Epigenetic inactivation of endothelin-2 and endothelin-3 in colon cancer.

Endothelin-1 (ET-1) and its receptors are overexpressed in human cancers, but much less is known about the roles of ET-2 and ET-3 in cancer etiology. We sought to examine human and rat colon tumors for dysregulation of ET-2 and ET-3 expression and determine the underlying mechanisms. Human primary colon cancers and carcinogen-induced rat colon tumors were subjected to real-time RT-PCR, immunoblotting and immunohistochemistry; EDN2 and EDN3 genes were examined by methylation-specific PCR, bisulfite sequencing and pyrosequencing; and forced expression of ET-2 and ET-3 was conducted in human colon cancer cells followed by real-time cell migration and invasion assays. Rat and human colon tumors had markedly reduced expression of ET-2 and ET-3 mRNA and protein compared with matched controls. Mechanistic studies revealed hypermethylation of EDN2 and EDN3 genes in human primary colon cancers and in a panel of human colon cancer cell lines. Forced expression of ET-2 and ET-3 attenuated significantly the migration and invasion of human colon cancer cells. We conclude that epigenetic inactivation of ET-2 and ET-3 occurs frequently in both rat and human colon cancers. Current therapeutic strategies target overexpressed members of the ET axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the re-expression of ET-2 and ET-3 as natural antagonists of ET-1 in colon cancer.

Aeromonas hydrophila PepO outer membrane endopeptidase activates human big endothelin-3 in vitro and induces skin ulcer in goldfish (Carassius auratus).

In Aeromonas hydrophila, the gram-negative bacterial fish pathogen, PepO constitutes the thermoregulated outer membrane M13 family zinc endopeptidase, which is expressed maximally at 16 degrees C and is down-regulated above 30 degrees C. Cultivation of A. hydrophila at 16 degrees C enabled it to activate big endothelin (ET), the vasoconstrictor and ulcerogenic peptide naturally secreted from human vascular endothelial cell (HUVEC) culture. Furthermore, A. hydrophila PepO in vitro shows strong enzymatic preference for human big ET-3 rather than big ET-1 and big ET-2. At water temperature of 16+/-1 degrees C, intramuscular infection of goldfish, Carassius auratus, with wild-type A. hydrophila led to development of a pathognomonic big ulcer at the injection site while the PepO deficient mutant strain lost both its big ET endopeptidase activity in vitro as well as its ulcerogenic property in vivo. This is the first report of expression, subcellular localization and functional analysis of PepO metalloendopeptidase in A. hydrophila.

Frequent loss of endothelin-3 (EDN3) expression due to epigenetic inactivation in human breast cancer.

INTRODUCTION: Endothelin (EDN) signalling plays a crucial role in cell differentiation, proliferation and migration processes. There is compelling evidence that altered EDN signalling is involved in carcinogenesis by modulating cell survival and promoting invasiveness. To date, most reports have focused on the oncogenic potential of EDN1 and EDN2, both of which are overexpressed in various tumour entities. Here, we aimed at a first comprehensive analysis on EDN3 expression and its implication in human breast cancer. METHODS: EDN3 mRNA expression was assessed by Northern blotting in normal human tissues (n = 9) as well as in matched pairs of normal and tumourous tissues from breast specimens (n = 50). EDN3 mRNA expression in breast cancer was further validated by real-time polymerase chain reaction (PCR) (n = 77). A tissue microarray was used to study EDN3 protein expression in breast carcinoma (n = 150) and normal breast epithelium (n = 44). EDN3 promoter methylation was analysed by methylation-specific PCR in breast cell lines (n = 6) before and after demethylating treatment, normal breast tissues (n = 17) and primary breast carcinomas (n = 128). EDN3 expression and methylation data were statistically correlated with clinical patient characteristics and patient outcome. RESULTS: Loss of EDN3 mRNA expression in breast cancer, as initially detected by array-based expression profiling, could be confirmed by Northern blot analysis (> 2-fold loss in 96%) and real-time PCR (> 2-fold loss in 78%). Attenuated EDN3 expression in breast carcinoma was also evident at the protein level (45%) in association with adverse patient outcome in univariate (P = 0.022) and multivariate (hazard ratio 2.0; P = 0.025) analyses. Hypermethylation of the EDN3 promoter could be identified as the predominant mechanism leading to gene silencing. Reversion of the epigenetic lock by 5-aza-2'-deoxycytidine and trichostatin A resulted in EDN3 mRNA re-expression in vitro. Furthermore, EDN3 promoter hypermethylation was detected in 70% of primary breast carcinomas with significant association to loss of EDN3 mRNA expression (P = 0.005), whilst normal matched breast tissues revealed no EDN3 promoter methylation. CONCLUSIONS: EDN3 is a frequent target of epigenetic inactivation in human breast cancer, potentially contributing to imbalanced EDN signalling commonly found in this disease. The clinical implication supports the view that EDN3, in contrast to EDN1 and EDN2, may act as natural tumour suppressor in the human mammary gland.

Endothelin-3 is produced by metastatic melanoma cells and promotes melanoma cell survival.

BACKGROUND: Endothelin-3 (ET-3) is an essential paracrine factor for the proliferation, migration, and survival of embryonic melanocytes during fetal development. Its expression is tightly regulated, being completely turned off in adult skin. OBJECTIVE: In this study, results are presented that demonstrate abnormal expression of ET-3 by metastatic melanoma cells in both tissue biopsies and cell culture. Further, in vitro experiments showed that metastatic melanoma cells have the capacity to respond to ET-3 stimulation by increasing survival. CONCLUSION: Therefore, an abnormal autocrine stimulation pathway involving ET-3 is present in metastatic melanoma cells. Blocking this signal transduction pathway may prove useful for the treatment of metastatic melanoma.

Endothelin-3 production by human rhabdomyosarcoma: a possible new marker with a paracrine role.

Several autocrine and paracrine growth factor circuits have been found in human rhabdomyosarcoma cells. In this study we show that endothelin-3 (ET-3), a vasoactive peptide, is produced by human rhabdomyosarcoma cell lines, whereas it is not expressed by human sarcoma cell lines of non-muscle origin. We did not find evidence of a significant autocrine loop; nevertheless ET-3 produced by rhabdomyosarcoma cells can act as a paracrine factor, since it promotes migration of endothelial cells. Moreover ET-3 is present in plasma of mice bearing xenografts of human rhabdomyosarcoma cells, and may be potential new marker of the human rhabdomyosarcoma to be studied further.

Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and endothelial dysfunction.

BACKGROUND: Endothelin (ET)-1 is a potent vasoconstrictor that contributes to vascular remodeling in hypertension and other cardiovascular diseases. Endogenous ET-1 is produced predominantly by vascular endothelial cells. To directly test the role of endothelium-derived ET-1 in cardiovascular pathophysiology, we specifically targeted expression of the human preproET-1 gene to the endothelium by using the Tie-2 promoter in C57BL/6 mice. METHODS AND RESULTS: Ten-week-old male C57BL/6 transgenic (TG) and nontransgenic (wild type; WT) littermates were studied. TG mice exhibited 3-fold higher vascular tissue ET-1 mRNA and 7-fold higher ET-1 plasma levels than did WT mice but no significant elevation in blood pressure. Despite the absence of significant blood pressure elevation, TG mice exhibited marked hypertrophic remodeling and oxidant excess-dependent endothelial dysfunction of resistance vessels, altered ET-1 and ET-3 vascular responses, and significant increases in ET(B) expression compared with WT littermates. Moreover, TG mice generated significantly higher oxidative stress, possibly through increased activity and expression of vascular NAD(P)H oxidase than did their WT counterparts. CONCLUSIONS: In this new murine model of endothelium-restricted human preproET-1 overexpression, ET-1 caused structural remodeling and endothelial dysfunction of resistance vessels, consistent with a direct nonhemodynamic effect of ET-1 on the vasculature, at least in part through the activation of vascular NAD(P)H oxidase.

Induction of melanoma phenotypes in human skin by growth factors and ultraviolet B.

Exposure to UV radiation likely plays a key role in melanoma development, whereas other etiologic agents remain unknown. Here we show that in normal human skin an increased expression of a combination of three growth factors, basic fibroblast growth factor, stem cell factor, and endothelin-3, along with exposure to UVB can transform normal melanocytes into a melanoma phenotype within 4 weeks. Invasion of melanoma lesions was found in skin from newborn donors, whereas melanomas in adult skin were of a noninvasive in situ type only. This suggests that susceptibility of skin to exogenous tumor promoters is dependent on age. This is the first report on human cancer initiation in vivo in which an imbalance of physiological factors combined with an environmental carcinogen can lead to transformation of normal tissue.

T cell phenotype in allergic asthma and atopic dermatitis.

BACKGROUND: T cells are key regulators of immunologic disease parameters. However, their contribution to the process of tissue remodeling is ill defined. In the present study, we investigated gene expression of allergy-characteristic, IL-4-rich T cell cDNAs to monitor expression of genes that might participate in the pathogenesis of allergic diseases. METHODS: cDNAs of freshly isolated and restimulated CD4+ T cells from patients with allergic asthma (AA) or atopic dermatitis (AD) and healthy subjects were analyzed on Nylon membrane-based DNA arrays. Three patients were selected for an allergy-characteristic T cell phenotype with high IL-4 expression (AA) or IL-13 expression (AD). RESULTS: Several gene families such as the TGF-beta family, chemokines and chemokine receptors were found to be upregulated. Matrix metalloproteinases and their inhibitors were also found to be expressed in an enhanced manner. Furthermore, factors regulating tissue turnover such as fibroblast growth factors and neurotrophic as well as vasoactive factors were found be expressed at a higher level in allergic patient compared to healthy donors. CONCLUSION: The present study reveals and confirms genes relevant for allergy and highlights an approach to applying a DNA array technique for diagnostic discrimination of allergic diseases.

Endothelin-1 production by prostate cancer cell lines is up-regulated by factors involved in cancer progression and down-regulated by androgens.

BACKGROUND: Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone. METHODS: We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1). RESULTS: Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression. CONCLUSIONS: In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.

Diabetes-induced vascular dysfunction in the retina: role of endothelins.

AIMS/HYPOTHESIS: Vasoactive factors like endothelins, by virtue of the microvascular regulation as well as by other effects, possibly play important parts in the pathogenesis of diabetic retinal microangiopathy. We investigated retinal vascular dysfunction in streptozotocin-induced diabetes and its relation with endothelins in short- and long-term diabetes. METHODS: Diabetic rats with or without an endothelin receptor antagonist (bosentan) treatment were investigated after 1 month and 6 months of follow-up. Retinal blood flow was measured and compared with age- and sex-matched non-diabetic control animals. Retinal tissues were analysed for endothelin-1, endothelin-3, endothelin A and endothelin B mRNA. Distribution of endothelin-1 and endothelin-3 was investigated by immunocytochemistry and that for endothelin receptors by ligand binding and autoradiography. RESULTS: Diabetic animals showed hyperglycaemia, glycosuria, elevated glycated haemoglobin values and reduced body weight gain. Retinal blood flow showed an increased resistivity index, an indicator of vasoconstriction, after 1 month of diabetes which was prevented by treatment with bosentan. This functional change in diabetes was eliminated after 6 months of follow-up. The retina from the diabetic animals showed increased mRNA expression for endothelin-1, endothelin-3 and endothelin A after one month. In addition, endothelin B mRNA expression was increased after 6 months. Furthermore, endothelin-1 and endothelin-3 immunoreactivity and endothelin receptor concentrations were increased in the retina of diabetic rats. CONCLUSION/INTERPRETATION: The results from this study indicate that the endothelin system is of importance in mediating retinal changes in diabetes although mechanisms of the endothelin system alteration as well as their effects might vary depending on the duration of diabetes. [Diabetologia (1999) 42: 1228-1234]

Induction of endothelin-1 synthesis by IL-2 and its modulation of rat intestinal epithelial cell growth.

Endothelin (ET), a vasoconstrictive peptide, is known to have a variety of biological actions. Although ET is released by vascular endothelial cells, other cell populations also have been reported to synthesize and release ET. In this study, we examined whether ET is synthesized by intestinal epithelial cells and whether it affects induction of epithelial cell proliferation by interleukin-2 (IL-2). Subconfluent monolayers of intestinal epithelial cells (IEC-6 and IEC-18) were maintained in serum-free medium before addition of rat IL-2. Both IEC-6 and IEC-18 cells released ET-1 into the medium under unstimulated conditions, as determined by a sandwich ELISA. IL-2 significantly enhanced ET-1 release in a time-dependent manner. ET-3 was not detectable in the culture media of either cell line. Expression of ET-1 and ET-3 mRNA in epithelial cells was assessed by competitive PCR. Both cell lines were shown to express ET-1 mRNA, but no ET-3 mRNA was detected. IL-2 treatment enhanced ET-1 mRNA expression by both IEC-6 and IEC-18 cells. Both cell lines also expressed mRNA for ETA and ETB receptor subtypes. When cell proliferation was assessed, exogenous ET-1 induced a slight proliferative response in both types of cells that was consistent and significant at low ET-1 concentrations; cell growth was inhibited at a higher concentration (10(-7) M). IL-2 produced a significant proliferative response in both cell lines. However, the addition of ET-1 (10(-7) M) to culture media attenuated the IL-2-induced increase in cell proliferation. ETA-receptor antagonists significantly enhanced cellular proliferation, suggesting involvement of the ETA receptor in modulation of IL-2-induced intestinal epithelial cell growth.

Selective expression of endothelin 1 mRNA in rat retina.

Endothelin is a potent vasoactive agent and three isoforms--endothelin 1 (ET-1), endothelin 2 (ET-2) and endothelin 3 (ET-3)-- have been found in ocular tissues. However its source has not been determined. Therefore we have investigated the ET mRNA in the rat retina using reverse transcriptase with polymerase chain reaction. For ET-1, three retina samples were positive. For ET-2 and ET-3, all samples were negative. ET-1 mRNA was abundantly expressed compared with ET-2 and ET-3 mRNA (p < 0.05). Our study provides direct evidence that ET-1 is abundantly synthesized in the rat retina. The ET-1 within the retina could contribute to the regulation of the retinal circulation through vessel contraction.

ETA and ETB receptor antagonists synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures.

Astrocytes produce and bind endothelins (ETs), suggesting that these cells have ET autoregulatory and eliminatory functions. To further investigate these functions in primary rat astrocytes, ET-1 levels in the cell culture media (RIA/HPLC) and intracellular content of ET-1 mRNA (RT PCR) were measured under basal and stimulated (thrombin, 2.2 U/ml) conditions in the presence and absence of ETA and ETB selective antagonists (BQ123 or LU135252, and BQ788, respectively). Neither basal nor stimulated ET-1 levels in astrocyte media were influenced by ETA or ETB antagonists alone, but were significantly increased by a combination of both. ir ET-3 levels were not affected by antagonist treatment. Exogenous ET-1, added to the cultures, was rapidly cleared from the supernatant; this clearance was markedly inhibited by a combination of BQ123 and BQ788. ET-1 mRNA levels were not altered by any treatment. To conclude, in primary rat astrocyte cultures, extracellular ET-1 is cleared by binding to ET-receptors, apparently involving both, ETA and ETB sites. Thus, a blockade of the astrocytic ET eliminatory function as a consequence of the in vivo application of non-selective ET receptor antagonists may lead to increased extracellular ET levels in the brain.

Ontogeny of endothelins-1 and -3, their receptors, and endothelin converting enzyme-1 in the early human embryo.

The targeted gene inactivation of endothelins-1 and -3 (ET-1 and ET-3) and of one of their receptors, ETB, in the mouse causes severe defects in the embryonic development. These defects, cardiovascular and craniofacial malformations for ET-1, and colonic agangliogenesis associated with skin pigmentation anomalies for ET-3 and the ETB receptor, reproduce pathological phenotypes due to natural mutations of the same genes in the mouse and the human. The mutant phenotypes have been causatively linked to deficient migration/proliferation/differentiation of neural crest cells, i.e., neurocristopathies. To bring new insight about the exact roles of ETs in development and the involvement of neural crest cells in these processes, we have explored, by in situ hybridization, the ontogeny in the early human embryo of the ET system (ET-1 and ET-3, ETA and ETB receptors, ET converting enzyme-1). ET receptor mRNA expression in neural crest cells starts at 3 wk of gestation and continues during the entire period studied (up to 6 wk of gestation). During this period, ETA expression progressively spreads to undifferentiated mesodermal components of various structures and organs (head and axial skeleton, lateral and ventral subdermal mesoderm), whereas ETB expression remains more restricted to fewer differentiated cells (neural tube, sensory and sympathetic ganglia, endothelium). Some of these tissues and structures that express either one of the receptors do not appear to be of neural crest origin. In the digestive tract and the cardiovascular area, the present observations on the sources of ETs and their target cells in the young embryo provide the basis for a dynamic interpretation of the results of gene targeting of the mouse and the human phenotypes, and point to other possible roles of ETs in other ontogenetic processes. The results support the concept of local, rather than hormonal, interactions between the sources and targets of ETs during development.