Luteal and hypophyseal expression of the canine relaxin (RLN) system during pregnancy: Implications for luteotropic function.
ABSTRACT: By acting through its receptors (RXFP1, RXFP2), relaxin (RLN) exerts species-specific effects during pregnancy; possible luteotropic effects through stimulation of prolactin (PRL) release have been suggested. In the domestic dog (Canis lupus familiaris) serum PRL increases in pregnant bitches shortly after RLN appears in the circulation, and a possible functional relationship between the RLN and the PRL systems in regulating progesterone secretion has been implied. Therefore, here (Study 1) the luteal expression and localization of the RLN system was investigated by immunohistochemistry using custom-made antibodies and semi-quantitative PCR, at selected time points during gestation: pre-implantation (d. 8-12), post-implantation (d. 18-25), mid-gestation (d. 35-40) and at normal and antigestagen-induced luteolysis. Further, (Study 2) hypophyseal expression of the RLN system and its spatial association with PRL was assessed. Luteal expression of RLN, but not of its receptors, was time-dependent: it increased significantly following implantation towards mid-gestation and decreased at prepartum. Antigestagen treatment resulted in downregulation of RLN and RXFP2. Whereas RLN was localized in steroidogenic cells, RXFP1 and RXFP2 also stained strongly in macrophages and vascular endothelial cells. The RLN system was detected in the canine adenohypophysis and was co-localized with PRL in hypophyseal lactotrophs. The intraluteal RLN seems to be involved in regulating the canine corpus luteum (CL) in a time-dependent manner. The presence of RLN family members in the adenohypophysis implies their possible involvement in regulating the availability of PRL and other pituitary hormones.
Project description:BACKGROUND: Endocrine mechanisms governing canine reproductive function remain still obscure. Progesterone (P4) of luteal origin is required for maintenance of pregnancy. Corpora lutea (CL) are gonadotrop-independent during the first third of dioestrus; afterwards prolactin (PRL) is the primary luteotropic factor. Interestingly, the increasing PRL levels are accompanied by decreasing P4 concentrations, thus luteal regression/luteolysis occurs in spite of an increased availability of gonadotropic support. PRL acts through its receptor (PRLr), the expression of which has not yet been thoroughly investigated at the molecular and cellular level in the dog. METHODS: The expression of PRLr was assessed in CL of non-pregnant dogs during the course of dioestrus (days 5, 15, 25, 35, 45, 65 post ovulation; p.o.) as well as in CL, the utero/placental compartments (Ut/Pl) and interplacental free polar zones (interplacental sites) from pregnant dogs during the pre-implantation, post-implantation and mid-gestation period of pregnancy and during the normal and antigestagen-induced luteolysis. Expression of PRLr was tested by Real Time PCR, immunohistochemistry and in situ hybridization. RESULTS: In non-pregnant CL the PRLr expression was significantly upregulated at day 15 p.o. and decreased significantly afterwards, towards the end of dioestrus. CL of pregnancy showed elevated PRLr expression until mid gestation while prepartal downregulation was observed. Interestingly, placental but not interplacental expression of PRLr was strongly time-related; a significant upregulation was observed towards mid-gestation. Within the CL PRLr was localized to the luteal cells; in the Ut/Pl it was localized to the fetal trophoblast and epithelial cells of glandular chambers. Moreover, in mid-pregnant animals treated with an antigestagen, both the luteal and placental, but not the uterine PRLr were significantly downregulated. CONCLUSIONS: The data presented suggest that the luteal provision of P4 in both pregnant and non-pregnant dogs may be regulated at the PRLr level. Furthermore, a role of PRL not only in maintaining the canine CL function but also in regulating the placental function is strongly suggested. A possible functional interrelationship between luteal P4 and placental and luteal PRLr expression also with respect to the prepartal luteolysis is implied.
Project description:The relaxin family peptides, although structurally closely related to insulin, act on a group of four G protein-coupled receptors now known as Relaxin Family Peptide (RXFP) Receptors. The leucine-rich repeat containing RXFP1 and RXFP2 and the small peptide-like RXFP3 and RXFP4 are the physiological targets for relaxin, insulin-like (INSL) peptide 3, relaxin-3 and INSL5, respectively. RXFP1 and RXFP2 have at least two binding sites--a high-affinity site in the leucine-rich repeat region of the ectodomain and a lower-affinity site in an exoloop of the transmembrane region. Although they respond to peptides that are structurally similar, RXFP3 and RXFP4 demonstrate distinct binding properties with relaxin-3 being the only peptide that can recognize these receptors in addition to RXFP1. Activation of RXFP1 or RXFP2 causes increased cAMP and the initial response for both receptors is the resultant of Gs-mediated activation and G(oB)-mediated inhibition of adenylate cyclase. With RXFP1, an additional delayed increase in cAMP involves betagamma subunits released from G(i3). In contrast, RXFP3 and RXFP4 inhibit adenylate cyclase and RXFP3 causes ERK1/2 phosphorylation. Drugs acting at RXFP1 have potential for the treatment of diseases involving tissue fibrosis such as cardiac and renal failure, asthma and scleroderma and may also be useful to facilitate embryo implantation. Activators of RXFP2 may be useful to treat cryptorchidism and infertility and inhibitors have potential as contraceptives. Studies of the distribution and function of RXFP3 suggest that it is a potential target for anti-anxiety and anti-obesity drugs.
Project description:Extensive uterine adaptations, including angiogenesis, occur prior to implantation in early pregnancy and are potentially regulated by the peptide hormone relaxin. This was investigated in two studies. First, we took a microarray approach using human endometrial stromal (HES) cells treated with relaxin in vitro to screen for target genes. Then we aimed to investigate whether or not relaxin deficiency in mice affected uterine expression of representative genes associated with angiogenesis and uterine remodeling, and also blood vessel proliferation in the pre-implantation mouse endometrium.Normal HES cells were isolated and treated with recombinant human relaxin (10 ng/ml) for 24 h before microarray analysis. Reverse transcriptase PCR was used to analyze gene expression of relaxin and its receptor (Rxfp1) in ovaries and uteri; quantitative PCR was used to analyze steroid receptor, angiogenesis and extracellular matrix remodeling genes in the uteri of wild type (Rln+/+) and Rln-/- mice on days 1-4 of pregnancy. Immunohistochemistry localized endometrial endothelial cell proliferation and mass spectrometry measured steroid hormones in the plasma.Microarray analysis identified 63 well-characterized genes that were differentially regulated in HES cells after relaxin treatment. Expression of some of these genes was increased in the uterus of Rln+/+ mice by day 4 of pregnancy. There was significantly higher vascular endothelial growth factor A (VegfA), estrogen receptor 1 (Esr1), progesterone receptor (Pgr), Rxfp1, egl-9 family hypoxia-inducible factor 1 (Egln1), hypoxia inducible factor 1 alpha (Hif1?), matrix metalloproteinase 14 (Mmp14) and ankryn repeat domain 37 (Ankrd37) in Rln-/- compared to Rln+/+ mice on day 1. Progesterone receptor expression and plasma progesterone levels were higher in Rln-/- mice compared to Rln+/+ mice. However, endometrial angiogenesis was not advanced as pre-implantation endothelial cell proliferation did not differ between genotypes.Relaxin treatment modulates expression of a variety of angiogenesis-related genes in HES cells. However, despite accelerated uterine gene expression of steroid receptor, progesterone and angiogenesis and extracellular matrix remodeling genes in Rln-/- mice, there was no impact on angiogenesis. We conclude that although relaxin deficiency results in phenotypic changes in the pre-implantation uterus, endogenous relaxin does not play a major role in pre-implantation angiogenesis in the mouse uterus.
Project description:BACKGROUND: Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors. METHODS: The presence of mRNA for Rxfp1 and Rxfp2 was investigated in testes, cultured Sertoli cells, epididymis, vas deferens, seminal vesicle, prostate, and spermatozoa by RT-PCR and Southern blot. Protein expression in the testis, vas deferens, primary culture of Sertoli cells, and spermatozoa was assessed by immunohistochemistry and immunofluorescence. The role of relaxin in the vas deferens was evaluated by contractility studies and radioimmunoassay of cAMP production. The effect of relaxin on mRNA levels for metalloproteinase-7 was measured by Northern blot. RESULTS: Transcripts for Rxfp1 and Rxfp2 were present in almost all parts of the male reproductive tract, with high levels in testis and vas deferens. Both receptors were immunolocalized in late stage germ cells but not in mature spermatozoa, although mRNAs for both receptors were also present in mature spermatozoa. Rxfp1 but not Rxfp2 was detected in cultured Sertoli cells. Strong immunostaining for Rxfp1 and Rxfp2 was seen in muscular and epithelial layers of the vas deferens and in arteriolar walls. Relaxin did not affect contractility and cyclic AMP production of the vas deferens, but increased the levels of mRNA for metalloproteinase-7. CONCLUSION: Rxfp1 and Rxfp2 are widely and similarly distributed throughout the male reproductive tract. Our results suggest that Rxfp1 on spermatids and Sertoli cells may be important in spermatogenesis. Relaxin in the vas deferens does not affect contractility, but may affect vascular compliance and collagen and matrix remodeling.
Project description:Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leucine-rich repeat (LRR)-containing modules. Although relaxin can bind and activate both RXFP1 and RXFP2, INSL3 can only bind and activate RXFP2. To investigate whether this difference is related to the nature of the high-affinity ECD binding site or to differences in secondary binding sites involving the receptor transmembrane (TM) domain, we created a suite of constructs with RXFP1/2 chimeric ECD attached to single TM helices. We show that by changing as little as one LRR, representing four amino acid substitutions, we were able to engineer a high-affinity INSL3-binding site into the ECD of RXFP1. Molecular modeling of the INSL3-RXFP2 interaction based on extensive experimental data highlights the differences in the binding mechanisms of relaxin and INSL3 to the ECD of their cognate receptors. Interestingly, when the engineered RXFP1/2 ECD were introduced into full-length RXFP1 constructs, INSL3 exhibited only low affinity and efficacy on these receptors. These results highlight critical differences both in the ECD binding and in the coordination of the ECD-binding site with the TM domain, and provide new mechanistic insights into the binding and activation events of RXFP1 and RXFP2 by their native hormone ligands.
Project description:Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit-human and guinea pig-human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing.
Project description:Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker's highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.
Project description:In most mammals the peptide hormone relaxin is a key physiological component regulating early pregnancy and birth. However, synteny analysis shows that the gene encoding ovarian relaxin-2 is deleted in cows and sheep. While, these ruminants appear to exhibit a relaxin-like physiology, as in other mammals, until now a molecular understanding of this has been lacking. Cloning and expression analysis of the cognate bovine receptor for relaxin, RXFP1, as well as of the structurally related receptor, RXFP2, in female tissues, shows that these are expressed in a similar way to other mammals. RXFP1 transcripts are found in ovarian theca cells, endometrium, and myometrium, whereas RXFP2 transcripts are expressed in ovarian theca cells, oocytes, as well as in myometrium. Transfection of receptor-expressing gene constructs into HEK293 cells indicates that bovine RXFP1 has a greater EC50 at 10-50 nM for porcine or human relaxin, compared to human RXFP1. For bovine RXFP2, in contrast, the EC50 is <1 nM for its cognate ligand, bovine INSL3, but also 10-30 nM for porcine or human relaxin. Functional analysis shows that bovine myometrial cells are able to respond to exogenous relaxin and INSL3 with a significant increase in cAMP. Although expressing mRNA for both RXFP1 and RXFP2, bovine follicular theca cells only respond to INSL3 with a dose-dependent increase in cAMP. Altogether the results suggest that the cow is able to compensate for the missing hormone, and moreover imply that relaxin analogs could offer an important therapeutic option in treating female ruminant infertility.
Project description:Relaxin, a heterodimeric polypeptide hormone, is a key regulator of collagen metabolism and multiple vascular control pathways in humans and rodents. Its actions are mediated via its cognate G-protein-coupled receptor, RXFP1 although it also "pharmacologically" activates RXFP2, the receptor for the related, insulin-like peptide 3 (INSL3), which has specific actions on reproduction and bone metabolism. Therefore, experimental tools to facilitate insights into the distinct biological actions of relaxin and INSL3 are required, particularly for studies of tissues containing both RXFP1 and RXFP2. Here, we chemically functionalized human (H2) relaxin, the RXFP1-selective relaxin analog H2:A(4-24)(F23A), and INSL3 to accommodate a fluorophore without marked reduction in binding or activation propensity. Chemical synthesis of the two chains for each peptide was followed by sequential regioselective formation of their three disulfide bonds. Click chemistry conjugation of Cy5.5 at the B-chain N-terminus, with conservation of the disulfide bonds, yielded analogs displaying appropriate selective binding affinity and ability to activate RXFP1 and/or RXFP2 in vitro. The in vivo biological activity of Cy5.5-H2 relaxin and Cy5.5-H2:A(4-24)(F23A) was confirmed in mice, as acute intracerebroventricular (icv) infusion of these peptides (but not Cy5.5-INSL3) stimulated water drinking, an established behavioral response elicited by central RXFP1 activation. The central distribution of Cy5.5-conjugated peptides was examined in mice killed 30 min after infusion, revealing higher fluorescence within brain tissue near-adjacent to the cerebral ventricle walls relative to deeper brain areas. Production of fluorophore-conjugated relaxin family peptides will facilitate future pharmacological studies to probe the function of H2 relaxin/RXFP1 and INSL3/RXFP2 signaling in vivo while tracking their distribution following central or peripheral administration.
Project description:Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin.