ß-arrestin-1 is a nuclear transcriptional regulator of endothelin-1-induced ß-catenin signaling.

Despite the fundamental pathophysiological importance of ß-catenin in tumor progression, the mechanism underlying its final transcriptional output has been partially elucidated. Here, we report that ß-arrestin-1 (ß-arr1) is an epigenetic regulator of endothelin (ET)-1-induced ß-catenin signaling in epithelial ovarian cancer (EOC). In response to ET A receptor (ETAR) activation by ET-1, ß-arr1 increases its nuclear translocation and direct binding to ß-catenin. This in turn enhanced ß-catenin nuclear accumulation and transcriptional activity, which was prevented by expressing a mutant ß-arr1 incapable of nuclear distribution. ß-arr1-ß-catenin interaction controls ß-catenin target gene expressions, such as ET-1, Axin 2, Matrix metalloproteinase 2, and Cyclin D1, by promoting histone deacetylase 1 (HDAC1) dissociation and the recruitment of p300 acetyltransferase on these promoter genes, resulting in enhanced H3 and H4 histone acetylation, and gene transcription, required for cell migration, invasion and epithelial-to-mesenchymal transition. These effects are abrogated by ß-arr1 silencing or by mutant ß-arr1, as well as by ß-catenin or p300 silencing, confirming that nuclear ß-arr1 forms a functional complex capable of regulating epigenetic changes in ß-catenin-driven invasive behavior. In a murine orthotopic model of metastatic human EOC, silencing of ß-arr1 or mutant ß-arr1 expression, as well as ETAR blockade, inhibits metastasis. In human EOC tissues, ß-arr1-ß-catenin nuclear complexes are selectively enriched at ß-catenin target gene promoters, correlating with tumor grade, confirming a direct in vivo ß-arr1-ß-catenin association at specific set of genes involved in EOC progression. Collectively, our study provides insights into how a ß-arr1-mediated epigenetic mechanism controls ß-catenin activity, unraveling new components required for its nuclear function in promoting metastasis.

Endothelin-1 induces tumor proteinase activation and invasiveness of ovarian carcinoma cells.

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinoma. In these cells, ET-1 acts as an autocrine mitogenic and angiogenic factor selectively through the ET(A) receptor (ET(A)R). We investigated at mRNA and protein levels whether ET-1 could affect the expression and activation of metastasis-related proteinases and whether this process was associated with ovarian tumor cell invasion. ELISA, gelatin zymography, Western blot, and reverse transcription-PCR analyses demonstrated that in two ovarian carcinoma cell lines (HEY and OVCA 433), the expression of matrix metalloproteinase (MMP) -2, -9, -3, -7, and -13 was up-regulated and activated by ET-1. Moreover we observed that ET-1 was able to enhance the secretion and activation of membrane-type metalloproteinase-1, a critical mediator of invasiveness. The secretion of tissue inhibitor of metalloproteinase-1 and -2 was decreased by ET-1, which increased the net MMP/tissue inhibitor of metalloproteinase balance and the gelatinolytic capacity. In addition, ET-1 induced overexpression of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 and -2. Finally, we demonstrated that, in HEY and OVCA 433 cells, ET-1 dose-dependently increased migration and MMP-dependent invasion through Matrigel. BQ123, an antagonist of the ET(A)R, inhibited the ET-1-induced tumor protease activity and subsequent increase in cell migration and invasion. These findings demonstrate that ET-1 promotes ovarian carcinoma cell invasion, acting through the ET(A)R by up-regulating secretion and activation of multiple tumor proteinases. Therefore, ET-1 may represent a key component of more aggressive ligand-induced invasiveness of ovarian carcinoma.

Endothelin receptor blockade inhibits proliferation of Kaposi's sarcoma cells.

Endothelin-1 (ET-1) has been shown to be mitogenic for endothelial and several tumor cells through an autocrine mechanism. In this study we evaluated whether the tumorigenic KS IMM cell line deriving from Kaposi's sarcoma (KS), a highly angiogenic tumor, is susceptible to ET-1 mitogenic activity. By reverse transcriptase-polymerase chain reaction, we detected ET-1 mRNA expression and both ET(A) receptor (ET(A)R) and ET(B)R mRNA transcripts in the KS IMM cells. High concentrations of ET-1 are released from the KS IMM cells and competition-binding studies demonstrated that these cells also express functional ET(A)R and ET(B)R with high affinity for ET-1 and ET-1/ET-3, respectively. Expression of ET-1 and cognate receptors could be detected by immunohistochemical method in vitro, in KS IMM xenograft, and in tissue sections of a human KS lesion. Furthermore ET-1 induces a marked and dose-dependent increase in [3H]thymidine incorporation comparable to that elicited by vascular endothelial growth factor. Addition of both selective ET(B)R antagonist (BQ 788) and ET(A)R antagonist (BQ 123), completely blocked ET-1-induced mitogenic response and reduced the basal growth rate of unstimulated cells, suggesting that both receptors mediated the proliferative signal. Such findings demonstrate that ET-1 participates on KS pathogenesis acting as an autocrine growth factor and that ET-1 receptor antagonists may thus be novel candidates for therapeutic intervention.

Endothelin-1 induces an angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.

The endothelial cell-derived endothelin-1 (ET-1) is a potent mitogen for endothelial cells, vascular smooth muscle cells, and tumor cells. In this study, we analyzed the role of ET-1 on human umbilical vein endothelial cell (HUVEC) phenotype related to different stages of angiogenesis. ET-1 promoted HUVEC proliferation, migration, and invasion in a dose-dependent manner. The ET(B) receptor (ET(B)R) antagonist, BQ 788, blocked the angiogenic effects induced by ET-1, whereas the ET(A)R antagonist was less effective. ET-1 stimulated matrix metalloproteinase-2 mRNA expression and metalloproteinase-2 production, as determined by reverse transcriptase-polymerase chain reaction and gelatin zymography. Furthermore ET-1 was able to enhance HUVEC differentiation into cord vascular-like structures on Matrigel. When tested in combination with vascular endothelial growth factor (VEGF), ET-1 enhanced VEGF-induced angiogenic-related effects on endothelial cells in vitro. Finally, using the Matrigel plug neovascularization assay in vivo, ET-1 in combination with VEGF stimulated an angiogenic response comparable to that elicited by basic fibroblast growth factor. These findings demonstrated that ET-1 induces angiogenic responses in cultured endothelial cells through ET(B)R and that stimulates neovascularization in vivo in concert with VEGF. ET-1 and its receptors acting as angiogenic regulators might represent new targets for anti-angiogenic therapy.

Role of endothelin-1 in neovascularization of ovarian carcinoma.

Endothelin-1 (ET-1) is overexpressed in ovarian carcinomas and acts, via ET(A) receptors (ET(A)R), as an autocrine growth factor. In this study we investigate the role of ET-1 in the neovascularization of ovarian carcinoma. Archival specimens of primary (n = 40) and metastatic (n = 8) ovarian tumors were examined by immunohistochemistry for angiogenic factor and receptor expression and for microvessel density using antibodies against CD31, ET-1, vascular endothelial growth factor (VEGF), and their receptors. ET-1 expression correlated with neovascularization and with VEGF expression. The localization of functional ET(A)R and ET(A)R mRNA expression, as detected by autoradiography and in situ hybridization, was evident in tumors and in intratumoral vessels, whereas ET(B)R were expressed mainly in endothelial cells. High levels of ET-1 were detected in the majority of ascitic fluids of patients with ovarian carcinoma and significantly correlated with VEGF ascitic concentration. Furthermore ET-1, through ET(A)R, stimulated VEGF production in an ovarian carcinoma cell line, OVCA 433, by an extent comparable to hypoxia. Finally, conditioned media from OVCA 433 as well as ascitic fluids caused an increase in endothelial cell migration and the ET-1 receptor blockade significantly inhibited this angiogenic response. These findings indicate that ET-1 could modulate tumor angiogenesis, acting directly and in part through VEGF.

Expression of endothelin 1 and endothelin A receptor in ovarian carcinoma: evidence for an autocrine role in tumor growth.

In the present study, we have investigated the expression of endothelin 1 (ET-1) and the ET(A) receptor (ET(A)R) and ET(B) receptor (ET(B)R) in primary (n = 30) and metastatic (n = 8) ovarian carcinomas and their involvement in tumor growth. By reverse transcription-PCR and Northern blot analysis, we detected ET-1 mRNA in 90% of primary and 100% of metastatic ovarian carcinomas. ET-1 mRNA expression was significantly higher in tumors than in normal ovarian tissues (n = 12; P < 0.01). ET(A)R mRNA was also detected in 84% of the carcinomas examined, whereas ET(B)R mRNA was expressed in 50% of the tumors. The in vivo presence of mature ET-1 and ET(A)R was confirmed by immunohistochemistry, demonstrating a higher expression in primary and metastatic cells. Ten primary cultures of ovarian tumors secreted ET-1 and were positive for ET-1 and ET(A)R mRNA, whereas only 40% expressed ET(B)R mRNA. Radioligand binding studies showed that ET-1-producing cells also expressed functional ET(A)R, whereas no specific ET(B)R could be demonstrated. ET-1 stimulated dose-dependent [3H]thymidine incorporation and enhanced the mitogenic effect of epidermal growth factor. The ET(A)R-selective antagonist BQ 123 strongly inhibited ET-1-stimulated growth and substantially reduced the basal growth rate of unstimulated cells, whereas the ET(B)R-selective antagonist BQ 788 had no effect. In conclusion, the present data demonstrate a novel mechanism in the growth control of ovarian carcinoma in vivo mediated by the ET-1 autocrine loop that selectively occurs via the ET(A)R.

The autonomous growth of human papillomavirus type 16-immortalized keratinocytes is related to the endothelin-1 autocrine loop.

Some human papillomaviruses (HPVs) such as HPV type 16 (HPV16) and HPV18 are involved in cervical carcinoma, and they can immortalize and transform keratinocytes. Endothelin-1 (ET-1) is produced in keratinocytes and has been shown to act through ETA receptors as an autocrine growth factor for keratinocytes. This study examines whether HPV16 alters the ET-1-mediated autocrine loop in human keratinocytes, providing a selective growth advantage for transformed cells. ET-1 is released in similar amounts from normal and HPV-transfected keratinocytes. All HPV-transfected cell lines express high-affinity ETA receptors. A two-fold increase in ET-1 binding sites is present in HPV16-immortalized keratinocytes, and this effect seems to be linked to the overexpression of mRNA for this receptor rather than to differences in the surface/internalized ratio of the receptors. ET-1 induces significant increases in [3H]thymidine incorporation and cell proliferation. Furthermore, HPV-transfected keratinocytes can proliferate in the absence of any growth factor added to the growth medium, and the ETA receptor antagonist BQ123 prevents this proliferation. These data suggest a new mechanism in the growth control of HPV-transformed cells mediated by the upregulation of ET-1 autocrine loop.

Activation of mitogenic signaling by endothelin 1 in ovarian carcinoma cells.

Endothelin 1 (ET-1) is produced in ovarian cancer cell lines and has been shown to act through ET(A) receptors as an autocrine growth factor to promote tumor cell proliferation in vitro. In OVCA 433 cells, the efficacy of ET-1 as a stimulus of [3H]thymidine incorporation was equivalent to that of epidermal growth factor. ET-1 also stimulated the rapid expression of c-fos, an action mediated by ET(A) receptors. The mitogenic action of ET-1 was not mediated by a pertussis toxin-sensitive G protein. An analysis of the effects of inhibition and depletion of protein kinase C (PKC) on mitogenic responses demonstrated that PKC was necessary but not sufficient for maximal stimulation by ET-1. In quiescent OVCA 433 cells, ET-1-induced stimulation of [3H]thymidine incorporation was prevented by two structurally distinct inhibitors of tyrosine kinase, herbimycin A and genistein. These results indicate that both PKC and protein tyrosine kinase participate in ET-1-stimulated mitogenic signaling. ET-1 rapidly stimulated tyrosine phosphorylation of several cellular proteins, among which p125FAK and p42 mitogen-activated protein kinase were identified. The additivity between the potent mitogenic actions of ET-1 and epidermal growth factor is consistent with the independence of their signal transduction pathways in ovarian cancer cells. These findings also indicate that intracellular signaling between the ET(A) receptor and a yet unidentified tyrosine kinase is involved in the mitogenic response to ET-1.

Autocrine actions of endothelin-1 as a growth factor in human ovarian carcinoma cells.

The production of endothelin 1 (ET-1) and its receptor-mediated actions on calcium signaling and growth responses were analyzed in human ovarian carcinoma cells. Immuno-reactive ET-1 was released from three of four ovarian tumor cell lines as a function of time in amounts ranging from 56 to 74 fmol/10(6) cells. Reverse-phase HPLC and radioimmuno-assay of conditioned media from tumor cells revealed a single peak coeluting with authentic ET-1. Radioligand binding studies showed that the ET-1-producing cell lines also expressed high-affinity ETA receptors (Kd < 0.1 nM) that ranged in abundance from 2,600 to 43,600 sites/cell. In fura-2-loaded ovarian carcinoma cells, ET-1 induced dose-dependent increases in cytoplasmic Ca2+ concentration. ET-1 also stimulated thymidine incorporation in the three cell lines that expressed ET receptors. In OVCA 433 cells, BQ 123 inhibited the stimulatory actions of ET-1 on thymidine incorporation and cell proliferation, and substantially reduced the basal growth rate of unstimulated ovarian tumor cells. These results demonstrate that ET-1 is produced in ovarian cancer cells and acts as an autocrine growth factor on ETA receptors to stimulate calcium signaling and proliferative responses. Such findings suggest that ET-1 participates in the progression of neoplastic growth in certain ovarian tumors.

Identification of the ETA receptor subtype that mediates endothelin induced autocrine proliferation of normal human keratinocytes.

Endothelin-1 has a wide range of pharmacological effects in various tissues and acts as autocrine/paracrine factor. The potential of ET-1 to function as an autocrine growth factor was evaluated in normal human keratinocytes. Radioligand binding studies showed that 125I-ET-1 bound to a single class of high-affinity-binding sites on the surface of the cells. The dissociation constant was 0.045 nM with receptor numbers of 1700 sites/cell. Treatment with serum caused increases in expression of binding sites (3500 sites/cell), with no change in binding affinity. ET-1 stimulated thymidine incorporation in these cells that expressed ET receptors. An ET antagonist selective for the ETA receptor subtype (BQ 123) inhibited DNA synthesis stimulated by ET-1 and reduced the basal growth rate of unstimulated cells. These data suggest that the ET-1 induced DNA synthesis is mediated by ETA receptor subtype and that endogenously produced ET-1 promotes the autocrine proliferation of keratinocytes.