RXFP1 Expression Research Articles

Biallelic RXFP2 variants lead to congenital bilateral cryptorchidism and male infertility, supporting a role of RXFP2 in spermatogenesis.

Does RXFP2 disruption impair male fertility? We identified biallelic variants in RXFP2 in patients with male infertility due to spermatogenic arrest at the spermatid stage, supporting a role of RXFP2 in human spermatogenesis, specifically in germ cell maturation. Since RXFP2, the receptor for INSL3, plays a crucial role in testicular descent during prenatal development, biallelic variants lead to bilateral cryptorchidism, as described in four families to date. While animal models have also suggested a function in spermatogenesis, the postnatal functions of RXFP2 and its ligand INSL3, produced in large amounts by the testes from puberty throughout adulthood, are largely unknown. A family with two male members affected by impaired fertility due to spermatogenic maturation arrest and a history of bilateral cryptorchidism underwent clinical, endocrinological, histological, genomic, in vitro cellular, and in silico investigations. The endocrinological and histological findings were correlated with publicly available single-cell RNA sequencing (scRNA-seq) data. The genomic defects have been characterized using long-read sequencing and validated with in silico modeling and an in vitro cyclic AMP reporter gene assay. An intragenic deletion of exon 1-5 of RXFP2 (NM_130806.5) was detected in trans with a hemizygous missense variant c.229G>A, p.(Glu77Lys). The p.(Glu77Lys) variant caused no clear change in cell surface expression or ability to bind INSL3, but displayed absence of a cAMP signal in response to INSL3, indicating a loss-of-function. Testicular biopsy in the proband showed a maturation arrest at the spermatid stage, corresponding to the highest level of RXFP2 expression in scRNA-seq data, thereby providing a potential explanation for the impaired fertility. Although this is so far the only study of human cases that supports the role of RXFP2 in spermatogenic maturation, this is corroborated by several animal studies that have already demonstrated a postnatal function of INSL3 and RXFP2 in spermatogenesis. This study corroborates RXFP2 as gene implicated in autosomal recessive congenital bilateral cryptorchidism due to biallelic variants, rather than autosomal-dominant cryptorchidism due to monoallelic RXFP2 variants. Our findings also support that RXFP2 is essential in human spermatogenesis, specifically in germ cell maturation, and that biallelic disruption can cause male infertility through spermatogenic arrest at the spermatid stage. Funding was provided by the Bellux Society for Pediatric Endocrinology and Diabetology (BELSPEED) and supported by a Research Foundation Flanders (FWO) senior clinical investigator grant (E.D.B., 1802220N) and a Ghent University Hospital Special Research Fund grant (M.C., FIKO-IV institutional fund). The authors declare no conflict of interest. N/A.

#1437 Renal expression pattern of the relaxin receptor, RXFP1, and effects of the RXFP1 agonist, AZD5462, on renin in non-human primates and humans

Abstract Background and Aims RXFP1 is a G protein-coupled receptor (GPCR) for the pregnancy hormone, relaxin. While preclinical studies, performed primarily in rodent, describe beneficial effects of this pleiotropic peptide across several organ systems, there has been a disappointing lack of success in translating these observations into clinical benefit. To provide a potential pharmacological explanation accounting for this translational gap, the aim of this study was thus to thoroughly evaluate RXFP1 expression across species and tissues as well as to evaluate the renal-intrinsic effects of the RXFP1 agonist, AZD5462. Method Unbiased bioinformatic analysis of several gene expression databases was used as a first approach to compare RXFP1 tissue-level expression between rodent and human. Experimental validation was subsequently performed using scRNAseq, RNAscope and immunohistochemistry on mouse, rat, non-human primate (NHP) and human kidney. The putative renal target engagement effects of RXFP1 agonism were assessed by determining the acute and chronic effects of the RXFP1-selective small molecule agonist, AZD5462, on plasma renin concentrations in both NHPs and in a Phase 1 randomized, single-blind, placebo-controlled study in healthy humans. Results RXFP1 was identified as one of the most highly expressed GPCRs in human and NHP kidney glomeruli. Specifically, by multiple assays, we identified glomerular endothelial cells as the principal cell type expressing RXFP1. This observation is in stark contrast to mouse and rat kidney, where RXFP1 was not readily detected. AZD5462 administered orally to NHPs and healthy human participants elicited an early and sustained dose-dependent increase in plasma renin concentration, and in humans an additional decline in serum creatinine. Conclusion Human RXFP1 kidney glomerular expression is conserved with NHPs but not rodent, thus highlighting the translational and pharmacological challenges in studying RXFP1/relaxin biology across species. The concordant ability of AZD5462 to induce elevations in renin and reduction in serum creatinine, together with relaxin's reported renal-hemodynamic activity in humans, may underlie a previously unappreciated renal-intrinsic RXFP1 target engagement mechanism driving its hemodynamic and renal effects. The potential therapeutic benefits of RXFP1 agonism are the subject of on-going clinical trials in heart failure patients.

Expression of Relaxin Family Peptide Receptors 1 and 3 in the Ovarian Follicle of Japanese Quail.

In our previous studies, we demonstrated that the primary source of relaxin 3 (RLN3) in Japanese quail is ovarian granulosa cells. Although several relaxin family peptide (RXFP) receptors have been sequenced, the intricacies of these receptors in avian species remain insufficiently clarified. Therefore, we assessed the expression of RXFP receptors, RXFP1 and 3, in Japanese quail. Using RT-PCR, we found that both RXFP1 and 3 were ubiquitously expressed. The expression level of RXFP1 is significantly higher in the ovarian theca layer, indicating that it is the primary receptor for RLN3 in the ovary. During follicular development, there was an elevation in thecal RXFP1 expression, but it declined after the luteinizing hormone (LH) surge. We found that the protease activity of the 60 kDa band increased after the LH surge, suggesting the involvement of RLN3 signaling in ovulation. These results suggest a paracrine role of RLN3, involving its binding with RXFP1 in ovarian theca cells. This interaction may elicit biological actions, potentially initiating ovulation after the LH surge.

Targeting RXFP1 by Ligustilide: A novel therapeutic approach for alcoholic hepatic steatosis

BackgroundLigustilide (Lig) is the main active ingredient of Umbelliferae Angelicae Sinensis Radix (Chinese Angelica) and Chuanxiong Rhizoma (Sichuan lovase rhizome). Lig possesses various pharmacological properties and could treat obesity by regulating energy metabolism. However, the impact and regulatory mechanism of Lig on alcoholic hepatic steatosis remains unclear. PurposeThis study aimed to explore the therapeutic effect of Lig on alcoholic hepatic steatosis and its related pharmacological mechanism. ResultsWith chronic and binge ethanol feeding, liver tissue damage and lipid accumulation in mice suffering alcoholic hepatic steatosis were significantly improved after Lig treatment. Lig effectively regulated the expression levels of lipid metabolism-related proteins in alcoholic hepatic steatosis. In addition, Lig reduced RXFP1 expression, inhibited the activation of NLRP3 inflammasome, and blocked NET formation. Lig reduced the infiltration of immune cells to the liver and the further prevented the occurrence of alcohol-stimulated inflammatory response in liver. Lig significantly regulated lipid accumulation in alcohol exposed AML12 cells via modulating PPARα and SREBP1. In MPMs, Lig decreased the expression of RXFP1, inhibited the activation of NLRP3 in macrophages stimulated by LPS/ATP, and slowed down the occurrence of inflammatory response. ConclusionLig sustained lipid metabolism homeostasis in alcoholic hepatic steatosis, through inhibiting the activation of NLRP3 inflammasomes and the formation of NETs, especially targeting RXFP1 in macrophages.

Runjing Decoction alleviated cyclophosphamide-induced oligoasthenospermia rats by inhibiting cell apoptosis via RXFP1/AKT/FOXO1 pathway.

Runjing Decoction (RJD) is a prescription of traditional Chinese medicine for the treatment of oligoasthenospermia. However, the molecular mechanism of RJD on oligoasthenospermia still remains unknown. A model of oligoasthenospermia was induced in 30 Sprague Dawley rats by intraperitoneal injection of cyclophosphamide at 35mg/kg per day for 5days and treated by intragastric RJD (13.5g/kg) or L-carnitine (100mg/kg) for 14days. The body weight, testis and epididymis weight, grade A spermatozoa, grade B spermatozoa, the percentage of sperm forward motility (PR%), the sperm activity rate and the sperm density of rats were evaluated before and after RJD treatment. The testis apoptosis was determined by TUNEL staining. The expressions of RXFP1, FoxO1, PI3K, Akt, Bax and Bcl-2 were determined by qRT-PCR and Western blot, respectively. After RJD treatment, the grade A spermatozoa, sperm PR%, sperm activity and sperm density were significantly increased relative to those in model rats. Cell apoptosis of testis tissue was reversed by RJD. RJD suppressed cell apoptosis, inhibited the expression of RXFP1, FOXO1, PI3K, AKT and Bax, and promoted the expression levels of Bcl-2 in testicular tissue of oligoasthenospermia rats. RJD could alleviate sperm quality and testis damage in oligoasthenospermia rats by inhibiting RXFP1/AKT/FOXO1 pathway.

Development of Relaxin‐Conjugated Nanoparticles to Target and Activate RXFP1 in Hepatic Stellate Cells

Background Hepatic fibrosis is a progressive disease which can result in end-stage cirrhosis, a leading cause of death in the US. It is caused by the activation of myofibroblasts (HSC), that produce excess collagen under perpetual injury. Relaxin, a member of the insulin/IGF/relaxin superfamily of peptide hormones, has been shown to play a role in deactivating HSC through its receptor, RXFP1. The deactivation of HSC reduces expression of collagen, therefore slowing the progression of fibrosis. Significance of Problem Relaxin has been shown to be antifibrotic in some rodent models of hepatic fibrosis, however it is unstable in circulation and requires long-term continuous treatment. By developing a stabilized transport system, it would improve the efficacy and provide an HSC-specific targeting system through RXFP1. Hypothesis Relaxin tethered to nanoparticles (NP) will direct the particles to the HSC and activate RXFP1, thereby inducing an antifibrotic response. Experimental Design NP were prepared from rhodamine-labled poly(lactide-co-glycolide) (PLGA 16kDa)-b-PEG (3.4kDa)-COOH copolymers to produce control NP (C-NP). Relaxin was attached to the carboxylic group by carbodiimide crosslinking (R-NP). The particles were subjected to hydrodynamic analysis to determine their size. Human HEK-293T cells stably expressing RXFP1 (HEK-RXFP1) were used for NP binding. RXFP1 activation in HEK-RXFP1 cells, control cells (HEK), and the hepatic stellate cell line, LX2, which endogenously expresses RXFP1, was determined by cAMP production using a TR-FRET assay. To determine the localization of NP in diseased liver, hepatic fibrosis was induced in mice by injection of carbon tetrachloride for 2 weeks. NP were injected via the tail vein, and 2 hours later the liver and other organs were removed, and fluorescence detected using an IVIS imager. Frozen sections were used to determine colocalization of NP with cell marker proteins by fluorescence immunohistochemistry. Results/Data The conjugation of relaxin caused a small increase in particle size. TR-FRET analysis showed R-NP increase cAMP production, indicative of RXFP1 activation. HEK cells showed no response to either NP, however HEK-RXFP1 cells produced a concentration-dependent response to R-NP. Similarly, R-NP induced a dose-dependent cAMP increase in LX2 cells. Contrary to the C-NP, there was increased binding of R-NP to HEK-RXFP1 cells. IVIS imaging showed that both NP homed primarily to the liver, however R-NP had increased homing to kidney and adipose tissue, other sites of RXFP1 expression. Immunohistochemistry revealed that C-NP primarily colocalized with macrophages in the liver, while R-NP had increased association with HSC. Conclusions This study showed that covalent attachment of relaxin to NP retains biological activity and allows for HSC targeting both in vitro and in vivo. This proof-of-concept shows that R-NP can be used as both a targeting moiety and antifibrotic agent. Future studies aim to optimize the configurations of the NP and load antifibrotic agents as cargo to be delivered to desired profibrotic cells.

Ligand-activated RXFP1 gene therapy ameliorates pressure overload-induced cardiac dysfunction

Recurrent episodes of decompensated heart failure (HF) represent an emerging cause of hospitalizations in developed countries with an urgent need for effective therapies. Recently, the pregnancy-related hormone relaxin (RLN) was found to mediate cardio-protective effects and act as a positive inotrope in the cardiovascular system. RLN binds to the RLN family peptide receptor 1 (RXFP1), which is predominantly expressed in atrial cardiomyocytes. We therefore hypothesized that ventricular RXFP1 expression might exert potential therapeutic effects in an invivo model of cardiac dysfunction. Thus, mice were exposed to pressure overload by transverse aortic constriction and treated with AAV9 to ectopically express RXFP1. To activate RXFP1 signaling, RLN was supplemented subcutaneously. Ventricular RXFP1 expression was well tolerated. Additional RLN administration not only abrogated HF progression but restored left ventricular systolic function. In accordance, upregulation of fetal genes and pathological remodeling markers were significantly reduced. Invitro, RLN stimulation of RXFP1-expressing cardiomyocytes induced downstream signaling, resulting in protein kinase A (PKA)-specific phosphorylation of phospholamban (PLB), which was distinguishable from β-adrenergic activation. PLB phosphorylation corresponded to increased calcium amplitude and contractility. In conclusion, our results demonstrate that ligand-activated cardiac RXFP1 gene therapy represents a therapeutic approach to attenuate HF with the potential to adjust therapy by exogenous RLN supplementation.

In Silico, In Vitro, and In Vivo Analysis Identifies Endometrial Circadian Clock Genes in Recurrent Implantation Failure.

Previous work has demonstrated the role of the circadian clock in ovarian steroid hormone synthesis and attributed embryo implantation failure associated with arrhythmic circadian clock genes to insufficient ovarian-derived progesterone synthesis. Research on expression of core circadian clock genes in the endometrium itself and possible roles in compromised endometrial receptivity and recurrent implantation failure (RIF) are limited. We aimed to assess the core circadian clock gene profiling in human endometrium across the menstrual cycle and the possible gene interaction networks in the endometrial receptivity of window of implantation (WOI) as well as RIF. The study was initially an in silico study, with confirmatory lab-based data from primary human endometrial stromal cells (hESCs) as well as endometrial biopsies obtained from 60 women undergoing gynecological surgery in a clinical research center. The study included 30 RIF women and 30 age-matched and body mass index-matched controls. Initial data mining and bioinformatics analysis of human endometrial microarray datasets across the menstrual cycle and between RIF women versus controls demonstrated the varied expression of core circadian clock genes across menstrual cycle, including the key role of PER2 in WOI and RIF. A PER2-centered network was investigated in the regulation of endometrial receptivity. We also confirmed the evidently increased mRNA expression of SHTN1, RXFP1, KLF5, and STEAP4 in the endometrium of RIF women, displaying the same trend as PER2 did, without any changes in MT1E and FKBP5. Treatment of PER2 siRNA in hESCs verified the positive regulation of PER2 to SHTN1, KLF5, and STEAP4. Aberrant expression of endometrial PER2 might contribute to impaired endometrial receptivity and development of RIF via regulating SHTN1, KLF5, and STEAP4.

Abstract 16878: Cardiac Overexpression of RXFP1 via AAV9 Gene Therapy Sensitizes Myocardial Inotropic Response to Serelaxin

Background: The pleiotropic hormone relaxin has been shown to mediate physiologic responses during pregnancy by facilitating increases in vascular compliance and cardiac output. While relaxin signaling has been attributed to increases in atrial inotropy, the hormonal effects on ventricular contractility have not been explored in vivo . In this study, we investigated the dose-response effects of recombinant human relaxin (serelaxin) on ventricular mechanics. Methods and Results: Adult male CD1 mice were anesthetized and mechanically ventilated prior to Millar cardiac catheterization. A pressure-volume catheter was advanced into the left ventricle (LV), and baseline systolic and diastolic pressure/volume (PV) loops were recorded (Pre). Mice were injected with either saline, 10 μg/kg (low dose) or 1 mg/kg (high dose) of serelaxin, and PV loops were obtained after stabilization (Post) . Mice injected with 1 mg/kg demonstrated significantly higher LV contractility ( Fig A ) independent of preload ( Fig B ). These responses were not observed with low dose. In order to further examine the role of RXFP1 in modulating these responses, healthy CD1 were injected with AAV9-RXFP1 or empty vector as control and receptor overexpression was confirmed four weeks later via qPCR ( Fig C ). PV loop analysis showed an increased inotropic response [ΔP Max , Δ(dP/dt) Max ] and improved relaxation kinetics [Δ(dP/dt) Min , Δτ] in RXFP1-overexpressing mice at a lower dose of serelaxin, when compared to empty vector mice ( Fig D ). Conclusions: Our data show a novel, dose-dependent correlation between serelaxin and ventricular inotropic and lusitropic responses in mice. These responses were observed at a lower drug dose in cardiac RXFP1-overexpressing mice. We propose that further investigation of this phenomenon could advance the therapeutic, cardioprotective effects associated with relaxin therapy in different etiologies of heart failure that may exhibit changes in RXFP1 expression.

BRAFV600E, hypothyroidism, and human relaxin in thyroid carcinogenesis.

BRAFV600E, a major driver of thyroid cancer, evaluated in the context of thyroid hormones and human relaxin. Immunohistochemical expressions of BRAFV600E, TSH, TSH receptor (TSHR), T4, T3 receptor (T3R), RLNH2, and its receptor, RXFP1, were evaluated in thyroid tumors from a retrospective U.S. population of 481 cancer cases diagnosed in 1983-2004. BRAFV600E was expressed in 52% of all thyroid tumors; expression of other markers ranged from 25% for T4 to 98% for RLNH2. Tumors predominantly exhibited hypothyroid-like conditions characterized by elevated TSH and TSHR and reduced T4. BRAFV600E prevalence was significantly higher in tumors expressing TSH, TSHR, T3R, and RXFP1 and lower in tumors expressing T4. The proportion of BRAFV600E mutation in classic papillary tumors significantly increased from 56 to 72% over the 21-year period of diagnoses, while expression of RXFP1, TSH, TSHR, and T3R decreased in non-tumor. Racial/ethnic differences were observed in thyroid hormone marker expression. Non-tumor expression of TSH, TSHR, and T3R were each associated with shorter overall survival, but did not remain significant after adjustment for demographic and clinical factors. Our study provides the first evidence of the potential interaction of BRAFV600E mutation, relaxin, and thyroid hormones in thyroid carcinogenesis. Moreover, our results suggest that hypothyroidism, influenced by RLNH2 activity, may underlie the development of the majority of thyroid cancers and mediate the role of BRAFV600E in thyroid carcinogenesis. BRAFV600E mutation is increasing in papillary thyroid cancers and may be contributing to the rising incidence of this malignancy.

Expression of RXFP2 receptor on human spermatozoa and the anti-apoptotic and antioxidant effects of insulin-like factor 3.

Insulin-like factor 3 (INSL3) has an important role in the human reproductive system; however, its detailed function is still mysterious. We aimed to investigate the possibility of expression of RXFP2 receptor on human spermatozoa and to determine the anti-apoptotic and antioxidant mechanism derived the binding of INSL3 and RXFP2. In this experimental study, the expression/location of the RXFP2 receptor was determined on the spermatozoa of fertile and infertile men. Twenty samples from 20 fertile men were collected and divided into 6 parts (control group, and five groups treated with INSL3 10, 100, 250, 500, 1,000ng/ml). DNA damage, active caspase, reactive oxygen species (ROS) and sperm parameters were evaluated by TUNEL, flow cytometry, optical microscope and computer-assisted sperm analysis. The expression of RXFP2 was confirmed by Western blot. Immunocytochemistry illustrated that this receptor is expressed in the posterior half of the spermatozoa's head. The INSL3 at concentrations of 500 and 1,000ng/ml reduced the active caspase and mitochondrial ROS, and also reduced DNA fragmentation at 1,000ng/ml. Besides, INSL3 500 and 1,000ng/ml significantly increased the sperm motility. This study confirmed the presence of RXFP2 receptor in fertile and infertile men's spermatozoa, indicating the highly dose-dependent efficacy of the INSL3, which may have promising impacts on the in-vitro fertilisation outcomes.

Characterizing relaxin receptor expression and exploring relaxin\u2019s effect on tissue remodeling/fibrosis in the human bladder

BackgroundRelaxin is an endogenous protein that has been shown to have antifibrotic properties in various organ systems. There has been no characterization of relaxin’s role in the human bladder. Our objective was to characterize relaxin receptor expression in the human bladder and assess relaxin’s effect on tissue remodeling/fibrosis pathways in bladder smooth muscle cells.MethodsRelaxin family peptide receptor 1 (RXFP1) and RXFP2 expression was assessed using quantitative reverse transcriptase-PCR (qRT-PCR) and immunohistochemistry (IHC) on primary bladder tissue. Primary human smooth muscle bladder cells were cultured and stimulated with various concentrations of relaxin. Western blot, qRTPCR, ELISA, and zymogram assays were used to analyze fibrosis/tissue remodeling pathway proteins.ResultsThere was universal mRNA transcript detection and protein expression of relaxin receptors in primary bladder specimens. Immunohistochemistry demonstrated RXFP1 and RXFP2 localizing to both urothelial and smooth muscle cell layers of the bladder. 24 h of in vitro relaxin stimulation did not affect mRNA expression of selected proteins in human bladder smooth muscle cells. However, 48 h of in vitro relaxin stimulation resulted in upregulation of active (p = 0.004) and latent (p = 0.027) MMP-2 in cell lysate, and upregulation of active MMP-2 in supernatant (p = 0.04). There was a dose dependent relationship with increasing expression of MMP-2 with increasing relaxin concentration. Relaxin stimulation resulted in decreased levels of active and total TGF-β1 in supernatant and extracellular matrix (p < 0.005 with 100 ng/mL relaxin stimulation).ConclusionsIn the human bladder, relaxin receptors are expressed at the dome and trigone and localize to the urothelium and smooth muscle cell layers. Stimulation of human bladder SMCs with relaxin in vitro affects expression of MMP-2 and TGF-β1.

Ocular Phenotype of Relaxin Gene Knockout (Rln-/-) Mice

ABSTRACT Purpose: To test if relaxin deficiency affects ocular structure and function we investigated expression of relaxin (Rln) and RXFP receptors (Rxfp1, Rxfp2), and compared ocular phenotypes in relaxin gene knockout (Rln−/− ) and wild type (Rln+/+ ) mice. Materials and Methods: Rln, Rxfp1 and Rxfp2 mRNA expression was detected in ocular tissues of Rln+/+ mice using RT-PCR. The eyes of 11 Rln−/− and 5 Rln+/+ male mice were investigated. Corneal and retinal thickness was assessed using optical coherence tomography. Intraocular pressure was measured using a rebound tonometer. Retinal, choroidal and sclera morphology and thickness were evaluated histologically. Eyes were collected and fixed for immunofluorescence staining or used for RNA extraction to evaluate mRNA expression using real-time PCR. Results: Rln mRNA was expressed only in the retina, whereas Rxfp1 transcripts were detected in the retina, cornea and sclera/choroid. Rxfp2 was only present in the cornea. None of these genes were expressed in the lacrimal gland, eyelid or lens. Intraocular pressure was higher and central cornea of Rln−/− mice was significantly thicker and had significantly larger endothelial cells and a lower endothelial cell density than Rln+/+ mice. Immunohistochemistry demonstrated no significant difference in AQP3 and AQP5 staining in the cornea or other regions between wildtype and Rln−/− mice. mRNA expression of Aqp4 was significantly higher in Rln−/− than in Rln+/+ corneas, whereas Col1a2, Mmp9, Timp1 and Timp2 were significantly decreased. Expression of Aqp1, Aqp4, Aqp5, Vim and Tjp1 was significantly decreased in Rln−/− compared to Rln+/+ uvea. No significant differences in these genes were detected in the retina. Retinal, choroidal and scleral thicknesses were not different and morphology appeared normal. Conclusion: The findings indicate that loss of Rln affects expression of several genes in the uvea and cornea and results in thicker corneas with altered endothelial cells. Many of the gene changes suggest alterations in extracellular matrix and fluid transport between cells.

LncRNA NKILA integrates RXFP1/AKT and NF-κB signalling to regulate osteogenesis of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are previously found to have potential capacity to differentiate into osteocytes when exposed to specific stimuli. However, the detailed molecular mechanism during this progress remains largely unknown. In the current study, we characterized the lncRNA NKILA as a crucial positive regulator for osteogenesis of MSCs. NKILA attenuation significantly inhibits the calcium deposition and alkaline phosphatase activity of MSCs. More interestingly, we defined that NKILA is functionally involved in the regulation of RXFP1/PI3K‐AKT and NF‐κB signalling. Knockdown of NKILA dramatically down‐regulates the expression of RXFP1 and then reduces the activity of AKT, a downstream regulator of RXFP1 signalling which is widely accepted as an activator of osteogenesis. Moreover, we identify NF‐κB as another critical regulator implicated in NKILA‐mediated osteogenic differentiation. Inhibition of NF‐κB can induce the expression of RUNX2, a master transcription factor of osteogenesis, in a HDAC2‐mediated deacetylation manner. Thus, this study illustrates the regulatory function of NKILA in osteogenesis through distinct signalling pathways, therefore providing a new insight into searching for new molecular targets for bone tissue repair and regeneration.

Abstract 870: Cardiac Gene Therapy With Relaxin Receptor 1 Overexpression Protects Against Acute Myocardial Infarction and Associated Adverse Remodeling

Background: Relaxin signaling confers cardioprotection against acute myocardial infarction (MI) and mitigates adverse cardiac remodeling. We investigated the role of relaxin receptor (RXFP1) overexpression via cardiotropic AAV9 vectors in attenuating MI-associated adverse remodeling. Methods and Results: Murine RXFP1 cDNA with CMV promoter was introduced into AAV cis plasmids for generation of AAV9-RXFP1 viral vectors. 1.0 x 10 11 viral genomes in 100μL saline were administered i.v. to 8-week-old CD1 male mice. Four weeks later, overexpression was confirmed via qPCR of RXFP1 mRNA in whole cardiac tissue lysates ( Fig A ) and primary cardiomyocytes ( Fig B ) versus control. Overexpressing (AAV9-RXFP1) mice were subjected to MI (30 minutes of coronary artery ligation followed by reperfusion for 24h or 7d). Immunohistochemical staining of cardiac sections 7d post-MI shows increased RXFP1 expression in AAV9-RXFP1 mice, as shown in Fig C . Infarct size was reduced at 24h post-MI (TTC staining, Fig D ) and fractional shortening (M mode echo) was preserved at 24h and 7d post-MI in AAV9-RXFP1 mice ( Fig E ); endocardial strain rate assessment (speckle tracking analysis) showed preserved function in the affected ventricular segments of AAV9-RXFP1 mice 7d post-MI ( Fig F ). Picrosirius staining showed reduced LV fibrosis at 7d post-MI in AAV9-RXFP1 mice ( Fig G ). Conclusion: Administration of AAV9-RXFP1 successfully upregulates RXFP1 in cardiac tissue and primary cardiomyocytes, reduces infarct size and fibrosis, and preserves cardiac function post MI. Targeting cardiac RXFP1 via gene therapy could be a novel strategy to prevent MI-related adverse remodeling.

SAT-035 Small Molecule Allosteric Agonist of Relaxin Receptor Ml290 Demonstrates Antifibrotic Properties in Liver Fibrosis

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological therapeutic options. The beneficial effects of relaxin peptide treatment have been demonstrated previously in clinically relevant animal models of liver disease. However, the low stability of the recombinant relaxin peptide in vivo requires continuous intravenous delivery for chronic therapeutic application. We have recently identified a first series of small molecule allosteric biased agonists of the human relaxin receptor RXFP1 which showed efficacy similar to relaxin in several functional assays in vitro. Here we investigated the therapeutic effects of small molecule RXFP1 agonist on activated hepatic stellate cells, the main source of excessive collagen production in liver fibrosis. We have demonstrated that RXFP1 expression is increased in fibrotic mouse liver, specifically in activated hepatic stellate cells. The lead compound, ML290, was selected based on its effects on the expression of the genes involved in fibrosis in primary human stellate cells. RNA-Seq analysis of TGFβ1-activated LX-2 hepatic stellate cells showed that about 500 genes were misregulated by ML290. Gene Ontology analysis demonstrated that ML290 treatment primarily affects extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment of human liver organoids with lipopolysaccharide-induced fibrotic phenotype resulted in dramatic reduction of type I collagen. The pharmacokinetics of ML290 in mice after multiple injections demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. The ML290 treatment of mice expressing human RXFP1 gene with carbon tetrachloride-induced liver fibrosis resulted in significantly reduced collagen content, alpha-smooth muscle actin expression and cell proliferation around portal ducts, and the decrease of pro-fibrotic genes’ expression. In summary, ML290, the small molecule agonist of relaxin receptor, has anti-fibrotic effects in liver fibrosis. Funding source: NIH/NIDDK

Relaxin 2/RXFP1 Signaling Induces Cell Invasion via the β-Catenin Pathway in Endometrial Cancer.

Relaxin is known to play an important role in animal pregnancies, including those of humans. It is suggested that relaxin induces aggressive cell growth and invasiveness in several types of cancer, including endometrial cancer. However, the mechanisms of relaxin remain largely unclear. In this study, we examined the effects of relaxin 2 (RLN2), the major circulating relaxin in humans, on human endometrial carcinoma cell lines. RLN2 treatment induced invasion in HEC-1B and Ishikawa cells. RLN2-induced cell invasion was significantly decreased by transfection of relaxin receptor 1 (RXFP1) siRNAs. The β-catenin inhibitor, XAV939, also significantly inhibited the RLN2-induced cell invasions. Both a decrease of cadherin expression and an increase of β-catenin phosphorylation were observed in response to the RLN2 treatment in HEC-1B and Ishikawa cells. We then examined RLN2 and RXFP1 expression in 80 human endometrioid endometrial carcinoma tissues. RLN2 immunoreactivity was detected in the human endometrial carcinoma cells and had a correlative tendency with histological grade and RXFP1. These results suggest that adherens junctions in cancer cells are weakened by the breakdown of the cadherin/catenin complex, which is induced by β-catenin phosphorylation via RLN2/RXFP1 signaling.

Receptor-independent modulation of TGF-β-induced pro-fibrotic pathways by relaxin-2 in human primary tubular epithelial cells.

Renal tubular epithelial cells actively contribute to the development of renal fibrosis and may be targeted by anti-fibrotic drugs. Relaxin-2 (RLX2) applied as recombinant protein is suggested to be renoprotective. Therefore, we investigated whether human primary tubular epithelial cells (hPTEC) obtained from various donors were target cells for the anti-fibrotic actions of RLX2. Treatment of hPTEC with RLX2 reduced the TGF-β1-induced secretion of the pro-fibrotic factor CTGF (connective tissue growth factor) and inhibited fibronectin synthesis and secretion. Furthermore, metalloproteinase MMP2 secretion was increased, with no effect on MMP9. Considerable differences were observed between hPTEC obtained from different donors. Therefore, expression of the relaxin family peptide receptor RXFP1, the major mediator of renal RLX2 effects, was analyzed. A validated antibody detected a double band of 80-90kDa in cellular homogenates by Western blotting. Expression of the detected protein was not altered by incubation with TGF-β1 and RLX2-induced modulation of CTGF expression did not correlate with the putative receptor expression. Therefore, relaxin family receptors RXFP1-4 were assessed by RNA-seq analysis. No evidence was found for mRNA expression of any of these receptors in several hPTEC preparations. Lack of RXFP1 mRNA was confirmed by qPCR using mRNA obtained from THP-1 cells as positive control. Our data thus provide evidence for primary renal human tubular epithelial cells as targets for the anti-fibrotic actions of RLX2. However, anti-fibrotic effects were observed at micromolar concentrations of RLX2 and shown to be independent of RXFP1 expression.

Whole-genome sequences of 89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization.

BackgroundAnimal domestication has been extensively studied, but the process of feralization remains poorly understood.ResultsHere, we performed whole-genome sequencing of 99 sheep and identified a primary genetic divergence between 2 heterogeneous populations in the Tibetan Plateau, including 1 semi-feral lineage. Selective sweep and candidate gene analysis revealed local adaptations of these sheep associated with sensory perception, muscle strength, eating habit, mating process, and aggressive behavior. In particular, a horn-related gene, RXFP2, showed signs of rapid evolution specifically in the semi-feral breeds. A unique haplotype and repressed horn-related tissue expression of RXFP2 were correlated with higher horn length, as well as spiral and horizontally extended horn shape.ConclusionsSemi-feralization has an extensive impact on diverse phenotypic traits of sheep. By acquiring features like those of their wild ancestors, semi-feral sheep were able to regain fitness while in frequent contact with wild surroundings and rare human interventions. This study provides a new insight into the evolution of domestic animals when human interventions are no longer dominant.

Alterations of relaxin and its receptor system components in experimental diabetic cardiomyopathy rats.

High glucose induces apoptosis of cardiomyocytes and fibrosis of cardiac fibroblasts, contributing to diabetic cardiomyopathy. In this work, we explore the production of relaxin alterations and the significance of their receptor system components in the hearts of experimental diabetic cardiomyopathy rats. We measured rat relaxin-1 (equivalent to human relaxin-2), relaxin-3, RXFP1 and RXFP3 mRNA expression in the hearts of experimental diabetic cardiomyopathy rats. Neonatal rat ventricular myocytes (NRVMs) and cardiac fibroblasts were treated with 5.5mmol/l normal glucose (NG) and 33mmol/l high glucose (HG) for 0, 6, 12, 24, 48 and 72h. Rat relaxin-1, relaxin-3, RXFP1 and RXFP3 mRNA expression were determined by real-time PCR. In the present study, we offer the first evidence that Relaxin-1 mRNA significantly increased and Relaxin-3 mRNA expression decreased at 4 and 8 weeks after STZ in the hearts of diabetic rats. In addition, significant down regulation of the mRNA expression of RXFP1 and RXFP3 was observed at 4 w after STZ; however, the mRNA expression levels of RXFP1 and RXFP3 were increased at 8 weeks after STZ. Apoptotic NRVMs induced by high glucose generate a decreased level of relaxin-1 and RXFP1. In HG-administered cardiac fibroblasts, Relaxin-1 mRNA was significantly increased and relaxin-3 mRNA was significantly decreased. Additionally, the mRNA expression of RXFP1 was decreased, and the mRNA expression of RXFP3 was increased. This results showed that an important role of relaxin-2, relaxin-3 and their receptors system in the regulation of diabetic cardiomyopathy.