endothelin-1 and Leiomyoma

The endothelin axis, comprising endothelin-1 (ET-1) and its receptors (ETA and ETB), is involved in the pathophysiology of different human tumors. Here we review conventional approaches and gene expression profiling indicating the association of ET-1 and its cognate receptors with human and rat leiomyomas, the most common benign tumors of myometrium. Specifically, ET-1/ETA interactions affect human and rat leiomyoma cell proliferation through protein kinase C and mitogen-activated protein kinase-dependent signaling pathways. Recent experiments demonstrate that the ET-1 axis exerts a potent antiapoptotic effect involving sphingolipid metabolism and prostaglandin-endoperoxide synthase 2/prostaglandin system in the rat Eker leiomyoma tumor-derived ELT3 cell line. Evidence supports that steroid hormones, growth factors, and extracellular matrix are key regulators of the leiomyoma growth. Interestingly, the ET-1 axis is under steroid hormones and can cooperate with these growth factors. Therefore, ET-1 alone or in association with these factors could contribute to the complex regulation of uterine tumor growth, such as proliferation, survival, and extracellular matrix production. This review summarizes current knowledge and emerging data on ET-1 in uterine leiomyoma pathology.

Topics: Animals; Cell Proliferation; Endothelin-1; Female; Humans; Leiomyoma; Models, Biological; Neoplasms, Hormone-Dependent; Rats; Receptors, Endothelin; Signal Transduction; Transcriptome; Uterine Neoplasms

Recent evidence suggests that repetitive hypoxia occurs during menstrual cycles due to vasoconstriction and myometrial contraction. It is unknown if hypoxia contributes to the development of uterine leiomyoma, the most common tumor of the female reproductive system. This study aims to characterize the response to hypoxia in leiomyoma and myometrial cells; and determine if an aberrant leiomyoma response to hypoxia may contribute to leiomyomatogenesis.. Primary and immortalized leiomyoma and myometrial cells were cultured under normoxic and hypoxic conditions. Expression levels of vascular endothelial growth factor-A (VEGF-A), adrenomedullin (ADM), endothelin-1 (ET-1), and hypoxia-inducible factor-1 alpha (HIF-1α) were measured by qRT-PCR, western blotting and ELISA. Cell proliferation was assessed using MTT assay and proliferating-cell-nuclear-antigen (PCNA) expression. KC7F2 (HIF-1α inhibitor) was used to examine the regulating mechanisms.. As expected, hypoxia induced HIF-1α expression in both leiomyoma and myometrial cells. However, hypoxia induced VEGF-A, ET-1 and ADM expression and VEGF-A secretion into the culture media in leiomyoma but not myometrial cells. MTT assay and PCNA expression showed that hypoxia induces proliferation in leiomyoma, but not myometrial cells. HIF-1α inhibitor abrogated the hypoxia-induced VEGF-A, ET-1, ADM, and PCNA expression in leiomyoma cells.. This study suggests an aberrant leiomyoma cellular response to hypoxia compared to myometrium. This differential response to menstruation-related repetitive hypoxia episodes may lead to selective proliferation of hypoxia-adaptive leiomyoma cells and contribute to leiomyoma growth. Thus, in addition to adding to our understanding of leiomyoma pathobiology, the study proposes angiogenic factors as a potential leiomyoma therapeutic target.

Topics: Adrenomedullin; Cell Hypoxia; Cell Line; Cell Proliferation; Endothelin-1; Female; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leiomyoma; Myometrium; Proliferating Cell Nuclear Antigen; Signal Transduction; Vascular Endothelial Growth Factor A

To determine a role for endothelin (ET) in progression of uterine fibroids.. An in vitro model of fibroid and myometrium cultivation.. A total of 32 women undergoing hysterectomies for uterine fibroids and 11 women undergoing hysterectomies for abnormal uterine bleeding (control population).. Women with uterine fibroids were hypertensive and displayed significantly greater circulating ET-1 compared to control patients. Secretion of ET-1 was greater from the fibroids compared to myometrium explants. Endothelin 1 secretion was attenuated with blockade of the angiotensin II type 1 or endothelinA receptors. Hypoxia stimulated ET-1 secretion from both myometrium and fibroid explants. Preproendothelin messenger RNA expression increased with hypoxia from fibroid explants compared to normoxic controls.. These data support the hypothesis that uterine fibroids are associated with hypertension and increased ET-1, which is exacerbated with hypoxia. These data suggest a possible link between mechanisms of blood pressure regulation and development of uterine leiomyoma.

Topics: Adult; Biomarkers, Tumor; Endothelin-1; Female; Humans; Hypertension; Leiomyoma; Middle Aged; Myometrium; Random Allocation; Tumor Cells, Cultured; Uterine Neoplasms

We recently reported that in ELT3 uterine leiomyoma cells, but not in normal myometrial cells, endothelin (ET)-1 exerts a survival effect insensitive to MAPK3/1(ERK1/2) inhibition. In the present work, we investigated the potential role of MAPK14 (p38) in this ET-1-mediated effect. We demonstrated that, in ELT3, but not in normal myometrial cells, ET-1 activated MAPK14. Data based on pharmacological and siRNA approaches indicate that ETA and ETB receptors contributed to the activation of MAPK14 by ET-1 through a mechanism involving Gi protein, but not PI3-kinase. The inhibition of MAPK3/1 by U0126 did not affect the activation of MAPK14 by ET-1. Conversely, the inhibition of MAPK14 by SB203580 and the down-regulation of MAP2K3/MAP2K6 (kinases upstream of MAPK14) by specific siRNA did not alter the activation of MAPK3/1. These data indicate that MAPK14 was activated by ET-1 independently from MAPK3/1. Furthermore, ET-1 increased protein expression of prostaglandin synthase 2 (PTGS2 or COX2), prostaglandin E2 (PGE2) production, and subsequent ELT3 cell survival. The inhibition of PTGS2 induction and subsequent survival induced by ET-1 required the coinhibition of MAPK14 and MAPK3/1. Our findings provide evidence that ET-1 activated MAPK14 only in ELT3 cells, but not in normal myometrial cells. This MAPK14 activation was required, in addition to MAPK3/1 in ET-1-mediated survival through the COX2/prostaglandin axis, and may explain the absence of ET-1 antiapoptotic effect in normal myometrial cells. Our data reinforce the role of ET-1 and associated signaling pathways in leiomyoma pathology.

Topics: Animals; Cell Survival; Cells, Cultured; Cyclooxygenase 2; Endothelin-1; Enzyme Activation; Enzyme Induction; Female; Leiomyoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 14; Mitogen-Activated Protein Kinase 3; Models, Biological; Myometrium; Rats; Receptor, Endothelin A; Receptor, Endothelin B; Uterine Neoplasms

Uterine leiomyoma are the most common benign tumors of the myometrium. We previously identified endothelin (ET)-1 as a proliferative and antiapoptotic factor in Eker rat-derived leiomyoma (ELT3) cells. A major role of ETB receptor in the prosurvival effect was revealed. Here we investigated, in ELT3 and myometrial cells, the respective contribution of ETA and ETB in the proliferative effect of ET-1. In myometrial cells, binding experiments show that ETA is almost exclusively expressed and stimulates phospholipase C (PLC) activity and ERK1/2 phosphorylation and proliferation. In ELT3 cells, ETB is expressed at about the same level as ETA, and the two receptors are differently coupled to Gi protein. The ETB agonist, sarafotoxin S6c, stimulates PLC activity 60% less than ET-1 but is as potent as ET-1 to increase ERK1/2 phosphorylation and induce proliferation. However, the ability of ETA to activate ERK1/2 is observed after ETB desensitization. Although ETA and ETB antagonists partially reduce ET-1 stimulated PLC activity, they are without effect on ET-1-induced ERK1/2 phosphorylation and proliferation. Only the simultaneous use of ETA and ETB antagonists reduces ET-1-triggered ERK1/2 activation. These unconventional properties of ETRs may reveal the existence of functional ETA-ETB heterodimers. Finally, treatment of ELT3 cells with ETB but not ETA-directed small interfering RNA reduces the proliferative effect of ET-1. All the data obtained in ELT3 cells strengthen the relation between ETB overexpression, which decreases the ETA to ETB ratio, and the ability of leiomyoma cells to highly proliferate and resist apoptosis.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; DNA; Endothelin-1; Endothelin-3; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Iodine Radioisotopes; Leiomyoma; Myometrium; Rats; Rats, Long-Evans; Receptor, Endothelin A; Receptor, Endothelin B; Type C Phospholipases; Uterine Neoplasms

Uterine leiomyomas, or fibroids, are the most common tumors of the myometrium. The ELT3 cell line, derived from Eker rat leiomyoma, has been successfully used as a model for the study of leiomyomas. We have demonstrated previously the potent mitogenic properties of the peptidic hormone endothelin (ET)-1 in this cell line. Here we investigated the antiapoptotic effect of ET-1 in ELT3 cells. We found that 1) serum starvation of ELT3 cells induced an apoptotic process characterized by cytochrome c release from mitochondria, caspase-3/7 activation, nuclei condensation and DNA fragmentation; 2) ET-1 prevented the apoptotic process; and 3) this effect of ET-1 was fully reproduced by ETB agonists. In contrast, no antiapoptotic effect of ET-1 was observed in normal myometrial cells. A pharmacological approach showed that the effect of ET-1 on caspase-3/7 activation in ELT3 cells was not dependent on phosphatidylinositol 3-kinase, ERK1/2, or phospholipase D activities. However, inhibitors of sphingosine kinase-1 (SphK1), dimethylsphingosine and threo-dihydrosphingosine, reduced the effect of ET-1 by about 50%. Identical results were obtained when SphK1 expression was down-regulated in ELT3 cells transfected with SphK1 small interfering RNA. Furthermore, serum starvation induced a decrease in SphK1 activity that was prevented by ET-1 without affecting the level of SphK1 protein expression. Finally, sphingosine 1-phosphate, the product of SphK activity, was as efficient as ET-1 in inhibiting serum starvation-induced caspase-3/7 activation. Together, these results demonstrate that ET-1 possesses a potent antiapoptotic effect in ELT3 cells that involves sphingolipid metabolism through the activation of SphK1.

Topics: Animals; Apoptosis; Culture Media, Serum-Free; Cytoprotection; Endothelin-1; Female; Leiomyoma; Myometrium; Phosphotransferases (Alcohol Group Acceptor); Rats; Tumor Cells, Cultured; Uterine Neoplasms

Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth.

Topics: Animals; Butadienes; Cell Proliferation; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Leiomyoma; Male; Nitriles; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Rats; Rats, Wistar; Signal Transduction; Uterine Neoplasms

The protein kinase C (PKC) isoenzyme expression pattern in human uterine leiomyoma was compared with that obtained in homologous myometrium distal from the tumor. The six PKC isoforms (PKCalpha, PKCbeta1, PKCbeta2, PKCdelta, PKCepsilon and PKCzeta) evidenced in the myometrium were found to be similarly expressed in leiomyoma. Quantitative immunoblotting revealed that all PKC isoforms were preferentially localized in the particulate fraction. To gain insight into the possible functional consequences of PKC expression patterns, subcellular redistribution in response to the mitogenic peptide endothelin-1 (ET-1) was studied. After stimulation with ET-1, differential redistribution occurred in leiomyoma and myometrium, suggesting a selective role of PKC isoforms in the myometrial growth process.

Topics: Adult; Blotting, Western; Endothelin-1; Female; Humans; Immunoblotting; Isoenzymes; Leiomyoma; Middle Aged; Phorbol 12,13-Dibutyrate; Protein Kinase C; Subcellular Fractions; Uterine Neoplasms; Uterus

Factors responsible for the abnormal proliferation of myometrial cells that accompanies leiomyoma formation are unknown, although steroid hormones and peptide growth factors have been implicated. We hypothesized that endothelin-1 (ET-1) is a physiological regulator of tumor growth.. In this study, we investigated the role of ET-1 on growth of human leiomyoma cells and its synergistic effect with growth factors, as well as the signaling pathway involved in this interaction.. Leiomyoma cell proliferation was assayed by [H]thymidine incorporation and cell number. Protein kinase C (PKC) isoforms were analyzed by Western blot using specific antibodies.. ET-1 on its own was unable to stimulate DNA synthesis but potentiated the leiomyoma cell growth effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), IGF-I and IGF-II. The failure of a protein tyrosine kinase (PTK) inhibitor, tyrphostin 51, to affect the potentiating effect of ET-1, supports the hypothesis of non-involvement of PTK in this process. The inhibition of PKC by calphostin C or its down-regulation by phorbol 12,13-dibutyrate (PDB) eliminated the potentiating effect of ET-1, but did not block cell proliferation induced by the growth factors alone. Five PKC isoforms (alpha, beta1, epsilon, delta and zeta) were detected in leiomyoma cells, but only phorbol ester-sensitive PKC isoforms (PKCalpha, epsilon and delta) contribute to the potentiating effect of leiomyoma cell growth by ET-1.. We have demonstrated that ET-1 potentiates leiomyoma cell proliferation to growth factors through a PKC-dependent pathway. These findings suggest a possible involvement of ET-1 in the pathogenesis of leiomyomas.

Topics: Blotting, Western; Cell Division; DNA, Neoplasm; Endothelin-1; Enzyme Inhibitors; Female; Growth Substances; Humans; Leiomyoma; Protein Kinase C; Protein-Tyrosine Kinases; Signal Transduction; Tumor Cells, Cultured; Uterine Neoplasms

The distribution of mRNAs for endothelinA and B (ET(A) and ET(B)) receptors and their binding properties was studied in human nonpregnant and pregnant term myometrium and in uterine leiomyomas. ET(A)- and ET(B)-receptors functionally coupled to phospholipase C (PLC) coexisted in myometrial tissues, but only the functional ET(A)-receptor subtype was detected in leiomyomas. ET(A)-receptor mRNA and three other spliced variants were distributed in all tissue studied. We reported an increase in the proportion of ET(A)-receptors coupled to PLC in term pregnant myometrium when compared to nonpregnant tissue. These results suggest that upregulation of the myometrial ET(A)-receptors may account for or contribute to the control of normal development and growth of human myometrium during pregnancy. They also support a pathological role for the endothelin-1 (ET-1)/ET(A)-receptor system in leiomyoma development.

Topics: Azepines; Endothelin-1; Female; Humans; Indoles; Leiomyoma; Myometrium; Oligopeptides; Piperidines; Pregnancy; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; RNA, Messenger; Uterine Neoplasms

To determine if there are endothelin receptors on human uterine leiomyomas.. Samples of leiomyomas from eight patients were analyzed for [iodine (I)-125]endothelin-1 binding. Several subtype-selective ligands were used to determine the endothelin receptor population.. Binding of [125I]endothelin-1 to uterine leiomyoma membranes was specific and saturable, with a mean +/- dissociation constant 85.5 +/- 8.4 pM. Competition binding studies showed that the order of potency was endothelin-1 > endothelin-3, which was consistent with the presence of the endothelinA receptor subtype. Binding of [125I]endothelin-1 was displaced by an endothelinA-selective antagonist, but not by sarafotoxin 6c, an endothelinB-selective agonist. An endothelins-selective ligand was not specifically bound to leiomyoma.. These results indicate that only endothelinA receptors are present in human uterine leiomyomas. We speculate that endothelin-1 may act through these endothelinA receptors to influence the development or regulation of hypertrophy and proliferation of the human myometrium during pregnancy and in uterine disorders like leiomyomas.

Topics: Adult; Endothelin-1; Female; Humans; Leiomyoma; Middle Aged; Radioligand Assay; Receptor, Endothelin A; Receptors, Endothelin; Uterine Neoplasms; Uterus