bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing the Functional Domains of Relaxin‐3 and the Creation of a Selective Antagonist for RXFP3/GPCR135 over Relaxin Receptor RXFP1/LGR7
Authors: Liu Changlu , Kuei Chester , Sutton Steven , Shelton Jonathan , Zhu Jessica , Nepomuceno Diane , Hossain Mohammed Akhter , Wade John D. , Bathgate Ross A. D. , Bonaventure Pascal , Lovenberg Timothy
Published: April 2009

Both relaxin‐3 and its receptor (RXFP3, also known as GPCR135) are predominantly expressed in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin‐3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin‐3/RXFP3 in the regulation of stress, feeding, and other potential functions remain to be studied. Since relaxin‐3 also activates the relaxin receptor (RXFP1, also known as LGR7), which is also expressed in the brain, selective RXFP3 agonists and antagonists are crucial for study of the physiological functions of relaxin‐3 and RXFP3 in vivo. The finding that the B chain of relaxin‐3 is an agonist for RXFP3 (albeit at low potency) but not RXFP1 suggests that the B chain of relaxin‐3 plays a dominant role for RXFP3 binding and activation. Chimeric peptide studies using the B chain from relaxin‐3 and the A chains from different members of the insulin and relaxin family have confirmed this hypothesis and led to the generation of R3/I5 (a chimeric peptide with relaxin‐3 B chain and INSL5 A chain) as a selective agonist for RXFP3 over RXFP1. Truncation of the C‐terminus of the B chain of R3/I5 results in a high‐affinity antagonist, R3(BΔ23–27)R/I5, for RXFP3 over RXFP1. R3(BΔ23–27)R/I5 has pA2 values of 9.15 and 9.6 for human and rat RXFP3, respectively, but has no affinity or agonistic activity for the human and rat RXFP1. Ongoing and future in vivo studies using the selective agonist and antagonist for RXFP3 will shed light on the physiological role of the relaxin‐3 system.
α1‐Adrenergic Activation Upregulates Expression of Relaxin Receptor RXFP1 in Cardiomyocytes
Authors: Moore Xiao‐Lei , Hong Alice , Du Xiao‐Jun
Published: April 2009

Cardiac RXFP1 was upregulated upon activation of the α1‐adrenergic receptor (AR) through signal pathways involving protein kinase A and C and ERK. In contrast, β1‐AR activation downregulated expression of cardiac RXFP1 and it further counteracted the α1‐AR‐mediated RXFP1 upregulation.
Relaxin in Endometriosis
Authors: Morelli Sara S. , Petraglia Felice , Weiss Gerson , Luisi Stefano , Florio Pasquale , Goldsmith Laura T.
Published: April 2009

Endometriosis is an important contributing factor to chronic pelvic pain and infertility. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) have each been implicated in the establishment of endometriotic lesions. Since relaxin regulates the expression of MMPs and VEGF in the endometrium, we tested the hypothesis that relaxin plays a role in endometriosis by comparing the expression of relaxin mRNA and its LGR7 (RXFP1) receptor mRNA in normal human endometrium to those in samples from patients with endometriosis.
Relaxin/RXFP1 Signaling in Prostate Cancer Progression
Authors: Feng Shu , Agoulnik Irina U. , Li Zhen , Han Hee Dong , Lopez‐Berestein Gabriel , Sood Anil , Ittmann Michael M. , Agoulnik Alexander I.
Published: April 2009

We found that relaxin peptide expression is significantly elevated in recurrent human prostate cancer. Stimulation with relaxin increased migration, invasiveness, proliferation, and adhesion of LNCaP and PC3 cells in vitro. Opposite effects on cellular phenotype were observed after suppression of endogenous relaxin/RXFP1 expression or signaling. We showed an accelerated progression of prostate cancer in TRAMP males with transgenic relaxin overexpression and a longer survival of TRAMP, RXFP1‐deficient males. Suppression of RXFP1 expression in PC3 xenografts in nude mice by intratumoral injections of short interfering RNA resulted in decreased tumor size, cell proliferation, and metastasis rate.
Relaxin: An Endogenous Renoprotective Factor?
Authors: Hewitson Tim D. , Samuel Chrishan S.
Published: April 2009

Fibrosis, so‐called scarring, is a key cause of pathology and dysfunction in a variety of organs. The biology of healing and scarring are similar, albeit with very different outcomes. Typical of this process in general, renal fibrosis is the result of a disproportionate accumulation of extracellular matrix that occurs in ongoing kidney disease. Renal scarring is both progressive and multifactorial, ultimately leading to end‐stage renal failure and the requirement for dialysis or kidney transplantation. Regardless of the underlying etiology, renal mesenchymal cells such as fibroblasts are the cellular basis of this process. Activity and differentiation of these cells are regulated by a number of profibrotic cytokines, growth factors, vasoactive mediators, and other signals, with transforming growth factor‐β being among the most important. However, we now also recognize that there are a number of endogenous antifibrotic factors that are renoprotective by counteracting these signals to limit matrix synthesis and organization and reduce net accumulation. Relaxin is one such factor. The absence of endogenous relaxin results in spontaneous fibrosis with aging and also accelerates the pathogenesis of progressive fibrosis after injury. Both effects are reversed by replenishment with recombinant H2 relaxin. This review summarizes recent developments in the understanding of the cellular and molecular mechanisms in fibrosis and how these are influenced by relaxin.
Relaxin‐3 and Its Role in Neuroendocrine Function
Authors: McGowan Barbara M. C. , Stanley Sarah A. , Ghatei Mohammad A. , Bloom Stephen R.
Published: April 2009

The hypothalamus plays a key role in the regulation of energy homeostasis and endocrine function. Relaxin‐3 is a hypothalamic neuropeptide that belongs to the insulin superfamily of peptides. It is expressed in the nucleus incertus of the brainstem, which has projections to the hypothalamus and is thought to act in the brain via the RXFP3 receptor, although the RXFP1 receptor may also play a role. RXFP3 and RXFP1 are present in the hypothalamic paraventricular nucleus, an area with a well‐characterized role in the regulation of energy balance. The paraventricular nucleus also modulates reproductive function by providing inputs to hypothalamic gonadotropin‐releasing hormone neurons. The physiological roles for relaxin‐3 remain to be established. Evidence for a role of relaxin‐3 as a hypothalamic orexigenic peptide will be reviewed, including its effects on the hypothalamo–pituitary–thyroid axis and energy expenditure. Studies pointing towards a putative role of relaxin‐3 in the hypothalamic–pituitary–gonadal axis will be discussed. Central endocrine effects of relaxin‐3 will be compared to relaxin. We conclude that relaxin‐3 may act as a hypothalamic signal to coordinate appetite, thyroid function, and reproductive status. Further studies will be required to determine whether these are physiological roles for relaxin‐3 and to determine the receptors involved.
Relaxin‐Induced Changes in Renal Function and RXFP1 Receptor Expression in the Female Rat
Authors: Bogzil Alsadek H. , Ashton Nick
Published: April 2009

Relaxin causes renal hyperfiltration, natriuresis, and diuresis in male rats. This study determined the effect of recombinant human relaxin (rhRLX) on renal function in the female rat. After 7 days of rhRLX treatment (0.4 μg/h) urinary excretion of sodium and chloride were significantly lower in the rhRLX‐treated group, but urine flow rate was not different from that in controls. Quantitative PCR showed that the RXFP1 receptor was expressed in both the renal cortex and medulla; immuno‐localization revealed expression in the proximal tubule and inner medullary collecting ducts. These data show that relaxin elicits pregnancy‐like changes in renal function in the female rat.
Relative Roles of the Epithelial and Stromal Tissue Compartment(s) in Mediating the Actions of Relaxin and Estrogen on Cell Proliferation and Apoptosis in the Mouse Lower Reproductive Tract
Authors: Yao Lijuan , Agoulnik Alexander I. , Cooke Paul S. , Meling Daryl D. , Sherwood O. David
Published: April 2009

Relaxin and estrogen are secreted by the ovary during the second half of pregnancy in rats and mice. Relaxin promotes marked growth of the lower reproductive tract in both species. Relaxin promotes accumulation of epithelial and stromal cells in the cervix and vagina by both stimulating cell proliferation and inhibiting apoptosis. Estrogen acting through estrogen receptor alpha (ERα) plays an essential permissive role in relaxin's actions. A fundamental step toward understanding the actions of relaxin and estrogen is to identify the tissue compartments that initiate their effects. Limited studies using either antibodies to human relaxin receptor (LGR7, RXFP1) or an IRES‐LacZ reporter cassette in the LGR7 gene revealed relaxin receptors in subepithelial stroma cells and smooth muscle cells but not in epithelial cells in rodent vaginal and/or cervical tissues. ERα has been reported in both stromal and epithelial compartments in the rodent reproductive tract. This chapter describes ongoing studies that use relaxin bioactivity as a means of identifying the tissue compartment(s) that initiates the actions of relaxin and estrogen on the lower reproductive tract. Specifically, a tissue separation–recombination methodology in combination with LGR7 knockout mice was initially used to obtain functional evidence that stromal LGR7 is both necessary and sufficient to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in mouse cervix and vagina. The tissue separation–recombination method is currently being used in conjunction with ERα knockout mice to determine if the obligatory permissive effect of estrogen on relaxin‐induced cell proliferation occurs through stromal and/or epithelial ERα.
Relaxin's Involvement in Extracellular Matrix Homeostasis
Authors: Cooney Timothy E. , Schober Justine M. , Lubahn John D. , Konieczko Elisa M.
Published: April 2009

Burgeoning evidence suggests that the hormone relaxin modulates collagen in the extracellular matrix of diverse tissues. In separate lines of study, we provide further substantiation of this hypothesis. Immunofluorescence was used to probe isolated fibroblasts derived from volar oblique ligament explant culture for vimentin, actin, RXFP1, and estrogen receptor beta. Ligaments were obtained as surgical waste from thumb reconstruction patients. Four specimens have been examined to date. Cells derived from these patients expressed vimentin and actin, consistent with fibroblast morphology. Putative fibroblasts derived from two of three female patients expressed RXFP1 receptors; the solitary male was negative. Given the small sample, however, the data are considered preliminary. Immunohistochemistry was used on frozen sections from 26 skin biopsies obtained from children undergoing genitoplasty. A subset of samples was also probed for transforming growth factor (TGF‐β1) and TGF‐β3. Appropriate controls were used. Finally, a subset of patient blood was assayed for relaxin by using an enzyme‐linked immunosorbent assay‐based method. The results showed RXFP1 receptor expression in the cells that populate the basement membrane in 96% of patients, regardless of gender. Most tissue expressed TGF‐β. Finally, serology suggested that relaxin was detectable in these children. Our two lines of research provide additional evidence for the diverse tissue tropism of relaxin. In particular, connective tissues as diverse as ligaments and basal lamina keratinocytes express RXFP1. These data lend support to our contention that relaxin affects ligament integrity and wound healing.
Relaxin as a Protective Substance in the Preserving Solution for Liver Transplantation
Authors: Boehnert Markus U. , Armbruster Franz Paul , Hilbig Heidegard
Published: April 2009

Ischemia reperfusion injury (IRI) is a problem in organ transplantation. Relaxin is known to have a protective effect against liver injury caused by IRI. Using a model of isolated perfused rat liver, the local oxygen supply in liver tissue was investigated by spectrophotometric in vivo imaging and compared to the protective effect of relaxin shown by immunohistochemical measurement of myeloperoxidase and malonyldialdehyde activities as determinants of oxidative stress. In relaxin‐treated liver tissue, spectrophotometry showed a better oxygen supply and decreased myeloperoxidase and malonyldialdehyde activities. Our data suggest that relaxin can influence the oxygen distribution in liver tissue and reduce cell damage caused by IRI.
Structural Properties of Relaxin Chimeras
Authors: Haugaard‐Jönsson Linda M. , Hossain Mohammed Akhter , Daly Norelle L. , Bathgate Ross A. D. , Wade John D. , Craik David J. , Rosengren K. Johan
Published: April 2009

Relaxin‐3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor–ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin‐3 B chain with the A chain from insulin‐like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three‐dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin‐3 B chain compared to native relaxin‐3, and the INSL5 A chain displays a relaxin/insulin‐like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure–activity data on relaxins.
An in Vitro Study of the Protective Effect of Relaxin on Brain Tissue under Ischemic Stress
Authors: Wilson Brian C. , Rappaport Rebecca
Published: April 2009

Organotypic brain slice culture was used to study the direct actions of relaxin on neural cells under ischemic stress. Cortical brain slices from neonatal rats were cultured for 14 days. Experimental slices were placed in a deoxygenated, glucose‐free balanced salt solution (BSS) for 1 h, one group with 10−7 M H2 relaxin in the medium and the other without. Control slices were transferred to oxygenated BSS with glucose. Slices were returned to normal culture conditions and analyzed 1, 4, 8, and 24 h post‐treatment using propidium iodide (PI) fluorescence to highlight cellular damage or death. The percentages of dead and dying cells in a slice were compared between groups. Relaxin treatment attenuated PI staining at 4 and 8 h postischemia, suggesting a direct action(s) on cells that improves their chance of survival during ischemia.
Nuclear Receptors, Testosterone, and Posttranslational Modifications in Human INSL3 Promoter Activity in Testicular Leydig Cells
Authors: Tremblay Jacques J. , Robert Nicholas M. , Laguë Éric
Published: April 2009

Insulin‐like peptide 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the mammalian testis. During embryonic life INSL3 is required for testicular descent, whereas in adults it is involved in bone metabolism and male germ cell survival. Despite these important roles, the molecular mechanisms regulating INSL3 expression remain poorly understood. So far, two transcription factors have been implicated in INSL3 transcription: the nuclear receptors SF1 and NUR77. Circumstantial evidence also points to a role for androgens. Using transient transfections in MA‐10 Leydig cells, we found that testosterone regulates in a time‐ and dose‐dependent manner the human INSL3 promoter. The INSL3 promoter, however, does not contain a classical androgen‐responsive element. Testosterone responsiveness was found to be mediated through an element located in the proximal INSL3 promoter, which also contains a NUR77‐SF1‐binding site. Furthermore, we found that posttranslational modifications, such as phosphorylation and acetylation, modulate transcription factor activity and therefore also contribute to INSL3 promoter activity in Leydig cells. All together, these data provide new insights into the molecular mechanisms regulating INSL3 expression in the mammalian testis.
Relaxin Family Peptide Receptor 1 Activation Stimulates Peroxisome Proliferator‐Activated Receptor Gamma
Authors: Singh Sudhir , Bennett Robert G.
Published: April 2009

Relaxin (RLX) has antifibrotic effects in a number of tissues. Many of these effects are similar to those induced by the activators of peroxisome proliferator‐activated receptor gamma (PPARγ), raising the possibility that a mechanism for RLX's antifibrotic effects may involve activation of the PPARγ pathway. This study investigated the effect of RLX on PPARs and their mechanism of upregulation. It shows that RLX stimulates ligand‐independent PPAR activation in a dose‐dependent manner. The combined effect of RLX and PPARγ agonists was superadditive, suggesting that both agents might be used together for an increased antifibrotic effect. RLX caused increased expression of the PPARγ target genes CD36 and LXRα. RLX's effect was mimicked by forskolin and partially blocked by pertussis toxin, suggesting that RLX works through a cAMP/protein kinase A pathway to activate PPARγ. A better understanding of this pathway might help in the amelioration of fibrotic diseases.
Conceptus Numbers Do Not Affect Blood Concentrations of Relaxin in the Rabbit
Authors: Fields Phillip , Fields Michael
Published: April 2009

On day 18 of pregnancy, conceptuses were surgically removed in order that rabbits would be left with seven, five, three, or one conceptus. During pregnancy, blood concentrations of relaxin were determined by radioimmunoassay. There was no difference between relaxin concentrations in rabbits carrying one versus seven conceptuses. The results indicate that conceptus number does not influence the blood concentration of relaxin in the rabbit.
Metabolic and Neuroendocrine Responses to RXFP3 Modulation in the Central Nervous System
Authors: Sutton Steven W. , Shelton Jonathan , Smith Craig , Williams John , Yun Sujin , Motley Timothy , Kuei Chester , Bonaventure Pascal , Gundlach Andrew , Liu Changlu , Lovenberg Timothy
Published: April 2009

Neuroanatomical studies have shown relaxin‐3 neurons, primarily found in the rodent nucleus incertus (NI), project widely into a large number of areas expressing the relaxin‐3 receptor (RXFP3), and these data suggest relaxin‐3/RXFP3 signaling modulates sensory, emotional, and neuroendocrine processing. The similar distribution of this receptor–ligand pair in the rat, mouse, and monkey brain suggests that experimental findings obtained in lower species will translate to higher species. A role for relaxin‐3 and RXFP3 in modulating stress responses is strongly suggested by the expression of corticotropin‐releasing factor R1 (CRF‐R1) by NI cells, increased relaxin‐3 expression in the NI after stress or CRF injection, and hormonal responses to intracerebroventricular (i.c.v.) relaxin‐3 injection. Recent data are consistent with a further role for this ligand–receptor pair in modulating memory. In addition, relaxin‐3 has been reported to modulate feeding and body weight control. Acute or chronic central (i.c.v. or intraparaventricular) injections of relaxin‐3 have shown a consistent stimulatory effect on food consumption while relaxin was inactive, suggesting the phagic effect of relaxin‐3 is mediated by RXFP3. We have confirmed the role of RXFP3 in modulating feeding and body weight by using a selective RXFP3 agonist (R3/I5) and antagonist [R3(Δ23–27)R/I5], collecting feeding, body weight, hormone, and body composition data. In addition, we have preliminary body weight and magnetic resonance imaging data from relaxin‐3 knockout mice, which on a 129S5:B6 background are smaller and leaner than congenic controls. These data suggest relaxin‐3, acting through RXFP3, is involved in coordinating stress, learning and memory, and feeding responses as predicted on the basis of neuroanatomy.
RXFP1 Couples to the Gαi3‐Gβγ‐PI3K‐PKCζ Pathway via the Final 10 Amino Acids of the Receptor C‐terminal Tail
Authors: Halls Michelle L. , Papaioannou Maria , Wade John D. , Evans Bronwyn A. , Bathgate Ross A. D. , Summers Roger J.
Published: April 2009

The relaxin family peptide receptors RXFP1 and RXFP2 are highly similar receptors that share approximately 80% amino acid sequence homology. Constitutively active receptors couple to increased cAMP accumulation, which is important for relaxin‐mediated decidualization and myometrial inhibition. Despite the high homology, the receptors couple to different G‐proteins to affect cAMP accumulation. This study aimed to determine the region of RXFP1 that directs coupling to the delayed Gαi3 pathway by using receptor mutagenesis. Receptor chimeras suggested that activation of this pathway by RXFP1 was dependent upon the membrane‐anchored domain of the receptor. Further receptor mutagenesis showed that activation of the Gαi3‐Gβγ‐PI3K‐PKCζ cAMP pathway by RXFP1 is dependent upon the C‐terminal 10 amino acids of the receptor and absolutely requires Arg752.
Addition of a Carboxy‐Terminal Green Fluorescent Protein Does Not Alter the Binding and Signaling Properties of Relaxin Family Peptide Receptor 3
Authors: Van Der Westhuizen Emma T. , Wade John D. , Sexton Patrick M. , Summers Roger J.
Published: April 2009

The relaxin family peptide receptor 3 (RXFP3) is the cognate receptor for the neuropeptide relaxin‐3. RXFP3 was tagged at the carboxy‐terminus with a variant of the green fluorescent protein (GFP2) for use in receptor localization studies. RXFP3‐GFP2 was examined to ensure it retained binding and signaling properties similar to untagged RXFP3. Competition for [125I]INSL5/H3 relaxin chimera binding to RXFP3 and RXFP3‐GFP2 indicated that the carboxy‐terminal tag did not affect receptor binding or receptor internalization. RXFP3‐GFP2 activated ERK1/2 with a similar potency to RXFP3 when transiently expressed in CHO‐K1 or HEK293T cells, suggesting that the GFP2 tag did not affect receptor function. This study demonstrated that addition of a carboxy‐terminal fusion protein to RXFP3 did not alter the binding or signaling properties of RXFP3, making RXFP3‐GFP2 a useful tool for future receptor localization and trafficking studies.
First Clinical Experience with Intravenous Recombinant Human Relaxin in Compensated Heart Failure
Authors: Dschietzig Thomas , Teichman Sam , Unemori Elaine , Wood Susy , Boehmer Julia , Richter Christoph , Baumann Gert , Stangl Karl
Published: April 2009

Relaxin is upregulated and plays a compensatory role in human heart failure. We therefore determined the safety of and dose response to human relaxin in stable patients with heart failure. Sixteen patients were treated with open‐label intravenous relaxin in three sequential dose cohorts and monitored hemodynamically during the 24‐h infusion and postinfusion periods. The safety demonstrated in group A (treatment for 8 h each with dosages equivalent to 10, 30, and 100 μg/kg/day) allowed escalation to group B (240, 480, and 960 μg/kg/day), and the highest safe dose, 960 μg/kg/day, was selected for a 24‐h dosing in group C. Relaxin showed no relevant adverse effects and produced hemodynamic effects consistent with systemic vasodilation, i.e., trends toward increases in the cardiac index and decreases in pulmonary wedge pressure, without inducing hypotension. The first therapeutic use of relaxin in human heart failure demonstrated favorable hemodynamic effects and indicated that it may be of value in the treatment of this widespread disease.
Relaxin and Maternal Lactocrine Programming of Neonatal Uterine Development
Authors: Bartol Frank F. , Wiley Anne A. , Bagnell Carol A.
Published: April 2009

In mammals, including the pig (Sus scrofa domesticus), structural patterning and functional programming of uterine tissues involve events that occur shortly after birth. Porcine endometrial development between birth (postnatal day 0 [PND 0]) and PND 15 is estrogen receptor (ER) dependent and estrogen sensitive. The endometrium is relaxin (RLX) receptor (RXFP1) positive and ERα negative at birth. Uterine expression of RXFP1 and ERα, detectable by PND 2, increases with age from PND 0 to 14. Estrogen exposure during this period sufficient to affect uterine developmental trajectory and adult uterine phenotype also alters uterine RXFP1 gene expression patterns in the neonatal uterus. Data implicate RXFP1 as an element of the uterine developmental program. Uterotrophic effects documented for both estrogen and RLX in the neonatal pig are age‐specific and most pronounced after onset of ERα expression. Patterns of inhibition of RLX effects on uterine development induced with ICI 182,780, an ER antagonist, indicate that cross talk between RLX and estrogen signaling systems evolve with age in the porcine uterus. Given that RLX administered from birth stimulates uterine ERα expression and that estrogen administered from birth stimulates RXFP1 expression by PND 2, a feed‐forward relationship between these signaling systems is envisioned. Evidence that RLX is present in porcine milk and in the circulation of nursing offspring supports the lactocrine hypothesis for maternal programming of uterine tissues in which milk‐borne RLX, acting via RXFP1, sustains ERα expression and porcine endometrial development in the neonate.
Biological Activity of Relaxin in Porcine Milk
Authors: Frankshun Amy‐Lynn , Ho Teh‐Yuan , Steinetz Bernard G. , Bartol Frank F. , Bagnell Carol A.
Published: April 2009

A lactocrine mechanism for delivery of maternally derived relaxin (RLX) into the neonatal circulation as a consequence of nursing has been proposed for the pig. Consistently, immunoreactive porcine RLX was detected in colostrum as well as in the serum of nursing pigs. Concentrations of porcine RLX in milk are highest during early lactation (9–19 ng/mL) and decline to less than 2 ng/mL by postnatal day 14. However, RLX bioactivity has not been described in porcine milk. Therefore, this study was designed to establish an assay for RLX bioactivity in porcine milk and to determine if milk RLX bioactivity was related to RLX concentrations in milk collected at parturition (lactation day 0) and on lactation day 14. To assess milk RLX bioactivity, an in vitro bioassay using human embryonic kidney (HEK293T) cells transfected with the human RLX receptor (LGR7) was developed. Milk RLX bioactivity was confirmed by documentation of a systematic increase in cAMP production by HEK293T‐LGR7 cells in response to increasing volumes of day 0 milk. Addition of lactation day 14 milk, porcine insulin, or human insulin‐like growth factor 1 to HEK293T‐LGR7 cells, or porcine RLX treatment of nontransfected HEK293T cells, failed to elicit a cAMP response. Western blot analysis of milk proteins revealed an 18‐kDa protein band, indicating that pro RLX is the primary form of bioactive RLX in porcine milk. Data support the lactocrine hypothesis and suggest a role for milk‐borne pro RLX in porcine neonatal development.
Investigations into the Inhibitory Effects of Relaxin on Renal Myofibroblast Differentiation
Authors: Samuel Chrishan S. , Mookerjee Ishanee , Halls Michelle L. , Summers Roger J. , Chew Elaine , Bathgate Ross A. D. , Tregear Geoffrey W. , Hewitson Tim D.
Published: April 2009

Derived from fibroblasts, myofibroblasts are the principal cells that are responsible for the synthesis and reorganization of excess matrix in renal interstitial fibrosis. Recognized from their de novo expression of α‐smooth muscle actin, myofibroblast differentiation and activity can be influenced by several factors, including a combination of growth factors and other soluble mediators, extracellular matrix components, and mechanical stress. Relaxin has previously been shown to inhibit renal myofibroblast differentiation in vitro, an effect partly mediated through its ability to interfere with the transforming growth factor‐β1 (TGF‐β1) pathway via inhibition of Smad2 phosphorylation and translocation. Furthermore, endogenous relaxin has been shown to protect the kidney from a myofibroblast‐mediated model of injury in vivo. However, the pathways involved in the interaction between relaxin and TGF‐β1 remain unknown. In this report, the inhibitory actions of relaxin on TGF‐β1‐induced renal myofibroblast differentiation are summarized to date, and the potential signaling pathways that are implicated in relaxin's inhibitory actions are discussed.
Relaxin Concentrations in Serum and Urine of Endangered and Crazy Mixed‐Up Species
Authors: Steinetz B. , Lasano S. , De Haas van Dorsser F. , Glickman S. , Bergfelt D. , Santymire R. , Songsassen N. , Swanson W.
Published: April 2009

The human population explosion has pushed many mammalian wildlife species to the brink of extinction. Conservationists are increasingly turning to captive breeding as a means of preserving the gene pool. We previously reported that serum immunoactive relaxin provided a reliable means of distinguishing between true and pseudopregnancy in domestic dogs, and this method has since been found to be a reliable indicator of true pregnancy in endangered Asian and African elephants and Sumatran rhinoceroses. Our canine relaxin radioimmunoassay (RIA) has now been adapted and validated to measure relaxin in the serum and urine of felids, including domestic and wild species. Moreover, a commercially available canine serum relaxin kit (Witness®Relaxin Kit; Synbiotics, San Diego, CA), has been adapted for reliable detection of relaxin in urine of some felid species. Our porcine relaxin RIA has also been utilized to investigate the role of relaxin in reproductive processes of the spotted hyena, a species in which the female fetuses are severely masculinizedin utero. Indeed, this species might well now be extinct were it not for the timely secretion of relaxin to enable copulation and birth of young through the clitoris. Additional studies have suggested relaxin may be a useful marker of pregnancy in the northern fur seal and the maned wolf (the former species has been designated as “depleted” and the latter as “near threatened”). Given appropriate immunoassay reagents, relaxin determination in body fluids thus provides a powerful tool for conservationists and biologists investigating reproduction in a wide variety of endangered and exotic species.
Mechanisms of Relaxin Receptor (LGR7/RXFP1) Expression and Function
Authors: Kern András , Bryant‐Greenwood Gillian D.
Published: April 2009

The LGR7/RXFP1 and LGR8/RXFP2 receptors are unique receptors among the G‐protein‐coupled receptors (GPCRs) in having a low‐density lipoprotein class A (LDL‐A) module. Their complex gene organization, among the intron‐richest of the GPCRs, suggests that alternative splicing is a common occurrence. We have therefore investigated the role of the LDL‐A module and shown the identity, expression, and functions of three LGR7 splice variants in the human decidua. Point mutations of conserved residues or complete deletion of the LDL‐A module resulted in loss of the cAMP response to relaxin. Its glycosylation also impacted LGR7 cell surface delivery and therefore receptor activation. The wild‐type (WT) LGR7 was expressed as both precursor and mature forms, but deletion of the LDL‐A module resulted in expression of only the mature form. Three new alternatively spliced variants of LGR7 were identified, all containing a truncated extracellular region. Their functional characterization showed them exerting dominant negative effects on the WT LGR7 by preventing its homodimerization, maturation, and subsequent trafficking to the cell surface, resulting in loss of function. In summary, different mechanisms have been identified for controlling the cell surface expression and function of the LGR7 protein which are likely to be significant for the role of relaxin in human parturition.
Role of Relaxin in Maternal Systemic and Renal Vascular Adaptations during Gestation
Authors: McGuane Jonathan T. , Debrah Julianna E. , Debrah Dan O. , Rubin J. Peter , Segal Mark , Shroff Sanjeev G. , Conrad Kirk P.
Published: April 2009

In this paper, the hemodynamic changes of normal pregnancy are reviewed and the underlying hormonal and molecular mechanisms are discussed. Among other findings related to these phenomena, our previous work has demonstrated the importance of relaxin in systemic and renal vascular as well as osmoregulatory changes during gestation. These findings are summarized, and new concepts related to the function of relaxin in blood vessels are presented. Finally, work in progress is briefly outlined.
Lysosomal Acid Hydrolases of the Cathepsin Family Are Novel Targets of INSL3 in Human Thyroid Carcinoma Cells
Authors: Bialek Joanna , Hombach‐Klonisch Sabine , Fiebig Barbara , Weber Ekkehard , Hoang‐Vu Cuong , Klonisch Thomas
Published: April 2009

Insulin‐like peptide 3 (INSL3) is present in hyperactive and neoplastic thyrocytes, but the functional role of this relaxin‐like peptide hormone during carcinogenesis in the thyroid gland is currently unknown. We generated new cell models of stable transfectants of the human follicular thyroid carcinoma cell line FTC‐133 expressing and secreting bioactive human INSL3. These transfectants displayed higher intracellular ATP levels, but INSL3 failed to act as a promoter of growth. The acquisition of an invasive tumor cell phenotype with local tissue invasion represents the beginning of a number of events leading to metastasis, the major cause of fatal outcome in cancer patients. Here we demonstrate a function of INSL3 in elastin degradation, which is considered an early step during basal membrane penetration and tissue invasion by tumor cells. INSL3 markedly increased the production of the lysosomal enzymes cathepsin‐L and cathepsin‐D. Enhanced secretion of the elastinolytic cathepsin‐L was associated with increased elastinolytic activity of FTC‐133–INSL3 transfectants. Thus, we provide the first evidence that the INSL3 peptide can promote early tumor cell invasiveness in human thyroid carcinoma cells by enhancing their metabolic activity and elastin‐degrading potential.
Structural Insights into the Function of Relaxins
Authors: Rosengren K. Johan , Bathgate Ross A. D. , Craik David J. , Daly Norelle L. , Haugaard‐Jönsson Linda M. , Hossain Mohammed Akhter , Wade John D.
Published: April 2009

The relaxin peptide hormones are members of the insulin superfamily and share a structural fold that is characterized by two peptide chains which are cross‐braced by three disulfide bonds. On this framework, various amino acid side chains are presented, allowing specific interactions with different receptors. The relaxin receptors belong to two unrelated classes of G‐protein‐coupled receptors, but interestingly they are not selective for a single relaxin peptide. Relaxin‐3, which is considered to be an extreme example of the relaxin family, can activate receptors from both classes and in fact interacts to some degree with all four receptors identified to date. To deduce how changes in the primary sequence can fine‐tune the overall structure and thus the ability to interact with the various receptors, we have studied a range of relaxin‐like peptides using solution nuclear magnetic resonance analysis. Three‐dimensional structures of relaxin‐3, insulin‐like peptide 3 (INSL3), and INSL5 were determined and revealed a number of interesting features. All peptides showed a significant amount of line‐broadening in certain regions, in particular around the intra‐A‐chain disulfide bond, suggesting that despite the disulfide bonds the fold is rather dynamic. Although the peptides share a common structural core there are significant differences, particularly around the termini. The structural data in combination with mutational studies provide valuable insights into the structure–activity relationships of relaxins.
Dimerization and Negative Cooperativity in the Relaxin Family Peptide Receptors
Authors: Svendsen Angela Manegold , Vrecl Milka , Knudsen Louise , Heding Anders , Wade John D. , Bathgate Ross A. D. , De Meyts Pierre , Nøhr Jane
Published: April 2009

Peptides of the relaxin family bind to the relaxin family peptide receptors or RXFPs, members of the G‐protein‐coupled receptor (GPCR) superfamily. For many years, ligand binding to GPCRs was thought to take place as monomeric complexes, ignoring early evidence of negative cooperativity. However, recent research has shown that most GPCRs form constitutive dimers or larger oligomers. The connection between dimerization and negative cooperativity has now been shown for several GPCRs, including the thyroid‐stimulating hormone, luteinizing hormone, and follicle‐stimulating hormone receptors, which like RXFP1 and ‐2 belong to the leucine‐rich repeat‐containing subgroup of class A GPCRs. We recently demonstrated homodimerization and negative cooperativity for RXFP1 and RXFP2 as well as their heterodimerization. Another study showed that RXFP1 has to homodimerize in order to be transported from the endoplasmic reticulum to the cell membrane.
Scientific Rationale and Design of a Phase I Safety Study of Relaxin in Women with Severe Preeclampsia
Authors: Unemori Elaine , Sibai Baha , Teichman Sam L
Published: April 2009

The pathophysiology of preeclampsia involves profound systemic vasoconstriction. Its etiology may be related to reduced blood flow to the placenta, leading to the elaboration of soluble, vasoactive factors which increase maternal systemic vascular resistance and cause renal dysfunction. Reduced bioactivity of vascular endothelial growth factor (VEGF) may play a central role in this pathophysiology. Previous clinical studies have strongly suggested that relaxin is a systemic and renal vasodilator. In nonclinical studies, relaxin administration to monkeys has been associated with increased vessel density in the endometrium, and in previous human trials relaxin administration has been highly correlated with increased menstrual bleeding in women. VEGF has been proposed as a mechanism for these endometrial effects. Together, these data suggest that relaxin may be able to relieve systemic and renal vasoconstriction and improve placental perfusion in women with preeclampsia. As a first step in the development program for relaxin in this indication, a multicenter, randomized, double‐blind, placebo‐controlled phase I safety study in women with severe preeclampsia has been launched in the USA. Three doses of relaxin, 3, 10, and 30 μg/kg of body weight/day, or placebo, will be administered for up to 72 h in women admitted to the hospital for management of their disease. Although the trial is primarily focused on safety, signs of efficacy, such as changes in blood pressure and renal markers, will also be assessed.
Development of Lanthanide‐Labeled Human INSL3 as an Alternative Probe to Radioactively Labeled INSL3 for Use in Bioassays
Authors: Shabanpoor Fazel , Hughes Richard A. , Bathgate Ross A. D. , Separovic Frances , Wade John D.
Published: April 2009

The use of radioisotope‐labeled peptides and proteins in bioassays is diminishing as a consequence of the emergence of fluorescent lanthanide‐labeled probes. We describe a simple, single‐step labeling of synthetic human insulin‐like peptide 3 (INSL3) with a commercially available activated europium chelator. The labeled peptide was then used for the development of a robust and highly reproducible receptor‐binding assay which is amenable to automated high‐throughput screening of ligands. Europium‐labeled INSL3 showed a similar binding profile to radioactively labeled INSL3.
INSL3/RXFP2 Signaling in Testicular Descent
Authors: Feng Shu , Ferlin Alberto , Truong Anne , Bathgate Ross , Wade John D. , Corbett Sean , Han Shuo , Tannour‐Louet Mounia , Lamb Dolores J. , Foresta Carlo , Agoulnik Alexander I.
Published: April 2009

Mutations of the insulin‐like peptide 3 (INSL3) hormone or its receptor, RXFP2, cause intraabdominal cryptorchidism in male mice. Specific RXFP2 expression in mouse gubernacula was detected at embryonic day 14.5 and markedly increased after birth in the developing cremaster muscle, as well as in the epididymis and testicular Leydig and germ cells. INSL3 treatment stimulated cell proliferation of embryonic gubernacular and Leydig cells, implicating active INSL3‐mediated signaling. The transcription factor SOX9, a known male sex determination factor, upregulated the activity of the RXFP2 promoter. INSL3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic–pituitary–gonadal axis signaling or Hoxa10, although these factors are important for inguinoscrotal testicular descent. Similarly, conditional ablation of the androgen receptor gene in gubernacular cells resulted in disruption of inguinoscrotal descent. We performed mutation screening of INSL3 and RXFP2 in human patients with cryptorchidism and control subjects from different populations in Europe and the USA. Several missense mutations were described in both the INSL3 and RXFP2 genes. A novel V39G INSL3 mutation in a patient with cryptorchidism was identified; however, the functional analysis of the mutant peptide did not reveal compromised function. In more than 2000 patients and controls analyzed to date, the T222P RXFP2 mutation is the only one strongly associated with the mutant phenotype. The T222P mutant receptor, when transfected into 293T cells, had severely decreased cell membrane expression, providing the basis for the functional deficiency of this mutation.
Relaxin Family Genes in Humans and Teleosts
Authors: Yegorov Sergey , Good‐Avila Sara V. , Parry Laura , Wilson Brian C.
Published: April 2009

Functions of the relaxin peptide family have been primarily investigated in mammals, and little attention has been paid to nonmammalian species. In this work, we performed phylogenetic and syntenic data analyses to identify the number and orthologous and paralogous relationships of relaxin family genes in teleosts and humans. Additionally, we performed reverse transcription PCR to determine the expression of three members of the relaxin family in zebrafish brain and gonads. We found evidence that teleosts harbor orthologs of the human INSL5, H2, H3, and INSL3 genes, so‐named RLND/RLNE, RLNC, RLN3a/RLN3b, and RLNF in teleosts, respectively. Moreover, the presence of mRNA for RLN3a, RLND, and RLNF in both male and female brain and gonads of zebrafish suggests that all three genes are expressed in these tissues of this teleost. Differential expression and/or regulation of these genes will be explored in future experimental work.
Relaxin Promotes Clustering, Migration, and Activation States of Mononuclear Myelocytic Cells
Authors: Figueiredo Kevin A. , Rossi Gina , Cox Michael E.
Published: April 2009

Monocytes are leukocytic precursors of macrophages, dendritic cells, and osteoclasts, with critical roles in inflammation and tumor biology. Tumors can elicit signals that activate monocytes to extravasate, infiltrate tumors, and differentiate into tumor‐associated macrophages (TAMs), which can modulate host immune surveillance. In order to assess whether relaxin can influence monocyte activation status, we assessed its ability to alter cell–cell clustering and cytokine expression of the monocytic cell line THP‐1. Here we report that relaxin can induce time‐ and substrate‐dependent homotypic cell–cell clustering of monocytes. In addition, we demonstrate that relaxin can suppress macrophage migration in an adenylate cyclase‐independent, nitric oxide synthase‐dependent fashion. We confirm relaxin‐induced upregulation of vascular endothelial growth factor expression and regulation of M1/M2 cytokine profiles. By stimulating monocyte activation and modulating inflammatory cytokine expression and migratory activity of resulting macrophages in response to endotoxin exposure, relaxin may be a critical regulator of the macrophage activation state that regulates the TAM phenotype.
Relaxin Supports Implantation and Early Pregnancy in the Marmoset Monkey
Authors: Einspanier Almuth , Lieder Kai , Husen Bettina , Ebert Katja , Lier Susanne , Einspanier Ralf , Unemori Elaine , Kemper Martina
Published: April 2009

To test the hypothesis that relaxin is an important factor supporting implantation, two approaches have been carried out using a human‐relevant animal model, the marmoset monkey. First, uterine mRNA transcription and protein expression during the implantation phase in the conceptive and nonconceptive cycles were examined. Second, functional parameters were analyzed to assess the in vivo effects of exogenous applied relaxin throughout implantation. Relaxin and its receptor, RXFP1, were highly upregulated shortly before and during the physical process of implantation, indicating that relaxin is an important factor for remodeling and immunotolerance. The action of relaxin on the uterus was accompanied by an increase of estrogen‐associated factors and macrophage infiltration, suggesting redundant systems necessary for successful implantation. The data from relaxin‐treated animals supported those obtained from naive tissues in terms of increases in angiogenesis as well as earlier and faster growth of the uterus and placenta in the relaxin‐treated marmoset monkey group, resulting in parturition 7–10 days earlier than the control group, but not pathological. In general, relaxin is very effective in preparing the endometrium for implantation. These findings should encourage further clinical research regarding introducing relaxin for pathological pregnancies, such as early pregnancy failure or insufficient placenta.
Scar Prevention and Cosmetically Enhanced Wound Healing Using Relaxin
Authors: Stewart Dennis R.
Published: April 2009

Relaxin has previously been tested in rodent wound healing models and been shown to promote angiogenesis and to speed healing. However, pigs have been shown to be a better model for human skin in dermatology studies, so juvenile pigs were selected for a study of scar reduction and cosmetic appearance. Twelve 20‐ by 6‐mm excisional wounds were created on the backs of all animals. Topical formulations of relaxin with 0, 0.5, or 2.5 mg/mL were applied twice daily for weeks 2–3 and then daily for weeks 3–6 in all animals. In addition, some animals received systemic relaxin, which was administered via infusion pumps at a rate of 125 μg/kg of body weight/day. Assessments of healing and cosmetic appearance were made by a dermatologist at weeks 2, 4, and 6. Wound sites were collected at 6 weeks and evaluated histologically for granulation tissue, inflammation, and collagen organization. Wounds in animals receiving systemic relaxin had an improved appearance with less redness, reduced granulation tissue, and lower amounts of inflammation. They showed a more‐well‐knit collagen structure compared to controls. Wounds treated with topical formulations did not show improvement over controls. The topical formulation used was found to have a short residence time, which likely limited penetration of relaxin. Reformulated relaxin preparations with improved penetration might be useful as a topical treatment for wounds to prevent or reduce scarring.