- Open Access
Immunoexpression of the relaxin receptor LGR7 in breast and uterine tissues of humans and primates
© Ivell et al; licensee BioMed Central Ltd. 2003
- Received: 29 October 2003
- Accepted: 24 November 2003
- Published: 24 November 2003
The receptor for the peptide hormone relaxin has recently been identified as the heptahelical G-protein coupled receptor, LGR7. In order to generate molecular tools with which to characterize both in vivo and in vitro expression of this receptor in human and primate tissues, specific monotypic antibodies have been generated and applied to a preliminary analysis of human and primate female reproductive tissues.
Three peptide sequences were identified from the proposed open reading frame of the cloned LGR7 receptor gene, representing both extracellular and intracellular domains. Two to three rabbits were immunized for each epitope, and the resulting sera subjected to a systematic validation using cultured cells transiently transfected with a receptor-expressing gene construct, or appropriate control constructs.
Human and monkey (marmoset, macaque) endometrium showed consistent and specific immunostaining in the stromal cells close to glands. Staining appeared to be more intense in the luteal phase of the cycle. Weak immunostaining was also evident in the endometrial epithelial cells of the marmoset. A myoma in one patient exhibited strong immunostaining in the circumscribing connective tissue. Uterine expression was supported by RT-PCR results from cultured primary endometrial and myometrial cells. Human breast tissue (healthy and tumors) consistently indicated specific immunostaining in the interstitial connective (stromal) tissue within the glands, but not in epithelial or myoepithelial cells, except in some tumors, where a few epithelial and tumor cells also showed weak epitope expression.
Using validated monotypic antibodies recognizing different epitopes of the LGR7 receptor, and from different immunized animals, and in different primate species, a consistent pattern of LGR7 expression was observed in the stromal (connective tissue) cells of the endometrium and breast, consistent also with the known physiology of the relaxin hormone.
- Keyhole Limpet Hemocyanin
- Endometrial Stromal Cell
- Human Breast Tissue
- Myometrial Cell
For many years the heterodimeric peptide hormone relaxin was considered only as a molecule involved in the periparturient widening of the pubic symphysis and softening of the cervix. More recently, it has been shown that relaxin, whose structure is similar to that of insulin, is also involved in endometrial differentiation associated with decidualization and embryo implantation [1, 2]. Relaxin acts on primary cultures of endometrial stromal cells also in vitro to induce the morphological and gene expression changes, known as decidualization , which are the essential prerequisite for implantation to occur. In fact, relaxin in this context appears to be more effective even than progesterone . In addition, altered levels of circulating relaxin, presumably of ovarian origin, are linked to early embryonic loss due to implantation failure [3, 4], and it has been shown that luteinizing ovarian granulosa cells from IVF treatments produce higher relaxin levels in association with good pregnancy outcome . Within the female reproductive system, relaxin has also been shown to suppress spontaneous contractions of the myometrium in the rat , and to modulate uterine connective tissue metabolism by regulating the expression of matrix metalloproteinases . Most recently, it has also been shown to positively influence uterine angiogenesis, probably by the induction of local VEGF [8, 9].
Relaxin is also known to be produced and have effects within the breast [10, 11]. Both H1 and H2 forms of human relaxin have been shown to be synthesized by the epithelial and myoepithelial cells of the human breast . Relaxin also influences growth parameters in breast cancer cell-lines, probably using local NO pathways [12, 13], and recently, it has been shown that significantly higher concentrations of circulating relaxin are associated with the formation of breast cancer metastases . Partly this appears to be due to an increased ability of breast cancer cells to invade extracellular matrix upon relaxin stimulation .
Two receptors responding to relaxin, LGR7 and LGR8, were cloned and described in 2002 [16, 17]. These are novel members of the G-protein-coupled receptor superfamily, with a heptahelical transmembrane domain and a large glycosylated ectodomain, and are distantly related to the receptors for the glycoproteohormones, such as LH or FSH. In transfected cell systems, these receptors are shown to respond to relaxin by causing an elevation of intracellular cAMP, presumably through a G-protein-dependent activation of adenylate cyclase . In human primary endometrial stromal cells, a similar elevation in cAMP is detected upon relaxin stimulation, but here inhibition of intracellular phosphodiesterases also appears to be playing an important role [18, 19]. Interestingly, pharmacological studies show that for the human LGR8 both relaxin and the recently identified peptide INSL3 (also called relaxin-like factor, RLF) can act as effective ligands, whereas LGR7 only responds to relaxin . In rodents, the equivalent receptors respond exclusively to either relaxin (LGR7) or INSL3 (LGR8), with no overlap in ligand specificity . Neither receptor is able to bind or respond to any of the other related peptide hormones, such as insulin, the IGF family, or the recently identifed INSL4 [16, 17, 20, 21]. Very recently, two further novel receptors, GPCR135 and GPCR142, have also been characterized as responding to a relaxin-like molecule, namely relaxin 3 [22, 23]. These receptors respond only very weakly to the porcine H2 relaxin homologue and appear not to be expressed in non-pregnant female reproductive tissues [22, 23].
Studies on receptor expression and localization in tissues have, until now, relied upon in situ relaxin-binding, using either radioactively labelled or biotinylated ligand [e.g. [24–26]]. These methods mostly appear unable to yield the sensitivity or specificity which can identify precisely the cell types where the relaxin receptors are expressed, and would be unable to distinguish receptors from other proteins interacting with the hormone at high specificity (analogous to the IGF-binding proteins). Alternatively, primary cells can be prepared and subjected to Scatchard analysis. Where this has been carried out, for example for endometrial stromal cells, relaxin receptors are shown to be expressed at a low concentration (ca. 1000 molecules per cell), though they have a high affinity for the peptide (Kd ~1 nM) .
In order to gain a better appreciation of the expression of the LGR7 receptor protein in both normal and pathological human tissues, we have generated a series of monotypic polyclonal antibodies, using as immunogens peptides from within different regions of the human LGR7 protein sequence. These antibodies have been systematically validated, and applied for the first time here to human breast tissue, and human and primate uterus.
Generation of antibodies
Three different peptides were designed using the published human LGR7 sequence as basis. Two peptides (L7-1: CFKNYHDLQKLYLQN AND L7-2: MRKNKINHLNENTFAC) were derived from the large ectodomain in regions which from preliminary folding bioinformatics suggested the sequences to be relatively hydrophilic and exposed on the surface of the protein, and not masked by neighbouring glycosylations at putative Asn-X-Thr/Ser sites. A further peptide epitope (L7-3: CSAITATEIRNQVKKEM) was derived from the sequence of the third intracellular loop, in a region which has been shown to generate good antibodies against other G-protein coupled receptors, for example, the oxytocin receptor . All peptide sequences were checked for uniqueness by searching the international databases. The peptides were then chemically synthesized and coupled via N- or C-terminal cysteine residues to keyhole limpet hemocyanin, before using as immunogens in rabbits, following a conventional immunization protocol . Peptide production and immunization was carried out by a commercial company (Pineda Antibody Services, Berlin, Germany). All rabbits were checked for non-specific binding of sera prior to immunization, and pre-immune sera were collected from all animals selected for immunization. Animals were bled following the second boost, and these sera used for all subsequent studies.
Cloning and transfection of human LGR7 and LGR8 expression constructs
Full-length cDNA constructs for human LGR7 and LGR8 were kindly provided by Professor Aaron Hsueh (Stanford University, CA). These were inserted into the pcDNA3.1-Zeo expression vector (Invitrogen, San Diego, CA) driven by a CMV viral promoter. In order to improve protein expression the natural signal sequences of the two receptors had been replaced by that of the human prolactin precursor polypeptide. In order to validate the specificity of the antibodies generated, the LGR7 and LGR8 expression constructs were transiently transfected into HEK293T cells, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, at a DNA concentration of 4 μg expression plasmid (encoding LGR7, LGR8 or the orphan G-protein coupled receptor, HE6) and 0.2 μg pEGFP-N1 (encoding enhanced green fluorescent protein; BD Biosciences, Heidelberg, Germany) per well. Cells were grown in 6-well plates (Nunc, Roskilde, Denmark) at a density of 106 cells per well in DMEM with phenol red (Sigma, Taufkirchen, Germany), 10% FCS Gold (PAA Laboratories, Linz, Austria), 2 mM L-Glutamine (Gibco Paisley, UK), without antibiotics. Transfection was performed after 24 h in serum-free OPTI-MEM medium (Gibco). After a further 24 hours, the transfected cells were seeded into T25 flasks (Nunc), and after overnight incubation were trypsinized, washed three times in PBS (Sigma) at 4°C, and transferred to cytospin chambers at a concentration of 50000 cells per chamber. After centrifugation at 800 rpm for 5 mins in a cytospin centrifuge, media was removed and the cells air-dried, and then fixed in 3% (w/v) paraformaldehyde for 10 mins, followed by 3 washes of 5 min each in PBS, and then permeabilization in 0.1% (v/v) Triton X-100 in PBS for 5 min. After further washing twice for 5 min in PBS, the cytospin preparations were subjected to immunohistochemistry. In order to validate the new antibodies generated, immune and the respective pre-immune sera from the same animals were diluted 1:4000, and applied to the permeablized cytospin preparations. Specific immunoreactions were visualized using the double PAP-ABC combination technique (see below) as previously described , but without additional nuclear counterstaining. As a further control, also an expression construct encoding HE6 (generous gift from Dr. C. Osterhoff, Hamburg, Germany), was similarly transfected into HEK293T cells. This receptor belongs to a discrete subfamily of heptahelical domain receptors from LGR7, though also possesses a large glycosylated ectodomain [31, 32]. The internal control transfections using the EGFP expression construct all indicated a high percentage (>60%) of cells transfected for all construct combinations (not shown).
Immunohistochemistry of human and primate tissues
Human tissues from four patients were collected in observance of the Helsinki Declaration, and with the authorization of the local ethical committee. Uterine tissue from two cynomolgus macaques (secretory phase) was a generous gift from Dr UF Habenicht (Schering AG, Berlin, Germany) and was also obtained with full ethical approval from the appropriate authority. Marmoset monkey tissues (five breeding females, 3–6 years old) were collected, with the approval of the local ethics commission on animal experiments, from a breeding colony at the German Primate Center in Göttingen, Germany, as previously described [4, 33]. Tissues were immersion-fixed overnight in buffered 10% (w/v) formaldehyde (human tissues), in 5% buffered formaldehyde for 3 h (marmosets), or in Bouin's solution overnight (macaque), before passaging through ascending ethanols to be embedded in paraffin. Human and macaque tissues were cut to 5–7 μm, and processed for immunohistochemistry as previously described , using the PAP(peroxidase-anti-peroxidase)-ABC(avidin-biotin-complex) combination method. Briefly, after passing through xylol and descending ethanols, entparafinized sections were washed 2 × 10 min in TBS (Tris-buffered saline; 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.4), then endogenous peroxidase was suppressed by incubation in 3% (v/v) H2O2 for 45 min at room temperature (RT). Sections were then blocked with 10% (v/v) normal goat serum for 1 h at RT. After brief rinsing in TBS, sections were incubated with the primary antiserum, diluted 1:4000 (L7-1, L7-2) or 1:5000 (L7-3) in antibody dilution buffer (ADB; TBS containing 2% (v/v) normal goat serum plus 0.05% (w/v) bovine serum albumin), overnight at 4°C. After rinsing 3 × 5 min in TBS, sections were then incubated with biotinylated goat-anti-rabbit IgG, diluted 1:500 in ADB for 1 h at RT. After 3 × 5 min rinsing in TBS, sections were then incubated in rabbit PAP-complex (Dianova, Hamburg, Germany), again diluted 1:500 in ADB, for 1 h at RT. After 3 × 5 min rinsing in TBS, these last two incubation steps were repeated, this time for only 30 min per step. After further brief rinsing in TBS, sections were then incubated with ABC-complex (Vector Laboratories, Burlingame, USA) for 1 h at RT. Specific signals were detected using DAB (Sigma) as chromogen, stopping the reaction by rapidly rinsing in tapwater, followed by conventional haemalaun counterstaining. Sections were then coverslipped in aqueous mounting medium. Marmoset tissues used the complete ABC-complex staining system (Vector Laboratories) as recommended by the manufacturer with AEC as chromogen. As controls for immunohistochemical specificity, all sections were treated in parallel with both immune sera and the pre-immune sera from the same animals at the same dilutions. All immunohistochemical analyses were completely repeated at least once for all tissues, with identical results. As a further control, some sections were additionally exposed to diluted primary antisera which had been subject to a preincubation for 2 h at room temperature with pure keyhole limpet hemocyanin (Sigma, Deisenhofen, Germany) in TBS at a concentration of 10 μg/ml.
RT-PCR analysis for LGR7 gene transcripts
Oligonucleotides used for the RT-PCR reactions.
(see Fig. 4C)
Production and validation of polyclonal anti-LGR7 antibodies
Polyclonal antisera were produced in rabbits against three discrete peptide epitopes from the same polypeptide molecule which represents the principal receptor for the peptide hormone relaxin, LGR7. For each epitope 2–3 different animals were immunized, each of which had had sera checked prior to immunization for low background non-specific immunostaining. Therefore, as controls for the validity of the antibodies, we are looking for positive immunostaining which agrees in cell-type specificity for all three epitopes from the same protein molecule, and which further agrees between different animals for the same epitope. As a second control system, all antisera are compared directly on parallel sections with the pre-immune sera taken from the same animals and used at the same dilution. Thirdly, it is possible that some antibodies will be generated against the keyhole limpet hemocyanin used as hapten, although this molecule is not expressed in any mammalian cell. Nevertheless, there remains the possibility that such antibodies could recognize a cell-specific mimetic epitope. In order to check for this, in further control experiments, appropriately diluted antisera were preincubated with pure keyhole limpet hemocyanin to saturate any such antibodies, before presenting them to the tissue sections.
Expression of LGR7 epitopes in primate and human endometrium
Expression of LGR7 epitopes in normal and cancerous human breast tissues
Antibodies represent one of the most sensitive instruments for the detection of receptor proteins in situ in tissue sections, and offer a high spatial resolution by comparison with radiolabelled ligand-binding or in situ mRNA hybridization. The use of biotinylated ligands can also offer a high resolution, but may be accompanied by problems of non-specific binding. In a recent study where biotinylated relaxin was used to detect binding proteins and possibly receptors in the marmoset uterus, a method was employed which also indicated the electrophoretic mobility of the binding moieties . Although some of the proteins detected might have represented receptors, other proteins of a diverse range of molecular weights were also observed, reinforcing the limited specificity of such methodology. We have made similar observations also for the human uterus (Y. Pohnke, unpublished observations). In the present study, polyclonal antibodies were raised against three quite different epitopes of the recently cloned LGR7 heptahelical domain relaxin receptor. For each epitope two or three rabbits had been immunized. Thus, as a first criterion of specificity, only those immunostaining features common to all three epitopes, and for more than one animal per epitope, can be considered as specific for the relaxin receptor. As a second criterion, all immunostaining was perfomed in parallel using as negative controls pre-immune sera from the same animals and at the same dilutions, under which conditions only the specific signals should not be detected. Thirdly, since all peptide epitopes were cross-linked to keyhole limpet hemocyanin (KLH) as a common hapten, it is possible that false positive immunostaining, meeting the above two criteria, could be obtained when antibodies recognizing the KLH cross-react with a mimetic epitope of some kind, though considerable experience of many scientists over the years suggests that this is a very unusual event for this hapten. To check for such source of error, antibodies were additionally pre-incubated with a large excess of pure KLH prior to applying them to tissue sections. The results showed that for the uterine sections of the cynomolgus macaque the staining in the epithelial cell layer may have been due to such false positive reactions. In contrast, the staining of the endometrial stroma remained quantitatively unaltered. In the human endometrium there was no such epithelial staining in the first place, reinforcing the view that, where this occurred in the macaque, this was indeed probably due to a false positive reaction. Finally, as positive controls, cytospin preparations were made of cell-lines having no endogenous relaxin receptors, as well as of similar cells transiently transfected with expression constructs encoding the LGR7 receptor, or alternatively the related receptors, LGR8 and HE6. All the LGR7 antibodies used showed clear positive staining only in those cells transfected by the LGR7 constructs, and neither in non-transfected cells nor in the cells expressing the other related receptors. In an independent study in the rat brain, the antibodies L7-2 and L7-3, whose immunogenic epitopes differ only slightly between humans and rats, indicate complete cellular identity between immunostaining, 33P-relaxin binding and in situ mRNA hybridization (T. Burazin, University of Melbourne, personal communication). Given these extensive controls, we can be confident that the immunostaining seen truly represents the relaxin receptor LGR7.
Within the uterus, marked specific immunostaining is seen in the endometrial stromal cells in human, macaque and marmoset uterus. The stroma is not uniformly stained but in patches within the functionalis but not in the basalis. Staining appears to be strongest in those stromal cells adjacent to, or circumscribing endometrial glands. The marmoset monkey offers an ideal model in which to study this expression, since tissues can be obtained from animals whose cycles are precisely synchronized by a luteolytic dose of prostaglandin-F2α , and hence at precise days of the cycle or pregnancy. Using such tissues, and in an independent laboratory, LGR7-immunostaining was observed essentially identical to that observed for the other species, with most being in the stromal cells close to endometrial glands. In the marmoset we can confirm the suggestion offered by the human tissues, that LGR7 immunoreactivity increases from the proliferative to the luteal pase of the cycle. It increases still further in early pregnancy, concomitant with a role in the decidualization process essential for implantation (not shown). The marmoset results were also fully coherent with the results of biotinylated-RLX binding , acting as a further control for the validity of the antibodies in these species.
The epithelial staining, observed consistently for some antibodies in the macaque, is attenuated by preincubaton of the antibodies with an excess of KLH and therefore probably represents unspecific false positive immunostaining. The weak but specific epithelial staining seen in the marmoset monkey appears to be truly specific and is not attenuated in KLH-suppression controls. Negligble immunostaining was observed in the myometrium in all three species. This may be due to true low level expression of the LGR7 receptor protein, though may represent a methodological problem; other receptor proteins in our hands have sometimes required a microwave antigen-retrieval protocol to be visualized in this tissue (MB, personal observations).
These observations of LGR7 immunoexpression in the uterus are very much in agreement with studies using purified human primary cell cultures. Relaxin has been shown to have an important effect in the induction of decidualization in endometrial stromal cells [1, 37]. This effect is mediated by well characterized relaxin receptors on the cell surface, expressed at a concentration of ca. 1000 molecules per cell, and having a Kd of ≤ 1 nM . Relaxin is also known to influence myometrial function by inhibiting spontaneous contractions, though these effects are most marked in rodents, and are considered not significant in the human . Relaxin-binding is also reported for endometrial epithelial cells , and functional responses to relaxin have been reported also for these cells [9, 39, 40].
Using RT-PCR we can detect LGR7 mRNA in all three uterine cell types. Since these are cultured cells, however, these experiments cannot provide information about the levels of transcripts or protein in the intact tissues. Furthermore, the relative levels might be distorted by the proportion of cells expressing the LGR7 transcripts. The immunohistochemistry emphasizes that not all stromal cells express LGR7, but only some of them, and this probably in a cycle-dependent manner. Depending on the ratio of expressing to non-expressing cells in the primary cultures, the observed mRNA concentrations in the extracted RNA samples could vary greatly. What is significant, however, is that known uterine effectors, like progesterone and relaxin, have no effect on the LGR7 gene expression in these cultured cells. This would imply that any cycle-dependent variation in vivo requires a more complex collection of factors and/or paracrine interactions. Transcripts for the shorter splice variant do not appear to be expressed in the cultured cells to any significant degree. Thus, although potentially this transcript could give rise to a free ectodomain, which has experimentally been shown to bind relaxin , there is still no evidence that such splice variants are expressed or translated in vivo, or that any such free ectodomain is exported from the cells.
Of particular interest, was the observation in one patient of fibroid nodules in the myometrium, which were clearly enveloped by positively immunostaining connective tissue cells. While this is possibly part of the normal encapsulation response towards such fibroids, it provokes the question of what role relaxin and its receptor could be playing in the etiology and pathology of this debilitating ailment.
In the normal and pathological human breast, LGR7 immunoreactivity is prominent in the stromal connective tissue between the glandular lobules. Epithelial cells of the healthy breast are unstained. Such epithelial staining does appear to occur sporadically in breast tumor tissue, which would be in agreement with the findings that the breast cancer cell-line MCF7, believed to be of epithelial origin, is also responsive towards relaxin . While the tumor tissue is mostly unstained, in some tumors weak and localized staining can be observed. Several studies have looked at a role for relaxin in human breast function, particularly in the context of cancer [11, 13]. It is reported that breast cancer cell lines can respond to relaxin by a change in growth parameters, probably involving local NO production . More interestingly, it has recently been shown that women with metastatic breast cancer have significantly higher serum relaxin concentrations than those with non-progressing cancer . Furthermore, breast cancer cells treated with relaxin show a significant increase in invasive potential, using a classic in vitro invasion assay . These studies thus emphasize the importance of relaxin on connective tissue remodelling in the breast particularly in relation to cancer progression and metastasis formation, and support well our observations using the new LGR7 antibodies that the relaxin receptor is prominently expressed on the connective tissue cells responsible for creating a capsule around a growing tumor, as well as on some tumor cells of epithelial origin in breast cancer patients. The similarity here to the situation for the uterine fibroids is striking.
The present article describes a new panel of polyclonal antibodies generated against the human LGR7 receptor. Using a combination of immunochemical, molecular biological and immunohistochemical techniques we here provide a first validation of these antibodies and demonstrate their value in studying the physiology and pathology of the human breast and uterus.
We are especially grateful to Professor Freimut Leidenberger and the Leidenberger-Müller-Foundation, Hamburg, for generous financial support of this project, as also to the German Research Council (DFG; grants Iv7/9-1 and Ei333/6-3), the Innovation Funds of the City of Hamburg, and the German Primate Centre, Göttingen, for also supporting parts of this study. We should like to thank Dr Tanya Burazin from the University of Melbourne for permission to cite her unpublished studies using our antisera, and Dr Caroline Osterhoff from the IHF, University of Hamburg, for the generous gift of the HE6 expression construct and HE6-specific antiserum used as controls. Our thanks also go to Dr Aaron Hsueh, Stanford University, for the generous provision of the LGR7 and LGR8 expression vectors used in the cytospin analyses, and Dr. O.D. Sherwood for kindly supplying purified porcine relaxin.
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