Publications by authors named "Alexander I Agoulnik"

74 Publications

Anti-apoptotic and Matrix Remodeling Actions of a Small Molecule Agonist of the Human Relaxin Receptor, ML290 in Mice With Unilateral Ureteral Obstruction.

Front Physiol 2021 7;12:650769. Epub 2021 Jul 7.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, United States.

Diseases, such as diabetes and hypertension, often lead to chronic kidney failure. The peptide hormone relaxin has been shown to have therapeutic effects in various organs. In the present study, we tested the hypothesis that ML290, a small molecule agonist of the human relaxin receptor (RXFP1), is able to target the kidney to remodel the extracellular matrix and reduce apoptosis induced by unilateral ureteral obstruction (UUO). UUO was performed on the left kidney of humanized RXFP1 mice, where the right kidneys served as contralateral controls. Mice were randomly allocated to receive either vehicle or ML290 (30 mg/kg) daily intraperitoneal injection, and kidneys were collected for apoptosis, RNA, and protein analyses. UUO significantly increased expression of pro-apoptotic markers in both vehicle- and ML290-treated mice when compared to their contralateral control kidneys. Specifically, expression and Erk1/2 activity were upregulated, accompanied by an increase of TUNEL-positive cells in the UUO kidneys. Additionally, UUO induced marked increase in myofibroblast differentiation and aberrant remodeling on the extracellular matrix. ML290 suppressed these processes by promoting a reduction of pro-apoptotic, fibroblastic, and inflammatory markers in the UUO kidneys. Finally, the potent effects of ML290 to remodel the extracellular matrix were demonstrated by its ability to reduce collagen gene expression in the UUO kidneys. Our data indicate that daily administration of ML290 has renal protective effects in the UUO mouse model, specifically through its anti-apoptotic and extracellular matrix remodeling properties.
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http://dx.doi.org/10.3389/fphys.2021.650769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8293094PMC
July 2021

INPP4B protects from metabolic syndrome and associated disorders.

Commun Biol 2021 03 26;4(1):416. Epub 2021 Mar 26.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA.

A high fat diet and obesity have been linked to the development of metabolic dysfunction and the promotion of multiple cancers. The causative cellular signals are multifactorial and not yet completely understood. In this report, we show that Inositol Polyphosphate-4-Phosphatase Type II B (INPP4B) signaling protects mice from diet-induced metabolic dysfunction. INPP4B suppresses AKT and PKC signaling in the liver thereby improving insulin sensitivity. INPP4B loss results in the proteolytic cleavage and activation of a key regulator in de novo lipogenesis and lipid storage, SREBP1. In mice fed with the high fat diet, SREBP1 increases expression and activity of PPARG and other lipogenic pathways, leading to obesity and non-alcoholic fatty liver disease (NAFLD). Inpp4b male mice have reduced energy expenditure and respiratory exchange ratio leading to increased adiposity and insulin resistance. When treated with high fat diet, Inpp4b males develop type II diabetes and inflammation of adipose tissue and prostate. In turn, inflammation drives the development of high-grade prostatic intraepithelial neoplasia (PIN). Thus, INPP4B plays a crucial role in maintenance of overall metabolic health and protects from prostate neoplasms associated with metabolic dysfunction.
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http://dx.doi.org/10.1038/s42003-021-01940-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998001PMC
March 2021

Diverse functions of insulin-like 3 peptide.

J Endocrinol 2020 10;247(1):R1-R12

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Miami, Florida, USA.

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.
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http://dx.doi.org/10.1530/JOE-20-0168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453995PMC
October 2020

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation.

PLoS Genet 2020 06 4;16(6):e1008810. Epub 2020 Jun 4.

Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.
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http://dx.doi.org/10.1371/journal.pgen.1008810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297385PMC
June 2020

Deletion of inositol polyphosphate 4-phosphatase type-II B affects spermatogenesis in mice.

PLoS One 2020 15;15(5):e0233163. Epub 2020 May 15.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, United States of America.

Inositol polyphosphate-4-phosphatase type II (INPP4B) is a dual-specificity phosphatase that acts as a tumor suppressor in multiple cancers. INPP4B dephosphorylates phospholipids at the 4th position of the inositol ring and inhibits AKT and PKC signaling by hydrolyzing of PI(3,4)P2 and PI(4,5)P2, respectively. INPP4B protein phosphatase targets include phospho-tyrosines on Akt and phospho-serine and phospho-threonine on PTEN. INPP4B is highly expressed in testes, suggesting its role in testes development and physiology. The objective of this study was to determine whether Inpp4b deletion impacts testicular function in mice. In testis, Inpp4b expression was the highest in postmeiotic germ cells in both mice and men. The testes of Inpp4b knockout male mice were significantly smaller compared to the testes of wild-type (WT) males. Inpp4b-/- males produced fewer mature sperm cells compared to WT, and this difference increased with age and high fat diet (HFD). Reduction in early steroidogenic enzymes and luteinizing hormone (LH) receptor gene expression was detected, although androgen receptor (AR) protein level was similar in WT and Inpp4b-/- testes. Germ cell apoptosis was significantly increased in the knockout mice, while expression of meiotic marker γH2A.X was decreased. Our data demonstrate that INPP4B plays a role in maintenance of male germ cell differentiation and protects testis functions against deleterious effects of aging and high fat diet.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233163PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228085PMC
August 2020

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

FASEB J 2019 11 16;33(11):12435-12446. Epub 2019 Aug 16.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-β1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability , as evidenced by the sustained concentrations of compound in the liver. In mice expressing human gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.
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http://dx.doi.org/10.1096/fj.201901046RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988856PMC
November 2019

INSL3 in the muscolo-skeletal system.

Mol Cell Endocrinol 2019 05 6;487:12-17. Epub 2019 Jan 6.

Unit of Endocrinology, Department of Clinical and Experimental Sciences, University of Brescia, Viale Europa 11, 25123, Brescia, Italy. Electronic address:

Bone and skeletal muscle are currently considered a unified functional unit, showing complementary regulation at mechanical, biochemical, paracrine and metabolic levels. This functional unit undergoes a central hormonal regulation which is mainly ascribed to sex steroids and, in particular, androgens. However, recent evidence suggest that another testicular hormone lines the classical anabolic effect of testosterone on bone and muscle, the insulin-like peptide 3 (INSL3) acting on its specific receptor RXFP2. This minireview focuses on the most recent findings describing the role of INSL3/RXFP2 axis on the muscolo-skeletal system, from the mechanistic insights to the phenotypic consequences. Pathophysiological and therapeutic widenings deriving from available data are also discussed.
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http://dx.doi.org/10.1016/j.mce.2018.12.021DOI Listing
May 2019

Targeting the relaxin/insulin-like family peptide receptor 1 and 2 with small molecule compounds.

Mol Cell Endocrinol 2019 05 24;487:40-44. Epub 2018 Dec 24.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, 33199, USA. Electronic address:

The peptide hormone relaxin has beneficial roles in several organs through its action on its cognate G protein-coupled receptor, RXFP1. Relaxin administration is limited to intravenous, subcutaneous, intramuscular, or spinal injection. Another drawback of peptide-based therapy is the short half-life, which requires continuous delivery of the drug to achieve efficient concentration in target organs. The discovery of a non-peptide small molecule agonist of RXFP1, ML290, provides an alternative to the natural ligand. This review summarizes the development of ML290 and its potential future therapeutic applications in various diseases, including liver fibrosis and cardiovascular diseases. We also discuss the development of small molecule agonists targeting the insulin-like 3 receptor, RXFP2, and propose the potential use of these small molecules in the context of bone and muscle remodeling.
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http://dx.doi.org/10.1016/j.mce.2018.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451876PMC
May 2019

Protective Role of Testicular Hormone INSL3 From Atrophy and Weakness in Skeletal Muscle.

Front Endocrinol (Lausanne) 2018 28;9:562. Epub 2018 Sep 28.

Department of Medicine, University of Padova, Padova, Italy.

Androgens are primarily involved in muscle growth, whilst disease-driven muscle wasting is frequently associated with hypogonadism. The Leydig cells of the testes also produce the peptide-hormone Insulin-like peptide 3 (INSL3). INSL3 displays anabolic activity on bone, a target tissue of androgens, and its plasma concentrations are diminished in male hypogonadism. Here we tested the role of INSL3 on muscle mass regulation, in physiological and pathological conditions. Studies on C2C12 cell line showed that INSL3, acting on his specific receptor RXFP2, promotes skeletal muscle protein synthesis through the Akt/mTOR/S6 pathway. Next, studies on mice showed that INSL3 is required to prevent excessive muscle loss after denervation. Mechanistically, denervated mice lacked the compensatory activation of the Akt/mTOR/S6 pathway and showed an abnormal ubiquitin-proteasome system activation. Lack of INSL3 activity resulted also in reduced contractile force. These findings underlie a role of INSL3/RXFP2 in protein turnover, contributing to muscle wasting in male hypogonadism.
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http://dx.doi.org/10.3389/fendo.2018.00562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172310PMC
September 2018

Optimization of the first small-molecule relaxin/insulin-like family peptide receptor (RXFP1) agonists: Activation results in an antifibrotic gene expression profile.

Eur J Med Chem 2018 Aug 7;156:79-92. Epub 2018 Jun 7.

NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850, USA. Electronic address:

A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293 cell line identified 2-acetamido-N-phenylbenzamides 1 and 3 with modest agonist activity. An extensive structure-activity study has been undertaken to optimize the potency, efficacy, and physical properties of the series, resulting in the identification of compound 65 (ML-290), which has excellent in vivo PK properties with high levels of systemic exposure. This series, exemplified by 65, has produced first-in-class small-molecule agonists of RXFP1 and is a potent activator of anti-fibrotic genes.
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http://dx.doi.org/10.1016/j.ejmech.2018.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102074PMC
August 2018

Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.

FASEB J 2018 Jun 8:fj201800437R. Epub 2018 Jun 8.

Department of Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands.

The pathophysiology of arteriovenous fistula (AVF) maturation failure is not completely understood but impaired outward remodeling (OR) and intimal hyperplasia are thought to be contributors. This adverse vascular response after AVF surgery results from interplay between vascular smooth muscle cells (VSMCs), the extracellular matrix (ECM), and inflammatory cells. Relaxin (RLN) is a hormone that acts on the vasculature via interaction with RLN/insulin-like peptide family receptor 1 (RXFP1), resulting in vasodilatation, ECM remodeling, and decreased inflammation. In the present study, we evaluated the consequences of RXFP1 knockout ( Rxfp1) on AVF maturation in a murine model of AVF failure. Rxfp1 mice showed a 22% decrease in vessel size at the venous outflow tract 14 d after AVF surgery. Furthermore, a 43% increase in elastin content was observed in the lesions of Rxfp1 mice and coincided with a 41% reduction in elastase activity. In addition, Rxfp1 mice displayed a 6-fold increase in CD45 leukocytes, along with a 2-fold increase in monocyte chemoattractant protein 1 (MCP1) levels, when compared with wild-type mice. In vitro, VSMCs from Rxfp1 mice exhibited a synthetic phenotype, as illustrated by augmentation of collagen, fibronectin, TGF-β, and platelet-derived growth factor mRNA. In addition, VSMCs derived from Rxfp1 mice showed a 5-fold increase in cell migration. Finally, RXFP1 and RLN expression levels were increased in human AVFs when compared with unoperated cephalic veins. In conclusion, RXFP1 deficiency hampers elastin degradation and results in induced vascular inflammation after AVF surgery. These processes impair OR in murine AVF, suggesting that the RLN axis could be a potential therapeutic target for promoting AVF maturation.-Bezhaeva, T., de Vries, M. R., Geelhoed, W. J., van der Veer, E. P., Versteeg, S., van Alem, C. M. A., Voorzaat, B. M., Eijkelkamp, N., van der Bogt, K. E., Agoulnik, A. I., van Zonneveld, A.-J., Quax, P. H. A., Rotmans, J. I. Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.
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http://dx.doi.org/10.1096/fj.201800437RDOI Listing
June 2018

Human Relaxin Receptor Is Fully Functional in Humanized Mice and Is Activated by Small Molecule Agonist ML290.

J Endocr Soc 2017 Jun 8;1(6):712-725. Epub 2017 May 8.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida 33199.

Relaxin, a small peptide hormone of the insulin/relaxin family, demonstrated antifibrotic, organ protective, vasodilatory, and proangiogenic properties in clinical trials and several animal models of human diseases. Relaxin family peptide receptor 1 (RXFP1) is the relaxin cognate G protein-coupled receptor. We have identified a series of small molecule agonists of human RXFP1. The lead compound ML290 demonstrated preferred absorption, distribution, metabolism, and excretion profiles, is easy to synthesize, and has high stability . However, ML290 does not activate rodent RXFP1s and therefore cannot be tested in common preclinical animal models. Here we describe the production and analysis of a mouse transgenic model, a knock-out/knock-in of the human () complementary DNA into the mouse () gene. Insertion of the vector into the locus caused disruption of and expression of . The transcriptional expression pattern of the allele was similar to . Female mice homozygous for showed relaxation of the pubic symphysis at parturition and normal development of mammary nipples and vaginal epithelium, indicating full complementation of gene ablation. Intravenous injection of relaxin led to an increase in heart rate in humanized and wild-type females but not in -deficient mice, whereas ML290 increased heart rate in humanized but not wild-type animals, suggesting specific target engagement by ML290. Moreover, intraperitoneal injection of ML290 caused a decrease in blood osmolality. Taken together, our data show humanized RXFP1 mice can be used for testing relaxin receptor modulators in various preclinical studies.
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http://dx.doi.org/10.1210/js.2017-00112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562169PMC
June 2017

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1.

Sci Rep 2017 06 7;7(1):2968. Epub 2017 Jun 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gα and Gα but weak coupling to Gα. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.
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http://dx.doi.org/10.1038/s41598-017-02916-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462828PMC
June 2017

Relaxin-like peptides in male reproduction - a human perspective.

Br J Pharmacol 2017 05 27;174(10):990-1001. Epub 2017 Feb 27.

School of Biosciences, University of Nottingham, Nottingham, LE12 5RD, UK.

The relaxin family of peptide hormones and their cognate GPCRs are becoming physiologically well-characterized in the cardiovascular system and particularly in female reproductive processes. Much less is known about the physiology and pharmacology of these peptides in male reproduction, particularly as regards humans. H2-relaxin is involved in prostate function and growth, while insulin-like peptide 3 (INSL3) is a major product of the testicular Leydig cells and, in the adult, appears to modulate steroidogenesis and germ cell survival. In the fetus, INSL3 is a key hormone expressed shortly after sex determination and is responsible for the first transabdominal phase of testicular descent. Importantly, INSL3 is becoming a very useful constitutive biomarker reflecting both fetal and post-natal development. Nothing is known about roles for INSL4 in male reproduction and only very little about relaxin-3, which is mostly considered as a brain peptide, or INSL5. The former is expressed at very low levels in the testes, but has no known physiology there, whereas the INSL5 knockout mouse does exhibit a testicular phenotype with mild effects on spermatogenesis, probably due to a disruption of glucose homeostasis. INSL6 is a major product of male germ cells, although it is relatively unexplored with regard to its physiology or pharmacology, except that in mice disruption of the INSL6 gene leads to a disruption of spermatogenesis. Clinically, relaxin analogues may be useful in the control of prostate cancer, and both relaxin and INSL3 have been considered as sperm adjuvants for in vitro fertilization.

Linked Articles: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
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http://dx.doi.org/10.1111/bph.13689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406299PMC
May 2017

Synthetic non-peptide low molecular weight agonists of the relaxin receptor 1.

Br J Pharmacol 2017 05 30;174(10):977-989. Epub 2016 Nov 30.

NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.

Relaxin is a small heterodimeric peptide hormone of the insulin/relaxin superfamily produced mainly in female and male reproductive organs. It has potent antifibrotic, vasodilatory and angiogenic effects and regulates the normal function of various physiological systems. Preclinical studies and recent clinical trials have shown the promise of recombinant relaxin as a therapeutic agent in the treatment of cardiovascular and fibrotic diseases. However, there are the universal drawbacks of peptide-based pharmacology that apply to relaxin: a short half-life in vivo requires its continuous delivery, and there are high costs of production, storage and treatment, as well as the possibility of immune responses. All these issues can be resolved by the development of low non-peptide MW agonists of the relaxin receptors which are stable, bioavailable, easily synthesized and specific. In this review, we describe the discovery and characterization of the first series of such compounds. The lead compound, ML290, binds to an allosteric site of the relaxin GPCR, RXFP1. ML290 shows high activity and efficacy, measured by cAMP response, in cells expressing endogenous or transfected RXFP1. Relaxin-like effects of ML290 were shown in various functional cellular assays in vitro. ML290 has excellent absorption, distribution, metabolism and excretion properties and in vivo stability. The identified series of low MW agonists does not activate rodent RXFP1 receptors and thus, the production of a RXFP1 humanized mouse model is needed for preclinical studies. The future analysis and clinical perspectives of relaxin receptor agonists are discussed.

Linked Articles: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
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http://dx.doi.org/10.1111/bph.13656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406302PMC
May 2017

Nuclear Receptor Corepressor 1 Expression and Output Declines with Prostate Cancer Progression.

Clin Cancer Res 2016 08 11;22(15):3937-49. Epub 2016 Mar 11.

Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas. Biomolecular Sciences Institute, School of Integrated Science and Humanity, Florida international University, Miami, Florida.

Purpose: Castration therapy in advanced prostate cancer eventually fails and leads to the development of castration-resistant prostate cancer (CRPC), which has no cure. Characteristic features of CRPC can be increased androgen receptor (AR) expression and altered transcriptional output. We investigated the expression of nuclear receptor corepressor 1 (NCOR1) in human prostate and prostate cancer and the role of NCOR1 in response to antiandrogens.

Experimental Design: NCOR1 protein levels were compared between matched normal prostate and prostate cancer in 409 patient samples. NCOR1 knockdown was used to investigate its effect on bicalutamide response in androgen-dependent prostate cancer cell lines and transcriptional changes associated with the loss of NCOR1. NCOR1 transcriptional signature was also examined in prostate cancer gene expression datasets.

Results: NCOR1 protein was detected in cytoplasm and nuclei of secretory epithelial cells in normal prostate. Both cytoplasmic and nuclear NCOR1 protein levels were lower in prostate cancer than in normal prostate. Prostate cancer metastases show significant decrease in NCOR1 transcriptional output. Inhibition of LNCaP cellular proliferation by bicalutamide requires NCOR1. NCOR1-regulated genes suppress cellular proliferation and mediate bicalutamide resistance. In the mouse, NCOR1 is required for bicalutamide-dependent regulation of a subset of the AR target genes.

Conclusions: In summary, we demonstrated that NCOR1 function declines with prostate cancer progression. Reduction in NCOR1 levels causes bicalutamide resistance in LNCaP cells and compromises response to bicalutamide in mouse prostate in vivo Clin Cancer Res; 22(15); 3937-49. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-1983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970919PMC
August 2016

Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists.

Biochemistry 2016 Mar 4;55(12):1772-83. Epub 2016 Mar 4.

NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, Rockville, Maryland 20850, United States.

The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone, including its tissue remodeling and antifibrotic effects. The peptide has a short half-life in plasma, limiting its therapeutic utility. However, small-molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach, especially for chronic diseases such as heart failure and fibrosis. The first small-molecule agonists of RXFP1 were recently identified from a high-throughput screening, using a homogeneous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here, we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small-molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seventh transmembrane domain (TM7) and the third extracellular loop (ECL3). Several residues were determined to play an important role in the agonist binding and receptor activation, including a hydrophobic region at TM7 consisting of W664, F668, and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule ML290 were compared with the cognate ligand, relaxin, providing valuable insights on the structural basis and molecular mechanism of receptor activation and selectivity for RXFP1.
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http://dx.doi.org/10.1021/acs.biochem.5b01195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137375PMC
March 2016

Constitutive Notch Signaling Causes Abnormal Development of the Oviducts, Abnormal Angiogenesis, and Cyst Formation in Mouse Female Reproductive Tract.

Biol Reprod 2016 Mar 3;94(3):67. Epub 2016 Feb 3.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas

The Notch signaling pathway is critical for the differentiation of many tissues and organs in the embryo. To study the consequences of Notch1 gain-of-function signaling on female reproductive tract development, we used a cre-loxP strategy and Amhr2-cre transgene to generate mice with conditionally activated Notch1 (Rosa(Notch1)). The Amhr2-cre transgene is expressed in the mesenchyme of developing female reproductive tract and in granulosa cells in the ovary. Double transgenic Amhr2-cre, Rosa(Notch1) females were infertile, whereas control Rosa(Notch1) mice had normal fertility. All female reproductive organs in mutants showed hemorrhaging of blood vessels progressing with age. The mutant oviducts did not develop coiling, and were instead looped around the ovary. There were multiple blockages in the lumen along the oviduct length, creating a barrier for sperm or oocyte passage. Mutant females demonstrated inflamed uteri with increased vascularization and an influx of inflammatory cells. Additionally, older females developed ovarian, oviductal, and uterine cysts. The significant change in gene expression was detected in the mutant oviduct expression of Wnt4, essential for female reproductive tract development. Similar oviductal phenotypes have been detected previously in mice with activated Smo and in beta-catenin, Wnt4, Wnt7a, and Dicer conditional knockouts, indicating a common regulatory pathway disrupted by these genetic abnormalities.
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http://dx.doi.org/10.1095/biolreprod.115.134569DOI Listing
March 2016

Long-Lasting Consequences of Testosterone Exposure.

Endocrinology 2015 Oct;156(10):3488-9

Departments of Cellular Biology and Pharmacology (I.U.A.) and Human and Molecular Genetics (A.I.A.), Herbert Wertheim College of Medicine, and Biomolecular Sciences Institute (I.U.A., A.I.A.), Florida International University, Miami, Florida 33199.

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http://dx.doi.org/10.1210/en.2015-1719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398639PMC
October 2015

Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290.

Front Endocrinol (Lausanne) 2015 17;6:128. Epub 2015 Aug 17.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University , Miami, FL , USA.

Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit-human and guinea pig-human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing.
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http://dx.doi.org/10.3389/fendo.2015.00128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538381PMC
September 2015

Conditional deletion of the relaxin receptor gene in cells of smooth muscle lineage affects lower reproductive tract in pregnant mice.

Biol Reprod 2015 Apr 25;92(4):91. Epub 2015 Feb 25.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas

Relaxin hormone secreted into the circulation during pregnancy was discovered through its effects on pubic symphysis relaxation and parturition. Genetic inactivation of the relaxin gene or its cognate relaxin family peptide receptor 1 (RXFP1) in mice caused failure of parturition and mammary nipple enlargement, as well as increased collagen fiber density in the cervix and vagina. However, the relaxin effect on discrete cells and tissues has yet to be determined. Using transgenic mice with a knockin LacZ reporter in the Rxfp1 allele, we showed strong expression of this gene in vaginal and cervical stromal cells, as well as pubic ligament cells. We produced a floxed Rxfp1 allele that was used in combination with the Tagln-cre transgene to generate mice with a smooth muscle-specific gene knockout. In pregnant females, the ROSA26 reporter activated by Tagln-cre was detected in smooth muscle cells of the cervix, vagina, uterine artery, and in cells of the pubic symphysis. In late pregnant females with conditional gene ablation, the length of pubic symphysis was significantly reduced compared with wild-type or heterozygous Rxfp1(+/-) females. Denser collagen content was revealed by Masson trichrome staining in reproductive tract organs, uterine artery, and pubic symphysis. The cervical and vaginal epithelium was less developed than in heterozygous or wild-type females, although nipple size was normal and the dams were able to nurse their pups. In summary, our data indicate that relaxin/RXFP1 signaling in smooth muscle cells is important for normal collagen turnover and relaxation of the pubic symphysis during pregnancy.
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http://dx.doi.org/10.1095/biolreprod.114.127209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643956PMC
April 2015

Genetic ablation of androgen receptor signaling in fetal Leydig cell lineage affects Leydig cell functions in adult testis.

FASEB J 2015 Jun 20;29(6):2327-37. Epub 2015 Feb 20.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA; and Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas, USA

It is commonly accepted that androgen-producing fetal Leydig cells (FLC) are substituted by adult Leydig cells (ALC) during perinatal testis development. The mechanisms influencing this process are unclear. We used mice with a retinoid acid receptor 2 promoter-Cre recombinase transgene (Rarb-cre) expressed in embryonic FLC precursors, but not in postnatal testis, and a dual fluorescent Cre recombinase reporter to label FLC and ALC in vivo. All FLC in newborn testis had the recombinant, whereas the majority of LC in adult testis had the nonrecombinant reporter. Primary LC cultures from adult testis had either recombinant (20%) or nonrecombinant (80%) cells, demonstrating that the FLC survive in adult testis and their ontogeny is distinct from ALC. Conditional inactivation of androgen receptor (AR) allele using the Rarb-cre transgene resulted in a 50% increase of AR-negative LC in adult testis. The mutant males became infertile with age, with all LC in older testis showing signs of incomplete differentiation, such as a large number of big lipid droplets, an increase of finger-like protrusions, and a misexpression of steroidogenic or FLC- and ALC-specific genes. We propose that the antiandrogenic exposure during early development may similarly result in an increase of FLC in adult testis, leading to abnormal LC differentiation.
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http://dx.doi.org/10.1096/fj.14-263632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137449PMC
June 2015

Normal fertility in male mice with deletion of β-catenin gene in germ cells.

Genesis 2014 Apr 31;52(4):328-32. Epub 2014 Jan 31.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida.

As a dual function protein, β-catenin affects both cell adhesion and mediates canonical Wnt/β-catenin cell signaling. β-Catenin is prominently expressed in somatic Sertoli cells in the testis and postmeiotic germ cells, suggesting an additional role in spermatogenesis. It was reported previously that Cre/loxP-mediated conditional inactivation of the β-catenin gene (Ctnnb1) in male gonads using a protamine promoter-driven Cre transgene (Prm-cre) resulted in partial infertility, reduced sperm count, and abnormal spermatogenesis. In this report, we demonstrated that the conditional deletion of Ctnnb1 using a germ cell specific Cre transgene (Stra8-icre) had no effect on male fertility. We have shown that the Stra8-icre transgene was highly efficient in generating deletion in early pre-meiotic and post-meiotic cells. No differences in anatomical or histological presentation were found in the mutant testis, the production of viable sperm was similar, and no abnormalities in DNA sperm content were detected. We concluded that β-catenin is fully dispensable in germ cells for spermatogenesis. The conflicting results from the earlier study may have been due to off-target expression of Prm-cre in testicular somatic cells. In future studies, the analysis of conditional mutants using several Cre-transgenes should be encouraged to reduce potential errors.
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http://dx.doi.org/10.1002/dvg.22742DOI Listing
April 2014

The fate of spermatogonial stem cells in the cryptorchid testes of RXFP2 deficient mice.

PLoS One 2013 3;8(10):e77351. Epub 2013 Oct 3.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077351PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789668PMC
April 2014

NOTCH1 gain of function in germ cells causes failure of spermatogenesis in male mice.

PLoS One 2013 30;8(7):e71213. Epub 2013 Jul 30.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA.

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icre transgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63 genes' expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071213PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728026PMC
April 2014

Identification and optimization of small-molecule agonists of the human relaxin hormone receptor RXFP1.

Nat Commun 2013 ;4:1953

NIH Chemical Genomics Center, Discovery Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, USA.

The anti-fibrotic, vasodilatory and pro-angiogenic therapeutic properties of recombinant relaxin peptide hormone have been investigated in several diseases, and recent clinical trial data has shown benefit in treating acute heart failure. However, the remodelling capacity of these peptide hormones is difficult to study in chronic settings because of their short half-life and the need for intravenous administration. Here we present the first small-molecule series of human relaxin/insulin-like family peptide receptor 1 agonists. These molecules display similar efficacy as the natural hormone in several functional assays. Mutagenesis studies indicate that the small molecules activate relaxin receptor through an allosteric site. These compounds have excellent physical and in vivo pharmacokinetic properties to support further investigation of relaxin biology and animal efficacy studies of the therapeutic benefits of relaxin/insulin-like family peptide receptor 1 activation.
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http://dx.doi.org/10.1038/ncomms2953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915074PMC
December 2013

Reduced androgen receptor expression accelerates the onset of ERBB2 induced breast tumors in female mice.

PLoS One 2013 8;8(4):e60455. Epub 2013 Apr 8.

Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Ar(fl)/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060455PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620158PMC
November 2013

Testicular cancer and cryptorchidism.

Front Endocrinol (Lausanne) 2013 20;4:32. Epub 2013 Mar 20.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University Miami, FL, USA.

The failure of testicular descent or cryptorchidism is the most common defect in newborn boys. The descent of the testes during development is controlled by insulin-like 3 peptide and steroid hormones produced in testicular Leydig cells, as well as by various genetic and developmental factors. While in some cases the association with genetic abnormalities and environmental causes has been shown, the etiology of cryptorchidism remains uncertain. Cryptorchidism is an established risk factor for infertility and testicular germ cell tumors (TGCT). Experimental animal models suggest a causative role for an abnormal testicular position on the disruption of spermatogenesis however the link between cryptorchidism and TGCT is less clear. The most common type of TGCT in cryptorchid testes is seminoma, believed to be derived from pluripotent prenatal germ cells. Recent studies have shown that seminoma cells and their precursor carcinoma in situ cells express a number of spermatogonial stem cell (SSC) markers suggesting that TGCTs might originate from adult stem cells. We review here the data on changes in the SSC somatic cell niche observed in cryptorchid testes of mouse models and in human patients. We propose that the misregulation of growth factors' expression may alter the balance between SSC self-renewal and differentiation and shift stem cells toward neoplastic transformation.
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http://dx.doi.org/10.3389/fendo.2013.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602796PMC
March 2013

Left-sided cryptorchidism in mice with Wilms' tumour 1 gene deletion in gubernaculum testis.

J Pathol 2013 May 12;230(1):39-47. Epub 2013 Mar 12.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA.

A significant number of patients with germline mutations in the Wilms' tumour 1 (WT1) gene, a transcriptional factor essential for early renal and gonadal development, display cryptorchidism or non-scrotal testis position. We show here that WT1 is expressed during development in the mouse gubernacular ligament connecting the testis to the abdominal wall. Conditional inactivation of Wt1 in the gubernaculum (GU-WT1KO animals) resulted in abnormal differentiation of the gubernacula during development and, in about 40% of adult males, unilateral, always left-sided, cryptorchidism. At birth the right testis was positioned above the processus vaginalis and eventually moved into the developing scrotal pouch. In affected mutants the left testis was displaced from the normal position and the left processus vaginalis failed to form. The analysis of testicular descent at different stages of postnatal development suggests that unilateral cryptorchidism might be caused by asymmetry in the positions of the abdominal organs providing a higher degree of mobility for the left testis. Spermatogenesis in GU-WT1KO animals was blocked in cryptorchid testes located in a high pararenal position, but was maintained in testes located in a low abdominal position. Conditional inactivation of both Wt1 and androgen receptor (Ar) genes in the gubernaculum led to a bilateral asymmetrical cryptorchidism in all mutant males, with the left testis again located higher than the right one. The malformations induced by WT1 and AR deficiency in the gubernaculum and processus vaginalis, in combination with mechanical constraints on testis descent, determine the final position of the testes. In summary, our data indicate that WT1 is directly involved in gubernaculum differentiation. Taken together, the results of the study underline the complex nature of testicular descent, with an involvement in this process of several genetic factors and developmental events.
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http://dx.doi.org/10.1002/path.4161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819214PMC
May 2013
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