H2 Relaxin Research Articles

Synthesis, Conformation, and Activity of Human Insulin‐Like Peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5–RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as Nα-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function.

The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of the Different Relaxin Family Peptide Receptors

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.

AP‐1 is activated by relaxin family peptides in cells expressing the relaxin family peptide receptor 3 (RXFP3)

Relaxin‐3 is a neuropeptide involved in appetite regulation and stress behaviour in rats. RXFP3 is the cognate receptor for relaxin‐3 that is coupled to cAMP inhibition and ERK1/2 activation in CHO‐K1 and HEK293 cell lines recombinantly expressing the receptor. Competition for [125I]‐INSL5/H3 relaxin chimera binding is observed with H3 relaxin and the B‐chain of H3 relaxin, but not with any other relaxin family peptides. We identified that H3 relaxin but also H2 relaxin and porcine relaxin activate transcription from AP‐1 elements in CHO and HEK cells transiently expressing human RXFP3 receptors, measured using a reporter gene. H2 relaxin was more potent at activating transcription than either H3 relaxin or porcine relaxin. Experiments with inhibitors of p38 MAPK (RWJ67657, 10 μM), MEK1/2 (PD98059, 20μM) and JNK (SP600125, 10μ M) showed that AP‐1 activation involved p38 MAPK in CHO‐K1 cells stimulated with H3 relaxin, H2 relaxin or porcine relaxin. In HEK293 cells, AP‐1 activation in response to H3 relaxin or porcine relaxin involved p38 MAPK, MEK1/2 and JNK, but, when stimulated with H2 relaxin, it involved only a MEK1/2 pathway. We propose that different relaxin family peptides interact at different sites on RXFP3, to activate distinct signalling pathways.This research was supported by National Health and Medical Research Council Project Grants 436713 and 454375

221. Localisation of relaxin receptors (Rxfp1) in the uterine artery and the effects of blocking circulating relaxin on passive mechanical wall properties in the uterine artery of late pregnant rats

During pregnancy, the uteroplacental circulation undergoes dramatic alterations to allow for the large increase in blood flow to the feto-placental unit. These alterations are achieved through several mechanisms including structural changes in the uterine artery wall and endothelium-dependent vasodilation. Small renal arteries of relaxin-deficient mice and rats have enhanced myogenic reactivity and decreased passive compliance, and are relatively vasoconstricted (Novak et al. 2001, 2006). To date, no study has identified relaxin receptors (Rxfp1) in arteries or investigated the effects of relaxin deficiency in pregnancy on uterine artery function. The aims of this current study were to: 1) localise Rxfp1 in the uterine arteries, 2) measure myogenic reactivity in small uterine arteries after relaxin treatment, and 3) test the hypothesis that blocking circulating relaxin in late pregnancy will increase uterine artery wall stiffness. We demonstrated that Rxfp1 is expressed in the uterine arteries of pregnant mice and rats. Brightfield immunohistochemistry and immunofluorescence using antibodies specific for rat Rxfp1, α-smooth muscle actin and CD31 localised Rxfp1 protein predominantly to the vascular smooth muscle in the uterine artery of pregnant rats. Administration of recombinant human H2 relaxin (4 ug/h) for 6 h or 5 days in intact and ovariectomised rats reduced myogenic reactivity of small uterine arteries in vitro. Pregnant rats were treated with a monoclonal antibody against circulating relaxin (MCA1) or control (MCAF) for 3 days (Days 17–19) and uterine arteries were mounted on a pressure myograph to assess passive mechanical wall properties. Neutralising circulating relaxin in late pregnancy resulted in a significant increase in uterine artery wall stiffness. These data demonstrate that relaxin acts on the vascular smooth muscle cells in the uterine artery and may be involved in the pregnancy-specific vascular remodelling of uterine arteries to increase vasodilation and blood flow to the uterus and placenta. (1) Novak J et al. (2001). J Clin Invest 107: 1469–75 (2) Novak J et al. (2006). FASEB J 20: 2352–62

Effects of recombinant H2 relaxin on the expression of matrix metalloproteinases and tissue inhibitor metalloproteinase in cultured early placental extravillous trophoblasts

Relaxin promotes softening of the uterine cervix and inhibits uterine contractility in rats, mice and pigs. Little information, however, is available about the role of relaxin in humans. In 2002, LGR7 and LGR8 were discovered to be receptors for relaxin and those receptors were identified in the human placenta. Thus, in this study, effects of recombinant H2 (rH2) relaxin on human early placental extravillous trophoblasts (EVTs) were examined. Isolation of EVTs from early placental trophoblasts was performed using the procedures established in our laboratory. After 48-h subculture, the presence of relaxin receptors in cultured EVTs was characterized by RT-PCR and immunoblotting. The cultured EVTs were treated with different doses (0.3-3 ng/ml) of rH2 relaxin for 24 h. The effects of rH2 relaxin on MMP-2, -3, -9 and TIMP-1 mRNAs levels were examined by real-time RT-PCR. RT-PCR and immunoblotting revealed that relaxin receptors are present in early placental EVTs. Treatment with rH2 relaxin increased MMP-2 and -9 mRNAs levels and decreased TIMP-1 mRNA levels in cultured EVTs, whereas rH2 relaxin did not affect MMP-3 mRNA levels. These results suggest that relaxin may promote the invasive potential of early placental EVTs through up-regulating MMP-2, -9 mRNAs and down-regulating TIMP-1 mRNA in EVTs.

A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation

In humans, the relaxin hormone family includes H1, H2 and H3 isoforms and insulin-like peptides 3 to 6. The ever-increasing interest in relaxin as potential new drug requires reliable methods to compare bioactivity of different relaxins. The existing bioassays include in vivo or ex vivo methods evaluating the organ-specific responses to relaxin and in vitro methods based on measurement of cAMP increase in relaxin receptor-bearing cells. We previously demonstrated that relaxin dose-dependently inhibits platelet aggregation. On this basis, we have developed a simple, reliable bioassay for relaxin used to compare purified porcine relaxin, assumed as reference standard, with two recombinant human H2 relaxins, H3 relaxin, insulin-like peptides 3 and 5. Pre-incubation of platelets with relaxins (3, 10, 30,100, 300 ng/ml; 10 min.) caused the inhibition of ADP-induced platelet aggregation. Within the 10–100 ng/ml range, porcine relaxin showed the highest effects and a nearly linear dose–response correlation. Lower peptide concentrations were ineffective, as were insulin-like peptides 3 and 5 at any concentration assayed. Platelet inhibition was mediated by specific RXFP1 relaxin receptor and cGMP, whose intracellular levels dose-dependently increased upon relaxin. For comparison, we stimulated THP-1 cells, a relaxin receptor-bearing cell line, with porcine relaxin, human H2 and H3 relaxins at the above concentrations (15 min.). We observed a dose-related increase of intracellular cAMP similar to the trend of platelet inhibition. Insulin like peptide 5 was ineffective. In conclusion, this study shows that inhibition of platelet aggregation may be used to assess bioactivity of relaxin preparations for experimental and clinical purposes.

Improved Chemical Synthesis and Demonstration of the Relaxin Receptor Binding Affinity and Biological Activity of Mouse Relaxin

The primary stored and circulating form of relaxin in humans, human gene-2 (H2) relaxin, has potent antifibrotic properties with rapidly occurring efficacy. However, when administered to experimental models of fibrosis, H2 relaxin can only be applied over short-term (2-4 week) periods, due to rodents mounting an antibody response to the exogenous human relaxin, resulting in delayed clearance and, hence, increased and variable circulating levels. To overcome this problem, the current study investigated the therapeutic potential of mouse relaxin over long-term exposure in vivo. Mouse relaxin is unique among the known relaxins in that it possesses an extra residue within the C-terminal region of its A-chain. To enable a detailed assessment of its receptor interaction and biological properties, it was chemically synthesized in good overall yield by the separate preparation of each of its A- and B-chains followed by regioselective formation of each of the intramolecular and two intermolecular disulfide bonds. Murine relaxin was shown to bind with high affinity to the human, mouse, and rat RXFP1 (primary relaxin) receptor but with a slightly lower affinity to that of H2 relaxin. When administered to relaxin-deficient mice (which undergo an age-dependent progression of organ fibrosis) over a 4 month treatment period, mouse relaxin was able to significantly inhibit the progression of collagen accumulation in several organs including the lung, kidney, testis, and skin (all p < 0.05 vs untreated group), consistent with the actions of H2 relaxin. These combined data demonstrate that mouse relaxin can effectively inhibit collagen deposition and accumulation (fibrosis) over long-term treatment periods.

1123: Defining the Signaling Mechanism and Relevance of H2 Relaxin in Prostate Cancer Progression

You have accessJournal of UrologyModerated Poster 33, Monday, May 21, 2007, 3:30 - 5:30 pm1 Apr 20071123: Defining the Signaling Mechanism and Relevance of H2 Relaxin in Prostate Cancer Progression Ruth L. Vinall, Kim Hwa, Shangqin Liu, Hsing-Jien Kung, and Ralph W. deVere White Ruth L. VinallRuth L. Vinall More articles by this author , Kim HwaKim Hwa More articles by this author , Shangqin LiuShangqin Liu More articles by this author , Hsing-Jien KungHsing-Jien Kung More articles by this author , and Ralph W. deVere WhiteRalph W. deVere White More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)31337-5AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "1123: Defining the Signaling Mechanism and Relevance of H2 Relaxin in Prostate Cancer Progression." The Journal of Urology, 177(4S), p. 371 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 177Issue 4SApril 2007Page: 371 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Ruth L. Vinall More articles by this author Kim Hwa More articles by this author Shangqin Liu More articles by this author Hsing-Jien Kung More articles by this author Ralph W. deVere White More articles by this author Expand All Advertisement PDF DownloadLoading ...

The Relaxin Family Peptide Receptor 3 Activates Extracellular Signal-Regulated Kinase 1/2 through a Protein Kinase C-Dependent Mechanism

Human gene 3 relaxin (H3 relaxin) is a member of the relaxin/insulin family of peptides. Neuropeptides mediate behavioral responses to stress and regulates appetite; however, the cell signaling mechanisms that control these events remain to be identified. The relaxin family peptide receptor 3 (RXFP3, formerly GPCR135 or SALPR) was characterized as the receptor for H3 relaxin, functionally coupled to the inhibition of cAMP. We have identified that RXFP3 stably expressed in Chinese hamster ovary (CHO)-K1 (CHO-RXFP3) and human embryonic kidney (HEK) 293 (HEK-RXFP3) cells activates extracellular signal-regulated kinase (ERK) 1/2 when stimulated with H3 relaxin and an H3 relaxin B-chain (dimer) peptide. Using inhibitors of cellular signaling proteins, we subsequently determined the mechanism of ERK1/2 activation by RXFP3. ERK1/2 phosphorylation requires the activation of G(i/o) proteins and seems to require receptor internalization and/or compartmentalization into lipid-rich environments. ERK1/2 activation also predominantly occurred via the activation of a protein kinase C-dependent pathway, although activation of phosphatidylinositol 3-kinase and Src tyrosine kinase were also involved to a lesser extent. The mechanisms underlying ERK1/2 phosphorylation were similar in both CHO-RXFP3 and HEK-RXFP3 cells, although some differences were evident. Phospholipase Cbeta and the transactivation of endogenous epidermal growth factor receptors both played a role in RXFP3-mediated ERK1/2 activation in HEK293 cells; however, they were not involved in RXFP3-mediated ERK1/2 activation in the CHO-K1 cell background. The pathways identified in CHO- and HEK-transfected cells were also used in the murine SN56 neuronal cell line, suggesting that these pathways are also important for RXFP3-mediated signaling in the brain.

Contrasting pharmacological profiles for functional responses and binding in cells expressing the relaxin family peptide receptor 3 (RXFP3)

Relaxin-3 is a neuropeptide involved in appetite regulation and stress behaviours in rats (Tanaka et al. 2005, McGowan et al. 2005), that is the cognate ligand for the Gi-coupled RXFP3 receptor. In CHO-K1 cells expressing human RXFP3 (CHO-RXFP3), [125I]-INSL5/H3 relaxin chimera (100pM) bound to a single binding site, with competition observed with H3 relaxin (pIC50: 8.42 ± 0.25) but not H2 relaxin, porcine relaxin or INSL3 (n=6). However, in the same cells, inhibition of forskolin (30μM, 3 min) stimulated cAMP accumulation was observed not only with H3 relaxin (pEC50: 9.01 ± 0.27), but also with H2 relaxin (pEC50: 7.14 ± 0.59), porcine relaxin (pEC50: 7.41 ± 0.67) and INSL3 (pEC50: 7.45 ± 0.43) (n=10). We also established that RXFP3 activates ERK1/2 when stimulated with H3 relaxin (6.57 ± 0.78 fold/basal) and weakly with H2 relaxin (2.13 ± 0.74 fold/basal) but not with porcine relaxin or INSL3. The activation of ERK1/2 closely reflects the binding site characterized with [125I]-INSL5/H3 relaxin chimera, which is distinct from the site that leads to cAMP inhibition. Further characterization of RXFP3 signalling using reporter genes, revealed that H2 relaxin and porcine relaxin (all 0.1μM, 24h) could activate AP1 reporter genes, whereas INSL3 activated NFκB reporter genes (n=6). We propose that different relaxin family peptides interact with different binding sites on RXFP3, to activate distinct signalling pathways.

Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth

Hormone antagonists can be effective tools to delineate receptor signaling pathways and their resulting downstream physiological actions. Mutation of the receptor binding domain (RBD) of human H2 relaxin (deltaH2) impaired its biological function as measured by cAMP signaling. In a competition assay, deltaH2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP-1 cells. In a flow cytometry-based binding assay, deltaH2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC-3 and LNCaP) were engineered to overexpress eGFP, wild-type (WT) H2, or deltaH2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing deltaH2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that deltaH2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of lentivectors engineered to express deltaH2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.

Endogenous Relaxin Is a Naturally Occurring Modulator of Experimental Renal Tubulointerstitial Fibrosis

Relaxin is a naturally occurring regulator of collagen turnover. In this study, we determined the role of endogenous relaxin in the pathogenesis of primary tubulointerstitial fibrosis after unilateral ureteric obstruction (UUO). Four- to 6-wk-old relaxin (RLX) gene-knockout (RLX(-/-)) and age-matched wild-type (RLX(+/+)) mice, with equivalent baseline collagen levels, were subjected to UUO. Obstructed and contralateral kidneys were collected at d 0, 3, and 10 after surgery and analyzed for changes in inflammatory and fibrosis-related markers. UUO was associated with a progressive increase in fibrosis in all obstructed, but not contralateral kidneys. The increase in total collagen (hydroxyproline analysis) was associated with more alpha-smooth muscle actin (alpha-SMA) staining (myofibroblasts) and interstitial collagen sub-types (SDS-PAGE; types I, III, and V), whereas gelatin zymography demonstrated increased expression of matrix metalloproteinase-2 after surgery. By d 10 after UUO, there was a 5-fold decrease in RLX mRNA expression (quantitative RT-PCR) in RLX(+/+) animals. Total collagen and alpha-SMA expression were significantly greater in the obstructed kidneys of RLX(-/-) mice 3 d after UUO (both P < 0.05 vs. RLX(+/+) D3 after UUO), but comparable to that in RLX(+/+) animals 10 d after UUO. Administration of recombinant H2 relaxin to RLX(-/-) mice 4 d before UUO ameliorated the increase in collagen and alpha-SMA expression (both P < 0.05 vs. untreated RLX(-/-) mice) by d 3 after UUO. Expression of monocyte chemoattractant protein-1 and macrophage infiltration (inflammation) in addition to that of matrix metalloproteinases was unaffected by genotype after UUO. These combined data demonstrate that endogenous RLX acts as a modulating factor in tubulointerstitial fibrosis, a hallmark of progressive renal disease. This is likely to be via direct effects on renal myofibroblast function.

Comparison of Signaling Pathways Activated by the Relaxin Family Peptide Receptors, RXFP1 and RXFP2, Using Reporter Genes

The receptors for H2 relaxin and insulin-like peptide 3, relaxin family peptide receptor (RXFP) 1 and RXFP2, respectively, were recently identified, but their signaling pathways are not yet well characterized. Although previous work has suggested that cAMP is a major signaling pathway activated by these receptors, RXFP1 has also been shown to activate a number of other signaling proteins. To this end, we examined the effect of stimulation of RXFP1 and RXFP2 receptors [expressed in human embryonic kidney (HEK) 293T cells] with human relaxin family peptides on a number of transcription factor-response elements coupled to reporter genes. Hence, reporter gene activity measured by enzyme activity in the cell media is a measure of the activation of a particular signaling pathway. Eight reporter genes were tested at both receptors as a screen to identify other signaling pathways activated by RXFP1 and RXFP2. The cAMP-response element reporter was strongly activated by both receptors. This effect was enhanced by preincubation with pertussis toxin (PTX), suggesting that Gs and inhibitory Gi/Go proteins mediate this response. Only activation of RXFP1 inhibited nuclear factor kappaB transcription, and this was reversed by PTX and the phosphoinositide-3-kinase inhibitor wortmannin. In addition, the glucocorticoid-response element was activated by RXFP1 but not by RXFP2 and was not activated in the parent HEK293T cells. Thus, the use of reporter genes enabled differences in signaling between these two receptors to be revealed and also threw light on the wide range of effects attributed to relaxin.

Regioselective Disulfide Solid Phase Synthesis, Chemical Characterization and In Vitro Receptor Binding Activity of Equine Relaxin

In the equine industry, pregnancy loss during the third trimester constitutes a large percentage of fetal and neonatal mortality and represents a major financial loss and time investment for the breeder. Early identification of placental insufficiency would, in some cases, make it possible to sustain the pregnancy through medical intervention. Recent work suggests that relaxin is a valuable clinical tool for diagnosing placental insufficiency and monitoring treatment efficacy in mares. Relaxin is a polypeptide member of the insulin superfamily that consists of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. It is typically produced in the ovary during pregnancy and has primary roles in maintaining mammalian pregnancy and facilitating the delivery of the young via remodelling of the reproductive tract. The placenta is the primary source of relaxin in the mare during pregnancy. Its primary structure has been determined and shown to be the smallest of the known mammalian relaxins. It consists of a 20 residue A-chain and a 28-residue B-chain. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken using regioselective disulfide formation methods. The synthetic equine relaxin showed typical α-helical structure under physiological conditions. The peptide was found to bind to the relaxin receptor, LGR7, in vitro, and its binding affinity was found to be higher than that of the “gold standard”, porcine relaxin, and similar to that of the human relaxin-2 (H2 relaxin).

Functional Expression of Mouse Relaxin and Mouse Relaxin-3 in the Lung from an Ebola Virus Glycoprotein-Pseudotyped Lentivirus via Tracheal Delivery

The peptide hormone relaxin is a known modulator of connective tissue and the extracellular matrix by virtue of its ability to regulate matrix metalloproteinases (MMPs). Relaxin knockout mice exhibit age-related pulmonary fibrosis, and delivery of recombinant human H2 relaxin ameliorates fibrotic-like conditions in the mouse lung. We investigated whether lentiviral vectors (LVs) engineering the expression of murine relaxins could induce MMP activity in the mouse lung. Mouse relaxin and mouse relaxin-3 peptides engineered by recombinant LVs were biologically active as shown by stimulation of cAMP from both THP-1 and 293T cells stably expressing relaxin receptor LGR7 and by up-regulation of MMP-2 activity from primary C57BL/6 lung cell cultures. To provide the virions with enhanced tropism for the lung, LVs were pseudotyped with the Zaire strain of the Ebola virus glycoprotein (EboZ GP) and delivered by endotracheal intubation. LVs engineering luciferase pseudotyped with EboZ GP, but not with vesicular stomatitis virus glycoprotein resulted in successful LV transduction and transgene expression in C57BL/6 mouse lung by as early as d 4. Mice treated via tracheal delivery with EboZ GP pseudotyped LVs that engineered expression of mouse relaxins exhibited increased MMP-2 and MMP-9 activity in lung tissue up until the end of our study at d 21. Taken together, this study provides proof-of- principle that relaxin gene expression targeted to the mouse lungs can result in enhanced MMP activity offering potential for alleviating disease conditions characterized by dysregulation of extracellular matrix protein accumulation.

The R273H p53 mutation can facilitate the androgen-independent growth of LNCaP by a mechanism that involves H2 relaxin and its cognate receptor LGR7

Mutations in p53 occur at a rate of approximately 70% in hormone-refractory prostate cancer (CaP), suggesting that p53 mutations facilitate the progression of CaP to androgen-independent (AI) growth. We have previously reported that transfection of p53 gain of function mutant alleles into LNCaP, an androgen-sensitive cell line, allows for AI growth of LNCaP in vitro. We herein confirm the in vivo relevance of those findings by demonstrating that the R273H p53 mutation (p53(R273H)) facilitates AI growth in castrated nude mice. In addition, we demonstrate that H2 relaxin is responsible for facilitating p53(R273H)-mediated AI CaP. H2 relaxin is overexpressed in the LNCaP-R273H subline. Downregulation of H2 relaxin expression results in significant inhibition of AI growth, whereas addition of recombinant human H2 relaxin to parental LNCaP promotes AI growth. Inhibition of AI growth was also achieved by blocking expression of LGR7, the cognate receptor of H2 relaxin. Chromatin immunoprecipitation analysis was used to demonstrate that p53(R273H) binds directly to the relaxin promoter, further confirming a role for H2 relaxin signaling in p53(R273H)-mediated AI CaP. Lastly, we used a reporter gene assay to demonstrate that H2 relaxin can induce the expression of prostate-specific antigen via an androgen receptor-mediated pathway.

Relaxin-3: Improved Synthesis Strategy and Demonstration of Its High-Affinity Interaction with the Relaxin Receptor LGR7 BothIn VitroandIn 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.

H2 relaxin overexpression increases in vivo prostate xenograft tumor growth and angiogenesis

Our study reports a preliminary investigation into the role of human H2 relaxin in prostate tumor growth. A luciferase-expressing human prostate cancer cell line, PC-3, was generated and termed PC3-Luc. PC3-Luc cells were transduced with lentiviral vectors engineering the expression of either enhanced green fluorescent protein (eGFP) or both H2 relaxin and eGFP in a bicistronic format. These transduced cells were termed PC3-Luc-eGFP and PC3-Luc-H2/eGFP, respectively. To gauge effects, PC3-Luc-H2/eGFP and PC3-Luc-eGFP cells were injected into NOD/SCID mice and monitored over 6 weeks. PC-3 tumor xenografts overexpressing H2 relaxin exhibited greater tumor volumes compared to control tumors. Circulating H2 relaxin levels in sera increased with the relative size of the tumor, with moderately elevated H2 relaxin levels in mice bearing PC3-Luc-H2/eGFP tumors compared to PC3-Luc-eGFP tumors. Zymographic analysis demonstrated that proMMP-9 enzyme activity was significantly downregulated in H2 relaxin-overexpressing tumors. An advanced angiogenic phenotype was observed in H2 relaxin-overexpressing tumors indicated by greater intratumoral vascularization by immunohistochemical staining of endothelial cells with anti-mouse CD31. Moreover, PC3-Luc-H2/eGFP tumors exhibited increased VEGF transcript by reverse-transcription PCR, compared to basal levels in control animals. Taken together, our study provides the first account of a potential role of H2 relaxin in prostate tumor development.

INSL3 in the benign hyperplastic and neoplastic human prostate gland

The human prostate gland is a well known source of H1 and H2 relaxin but information is lacking on the expression and potential role of the INSL3 peptide hormone within the prostate gland. In the present study we have investigated the expression of human INSL3 in patients with benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma tissues. Of the prostate epithelial cells, strongest INSL3 expression was detected in the basal epithelial cell compartment. Weaker INSL3 mRNA expression and immunoreactive INSL3 production were observed in secretory epithelial cells and in interstitial smooth muscle cells. Prostate epithelial cells were also a source for transcripts encoding the INSL3 receptor LGR8 suggesting the presence of an autocrine/paracrine INSL3-LGR8 ligand-receptor system within the human prostate. Three human prostate carcinoma cell lines displayed differential gene activity for INSL3 and LGR8. While LNCaP was devoid of INSL3, the androgen-insensitive PC-3 and the stromal prostate cell line hPCP co-expressed INSL3 and LGR8 transcripts. In addition to expressing INSL3 mRNA, the LGR8-negative DU-145 also expressed an INSL3 splice form formerly demonstrated in thyroid carcinoma cells. When incubated with recombinant INSL3, PC-3 cells showed significantly increased intracellular cAMP levels indicating functional LGR8 receptors. INSL3 did not alter the proliferative or metabolic activity of PC-3 carcinoma cells. Instead, PC-3 responded to INSL3 with significantly enhanced tumor cell motility and a transcriptional down-regulation of ErbB receptors and EGF. All-trans-retinoic acid was demonstrated in PC-3 to up-regulate LGR8 gene activity in a dose- and time-dependent manner while having no effect on INSL3 gene activity. In conclusion, we have identified a functional INSL3-LGR8 ligand-receptor system in human prostate carcinoma cells.

Human Medullary Thyroid Carcinoma: A Source and Potential Target for Relaxin‐Like Hormones

We investigated the expression of H1, H2 relaxin and INSL-3, mRNA and protein, and LGR7 and LGR8 transcripts in human C-cell hyperplasia, primary medullary thyroid carcinoma (MTC) tissues, MTC metastases, and the human MTC-TT and mouse MTC-M cell lines. Relaxin-like peptide hormones were detected in C-cell hyperplasia and in MTC tissues, but were absent in human normal parafollicular C-cells of benign goiter tissues. In contrast to calcitonin, mRNA, and immunoreactive protein, no differences in the expression of relaxin and INSL3 were observed in MTC tissues of different pTNM classification or between primary and metastatic MTC tissues studied. All MTC tissues constitutively expressed LGR7 and LGR8 transcripts. Thus, relaxin-like hormones appear to be present early during C-cell hyperplasia and potentially functional relaxin/INSL3 ligand-receptor systems are present in human MTC tissues and in MTC cell lines.