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Regional and cellular distribution of Lgr8 mRNA in frozen sections of adult kidney from male rats. (A) In situ hybridization of 35 S-labelled oligonucleotide probes revealed that Lgr8 mRNA was present in renal cortex ('C') in a specific pattern consistent with an association with glomeruli, while no hybridization signal was detected in the renal medulla ('Me'). (A) In a competition experiment, the hybridization signal was eliminated by a 100-fold excess of unlabelled oligonucleotides added to the hybridization mixture containing labelled probes, leaving only 'non-specific hybridization' ('NSH'). (B) Nuclear-emulsion autoradiograms confirmed that Lgr8 mRNA was associated with cortical glomeruli ('G', *). (C) At high magnification, Lgr8 mRNA-associated grains were observed over individual cells putatively identified as mesangial cells. Scale bar=2·5 mm (A, A); 7·5 m (B); 2·5 m (C).
Source publication
Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the...
Contexts in source publication
Context 1
... sections from adult (8-to 10-week-old) male rats were subjected to in situ hybridization histochemistry for Lgr8 mRNA using 35 S-labelled oligonucleotides. Relatively low-resolution film autoradiograms revealed the expression of Lgr8 mRNA restricted to the renal cortex, in a pattern consistent with labelling of cells within the glomeruli (Fig. 1A). The specificity of the Lgr8 mRNA signal detected was confirmed by several methods. In competition experiments, the signal was abolished when a 100-fold excess of unlabelled oligo- nucleotides was added to the incubation (Fig. 1A). Secondly, sections hybridized with the three individual oligonucleotide probes displayed the same ...
Context 2
... of Lgr8 mRNA restricted to the renal cortex, in a pattern consistent with labelling of cells within the glomeruli (Fig. 1A). The specificity of the Lgr8 mRNA signal detected was confirmed by several methods. In competition experiments, the signal was abolished when a 100-fold excess of unlabelled oligo- nucleotides was added to the incubation (Fig. 1A). Secondly, sections hybridized with the three individual oligonucleotide probes displayed the same distribution of labelling (data not shown), and finally the oligonucleotide probes used produced strong, specific labelling of sections of gubernaculum from a 17-day rat embryo and adult testis (data not shown; Scott et al. 2005). ...
Context 3
... specific labelling of sections of gubernaculum from a 17-day rat embryo and adult testis (data not shown; Scott et al. 2005). Microscopic analysis of high-resolution nuclear emulsion autoradio- grams from counterstained kidney sections indicated that Lgr8 mRNA expression was restricted to putative mesangial cells within the adult glomeruli (Fig. 1B and C), with no obvious specific hybridization observed in glomerular podocyte or epithelial cells ( Hoffmann et al. 2004). Notably, parallel in situ hybridization studies with Lgr7-directed oligonucleotides ( Burazin et al. 2005) failed to detect Lgr7 mRNA-positive cells in rat kidney (data not ...
Citations
... Given the expression of RXFP2 in organs that are important in the regulation of blood pressure, and the previously described functions of INSL3/RXFP2 in the brain 16 , renal glomerular cells 11 , and androgen secretion 14 , we cannot rule out non-adrenal mechanisms for RXFP2 in the regulation of blood pressure. During development, androgens increase RXFP2 expression in gubernaculum which mediates testis descent 53 . ...
Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.
... In addition, INSL3 could play a subtle endocrine function in the kidney, particularly on maturation and regulation of glomeruli and of adult mesangial cell density [17]. In the brain, INSL3 might also be involved in the modulation of basic functions like arousal, feeding and sexual behaviour [18]. ...
This article reviews the role of INSL3 as biomarker of Leydig cell function and its systemic action in testis-boneskeletal muscle crosstalk in adult men. Insulin-like factor 3 (INSL3) is a peptide hormone secreted constitutively in a differentiation-dependent mode by testicular Leydig cells. Besides the role for the testicular descent, this hormone has endocrine anabolic functions on the bone-skeletal muscle unit. INSL3 levels are low in many conditions of undifferentiated or altered Leydig cell status, however the potential clinical utility of INSL3 measurement is not yet well defined. INSL3 levels are modulated by the long-term cytotropic effect of the hypothalamic- pituitary-gonadal axis, unlike testosterone that is acutely sensitive to the stimulus by luteinizing hormone (LH). INSL3 directly depend on the number and differentiation state of Leydig cells and therefore it represents the ideal marker of Leydig cell function. This hormone is more sensitive than testosterone to Leydig cell impairment, and the reduction of INSL3 in adult men can precociously detect an endocrine testicular dysfunction. Low INSL3 levels could cause or contribute to some symptoms and signs of male hypogonadism, above all sarcopenia and osteoporosis. The evidence provided, suggested that the measurement of INSL3 levels should be considered in the clinical management of male hypogonadism and in the evaluation of testicular endocrine function. The monitoring of INSL3 levels could allow an early detection of Leydig cell damage, even when testosterone levels are still in the normal range.
... A mouse corneal ulcer model was used to test the topical application of INSL3, which was determined to be effective in re-epithelialization and healing of corneal wounds (Hampel et al. 2013). INSL3 radioligand binding was also detected in the glomeruli of the renal cortex of post-natal and adult rats (Fu et al. 2006). It has been proposed that INSL3 in the kidney inhibits glomerular cell proliferation, which may be beneficial in targeting glomerular diseases that are associated with uncontrolled mesangial cell proliferation (Fu et al. 2006). ...
... INSL3 radioligand binding was also detected in the glomeruli of the renal cortex of post-natal and adult rats (Fu et al. 2006). It has been proposed that INSL3 in the kidney inhibits glomerular cell proliferation, which may be beneficial in targeting glomerular diseases that are associated with uncontrolled mesangial cell proliferation (Fu et al. 2006). ...
Insulin-like 3 peptide (INSL3) is a member of the insulin-like peptide superfamily and is the only known physiological ligand of relaxin family peptide receptor 2 (RXFP2), a G protein-coupled receptor (GPCR). In mammals INSL3 is primarily produced both in testicular Leydig cells and in ovarian theca cells, but circulating levels of the hormone are much higher in males than in females. The INSL3/RXFP2 system has an essential role in the development of the gubernaculum for the initial transabdominal descent of the testis and in maintaining proper reproductive health in men. Although its function in female physiology has been less well-characterized, it was reported that INSL3 deletion affects antral follicle development during the follicular phase of the menstrual cycle and uterus function. Since the discovery of its role in the reproductive system, the study of INSL3/RXFP2 has expanded to others organs such as skeletal muscle, bone, kidney, thyroid, brain, and eye. This review aims to summarize the various advances in understanding the physiological function of this ligand-receptor pair since its first discovery and elucidate its future therapeutic potential in the management of various diseases.
... Significant RXFP2 expression has also been found in the kidney (Hsu et al., 2002), in particular on the mesangial cells of the glomeruli (Fu et al., 2006). In vitro experiments suggest that INSL3 may have an effect on mesangial cell proliferation (Fu et al., 2006). ...
... Significant RXFP2 expression has also been found in the kidney (Hsu et al., 2002), in particular on the mesangial cells of the glomeruli (Fu et al., 2006). In vitro experiments suggest that INSL3 may have an effect on mesangial cell proliferation (Fu et al., 2006). Intriguingly, phenotypic analysis of RXFP2-disrupted mice indicates a significantly altered circulating electrolyte profile with increased levels of sodium, calcium and chloride besides an elevated haematocrit (http://www. ...
Background:
Insulin-like peptide 3 (INSL3) is a member of the relaxin family of neohormones which has evolved to address specifically mammalian aspects of reproduction related to viviparity and internal fertilization. It was originally identified as a major product of testicular Leydig cells and has proved to be an important biomarker of Leydig cell functional capacity. However, INSL3 is also produced by theca interna cells of growing antral follicles and is secreted into the bloodstream in phases corresponding to the number and health of the follicles. Moreover, gene silencing experiments have shown that INSL3 is essentially required for androstenedione synthesis, which is the major steroid precursor for the granulosa cells of antral follicles to produce oestrogens. Knockout studies in mice confirm that loss of INSL3 or its receptor in females leads to partial infertility, with reduced follicle numbers, ovulations and litter size. Circulating INSL3 concentration corresponds to the reproductive lifespan, beginning with puberty and declining at the menopause, and thus may contribute to the physiology of other organ systems, particularly those relevant for hormone replacement strategies.
Search methods:
A literature review was carried out by exhaustive searching of literature databases (PubMed and Google Scholar) with the search terms INSL3, RLF, Ley-IL and RXFP2.
Objective and rationale:
We present the first comprehensive review of INSL3 and its specific receptor RXFP2, and their roles in the context of female reproductive physiology. Moreover, we highlight the potential involvement of INSL3 in female reproductive pathology, such as PCOS, its clinical application as a valuable biomarker of reproductive processes, and its potential for therapeutic interventions.
Outcomes:
In the female mammal, INSL3 is largely produced by the theca interna cells of growing antral follicles during the follicular phase of the menstrual (oestrous) cycle. Within the follicle, INSL3 acts via its G-protein-coupled receptor, RXFP2, in an autocrine/paracrine manner to orchestrate and drive the production of the major steroid precursor androstenedione and its conversion by granulosa cells into oestrogens. These in turn create a positive feedback loop promoting the expression of more theca cell INSL3. This is countered by the follicular production of bone morphogenetic proteins and by the LH surge. Thus, the activity of the theca cell INSL3-RXFP2 system effectively determines the production of estradiol within an antral follicle through the follicular phase. INSL3 is also secreted into the circulation where it acts as a valuable biomarker to monitor the growth of antral follicles; it is consequently increased in PCOS and decreased in women with premature ovarian failure (POF). As an endocrine factor, INSL3 may also influence bone metabolism and kidney function. Additionally, INSL3 or its analogues may prove valuable as an adjunct in hormone replacement therapy or to monitor or influence IVF protocols.
Wider implications:
The INSL3-RXFP2 system represents a new regulatory pathway essential for the proper functioning of growing antral follicles. We still know very little about its involvement in pathologies such as PCOS or POF, and its role as a new biomarker of female function needs to be explored more widely to improve diagnosis and treatment of ovarian dysfunction. We need to examine how INSL3 might be used to improve IVF protocols and outcomes. Opportunities should also be investigated in regard to the systemic application of INSL3 as a rejuvenant therapy, with positive effects on bone or kidney function, and possibly also for fertility regulation. Most research to date has involved animal models; this now needs to be extended to include more human studies.
... In the kidney, RXFP2 is expressed in mesangial cells in mature glomeruli and inhibits proliferation of cultured primary glomerular cells, suggesting that INSL3 and RXFP2 influence the genesis or maturation of renal glomeruli and regulate mesangial cell density (Fu et al. 2006). Pod1 is a transcription factor involved in glomerulogenesis (Quaggin et al. 1999), and an E-box consensus sequence capable of binding Pod1 and other helix loop helix transcription factors is located upstream of the gene for RXFP2 (Funato et al. 2003). ...
... RXFP2 mRNA expression is highest in rat kidney at late stage gestation and decreases dramatically at birth with lowest levels in adulthood (Fu et al., 2006). RXFP2 is expressed in mesangial cells in mature glomeruli and inhibits proliferation of cultured primary glomerular cells, suggesting that INSL3 and RXFP2 influence the genesis or maturation of renal glomeruli and regulate mesangial cell density (Fu et al., 2006). ...
... RXFP2 mRNA expression is highest in rat kidney at late stage gestation and decreases dramatically at birth with lowest levels in adulthood (Fu et al., 2006). RXFP2 is expressed in mesangial cells in mature glomeruli and inhibits proliferation of cultured primary glomerular cells, suggesting that INSL3 and RXFP2 influence the genesis or maturation of renal glomeruli and regulate mesangial cell density (Fu et al., 2006). Pod1 is one of the main transcription factors involved in glomerulogenesis (Quaggin et al., 1999), and an E-box consensus sequence capable of binding Pod1 in conjunction with other helix loop helix transcription factors is located upstream of the gene for RXFP2 (Funato et al., 2003). ...
Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1-4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gαs, Gαi, and Gαo proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gαs- and Gαo-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gαi/Gαo proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and β-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1-4, the challenges facing the field, and current prospects for new therapeutics.
Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
... Indeed, following the report of abnormal differentiation of the gubernaculae (ligaments that control testicular descent) and consequent abdominal cryptorchidism in mice deficient in RXFP2 or in its testes-secreted ligand, INSL345464748, these genes have generally been studied in the context of the development of the reproductive tract of male eutherian mammals. Investigation in other contexts has just begun and new roles of INSL3-RXFP2 signaling have recently been reported in kidney differentiation [49] and bone metabolism505152. A possible involvement of RXFP2 (also known as LGR8) in skin differentiation, in general, and in horn bud differentiation, in particular, is also supported by the function of its second type of ligands, the relaxins. ...
Despite massive research efforts, the molecular etiology of bovine polledness and the developmental pathways involved in horn ontogenesis are still poorly understood. In a recent article, we provided evidence for the existence of at least two different alleles at the Polled locus and identified candidate mutations for each of them. None of these mutations was located in known coding or regulatory regions, thus adding to the complexity of understanding the molecular basis of polledness. We confirm previous results here and exhaustively identify the causative mutation for the Celtic allele (PC) and four candidate mutations for the Friesian allele (PF). We describe a previously unreported eyelash-and-eyelid phenotype associated with regular polledness, and present unique histological and gene expression data on bovine horn bud differentiation in fetuses affected by three different horn defect syndromes, as well as in wild-type controls. We propose the ectopic expression of a lincRNA in PC/p horn buds as a probable cause of horn bud agenesis. In addition, we provide evidence for an involvement of OLIG2, FOXL2 and RXFP2 in horn bud differentiation, and draw a first link between bovine, ovine and caprine Polled loci. Our results represent a first and important step in understanding the genetic pathways and key process involved in horn bud differentiation in Bovidae.
... Indeed, following the report of abnormal differentiation of the gubernaculae (ligaments that control testicular descent) and consequent intra- abdominal cryptorchidism in mice deficient in RXFP2 or in its testes-secreted ligand, INSL3 [45][46][47][48], these genes have generally been studied in the context of the development of the reproductive tract of male eutherian mammals. Investigation in other contexts has just begun and new roles of INSL3-RXFP2 signaling have recently been reported in kidney differentiation [49] and bone metabolism [50][51][52]. ...
Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access
... To determine which relaxin receptors these cells were expressing, rat renal myofibroblasts were plated in 6-well plates (at a density of 1-2ϫ10 5 cells/well) and grown to confluency in DMEM-FBS. RNA extraction and RT-PCR of RXFP1 (the primary relaxin receptor) and RXFP2 [the insulin-like peptide 3 (INSL3) receptor, which also weakly binds H2 RLX; ref. 23] mRNA was performed as described previously (11,24), using specific primers and PCR conditions for rat RXFP1 (11) and RXFP2 (24). cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in separate PCR reactions to control for quality and equivalent loading. ...
... To determine which relaxin receptors these cells were expressing, rat renal myofibroblasts were plated in 6-well plates (at a density of 1-2ϫ10 5 cells/well) and grown to confluency in DMEM-FBS. RNA extraction and RT-PCR of RXFP1 (the primary relaxin receptor) and RXFP2 [the insulin-like peptide 3 (INSL3) receptor, which also weakly binds H2 RLX; ref. 23] mRNA was performed as described previously (11,24), using specific primers and PCR conditions for rat RXFP1 (11) and RXFP2 (24). cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in separate PCR reactions to control for quality and equivalent loading. ...
The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF-beta1/Smad2 signaling. However, the pathways involved in the relaxin-TGF-beta1/Smad2 interaction remain unknown. This study investigated the signaling mechanisms by which human gene-2 (H2) relaxin regulates myofibroblast differentiation in vitro by examining its effects on mixed populations of fibroblasts and myofibroblasts propagated from injured rat kidneys. Cultures containing approximately 60-70% myofibroblasts were used to determine which relaxin receptors, G-proteins, and signaling pathways were involved in the H2 relaxin-mediated regulation of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblast differentiation). H2 relaxin only inhibited alpha-SMA immunostaining and collagen concentration in the presence of relaxin family peptide receptor 1 (RXFP1). H2 relaxin also induced a transient rise in cAMP in the presence of G(i/o) inhibition, and a sustained increase in extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Furthermore, inhibition of neuronal nitric oxide synthase (nNOS), NO, and cGMP significantly blocked the inhibitory effects of relaxin on alpha-SMA and Smad2 phosphorylation, while the NO inhibitor, L-nitroarginine methyl ester (hydrochloride) (L-NAME) significantly blocked the inhibitory actions of relaxin on collagen concentration in vivo. These findings suggest that relaxin signals through RXFP1, and a nNOS-NO-cGMP-dependent pathway to inhibit Smad2 phosphorylation and interfere with TGF-beta1-mediated renal myofibroblast differentiation and collagen production.
... The members of this subgroup are conserved in evolution (25). Some members of LGR subfamily have been shown to be essential for mouse kidney development (26,27). ...
The purpose of the present study was to screen the autoantibody signature of colon cancers to develop serum markers for colon cancer detection.
A phage cDNA expression library of colon cancer was built. The library was sequentially screened by a pool of 10 colon cancer sera, goat antihuman IgG, and a pool of two healthy sera to identify phage-expressed antigens recognized by tumor-associated antibodies. The clones picked out by these screening were subjected to a training set with 24 colon cancer sera and 24 healthy sera. The antigen combination, which got the most satisfactory classification, was tested by an independent set of 24 colon cancer sera with equal number of sera from normal donors. The carcinoembryonic antigen (CEA) level of these sera was detected for the additional classification analysis with or without the antigen combination.
A cDNA expression library consisting of 2 x 10(6) primary clones was prepared. After three turns of screening, 24 antigens recognized by tumor-associated antibodies were picked out for serum marker identification. The training set showed that a six-marker combination got the most satisfactory classification in a logistic regression model; leave-one-out validation achieved 91.7% sensitivity and 91.7% specificity. In a testing set with this marker panel, we correctly predicted 85% of the samples. Although according to CEA level alone, we correctly predicted 75% of the samples with 42% of cancer patients misclassified. When CEA was combined with the six markers, the sensitivity and specificity increased to 91.7% and 95.8%, respectively. The six antigen sequences in the phage display system are relatively short peptides. Only two of them showed homology to known protein sequences.
Autoantibodies against phage-expressed antigens derived from colon cancer tissues could be used as serum markers for the detection of colon cancer.
