Developmental expression and serotonergic regulation of relaxin 3/INSL7 in the nucleus incertus of rat brain

Relaxin 3 or insulin like peptide 7 has been identified as a new member of the insulin/relaxin superfamily. We recently reported that relaxin 3 was dominantly expressed in the brain, particularly in neurons of the nucleus incertus (NI) of the median dorsal tegmental pons and that it might act as a neurotransmitter. In the present study we investigated the developmental expression and serotonergic regulation of relaxin 3 gene in the rat brain. Relaxin 3 mRNA appeared at embryonic day 18 in the near region of the fourth ventricle, and was shown to have increased its density and the number of expressing neurons by in situ hybridization and RT-PCR examination. Relaxin 3 peptide was detected after birth by immunocytochemistry. Since the NI is located just caudal to the dorsal raphe nucleus where abundant serotonin (5-HT) neurons are present, we examined if 5-HT effects on the expression of relaxin 3. Relaxin 3 gene expression in the NI significantly increased after 5-HT depletion by p-chlorophenylalanine (PCPA) administration. We also observed the 5-HT1A receptor localization in relaxin 3 positive neurons of the NI. This result suggests that 5-HT negatively regulates the expression of relaxin 3 gene in the NI. The function of relaxin 3 neurons in the brain is influenced by the serotonergic activity.     

Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.     

Searching the human genome database for novel relaxin- and insulin-like peptides

Summary In recent times, new members of the insulin/relaxin peptide superfamily have been identified by both differential cloning strategies as well as bioinformatic searching of the EST databases. We have used the public and Celera Genomics databases to search for novel members of this peptide family. No new members of the insulin/relaxin family were identified although the human (H3) and mouse (M3) relaxin 3 genes that we recently discovered in the Celera Genomics database were identified in the public database. We were able to confirm that there are no mouse equivalents of human INSL-4 or human gene 1 relaxin. Hence, as the two human relaxin genes (H1 and H2) are localized together with INSL6 and INSL4 on chromosome 9 it is probable that INSL4 and H1 relaxin are the result of a gene duplication which did not occur in non-primates. The discovery of a full relaxin 3 sequences in a new Zebrafish brain EST library, which retains a high homology in both A and B chain peptide sequence with the H3 peptide, indicate that this novel peptide has important conserved functions.     

The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist

Relaxin is a pleiotropic hormone which exerts its biological functions through its G-protein coupled receptor, RXFP1. While relaxin is well known for its reproductive and antifibrotic roles, recent studies suggest that it is produced by cancer cells and acts on RXFP1 to induce growth and metastasis. Furthermore, more recently Silvertown et al. demonstrated that lentiviral production of a human gene-2 (H2) relaxin analog reduced the growth of prostate xenograft tumors. The authors proposed that the lentivirally produced peptide was an RXFP1 antagonist; however, the processed form of the peptide produced was not demonstrated. In this study, we have chemically synthesized the H2 relaxin analog, B-R13/17K H2 relaxin, and subjected it to detailed chemical characterization by HPLC, MALDI-TOF mass spectrometry, and amino acid analysis. The biological activity of the synthetic peptide was then tested in three different cell lines. It was found to bind with 500-fold lower affinity than H2 relaxin to RXFP1 receptors over-expressed in HEK-293T cells where it acted as a partial agonist. However, in cells which natively express the RXFP1 receptor, rat renal myofibroblasts and MCF-7 cancer cells, it acted as a full antagonist. Importantly, it was able to significantly inhibit cell invasion induced by H2 relaxin in MCF-7 cells consistent with the results of the lentiviral-driven expression in prostate cancer cells. The relaxin analog, B-R13/17K H2, can now be used as a tool to further understand RXFP1 function, and serve as a template for drug design for a therapeutic to treat prostate and other cancers.     

Relaxin activity in the pregnant cat

Plasma relaxin activity was measured by radioimmunoassay (RIA) in the domestic cat utilizing two different antisera developed against highly purified porcine relaxin. One was the 5858 antiserum from our laboratory and the other was the R6 antiserum of Dr. Bernard Steinetz. Relaxin activity could not be detected during the estrous cycle or during pseudopregnancy. Relaxin immunoactivity during early gestation was not detected by either antiserum. Plasma relaxin immunoactivity was first detected by both antisera on about Day 25 of gestation. Relaxin concentrations then increased rapidly, with a plateau reached between Days 30 and 35 that was maintained until 10-15 days prepartum. Relaxin concentrations then declined gradually until parturition. No prepartum increase was observed. Relaxin concentrations were undetectable within 24 h of delivery. Although amounts of immunoactivity measured with the R6 antiserum were consistently higher than measurements with the 5858 antiserum, the patterns of secretions observed were similar for both antisera.     

Relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts

Relaxin is believed to play a role in connective tissue remodeling during pregnancy (Bell, R.J., Eddie, L. W., Lester, A. R., Wood, E. C., Johnston, P.D., and Niall, H. D. (1987) Obstet. Gynecol. 69, 585-589; MacLennan, A. H. (1983) Clin. Reprod. Fertil. 2, 77-95). In the present study, normal human fibroblasts exposed to concentrations of a synthetic bioactive relaxin peptide from 0.1 to 10 ng/ml synthesized and secreted the metalloproteinase procollagenase, which was immunoprecipitable as a doublet of 52 and 57 kDa by a monoclonal antibody to human collagenase. The stimulation in procollagenase protein expression was reflected in an elevation in procollagenase mRNA levels. Media conditioned for 48 h by relaxin-treated fibroblasts (0.1 ng/ml) contained 1.7 units/ml activatable collagenase compared with 0.2 units/ml by untreated fibroblasts. In addition, relaxin caused a modest decrease in the levels of tissue inhibitor of metalloproteinases, as detected by reverse zymography and Northern analysis. Relaxin was also a potent modulator of the collagen secretory phenotype of these fibroblasts. Relaxin at 100 ng/ml down-regulated collagen secretion by 40%. When fibroblasts were treated simultaneously with cytokines such as transforming growth factor beta or interleukin 1 beta, which stimulated collagen synthesis to at least 9-fold of basal levels, relaxin at 100 ng/ml was able to down-regulate collagen expression by up to 88%. This decrease was reflected by changes at the mRNA level. These results indicate that relaxin can cause significant collagen turnover both by stimulating collagenase expression and by down-modulating collagen synthesis and secretion.     

Relaxin stimulates bronchial epithelial cell PKA activation,migration, and ciliary beating

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12 hr (1 micro M). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3-4 fold by approximately 4 hr, with a return to baseline levels by 8-10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) beta-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.     

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.     

H3 relaxin demonstrates antifibrotic properties via the RXFP1 receptor

Human gene 3 (H3) relaxin is the most recently discovered member of the relaxin peptide family and can potentially bind all of the defined relaxin family peptide receptors (RXFP1-4). While its effects as a neuromodulator are being increasingly studied through its primary receptor, RXFP3, its actions via other RXFPs are poorly understood. Hence, we specifically determined the antifibrotic effects and mechanisms of action of H3 relaxin via the RXFP1 receptor using primary rat ventricular fibroblasts in vitro, which naturally express RXFP1, but not RXFP3, and a mouse model of fibrotic cardiomyopathy in vivo. Transforming growth factor β1 (TGF-β1) administration to ventricular fibroblasts significantly increased Smad2 phosphorylation, myofibroblast differentiation, and collagen deposition (all p < 0.05 vs untreated controls), while having no marked effect on matrix metalloproteinase (MMP) 9, MMP-13, tissue inhibitor of metalloproteinase (TIMP) 1, or TIMP-2 expression over 72 h. H3 relaxin (at 100 and 250 ng/mL) almost completely abrogated the TGF-β1-stimulated collagen deposition over 72 h, and its effects at 100 ng/mL were equivalent to that of the same dose of H2 relaxin. Furthermore, H3 relaxin (100 ng/mL) significantly inhibited TGF-β1-stimulated cardiac myofibroblast differentiation and TIMP-1 and TIMP-2 expression to an equivalent extent as H2 relaxin (100 ng/mL), while also inhibiting Smad2 phosphorylation to approximately half the extent of H2 relaxin (all p < 0.05 vs TGF-β1). Lower doses of H3 (50 ng/mL) and H2 (50 ng/mL) relaxin additively inhibited TGF-β1-stimulated collagen deposition in vitro, while H3 relaxin was also found to reverse left ventricular collagen overexpression in the model of fibrotic cardiomyopathy in vivo. These combined findings demonstrate that H3 relaxin exerts antifibrotic actions via RXFP1 and may enhance the collagen-inhibitory effects of H2 relaxin.     

Relaxin Treatment in an Ang-II-Based Transgenic Preeclamptic-Rat Model

Relaxin is a peptide related to pregnancy that induces nitric oxide-related and gelatinase-related effects, allowing vasodilation and pregnancy-related adjustments permitting parturition to occur. Relaxin controls the hemodynamic and renovascular adaptive changes that occur during pregnancy. Interest has evolved regarding relaxin and a therapeutic principle in preeclampsia and heart failure. Preeclampsia is a pregnancy disorder, featuring hypertension, proteinuria and placental anomalies. We investigated relaxin in an established transgenic rat model of preeclampsia, where the phenotype is induced by angiotensin (Ang)-II production in mid pregnancy. We gave recombinant relaxin to preeclamtic rats at day 9 of gestation. Hypertension and proteinuria was not ameliorated after relaxin administration. Intrauterine growth retardation of the fetus was unaltered by relaxin. Heart-rate responses and relaxin levels documented drug effects. In this Ang-II-based model of preeclampsia, we could not show a salubrious effect on preeclampsia.     

Relaxin concentrations in endometrial, placental, and ovarian tissues and in sera from ewes during middle and late pregnancy

These studies were designed to determine the tissue source of ovine relaxin and to determine the feasibility of using the pregnant ewe for study of relaxin production and secretion. On Day 4 of gestation, ewes were laparotomized, the nonpregnant uterine horn was ligated, and the ovary not containing the corpus luteum was removed. During a second surgery at Day 45 (n = 8) or 140 (n = 9) of gestation, 10-ml blood samples were drawn from a uterine artery, the ovarian vein, and veins draining the pregnant and nonpregnant uterine horns. Endometrial, placental, and luteal tissues were obtained for immunocytochemistry and extraction. Relaxin was detected by a heterologous porcine radioimmunoassay (RIA) in 3 of 54 serum samples (701.3 +/- 25.4 pg/ml, mean +/- SEM). Relaxin was not detected in crude tissue extracts, but low quantities were detected by RIA following Sephadex G-50 column chromatography of tissue extracts. Total relaxin activity for all tissues was equivalent to 0.57 +/- 0.13 ng of porcine relaxin/g tissue (w.w.). Relaxin was not detected immunocytochemically by light or electron microscopy. These data indicate that low quantities of relaxin are present in tissues and sera of pregnant ewes.     

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

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.     

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin

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.     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.