The effect of relaxin on cell proliferation in mouse cervix requires estrogen receptor {alpha} binding to estrogen response elements in stromal cells.

The study objective was to determine whether stromal and/or epithelial estrogen receptor-alpha (ERalpha) is required for relaxin to promote proliferation of stromal and epithelial cells in the mouse cervix. Four types of tissue recombinants were prepared with cervical stroma (St) and epithelium (Ep) from wild-type (wt) and ERalpha knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing 17beta-estradiol (treatment d 1). Animals were injected sc with relaxin or vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before tissue recombinants were collected at 1000 h on d 10. Relaxin promoted marked proliferation of both epithelial and stromal cells in tissue recombinants containing wt St (P < 0.001) but far lower proliferation in recombinants prepared with ko St, regardless of whether Ep was derived from wt or ko mice. An additional experiment using mice expressing wt ERalpha, a mutant of ERalpha that selectively lacks classical signaling through estrogen response element binding, or no ERalpha demonstrated that ERalpha must bind to an estrogen response element to enable relaxin's proliferative effects. In conclusion, this study shows that ERalpha-expressing cells in St, using a classical signaling pathway, are necessary for relaxin to promote marked proliferation in both stromal and epithelial cells of the mouse cervix.

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.

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 (ERalpha) 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. ERalpha 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 ERalpha knockout mice to determine if the obligatory permissive effect of estrogen on relaxin-induced cell proliferation occurs through stromal and/or epithelial ERalpha.

Relaxin acts on stromal cells to promote epithelial and stromal proliferation and inhibit apoptosis in the mouse cervix and vagina.

The objective of this study was to determine whether stromal and/or epithelial relaxin receptor (LGR7) is required for relaxin to promote proliferation and inhibit apoptosis of stromal and epithelial cells in the mouse cervix and vagina. Tissue recombinants were prepared with stroma (St) and epithelium (Ep) from wild-type (wt) and LGR7 knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep, and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of intact syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing progesterone and estradiol-17beta (designated d 1 of treatment). Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10 of treatment. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before cervices and vaginas were collected at 1000 h on d 10. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end labeling was used to quantify apoptosis. Relaxin markedly increased proliferation and decreased apoptosis of epithelial and stromal cells in tissue recombinants containing wt stroma (P < 0.01) but had no effect on tissue recombinants prepared with ko stroma, regardless of whether epithelium was derived from wt or ko mice. In conclusion, this study shows that LGR7-expressing cells in the stroma are both necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells of cervix and vagina, whereas epithelial LGR7 does not affect these processes.

International Union of Pharmacology LVII: recommendations for the nomenclature of receptors for relaxin family peptides.

Although the hormone relaxin was discovered 80 years ago, only in the past 5 years have the receptors for relaxin and three other receptors that respond to related peptides been identified with all four receptors being G-protein-coupled receptors. In this review it is suggested that the receptors for relaxin (LGR7) and those for the related peptides insulin-like peptide 3 (LGR8), relaxin-3 (GPCR135), and insulin-like peptide 5 (LGPCR142) be named the relaxin family peptide receptors 1 through 4 (RXFP1-4). RXFP1 and RXFP2 are leucine-rich repeat-containing G-protein-coupled receptors with complex binding characteristics involving both the large ectodomain and the transmembrane loops. RXFP1 activates adenylate cyclase, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase, and extracellular signaling regulated kinase (Erk1/2) and also interacts with nitric oxide signaling. RXFP2 activates adenylate cyclase in recombinant systems, but physiological responses are sensitive to pertussis toxin. RXFP3 and RXFP4 resemble more conventional peptide liganded receptors and both inhibit adenylate cyclase, and in addition RXFP3 activates Erk1/2 signaling. Physiological studies and examination of the phenotypes of transgenic mice have established that relaxin has roles as a reproductive hormone involved in uterine relaxation (some species), reproductive tissue growth, and collagen remodeling but also in the cardiovascular and renal systems and in the brain. The connective tissue remodeling properties of relaxin acting at RXFP1 receptors have potential for the development of agents effective for the treatment of cardiac and renal fibrosis, asthma, and scleroderma and for orthodontic remodelling. Agents acting at RXFP2 receptors may be useful for the treatment of cryptorchidism and infertility, whereas antagonists may be used as contraceptives. The brain distribution of RXFP3 receptors suggests that actions at these receptors have the potential for the development of antianxiety and antiobesity drugs.

The effects of blocking the actions of estrogen and progesterone on the rates of proliferation and apoptosis of cervical epithelial and stromal cells during the second half of pregnancy in rats.

Serum levels of the ovarian hormones relaxin, estrogen, and progesterone are elevated during the second half of 23-day rat pregnancy when dramatic growth of the cervix occurs. Recently, we demonstrated that relaxin contributes to cervical growth by both promoting cell proliferation and inhibiting apoptosis of cervical cells during late pregnancy. The objective of this study was to determine the influence of estrogen and progesterone on the rates of proliferation and apoptosis of cervical cells at 3-day intervals during the second half of rat pregnancy. The actions of estrogen and progesterone were blocked with s.c. injections of estrogen antagonist ICI 182,780 and progesterone antagonist RU486, respectively. To evaluate cell proliferation, 5'-bromo-2'-deoxyuridine was injected s.c. 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling was used to detect apoptotic cells. Proliferating and apoptotic cells were identified by immunohistochemistry, and the rates at which these processes occurred were determined by morphometric analysis. Blocking the actions of estrogen and progesterone decreased the rates of proliferation and increased the rates of apoptosis of both cervical epithelial and stromal cells during late pregnancy. However, blocking the actions of progesterone had the opposite effects on apoptosis of both cervical epithelial and stromal cells during the middle of pregnancy. In conclusion, this study provides evidence that estrogen and progesterone, like relaxin, contribute to the increase in the cervical cell content during late pregnancy by promoting proliferation and inhibiting apoptosis of cervical cells.

Receptors for relaxin family peptides.

Recent studies have identified four receptors that are the physiological targets for relaxin family peptides. All are class I (rhodopsin like) G-protein-coupled receptors with LGR7 (RXFP1) and LGR8 (RXFP2) being type C leucine-rich repeat-containing receptors, whereas GPCR135 (RXFP3) and GPCR142 (RXFP4) resemble receptors that respond to small peptides such as somatostatin and angiotensin II. The cognate ligands for the receptors have been identified: relaxin for RXFP1; INSL3 for RXFP2; relaxin 3 for RXFP3 and INSL5 for RXFP4. RXFP1 and RXFP2 receptors produce increases in intracellular cAMP levels upon stimulation, although the response is complex and contains a component sensitive to PI-3-kinase inhibitors. There is also evidence that RXFP1 can activate Erk1/2 and nitric oxide synthase, and relaxin has been reported to enter cells and activate glucocorticoid receptors. In contrast, RXFP3 and RXFP4 couple to Gi by a pertussis toxin-sensitive mechanism to cause inhibition of cAMP production. Now that the receptors for relaxin family peptides and their cognate ligands have been identified, we suggest a nomenclature for both the peptides and the receptors that we hope will be helpful to researchers in this rapidly advancing field.

Immuno-neutralization of circulating relaxin does not alter the breast cancer-protective action of parity in MNU-treated rats.

Early pregnancy and childbirth protects women against future development of breast cancer by an unknown mechanism. Parity likewise reduces mammary cancer incidence in rats exposed to the carcinogen, N-methyl-N-nitrosourea (MNU), providing a model for the human phenomenon. We hypothesized that relaxin, a 6KD luteal mammotropic hormone of pregnancy, might be the anti-cancer pregnancy factor, and that induced relaxin deficiency during rat gestation would restore carcinogen sensitivity. Forty-one pregnant (age 50 days) and 25 age-matched virgin Sprague-Dawley rats were used. Relaxin deficiency was induced by injecting mouse monoclonal anti-rat relaxin antibody (MCA1) days 12-18 of gestation. Pregnant controls were injected with vehicle or mouse IgG on the same schedule. Because MCA1 disrupts parturition, all rats underwent cesarean section on day 22. At age 100 days, all rats were injected i.v. with MNU (50mg/Kg) and examined daily for tumors until euthanized at age 240 days. Mammary tumor incidence and frequency were significantly (p<0.01) reduced and tumor latency was increased (p<0.001) in primiparous as compared with virgin rats. However, tumor incidence, type, size and latency were similar in MCA1-treated and control primiparous rats. Thus, luteal relaxin does not appear to be the factor responsible for resistance to breast cancer.

Relaxin's physiological roles and other diverse actions.

Relaxin has vital physiological roles in pregnant rats, mice, and pigs. Relaxin promotes growth and softening of the cervix, thus facilitating rapid delivery of live young. Relaxin also promotes development of the mammary apparatus, thus enabling normal lactational performance. The actions of relaxin on the mammary apparatus vary among species. Whereas relaxin is required for development of the mammary nipples in rats and mice, it is essential for prepartum development of glandular parenchyma in pregnant pigs. During pregnancy relaxin also inhibits uterine contractility and promotes the osmoregulatory changes of pregnancy in rats. Recent studies with male and nonpregnant female rodents revealed diverse therapeutic actions of relaxin on nonreproductive tissues that have clinical implications. Relaxin has been reported to reduce fibrosis in the kidney, heart, lung, and liver and to promote wound healing. Also, probably through its vasodilatory actions, relaxin protects the heart from ischemia-induced injury. Finally, relaxin counteracts allergic reactions. Knowledge of the diverse physiological and therapeutic actions of relaxin, coupled with the recent identification of relaxin receptors, opens numerous avenues of investigation that will likely sustain a high level of research interest in relaxin for the foreseeable future.

Induction of labor with RU 486 (mifepristone) in relaxin-deficient rats: antepartum administration of relaxin facilitates delivery and increases pup survival.

OBJECTIVE: This study was conducted to determine whether antepartum administration of relaxin improves RU 486-induced delivery at term in rats that lack circulating endogenous relaxin. STUDY DESIGN: Pregnant rats were modified two ways to obtain circulating levels of relaxin and progesterone that resemble those of pregnant humans: relaxin was immunoneutralized throughout the second half of the 23-day pregnancy and high progesterone levels were sustained until term by inserting progesterone implants on day 20. Porcine relaxin was administered subcutaneously from 8 AM on day 20 until delivery. Labor was induced by administering RU 486 subcutaneously at 4 AM on day 22. RESULTS: After induction of labor with RU 486, labor and delivery were faster, and the incidence of live births was higher when rats were also administered relaxin during the antepartum period. CONCLUSION: Antepartum administration of relaxin in combination with RU 486 has beneficial effects on delivery in relaxin-deficient rats.

Relaxin signaling in reproductive tissues.

The insulin/relaxin peptide family includes insulin, IGFs, relaxin1-3, INSL3/RLF, INSL4, INSL5/RIF2 and INSL6/RIF1, many without functional characterization. Based on analysis of transgenic phenotypes and phylogenetic profiling, we have discovered that two orphan leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8, are cognate receptors for relaxin whereas INSL3 is a specific ligand for LGR8. With the identification of the relaxin receptors, it is now possible to investigate specific cells and tissues that are responsive to relaxin in diverse physiological and pathological conditions as well as to develop agonists and antagonists for LGR7 and LGR8 as therapeutics to treat different labor disorders. Furthermore, future functional characterization of the specificity of these pluripoentent receptors with peptide ligands could lead to the understanding of related orphan ligands and receptors.

Myogenic reactivity is reduced in small renal arteries isolated from relaxin-treated rats.

Administration of the ovarian hormone relaxin to nonpregnant rats vasodilates the renal circulation comparable to pregnancy. This vasodilation is mediated by endothelin (ET), the ET(B) receptor, and nitric oxide. Furthermore, endogenous relaxin mediates the renal vasodilation and hyperfiltration that occur during gestation. The goal of this study was to investigate whether myogenic reactivity of small renal and mesenteric arteries is reduced in relaxin-treated rats comparable to the pregnant condition. Relaxin or vehicle was administered to virgin female Long-Evans rats for 5 days at 4 microg/h, thereby producing midgestational blood levels of the hormone. The myogenic responses of small renal arteries (200-300 microm in diameter) isolated from these animals were evaluated in an isobaric arteriograph system. Myogenic reactivity was significantly reduced in the small renal arteries from relaxin-treated compared with vehicle-treated rats. The reduced myogenic responses were mediated by the ET(B) receptor and nitric oxide since the selective ET(B) receptor antagonist RES-701-1 and the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester restored myogenic reactivity to virgin levels. The influence of relaxin was not limited to the renal circulation because myogenic reactivity was also reduced in small mesenteric arteries isolated from relaxin-treated rats. Thus relaxin administration to nonpregnant rats mimics pregnancy, insofar as myogenic reactivity of small renal and mesenteric arteries is reduced in both conditions.

Activation of orphan receptors by the hormone relaxin.

Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3',5'-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.