Molecular Human Reproduction, Vol. 7, No. 7, 655-664,
July 2001
© 2001 European Society of Human Reproduction and Embryology
Uterine physiology |
Differential and cell-specific expression of calcitonin receptor-like receptor and receptor activity modifying proteins in the human uterus
1 Nuffield Department of Obstetrics and Gynaecology and 2 Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU and 3 Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK
Abstract
The calcitonin receptor-like receptor (CRLR) can function as a receptor for either calcitonin gene-related peptide (CGRP) or adrenomedullin (AM), depending upon co-expression with members of a novel family of receptor activity-modifying proteins (RAMP). RAMP1 presents the CRLR at the cell surface as a CGRP/AM receptor. RAMP2- and RAMP3-transported CRLR receptors act as AM-specific receptors. However, it is still unknown if this signalling system operates in vivo. Of particular interest is the uterus, where both peptides and their binding sites are known to be present and where both mitogenic and vasodilatory responses to AM and CGRP have been demonstrated. In this study, we examined whether CRLR and RAMP are co-expressed in the same populations of cells in human uterine tissue. Analysis by in-situ hybridization and immunocytochemistry revealed a heterogeneous and cell type-specific distribution of components of this AM/CGRP signalling system. Adrenomedullin mRNA was expressed and evenly distributed across all cell types. CRLR mRNA was predominantly found in blood vessels. RAMP1 expression was specific to myometrial myocytes and vascular smooth muscle cells in uterine arteries. RAMP2 and RAMP3 mRNA were not detectable by in-situ hybridization. The pattern of differential and cell-specific expression of CRLR and RAMP suggests the involvement of CRLR/RAMP1 in the processes of vasodilation, smooth muscle relaxation and angiogenesis in response to AM and CGRP in the human uterus, but also indicates that other receptors may be implicated.
adrenomedullin/CGRP/CRLR/RAMP/uterus
Introduction
Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP), together with calcitonin and amylin, form a family of structurally and biologically related polypeptides (Muff et al., 1995
; Hinson et al., 2000). The members of this family are characterized by a six or seven amino acid ring structure linked by a disulphide bridge and an amidated C-terminus. Calcitonin gene-related peptide is a 37 amino acid neurotransmitter that is produced as a result of alternative processing of the RNA transcribed from the calcitonin gene (Amara et al., 1982; Rosenfeld et al., 1983). Adrenomedullin is a 52-amino acid peptide, which was originally isolated from human pheochromocytoma cells (Kitamura et al., 1993). Adrenomedullin and CGRP immunoreactivity have been demonstrated in human reproductive organs (Samuelson et al., 1985; Happola and Lakomy, 1989; Reinecke et al., 1989; Shew et al., 1992; Papka et al., 1996; Casey et al., 1997; Upton et al., 1997; Cameron and Fleming, 1998; Zhao et al., 1998; Michishita et al., 1999; Nikitenko et al., 2000a). CGRP-containing nerve fibres are believed to play a role in the regulation of motor activity (Samuelson et al., 1985), and are involved in myometrial contractility in rodents (Shew et al., 1992) and humans (Samuelson et al., 1985). Adrenomedullin also attenuates the contractile response in rat uterus (Upton et al., 1997) and human myometrial cells (Casey et al., 1997). Both peptides are also thought to be involved in the local regulation of blood flow in the female reproductive tract, as they are known vasodilators (Brain et al., 1985; He et al., 1995). CGRP has a potent vasodilating effect in uterine artery segments from non-pregnant and pregnant patients (Bodelsson et al., 1992; Nelson et al., 1993a,b). In addition, the blood pressure in CGRP-
knockout mice is significantly raised (Gangula et al., 2000). Transgenic mice that overexpress AM mainly in their vascular endothelial and smooth muscle cells exhibit significantly lower blood pressure than their wild-type littermates (Shindo et al., 2000). Furthermore, AM is a growth factor for endometrial endothelium (Nikitenko et al., 2000a) and has angiogenic activity in vivo (Zhao et al., 1998).
The calcitonin family of peptides exhibit overlapping pharmacological actions (Muff et al., 1995; Hinson et al., 2000), and these peptides are thought to act through seven-transmembrane domain, G-protein-coupled receptors. Radioligand binding studies suggest that there may be subtypes of CGRP receptors (Dennis et al., 1989, 1990). Molecular characterization has recently shown that AM and CGRP receptors may be related, as cross-reaction of these peptides with the same receptor has been reported (Casey et al., 1997).
An orphan receptor, named calcitonin receptor-like receptor (CRLR), was initially isolated from rat lung (Njuki et al., 1993). CRLR has 55% identity with the calcitonin receptor. It was shown that CRLR acts as either a CGRP or an AM receptor, depending on which receptoractivity-modifying proteins (RAMP) are co-expressed with CRLR (McLatchie et al., 1998). Human (McLatchie et al., 1998), rat (Nagae et al., 2000) and mouse (Husmann et al., 2000) RAMP were recently cloned and shown to be type I transmembrane proteins, with an extracellular N-terminus and a cytoplasmic C-terminus (McLatchie et al., 1998). RAMP are required to transport CRLR to the plasma membrane. At the cell surface, a CRLR/RAMP1 complex acts as a CGRP receptor, whilst RAMP2- and RAMP3- transported CRLR acts as an AM receptor (McLatchie et al., 1998). CGRP binding in rat tissues correlates well with RAMP1 mRNA levels, whilst AM binding shows a tendency to vary with RAMP2 mRNA levels (Chakravarty et al., 2000). However, this hypothesis may not apply to all cell types and may be species specific.
Little is currently known about AM and CGRP receptors in the human uterus, where both peptides are present (Reinecke et al., 1989; Zhao et al., 1998). Adrenomedullin binding sites are present in the rat uterus (Upton et al., 1997) and CGRP receptors are possibly localized to the myometrial cells (Yallampalli et al., 1999). It is still unclear, however, whether the recently discovered G-protein-coupled receptor complexes for AM and CGRP are present in uterine tissue. This study was therefore undertaken to investigate the localization of AM, CRLR and RAMP in human uterus.
Materials and methods
Tissue collection
Uteri (n = 22: seven proliferative, 11 secretory, four menstrual phase of the cycle) were obtained at hysterectomy from non-pregnant women of reproductive age (30–49 years) who had a history of regular menstrual cycles (26–30 days) and had used neither oral nor intrauterine contraception nor had received any hormonal treatment for at least 6 months prior to surgery. Hysterectomy was undertaken for a subjective complaint of menorrhagia. No pelvic pathology was seen at operation. This was confirmed by histological examination performed by an independent pathologist. Uteri were bisected: one half was used for preparation of frozen samples, the other half was processed for histological examination. The frozen samples of morphologically separated endometrium and myometrium, alongside samples of corpus luteum (n = 1) and placenta (n = 3) (obtained at term from apparently healthy pregnancies), were snap-frozen in liquid nitrogen. All tissues were collected in accordance with the requirements of the Central Oxfordshire Research Ethics Committee.
Isolation of RNA and reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA were isolated from frozen specimens by the guanidinium isothiocyanate method (Chomczynski and Sacchi, 1987). The RNA pellets were dissolved in sterile distilled water and quantified by optical density at 260 nm.
cDNA were synthesized from 1 µg of total RNA using the Reverse-iTcDNA synthesis kit (Advanced Biotechnologies Ltd, Surrey, UK). The reaction mixture had a final volume of 20 µl, and contained reaction buffer with addition of 5 U of RNase inhibitor, 25 U M-MLV reverse transcriptase (RTase), 500 µmol/l of each dNTP, and 0.5 µg anchored oligo dT primer. Annealing and primer extension were performed at 42°C for 60 min. RTase was inactivated by incubation at 75°C for 10 min. Then PCR was performed on an aliquot (1 µl) of this mixture using Expand High Fidelity PCR system (Roche, Mannheim, Germany). The reaction mixture had a final volume of 20 µl containing reaction buffer with 1.5 mmol/l MgCl2, 300 nmol/l of each specific primer (Table I), 200 µmol/l of each dNTP and 0.5 U Expand High Fidelity PCR System enzyme mix (Taq DNA and Pwo polymerases) (Barnes, 1994). Final mixture was overlaid with 30 µl mineral oil. Amplifications were routinely performed using 30 cycles in the Perkin Elmer Gene Amp PCR System 2400 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control, adrenomedullin, CRLR, RAMP1, RAMP2 and RAMP3 using primers designed for their specificity (Table I). The thermal cycling protocol was 94°C for 1 min, 58°C for 1 min, 72°C for 1 min. During the first cycle, the 94°C step was extended to 3 min, and on the final cycle the 72°C step was extended to 11 min.
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In-situ hybridization
Cryostat sections
Samples of uterus, containing both endometrium and myometrium, were snap-frozen in liquid nitrogen and stored at –70°C. Frozen sections 8–10 µm thick were cut on a Leica Kryostat 1720 Digital cryostat, thaw-mounted onto poly-L-lysine (Sigma, Poole, UK)-coated or Superfrost Plus slides (BDH, Poole, UK) and allowed to dry for at least 30 min at room temperature before being fixed for 5 min in 4% ice-cold paraformaldehyde solution in 1xPBS, washed three times for 2 min in 1xPBS, and dehydrated in a series of graded ethanol concentrations and stored in 95% (v/v) ethanol at 4°C until required.
Oligonucleotide probes for in-situ hybridization
Oligonucleotides (35–40 mer), to be used as sense and antisense probes, were designed for the in-situ detection of human AM, CRLR, RAMP1, RAMP2 and RAMP3 mRNA (Table II) with the aid of the computer program Oligo-4. Their sequences were checked against the GenBank database to eliminate any probes which inadvertently possessed similarity to other known mRNA. Sense oligodeoxyribonucleotide probes exactly complementary to the antisense probes were used as negative controls to test the specificity of the in-situ hybridization.
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For in-situ hybridization, oligonucleotides were labelled at their 3' end with [
-35S]dATP using terminal deoxynucleotidyl transferase (Amersham–Pharmacia, Burlingame, USA). Unincorporated radioactivity was removed by centrifuging the reaction mixture through a Sephadex G-50 column and radioactivity in the excluded fraction was determined using a liquid scintillation spectrometer. To increase the sensitivity of the in-situ hybridization signal, cocktails of probes were used as suggested (Trembleau and Bloom, 1995) for detection of low abundance mRNA.
Hybridization
In-situ hybridization was performed as previously described (Nikitenko et al., 2000b). In brief, all solutions for in-situ hybridization were prepared using double-distilled water treated with 0.1% (v/v) diethylpyrocarbonate (DEPC) and then autoclaved. Radioactively labelled oligonucleotide probes containing ~3x105 d.p.m. (5–15 ng DNA) were added to each slide in 100 µl hybridization buffer and hybridization was performed overnight at 42°C. The slides were then washed, dehydrated, air-dried and then dipped in Ilford K-5 emulsion and left at room temperature in the dark for 1–2 weeks. Emulsion-coated sections were developed and then counterstained with haematoxylin and eosin, dehydrated, cleared and mounted in Biomount (British Bio Cell International, Cardiff, UK). In-situ hybridization specificity was tested by omitting the antisense probe and by using the sense control. Sections were examined under bright and dark field using a Leitz Diaplan microscope and photomicrographs were taken using a Leica Wild MPS 52 rollfilm camera.
Northern blotting
Total RNA were prepared as described above. Samples (10 µg) of total RNA were subjected to formaldehyde–agarose gel electrophoresis prior to transfer onto Nylon membranes (Sambrook et al., 1989). RNA was fixed onto the membranes by UV irradiation or by baking the filter at 75°C for 2 h. Northern hybridization was carried out overnight at 42°C using standard methods (Sambrook et al., 1989; Nikitenko et al., 2000b). The probes used were oligonucleotides labelled with [-32P]dATP at their 3' end by terminal deoxynucleotidyl transferase (Amersham-Pharmacia, Little Chalfont, UK) and then purified as for in-situ hybridization. Cocktails of probes were used to determine the specificity of binding to mRNA of interest. After hybridization, filters were washed in 1x 150 mmol/l NaCl, 10 mmol/l NaH2PO4, 50 mmol/l EDTA (SSPE), 0.1% (w/v) sodium dodecyl sulphate (SDS) at 65°C. Hybridization signals were detected by autoradiography using Fuji film.
Western blotting and immunoprecipitation
Frozen samples of endometrium, myometrium and placental tissue were finely minced with a scalpel, then pulverized under liquid nitrogen in a chilled pestel and mortar. The powdered tissue was suspended in ice-cold homogenization buffer using 20 ml buffer per 1 g tissue. Homogenization buffer consisted of 20 mmol/l HEPES pH 7.4, 1.5 mmol/l EDTA, 0.5 mmol/l PMSF, 0.5 mmol/l benzamidine and 10 µg/ml ovomucoid trypsin inhibitor (Sigma–Aldrich, Poole, Dorset, UK). The tissue powder was then homogenized for 30 s with an IKA ultraturrax homogenizer (Janke & Kunkel, Staufen, Germany) and centrifuged for 20 min at 3000 g to remove particulates. The protein content of the lysates was determined by the bicinchonic acid protein assay (Pierce & Warriner, Chester, UK). Lysates were equalized for protein content in homogenization buffer, then heated to 95°C for 10 min after addition of a 6x sample buffer (0.35 mol/l Tris–HCl pH 6.8, 30% glycerol, 10% SDS, 25% ß-mercaptoethanol, 93 mg/ml dithiothreitol, 0.12 mg/ml bromophenol blue). Protein lysates (6.5 µg) were separated by electrophoresis on a 15% acrylamide gel, and transferred in buffer (25 mmol/l Tris, 200 mmol/l glycine, 17% methanol) to an `Immobilon-P' membrane (Millipore, Watford, UK) using semi-dry blotting equipment (W.E.P. Company, Seattle, WA, USA) for 1 h at 500 mA. The membrane was blocked in PBS (phosphate buffered saline pH 7.2; 137 mmol/l NaCl, 3.35 mmol/l KCl, 10 mmol/l Na2HPO4, 1.84 mmol/l KH2PO4), containing 0.1% Tween-20 and 5% non-fat milk for 1 h at 23°C, then probed overnight at 4°C with a primary goat polyclonal antibody (Table I
) at 1:1000 dilution (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), followed by a rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody at 1:2000 dilution (Dako, Ely, UK). Proteins were visualized using either the `Renaissance' enhanced luminol system (New England Nuclear, Hounslow, UK) or ECL Western blot detection reagents (Amersham–Pharmacia Biotech) and were then exposed to `Hyperfilm' (Amersham–Pharmacia Biotech). The membrane was then re-probed for ß-tubulin (Gozes and Barnstable, 1982) to confirm equal loading of samples. Antibodies were stripped from the membrane at room temperature by washing for 5 min in distilled water, followed by two 5 min washes in 200 mmol/l sodium hydroxide and two 5 min distilled water washes. The membrane was re-blocked in PBS containing 0.1% Tween-20 and 5% non-fat milk, and then probed overnight at 4°C with an anti-ß-tubulin mouse monoclonal antibody at 1:2000 dilution (Sigma–Aldrich, Dorset, UK), followed by a goat anti-mouse HRP-conjugated secondary antibody at 1:2500 dilution (Dako). ß-Tubulin was then visualized as above.
For immunoprecipitation, equal amounts of protein (150–500 µg) were mixed with 5 µg of appropriate goat polyclonal antibodies (Santa Cruz Biotechnologies). The lysates and antibodies were gently mixed for 24 h at 4°C. The immune complexes were collected with protein A/G agarose (Santa Cruz Biotechnologies) and were washed three times with lysis buffer. Proteins were separated by electrophoresis on 7.5% polyacrylamide gel and transferred to PVDF membrane (all using Phast System; Pharmacia, Little Chalfont, UK). The membrane was blocked in 5% non-fat milk in Tris-buffered saline containing 0.5% Tween-20. Blots were probed with the same goat polyclonal antibodies (Santa Cruz Biotechnologies) used for precipitation, following the procedure described for Western blotting.
Immunohistochemistry and double-staining
Antibodies
The localization of RAMP was determined according to the immunoreactivity staining pattern obtained with the anti-RAMP1, -RAMP2 and -RAMP3 polyclonal antibodies (all from Santa Cruz Biotechnology). Immunophenotyping was performed on adjacent sections immunostained using anti-smooth muscle actin monoclonal (clone 1A4), anti-Von Willebrand factor polyclonal and anti-keratins monoclonal (clone LP34) antibodies (all from Dako).
Secondary antibodies were biotinylated horse anti-mouse, anti-rabbit and anti-goat (Vector) and HRP-conjugated swine anti-rabbit (Dako).
Immunohistochemical staining
Sections (8–10 µm thick) of frozen tissue were prepared in the same way as for in-situ hybridization. Before immunohistochemistry, frozen sections were dried, washed for 5 min in distilled water and rinsed in buffer 1 (100 mmol/l Tris–HCl, 150 mmol/l NaCl pH 7.5) for 5 min. Serial sections were cut and placed onto the slides so that corresponding areas could be studied as mirror images. After rinsing in buffer 1, sections were incubated for 30 min in buffer 2 (buffer 1 + 2% horse serum + 0.1% Triton X-100), followed by incubation with primary antibody (or a mix of primary antibodies in the case of double-staining) diluted as required in buffer 3 (buffer 1 + 1% horse serum + 0.1% Triton X-100) and incubated overnight at 4°C. Sections were then washed twice in buffer 1 for 5 min and incubated for 60 min with the biotin-labelled secondary antibody (or a mix of two different appropriate secondary antibodies labelled with either biotin or HRP in the case of double staining). This procedure was followed by two 5 min washes in Buffer 1. When biotinylated secondary antibodies were used, sections were washed twice in buffer 1, followed by application of streptavidin–alkaline phosphatase complex, using Vectastain ABC-AP Kit (Vector). Slides were then washed twice in buffer 1, and briefly washed in buffer 4 (100 mmol/l Tris–HCl, 100 mmol/l NaCl, 50 mmol/l MgCl2, pH 9.5). Signal was revealed by colour development using nitroblue tetrazolium chloride and X-phosphate, 4-toluidine salt (Boehringer Mannheim, UK). HRP visualization was performed with diaminobenzidine (Vector). Controls included: omission of primary antibodies, substitution of primary antibody with non-immune serum from corresponding species (Vector) or blocking of primary antibodies with corresponding peptides according to the manufacturer's instructions. Sections were then counterstained with Mayer's haematoxylin, rinsed in tap water and PBS, and finally embedded in Hydromount (BDH).
Visualization was performed on a Leitz Diaplan microscope. Evaluation of staining was performed by a semi-quantitative method using a scale from – (no staining) to +++ (very strong staining).
Results
Evaluation of expression of adrenomedullin, CRLR and RAMP mRNA by RT–PCR
To characterize the expression patterns of adrenomedullin, CRLR and RAMP at the mRNA level in human uterus, we performed RT–PCR using specific primers (Table I). Total RNA isolated from placenta served as a positive control, as the expression of AM mRNA is known to be high in this tissue. Adrenomedullin expression in placenta was found to be higher than that in endometrium and myometrium (Figure 1). CRLR mRNA was detected at low levels in uterus and was virtually absent from placenta. Expression of RAMP1 mRNA was much higher in endometrium and myometrium than in placenta. RAMP2 and RAMP3 mRNA expression was similar in all tissues examined and was always lower than RAMP1 expression.
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Localization of adrenomedullin, CRLR and RAMP mRNA by in-situ hybridization
Cell-specific expression of AM, CRLR and RAMP at the mRNA level was examined by in-situ hybridization with specific antisense oligonucleotides (Tables II and III
). No distinct changes in the distribution of these mRNA were observed when examining uterine samples obtained at different stages of the menstrual cycle.
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Adrenomedullin mRNA is evenly expressed across all uterine cell types
Placenta and corpus luteum were used as positive controls, since they are known to have high levels of AM mRNA. Northern blot analysis (Figure 2
) and in-situ hybridization controls using sense oligonucleotide probes (Figure 3j–l) confirmed that hybridization of antisense oligonucleotides is specific.
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In the uterus, AM mRNA was present at a low level and was evenly expressed across all cell types (Figure 3a–c
). In comparison, AM mRNA expression was higher in corpus luteum, and mRNA transcripts were found predominantly in granulosa cells (Figure 3d–f). Extravillous cytotrophoblast is the major source of AM mRNA in placenta (Figure 3g–i). Our in-situ hybridization results concur with the data obtained from RT–PCR (Figure 1) demonstrating a lower expression of AM mRNA in uterus than in placenta and corpus luteum (Figure 3).
CRLR mRNA is highly expressed in endometrial and myometrial blood vessels
The results of in-situ hybridization with a cocktail of antisense CRLR oligonucleotide probes (Table II) in uterine sections are shown in Figure 4 and summarized in Table III. High levels of CRLR mRNA expression were observed in endometrial (Figure 4c,d) and myometrial (Figure 4e,f) blood vessels. CRLR expression in endometrial epithelium, stroma and myometrial myocytes (Figure 4a–f) was extremely low. Controls (Figure 4g,h) confirmed the specificity of hybridization.
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Expression pattern of RAMP1, -2 and -3 mRNA in endometrium and myometrium
Hybridization of 35S-labelled RAMP1 antisense oligonucleotide probes to uterine sections (Figure 5a,b
) showed that RAMP1 expression was confined to stromal and vascular cells in endometrium (Figure 5c,d) and myocytes in myometrium (Figure 5e,f). No transcripts were detectable in the endometrial epithelium or in other myometrial cell types. Controls (Figure 5g,h) confirmed the specificity of hybridization. RAMP2 and RAMP3 mRNA transcripts were undetectable in both endometrium and myometrium (Table III), even when using cocktails of specific oligonucleotide probes (Table II).
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Detection of localization of RAMP in human uterus by immunocytochemistry
Tissue- and cell-specific expression of RAMP was examined by Western blotting (Figure 6
) and immunocytochemistry (Figure 7). The results of immunohistochemistry on frozen uterine sections, using goat polyclonal antibodies raised against specific peptides for RAMP1, RAMP2 and RAMP3, are shown in Table III.
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RAMP1 protein was detected by Western blotting in lysates from myometrial but not endometrial tissues (Figure 6
). This tissue-specific pattern was confirmed by immunocytochemistry showing RAMP1 immunoreactivity predominantly in myometrium, where it was present in myocytes (Figure 7f) and, to a lesser extent, in vascular smooth muscle cells. In endometrium, RAMP1 immunoreactivity was seen only in vascular smooth muscles (Figure 7d,e). No RAMP1 immunoreactivity was detected in the endometrial epithelium and stroma and no alterations in RAMP1 immunoreactivity was found throughout the menstrual cycle. In parallel control sections, no immunoreactivity was detected when incubated with non-immune serum or with antibodies pre-adsorbed with appropriate blocking peptides (Figure 7b,c). No specific immunoreactivity was detected by Western blotting, immunocytochemistry or immunoprecipitation using antibodies raised against peptides for RAMP2 and RAMP3.
Discussion
The expression of CGRP and CGRP-binding sites in both uterus (Upton et al., 1997) and smooth muscle cells of the gastrointestinal tract (Gates et al., 1999) suggests that CGRP acts as a neurotransmitter not only in the brain (Van Rossum et al., 1997), but also in peripheral tissues. The related peptide adrenomedullin (Zhao et al., 1998), and its binding sites (Upton et al., 1997), are also present in uterus. Adrenomedullin is thought to contribute to the processes of vasodilation (Nelson et al., 1993b; Gangula et al., 2000), smooth muscle relaxation (Upton et al., 1997) and angiogenesis (Zhao et al., 1998; Nikitenko et al., 2000a). The present study was therefore undertaken to determine the localization of components of a novel AM/CGRP–CRLR/RAMP ligand–receptor system in the human uterus. Our results show expression of AM mRNA across all cell types, cell-specific localization of CRLR in uterine blood vessels and RAMP1 in endometrial vascular smooth muscle cells and myometrial myocytes.
The distribution of AM mRNA in endometrium and myometrium was examined as a prelude to studying the localization of the AM/CGRP receptor system. Placenta and corpus luteum were used as control tissues to study the localization of AM mRNA, as it has been shown by Northern blotting that the placenta is a source of AM mRNA (Minegishi et al., 1999; Kobayashi et al., 2000). In-vitro studies show that the AM gene is expressed in decidua and fetal membranes of human placenta (Morrish et al., 1996; Kobayashi et al., 2000) and in the granulosa cells in corpus luteum (Abe et al., 1998; Moriyama et al., 2000). We have shown for the first time in vivo that AM mRNA is predominantly expressed in granulosa cells of the corpus luteum and in extravillous cytotrophoblast cells of term placenta. However, there were no distinct cell type(s) expressing AM mRNA in non-pregnant endometrium and myometrium. Instead, AM mRNA was expressed at a very low level across all cells. These data confirm earlier findings concerning the presence of AM immunoreactivity in stromal, epithelial and endothelial cells in human endometrium (Zhao et al., 1998; Michishita et al., 1999; Nikitenko et al., 2000a).
Data regarding the localization of CRLR mRNA are limited. In rat tissues CRLR mRNA is predominantly found in the lung, blood vessels, liver, midgut, rectum and urethra during development (Fluhmann et al., 1997) and also at a very low level in the central nervous system (Oliver et al., 1998). In addition, CRLR is localized in the blood vessels of other organs (Njuki et al., 1993; Fluhmann et al., 1997). We have found by in-situ hybridization that the expression of CRLR is low in uterus, and is localized predominantly in blood vessels. The presence of CRLR mRNA in uterine blood vessels may be important, as AM is mitogenic for endometrial endothelium (Nikitenko et al., 2000a). It could also contribute to the vasodilatory effect of CGRP on uterine (Shew et al., 1992; Gangula et al., 2000) and possibly cervical (Hansen et al., 1988) arteries.
We have demonstrated by in-situ hybridization that RAMP1 mRNA is expressed in myometrial myocytes and endometrial stroma and vasculature, but were unable to detect it in epithelial cells. Immunohistochemistry, however, revealed the presence of RAMP1 immunoreactivity predominantly in myometrial myocytes and uterine vascular smooth muscle cells. The lack (or low level) of translation of RAMP1 protein in endometrial stroma might indicate that additional factors are required for the activation of the CRLR/RAMP1 receptor system in this cell type. As RAMP1 is involved predominantly in the generation of the CGRP receptor, our results concerning RAMP1 localization in the vascular smooth muscle cells of endometrial spiral arteries therefore concur with the finding that endothelium is not involved in CGRP-induced relaxation of human uterine arteries (Nelson et al., 1993b). The data obtained from immunocytochemistry and in-situ hybridization are in general agreement with the Western blotting results. Immunoblotting confirmed tissue-specific expression of RAMP1 immunoreactivity in myometrium, but not in endometrial tissue, and demonstrated the presence of a distinct band of ~17 kDa after SDS–polyacrylamide gel electrophoresis under reducing conditions. However, even under reducing conditions, a band of ~34–37 kDa could be detected by both Western blotting and immunoprecipitation (data not shown). These findings are in accordance with data about the presence of a RAMP1 multimer when the protein is expressed in vitro (McLatchie et al., 1998). The appearance of a RAMP1 multimer might be explained by the presence of active CRLR glycosylation in these cells in vivo, as suggested by in-vitro experiments (McLatchie et al., 1998; Muff et al., 1998). Thus we speculate that the cell-specific expression of RAMP1 and the presence of multimers are possibly due to the local activation of the CRLR/RAMP1 complex, which results in the generation of an active CGRP receptor in myometrial myocytes and uterine vascular smooth muscle cells, under intimate contact with CGRP-immunoreactive nerve fibres. These nerve fibres innervate different target cell types in human uterus and are found in muscular layers and around blood vessels (Samuelson et al., 1985; Reinecke et al., 1989). Additional in-vivo and in-vitro studies are necessary to support this hypothesis. In addition, despite the presence of AM immunoreactivity and AM mRNA, RAMP2 and RAMP3 immunoreactivity were undetectable in uterine tissues. This is in accordance with the results obtained by in-situ hybridization and RT–PCR.
The differential expression of CRLR and RAMP1 mRNA is of interest. Although CGRP binding in rat tissues correlates well with the RAMP1 mRNA level (Chakravarty et al., 2000), it is still not clear whether the CRLR and RAMP are co-expressed or produced by different cell types in vivo in various tissues. The lack of strong expression of CRLR in myometrial myocytes, where RAMP1 is abundant, is either due to the control of its activity by RAMP (McLatchie et al., 1998; Muff et al., 1998) or to the possible interaction of RAMP1 with a different type of CGRP receptor, which also could be involved in uterine smooth muscle relaxation. The absence (or very low level) of RAMP2 and RAMP3 expression in the human uterus might indicate that CRLR/RAMP1 complex plays a main role in the vasodilatory and angiogenic response of uterine blood vessels to AM and CGRP. On the other hand, the CRLR/RAMP2 complex may be functional under certain conditions in endometrial endothelium, resulting in AM-induced mitogenesis (Nikitenko et al., 2000a). This has been seen in other endothelia (Kamitani et al., 1999) where RAMP1 and RAMP2 are considered to regulate the activity of a CRLR receptor (Muff et al., 1998). Alternatively, CRLR might also interact with other receptors (possibly other members of the RAMP family), generating CGRP/AM receptors in uterine blood vessels.
Thus the differential expression of CRLR and RAMP and the possibility of alternative systems/complexes for these receptors might explain data showing significant levels of CGRP receptors in some tissues where the expression of mRNA encoding CRLR is very low (Fluhmann et al., 1997), whilst in other tissues high levels of CRLR mRNA coexist with low levels of CGRP (Njuki et al., 1993; Aiyar et al., 1996). In summary, AM/CGRP signalling pathways other than the CRLR/RAMP system might operate in individual cell types in human uterine tissue. All of these pathways are thought to contribute to the processes of smooth muscle relaxation, vasodilation and endometrial angiogenesis.
Acknowledgements
This work was supported through the Johnson & Johnson Focused Giving Program and the Imperial Cancer Research Fund. We would like to thank Dr Hua-Tang Zhang (Angiogenesis Laboratory, Department of Pharmacology, University of Cambridge) for helpful advice; Dr Robin Roberts–Gant (Medical Informatics Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, UK) for assistance with formatting figures; Dr Katya Chobotova for help with immunoprecipitation technique and Mrs Janet Carver for assistance with sectioning frozen material used in this study (both of Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford); Dr John Coadwell, Mr Neil Brew (Babraham Institute, Cambridge); Dr Richard Giles (School of Biological Sciences, University of Liverpool) for general advice in design of nucleotides; and the operating theatre personnel in the Women's Centre, John Radcliffe Hospital, for the collection of hysterectomy specimens.
Notes
4 To whom correspondence should be addressed. E-mail: margaret.rees{at}obs-gyn.ox.ac.uk 
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Submitted on December 19, 2000; accepted on April 27, 2001.
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