described previously (Slayden et al., 1993). Briefly, sexually mature rhesus macaques (Macaca mulatta) were housed and cared for by the veterinary staff of the Oregon Regional Primate Research Center in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Ovariectomy was performed by standard techniques. After ovariectomy, the monkeys were either left untreated to serve as controls, treated for 4 weeks with estradiol-17β (E2) to provide estrogenized samples, or treated with E2 for 2 weeks, then E2 ⫹ progesterone for 2 subsequent weeks to provide artificial luteal phase samples. Tissues collected for Western blotting were cut freehand with a razor blade (2 mm thick), frozen in liquid propane and stored at –70°C until use. The frozen tissue samples were pulverized in liquid N2, then immediately homogenized in 5 volumes of RIPA lysis buffer [150 mmol/l NaCl, 0.1% (sodium dodecyl sulphate) SDS, 1% CHAPS, 1 mmol/l EDTA and 10 mmol/l Tris–HCl at pH 7.4] containing a cocktail of protease inhibitors (0.5 mmol/l PMSF, 10 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin; Sigma, St Louis, MO, USA). After sonication with a Branson Sonifier (Ultrasonics Inc., Plain View, NY, USA), the samples were cleared by centrifugation at 10 000 g for 20 min. Aliquots of the clear supernatant were stored at –70°C until use. Tissues collected for histology and immunohistochemistry were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 6 µm thickness and mounted on slides. Monkey prostate tissues were obtained from normal adult males during regularly scheduled Primate Center necropsies. Monkey milk was obtained from lactating females with a milk pump by standard procedures. Nuclear protein preparation and EMSA Nuclear protein enriched with ERα from human endometrial carcinoma RL95-2 cells (RL95-2) was prepared as previously described (Yang et al., 1996). ERα was overexpressed in RL95-2 cells with expression vector (HEGO, a gift from Pierre Chambon, Paris, France) for 48 h before harvesting the cells and extracting the nuclear protein. Baculovirus expressed ERα (BV-ERα) was a gift from Malcom Parker (London, UK). The probes that were used in the electrophoresis mobility shift assay (EMSA) have been characterized (Teng et al., 1992; Yang and Teng, 1994) and the EMSA was performed as previously described (Liu and Teng, 1992). The antibody to ERα was obtained from Abbott Laboratories (Abbott ER-ICA monoclonal H222 kit, Chicago, IL, USA). Western blot analysis Tissue extracts containing 25 µg of protein (BioRad Protein Assay Kit) from the prostate or endometrial samples of the human and

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monkey were examined in Western blot analysis. For antibody characterization, mouse lactoferrin (mLF) was isolated from the uterine fluid of an estrogen-treated mouse (Teng et al., 1986). Bovine milk lactoferrin (bLF), human milk lactoferrin (hLF), and mouse transferrin (mTF) were obtained commercially (Sigma). The rabbit polyclonal antibodies, raised against mouse lactoferrin (mLF 8344), human lactoferrin (hLF 12484) and mouse transferrin (mTF), have been previously described (Teng et al., 1986; Panella et al., 1991). Proteins were loaded onto precast 4–12% Bis–Tris NuPAGE gels and electrophoresed in a 1⫻ MOPS buffer at 120 mA and 200 V for 1.25 h in a Novex XCell II Mini-Cell system (Novex, San Diego, CA). Proteins were transferred from the gel onto an Immobilon-P polyvinylidene difluoride (PDVF) membrane (Millipore) at 200 mA and 25 V for 2 h in a 1⫻ NuPAGE transferring buffer containing 20% methanol. Immunodetections were carried out with an enhanced chemiluminescence (ECL) kit (Amersham) according to the manufacturer’s specifications. Primary antibodies were diluted ⫻10 000 and incubated with the protein blots overnight in the cold and with constant shaking. (For the transferrin antibody neutralization experiments, 10 µg/ml of transferrin from either human or rat was included in the primary antibody interaction step.) After washing, the blots were incubated with diluted (⫻10 000) second antibody (donkey anti-rabbit IgG linked with horseradish peroxidase) for another hour before ECL detection. X-ray film was exposed to the blot for 1 min and developed in Konica SRX-101 developer (Tokyo, Japan).

the receptor (lane 4). Furthermore, the receptor expressed in the baculovirus and in RL95-2 cells (NPE) binds ERE in the context of the human lactoferrin gene (hLF –417/–336) promoter (Figure 2B). The specific binding of ERα (lane 1 and 5) could be competed off by 50⫻ excess cold ERE oligonucleotides (lane 3), and the ERα antibody (H222) retarded the mobility of the complexes in EMSA (lanes 2 and 4).

Results

Lactoferrin gene expression in human endometrium during the menstrual cycle Endometrial samples collected from women during early proliferative (EP), mid proliferative (MP), mid luteal (days 20–22), and late luteal (days 26–28) phases of the cycle were examined by Western blotting analysis with human lactoferrin antiserum, hLF 12484 (Figure 3A). Two sets of human endometrial samples were evaluated. With Set 1 (100297; lanes 1–4), lactoferrin was found in the early proliferative phase (lane 1) but not in other phases (lanes 2–4). With Set 2 (102897; lanes 6–9), lactoferrin was mainly found in early (lane 6) and mid (lane 7) proliferative. The initial characterization of the antibody indicated that this lot of anti-hLF antibody cross-reacted weakly with the mouse transferrin (Figure 4, top panel). Therefore, the faint band that ran faster than the milk lactoferrin (Figure 3A, lane 10) and was detected by the antibody in all endometrial samples could be transferrin or a related family member. To verify the presence of transferrin in the endometrial samples, we stripped the lactoferrin antibody from the membrane and re-blotted with antibody to the mTF. Indeed, a transferrin band was detected in every sample (Figure 3A, lanes 11–14 and 16–19) except the milk sample that contains a high level of lactoferrin but no transferrin (lane 15). For this reason, we repeated the Western blotting experiment of Set 2 (102897) and included 10 µg/ml of human transferrin in the primary antibody to neutralize the immunoreactivity of transferrin (Figure 3B). As expected, the transferrin antibody activity was completely blocked (lanes 1–4) whereas lactoferrin immunoreactive protein was detected in the early and mid proliferative phase endometrial samples (lanes 5 and 6). As a loading control, a duplicated gel was stained with colloidal blue (Figure 3C, lanes 1–4) to verify the equal loading. These results demonstrated that human lactoferrin gene in the endometrium is highly expressed during the early and mid proliferative phases of the menstrual cycle, when estrogen is the dominant hormone.

ERα binding to the imperfect ERE of human lactoferrin gene Multiple DNA elements in the human lactoferrin gene promoter are involved in estrogen regulation (Figure 1). The imperfect palindromic ERE (–362 and –336) has been shown to mediate the estrogen action (Teng et al., 1992) and a stronger estrogen response can be obtained if the SFRE (–390 and –379) is also included (Yang et al., 1996). Whether ERα binds the ERE in the context of human lactoferrin gene was investigated in the current study. By EMSA we demonstrated that the BV- ERα binds to the ERE in vitro and forms two complexes (Figure 2A, lanes 2 and 3). To authenticate the binding, we showed that both complexes were supershifted by antibody specific to

Estrogen effect on lactoferrin expression in rhesus monkey endometrium To explore whether lactoferrin in monkey endometrium is regulated by estrogen, we first evaluated the species specificity of the rabbit polyclonal anti-lactoferrin sera. Among the ten rabbit antisera that we analysed, the hLF 12484 and the mLF 8344 antisera were used in the present study (Figure 4). The hLF 12484 was very specific to the hLF and did not crossreact with lactoferrin of other species (Figure 4, top panel), whereas the mLF 8344 cross-reacted with lactoferrin from other species as well as with the mTF although with less intensity (middle panel). The antibody to mTF reacted to all

IgG purification and immunohistochemistry staining IgG fractions of the hLF 12484 and mLF 8344 were purified from the rabbit antiserum according to the IgG purification protocol from ImmunoPure™ IgG (Protein A) Purification Kit (Pierce, Rockford, IL. USA). The immunohistochemistry protocol was derived from the Histostain SP Kit for rabbit polyclonal antibody (Zymed Laboratories Inc., San Francisco, CA, USA). Briefly, the deparaffinized tissue sections were first circled with a PAP to define the area and all subsequent steps were carried out in a dark and humidified box. The tissue sections were blocked by two different blocking solutions: first the Pierce Peroxidase Suppressor to block the endogenous peroxidase activity and then another blocking solution supplied with the Kit to block non-specific interactions. The primary antibody, mLF 8344 IgG at a concentration of 10 µg/ml in phosphate-buffered saline containing either 1 mg/ml of bovine serum albumin (BSA) or rat transferrin, was applied to the tissue sections overnight at room temperature. The secondary antibody, Pharmingen goat anti-rabbit biotin, was diluted 1:1500 before use. The colour reaction was then developed with horseradish peroxidase–steptavidin and aminoethyl carbazole, and counter-stained with a modified Harris haematoxylin (Sigma).

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Figure 1. Diagram of estrogen response modules of the human lactoferrin gene promoter. The numbers indicate the position upstream from the start site. COUP/SFRE ⫽ overlapping COUP-TF (cross-hatched box) and SF-1 binding elements (shaded box); COUP/ ERE ⫽ overlapping COUP-TF (cross-hatched box) and ERα binding elements (shaded box); GATA ⫽ the consensus GATA1 binding element (clear box).

Figure 2. Electrophoresis mobility shift assay (EMSA) detection of ERα binding to the human lactoferrin (hLF) estrogen response element (ERE). (A) Baculovirus expressed ERα (BV-ERα) binds the ERE. Concentrations of the BV-ERα used to bind ERE are 0.6 and 3.2 ng protein. (B) BV-ERα (3.2 ng) and ERα in the nuclear extract of RL95-2 cells (NPE, 4 µg) bind the ERE in the context of lactoferrin estrogen response module (–417/–336) H222, antibody to ERα. Double arrows: the receptor–DNA complexes; single arrow: ERα antibody supershifted complex.

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Figure 3. Lactoferrin expression in human endometrium during the proliferative phase of the menstrual cycle. (A) Western blotting of endometrium extract with human anti-lactoferrin 12484 (hLF) or mouse transferrin (mTF) antiserum. Two sets of samples were evaluated, Set 1 (100297) and Set 2 (102897). Equal amounts of human endometrium extract (25 µg protein) were loaded and analysed by Western blotting with enhanced chemiluminescence detection. EP ⫽ early proliferative phase; MP ⫽ mid proliferative phase; L20–22 ⫽ luteal phase at day 20–22 of the cycle; L26–28 ⫽ luteal phase at day 26–28 of the cycle; ST ⫽ standard mol. wt markers; h milk ⫽ human milk. (B) Samples from Set 2 study were Western-blotted with hLF or mTF in the presence of 10 µg/ml human transferrin. The arrow indicates the lactoferrin and the mol. wts are marked. (C) Colloidal blue staining of the protein samples from B.

transferrin tested (data not shown) and to the bLF slightly (bottom panel). To detect the immunoreactive protein by Western blotting, monkey endometrial tissue extracts were prepared and analysed (Figure 5A). Milk, which contains a high level of lactoferrin but no transferrin, and prostate tissue extracts, which contain both lactoferrin and transferrin, were used as controls. Both human and monkey samples were used. The tissue extracts and the control samples were examined by antisera to mLF 62

(lanes 1–6), hLF (lanes 7 and 8) and mTF (lanes 9–14). The 84 kDa immunoreactive band detected in the samples of monkey milk and prostate (lane 1 and lane 2, upper band) is likely to represent a form of monkey lactoferrin, based on the lack of reactivity to the anti-mTF antiserum. The size of this protein contrasts with the 76 kDa protein detected in human milk (lanes 5 and 8) and prostate (lane 6) lactoferrin. Interestingly, the monkey prostate and endometrial tissue extracts contained a 70 kDa lactoferrin-immunoreactive protein, which


Figure 4. Characterization of the rabbit polyclonal antisera by Western blotting. Sources of the purified protein are indicated in the Materials and methods section. The antibody, hLF 12484, mLF 8344 or mTF used in the Western blots is indicated at the bottom of each panel. Concentrations of the protein loaded in each lane are indicated at the top and the mol. wt marker is shown. The primary antibody was diluted 10 000-fold and the secondary antibody was diluted 10 000-fold. mTF ⫽ mouse transferrin; bLF ⫽ bovine lactoferrin; hLF ⫽ human lactoferrin; mLF ⫽ mouse lactoferrin.

Figure 5. Detection of lactoferrin in the monkey endometrium. (A) Western blotting of tissue extracts from monkey (MK) and human (HU) prostate (pros) and monkey endometrium (endo) treated with estrogen alone (endo E) or estrogen plus progesterone (endo E ⫹ P). Milk was included as the control. The mouse anti-lactoferrin 8344 (mLF), human anti-lactoferrin 12484 (hLF) and mouse antitransferrin (mTF) antisera were examined. (B) Western blotting of the monkey samples with mLF in the presence of 10 µg/ml of rat transferrin. The double arrow indicates the location of the lactoferrin.

was distinct from the 84 kDa milk lactoferrin (lanes 2–4, lower band). Transferrin ran slightly ahead of the lactoferrin on a SDS–polyacrylamide gel electrophoresis as shown earlier (Figure 3). Therefore, the size of monkey transferrin was

~78 kDa (Figure 5A, lanes 10–12, the upper band) and the hTF was ~74 kDa (lane 14). As expected, the transferrin was not detected in either monkey (lane 9) or human (lane 13) milk, and the hLF antiserum only reacted to hLF (lane 8) and 63

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not with monkey lactoferrin (lane 7). There was some crossreactivity between the mTF and the monkey lactoferrin-reactive 70 kDa protein (lanes 10–12, lower band). The monkey endometrial extracts of E2 and E2 ⫹ progesterone consisted of heterogenous cell populations from various zones of the endometrium and infiltrating neutrophils which have a high content of lactoferrin. For this reason, Western blot experiments of the monkey endometrium can only provide qualitative but not quantitative information on lactoferrin. Therefore, the lactoferrin-reactive protein from E2 and E2 ⫹ progesterone samples showed equal intensity (Figure 5A, lanes 3 and 4). We also performed Western blotting on samples of monkey prostate and endometrium with human and rhesus monkey milk as control, in which 10 µg/ml rat transferrin was added to the mLF 8344 antibody. Comparing the amino acid sequence of the transferrin protein from mouse and rat (GeneBank, ratd38380 and mouse tf- bco12310), a 90% sequence homology was found. Therefore, the presence of neutralizing rat transferrin with the mLF antibody could block most of the antibody epitopes that recognize mTF. Under these conditions the mLF antibody detected only the 70 kDa lactoferrin-like protein in monkey endometrium (Figure 5B). The fast-moving bands apparent in the monkey milk and prostate samples may be degradation products of lactoferrin. To immunolocalize the lactoferrin protein in the monkey endometrium, we isolated the IgG fractions from the mLF 8344 antiserum and performed immunohistochemistry with the mLF 8344 IgG purified fraction (Figure 6). The endometrial samples examined in this study were from ovariectomized monkeys either untreated or treated with E2 or E2 ⫹ progesterone as described in Materials and methods. In preliminary work, high background was seen in the stroma of E2 ⫹ progesterone samples. This background persisted even with the purified mLF 8344 IgG fraction whereas the hLF 12484 antiserum or IgG fraction was negative on all samples (data not shown). It is possible that the high level of serum transferrin in the tissue could be recognized by the mTF epitopes of the mLF. To minimize the interference of monkey transferrin on the immunohistochemistry study, we added 1 mg/ml of rat transferrin to the mLF 8344 IgG during the overnight primary antibody-staining step. Inclusion of transferrin greatly reduced all background staining and revealed specific lactoferrin staining in the endometrial glands. The residual positive staining in the stroma could be due to lactoferrin synthesized by an unknown cell type or released from the secondary granules of the infiltrating neutrophils (Masson et al., 1969) during hormonal treatment. The staining was absent in the untreated animals, strong in the glands of E2-treated animals, and greatly suppressed in the glands of the E2 ⫹ progesterone-treated rhesus macaque monkeys (Figure 6, compare A, B and C). These results demonstrated that estrogen induces, and sequential progesterone suppresses, lactoferrin gene expression in the glands of monkey endometrium (Figure 6D–F).

Discussion Estrogen regulation of lactoferrin gene expression in the mouse uterus is well established (Teng, 1999). However, there are 64

inconsistent reports on estrogen regulation of the lactoferrin gene in human endometrium (Tourville et al., 1970; Teng et al., 1992; Walmer et al., 1995; Kelver et al., 1996). The present study demonstrated that the estrogen receptor binds to the imperfect ERE of human lactoferrin gene both in isolation and in the context of the gene promoter region. This finding supported our previous observations that the human lactoferrin gene is activated in transiently transfected human endometrial carcinoma cells (RL95-2) through an ER-mediated process and that the imperfect ERE of the gene is required (Teng et al., 1992). In addition to the in vitro physical interaction between ER and the ERE of the human lactoferrin gene, we demonstrated that lactoferrin gene expression in the endometrium fluctuates during the menstrual cycle. In Western blotting experiments, lactoferrin was detected in the proliferative phase but not in the secretory phase, suggesting that estrogen induces and progesterone suppresses lactoferrin gene expression in vivo. It is unlikely that lactoferrin from neutrophils confounded these results, as more are present in the endometrium during the secretory phase when lactoferrin is marginally detectable on the Western blots. There are two major differences between the current study and the previous studies on the same subject matter (Tourville et al., 1970; Teng et al., 1992; Walmer et al., 1995; Kelver et al., 1996). First, the tissue samples were collected by carefully scraping the functionalis endometrium off the basalis zone and the myometrium. This provides a relatively homogeneous cell population. Second, the epitopes of the antiserum that recognizes transferrin were neutralized by a high level of transferrin protein incorporated in the blotting procedure. This step significantly reduced any interference caused by close family members of lactoferrin. In the present study, we also investigated the use of a nonhuman primate model to study estrogen regulation of lactoferrin gene expression in the endometrium. The lactoferrin of rhesus monkey has high similarity to the human protein in both amino acid composition and carbohydrate moiety (Davidson and Lonnerdal, 1986). Moreover, monkey lactoferrin, like human lactoferrin, has an unusual amino acid sequence at the Nterminus which is essential for binding to bacterial lipopolysaccharide and to the mammalian lactoferrin receptor (Wu et al., 1995; van Berkel et al., 1997), indicating that monkey lactoferrin could function similarly to the human protein. The reproductive physiology of the female rhesus macaque monkey is also highly comparable to that of women (Slayden et al., 1993). However, the polyclonal antibody produced against human lactoferrin did not cross-react with the monkey milk lactoferrin nor did it detect any immunoreactive protein in the endometrial tissue extracts or in the immunostaining study (data not shown). However, the rabbit anti-mouse lactoferrin serum, mLF8344, reacted to an 84 kDa lactoferrin in the monkey milk and to a 70 kDa protein, presumed to be lactoferrin, in the endometrium. With this antibody, we were able to show that lactoferrin-reactive protein in monkey endometrium is up-regulated by estrogen. This finding is in agreement with the human study showing that lactoferrin gene expression is increased under the influence of estrogen. A reduced immunostaining for lactoferrin in the endometrial glands after E2 ⫹ progesterone treatment suggests that a high


Figure 6. Effect of estrogen on lactoferrin expression in the monkey endometrium. Ovariectomized monkeys were either untreated or treated with estrogen only or estrogen plus progesterone (E ⫹ P). (A) Untreated. (B) Treated with estrogen for 14 days. (C) Treated with estrogen for 14 days plus another 14 days of estrogen and progesterone. The original magnification of untreated endometrium was ⫻100 (A) and the other two samples were ⫻40 (B and C). (D–F) Higher magnifications (original magnification ⫻400) of the glands from each sample are shown. Rat transferrin (1 mg/ml) was included with the mLF 8344 IgG (10 µg/ml) in the immunostaining procedure. The pink stain indicates the presence of lactoferrin. The tissue sections were counter-stained with haematoxylin. Scale bars ⫽ 100 µm. En ⫽ endometrium; My ⫽ myometrium.

level of progesterone may block the affects of E2 on lactoferrin, probably by suppressing glandular estrogen receptors, as previously reported (West and Brenner, 1985). Nonetheless, in the basal glands of the endometrium, the intensity of lactoferrin staining was similar in both estrogen- and progesterone-dominant stages. This observation is consistent with the finding that 2–56% of the basal glands in the human endomet-

rium at any give time during the menstrual cycle are lactoferrin positive (Walmer et al., 1995). Whether lactoferrin gene expression in the human and monkey endometrium exhibit regional specificity is under investigation. Based on the organization of the estrogen response module and the transcription factors that are involved in estrogen action (Teng et al., 1992; Yang et al., 1996; Teng, 1999), the molecular mechanisms 65

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of estrogen action in the human and non-human primate endometrium could be very different from that in the mouse uterus. Multiple levels of control may be required to fine-tune the estrogenic effect in human endometrial cells. The 70 kDa immunoreactive band detected in the monkey endometrium and presumed to be lactoferrin, is smaller than the milk protein. This 70 kDa smaller form of lactoferrin was also detected in the prostate. Interestingly, lactoferrin in the human prostate is also smaller than that in the milk. Protein modification and presence of isoforms could contribute to the size differences in various tissues. Isoforms of lactoferrin have been identified in the human milk (Furmanski and Fortuna, 1989), granulocytes (Furmanski and Li, 1990) and seminal plasma (Sorrentino et al., 1999). These isoforms share physical, chemical and antigenic properties with lactoferrin, yet differ in functions. Recently, a subset of cytotrophoblasts of the human placenta was reported to be recognized by a lactoferrin monoclonal antibody but not by several other polyclonal and monoclonal antibodies to human lactoferrin, suggesting that the cytotrophoblasts express a unique epitope of lactoferrin (Thaler et al., 1999). In addition, an alternative form of human lactoferrin mRNA that is expressed in adult and fetal tissues but not tumour-derived cell lines has been described (Siebert and Huang, 1997). The biological significance of lactoferrin and its isoforms in these tissues is not known. It is well documented that estrogen regulates the mucosal immune system of the female rodent reproductive tract (Wira and Stern, 1992; Wira and Kaushic, 1996) and it is therefore likely that lactoferrin, an immunomodulator, could participate in the mucosal immunity of the primate endometrium.

Acknowledgements We thank Malcom Parker, Pierre Chambon and Nenyu Yang for the reagents used in this study and we appreciate Barbara Davis and Sylvia Hewitt for reading the manuscript.

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