The immunosuppressive role of Edn3 overexpression in the melanoma microenvironment.

Endothelins are cytokines expressed in the microenvironment of several tumors. To identify which stromal cells in the melanoma microenvironment respond to endothelin, we injected murine melanoma cell lines B16F10, YUMM1.7, and YUMMER1.7 in a transgenic mouse that overexpresses endothelin 3 (Edn3) under the control of the keratin 5 promoter in the skin (K5-Edn3). All cell lines developed larger tumors in K5-Edn3 mice than in control animals. In YUMM1.7 tumors, the Edn3 receptor, endothelin receptor B (Ednrb), was expressed in several stromal cell types including immune cells. This result was validated by the identification of Ednrb-positive stromal cells in human melanoma from previously published RNA-seq data. Regulatory T cells (Tregs) and dendritic cell numbers were significantly higher in K5-Edn3 tumors when compared to control tumors. Edn3 increased Treg proliferation in vitro and the expression of FOXP3. YUMM1.7-GFP tumors in K5-Edn3 mice were sensitive to immune checkpoint inhibitor (anti-CTLA-4) as well as to Ednrb blockage (BQ-788). Our results indicate that Ednrb signaling has an important role in the melanoma microenvironment where it mediates immunosuppression resulting in escape from tumor immunity.

Enterocolitis causes profound lymphoid depletion in endothelin receptor B- and endothelin 3-null mouse models of Hirschsprung-associated enterocolitis.

Potentially life-threatening enterocolitis is the most frequent complication in children with colonic aganglionosis (Hirschsprung disease, HSCR), and little is known about the mechanisms leading to enterocolitis. Splenic lymphopenia has been reported in the Endothelin Receptor B (Ednrb)-null mouse model of HSCR that develops enterocolitis. In this study, we sought to identify molecular mechanisms underlying this immune phenotype. We employed the Ednrb(-/-) mouse, and the knockout of its ligand, Edn3 (Edn3(-/-)). The major finding is that enterocolitis in the Ednrb(-/-) and Edn3(-/-) mice lead to thymic involution, splenic lymphopenia, and suppression of B lymphopoiesis as a consequence of colonic aganglionosis, not an intrinsic Edn3-Ednrb signaling defect directly affecting the lymphoid organs. We showed that adoptive transfer of Ednrb(-/-) marrow repopulated the RAG2-null mice marrow, thymus and spleen without development of enterocolitis. We identified the glucocorticoid corticosterone, as a potential mediator of the immune phenotype. This previously unrecognized pattern of immune abnormalities in mouse is nearly identical to lymphoid depletion in neonatal sepsis during severe physiological stress, suggesting that the mouse model used here could be also used for sepsis studies.

Endothelin is a potent inhibitor of matrix metalloproteinase-2 secretion and activation in rat mesangial cells.

We examined the effects of endothelin (ET) on the activity of matrix metalloproteinase-2 (MMP-2) in cultured MCs. Addition of the ET(A) receptor antagonists or neutralizing anti-endothelin antibody into MC cultures markedly augmented the secretion and activation of MMP-2. On the contrary, addition of the exogenous ET-1 into MC culture significantly inhibited the synthesis of MMP-2 in both basal and cytokines (tumor necrosis factor-alpha and interferon-gamma) plus lipopolysaccharide-stimulated conditions. Furthermore, pretreatment of cells with exogenous ET-1 obviously prevented cytochalasin D-elicited activation of MMP-2, an effect that was completely abolished by ET(A) receptor antagonist, FR139317. In addition, ET-1 was found to be able to suppress the expression of membrane type-1 MMP (MT1-MMP) and promote the conversion of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) from cell associated form to secreted form. The addition of recombinant TIMP-2 into the culture abrogated dose-dependently the cytochalasin D-elicited activation of MMP-2. These results suggest that ET is a potent inhibitor of MMP-2 secretion and activation in MCs. These novel findings may help us understand the subtle regulation of the synthesis and activation of MMP-2 in MCs. It also provides us with further insight into the pathophysiological mechanisms involving ET in the regulation of matrix turnover in glomerulus.

Endothelins are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in serum-free primary culture.

Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free melanoblast-defined medium (MDM). After 14 d, almost all keratinocytes that existed predominantly in the early stage of primary culture died, and pure cultures of melanoblasts were obtained. Epidermal melanoblasts dramatically increased in number in MDMDF consisting of MDM supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). Epidermal melanocytes increased in number in MDMD consisting of MDM supplemented with DBcAMP. On the other hand, epidermal melanocytes were induced to differentiate in MDMM consisting of MDM supplemented with alpha-melanocyte-stimulating hormone (MSH). Pure cultured primary melanoblasts or melanocytes in MDMDF or MDMD were further cultured with MDMDF or MDMD supplemented with endothelin (ET)-1, -2, or -3 from 14 d. A dramatic increase in the number of melanoblasts or melanocytes was observed after 7 d; however, no increase in the number of melanoblasts or melanocytes was observed in the absence of ET-1, -2, or -3. The increase in the number of melanoblasts or melanocytes was comparable with that of melanoblasts or melanocytes cocultured with secondary keratinocytes in MDMDF or MDMD. Also, pure cultured primary melanoblasts in MDM were further cultured with MDMM supplemented with ET-1, -2, or -3 from 14 d. A dramatic increase in the percentage of melanocytes in the melanoblast-melanocyte population was observed after 7 d; however, no increase in the percentage of melanocytes was observed in the absence of ET-1, -2, or -3. The increase was comparable with that of melanocytes cocultured with secondary keratinocytes in MDMM. Moreover, anti-ET-1, -2, and -3 antibodies inhibited both the proliferation of melanoblasts or melanocytes in MDMDF or MDMD and the differentiation of melanocytes in MDMM in primary culture. These results suggest that ET-1, -2, and -3 are one member of the keratinocyte-derived factors that are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in primary culture.

Placental endothelin gene expression and endothelin concentration in fetal fluids of the first trimester gestational sac.

We have investigated the distribution of immunoreactive endothelins (irET) in fetal fluids and expression of ET precursor genes in villous tissue during the first trimester. Samples of maternal plasma (n = 6), coelomic fluid (n = 28), amniotic fluid (n = 23) and villous tissue (n = 3) were obtained from 30 pregnancies immediately before surgical termination at 7-12 weeks gestation. irET concentration was measured in plasma and fluids using two different radioimmunoassay kits, i.e. RPA 545 and RPA 555 and high performance liquid chromatography (HPLC). Total RNA was extracted and purified from villous tissue, reverse transcription and polymerase chain reaction (RT-PCR) were performed to evaluate the expression of ET-related genes. The irET concentration as evaluated by both kits was significantly higher (P<0.005) in maternal plasma than in coelomic or amniotic fluid and significantly higher (P<0.005) in coelomic fluid than in amniotic fluid using the RPA 555 kit. The profile of ET obtained by the HPLC- radioimmunoassay (RPA 555 kit) method confirmed significantly (P<0.005) higher ET concentration in coelomic than in amniotic fluid, although a similar distribution pattern for the three ET was observed in both embryonic fliud cavities. ET-3 was the predominant isoform in both fluids, reaching 19.4+/-2.0 pg/ml and 6.3+/-1.6 pg/ml in coelomic and amniotic fluid, respectively. Coelomic or amniotic fluid irET concentration did not change with gestational age irrespective of the kit used. RT-PCR demonstrated that first trimester placenta expresses the genes encoding for prepro-ET-1, -ET-2 and -ET-3. The similar ET distribution pattern in both fluid cavities could reflect their origin from the villous tissue and suggests that ET may play a role in the development of placenta and other fetal organs during organogenesis.

Specific sandwich-type enzyme immunoassays for smooth muscle constricting novel 31-amino acid endothelins.

We established highly sensitive and specific sandwich-enzyme immunoassays (EIAs) for three newly discovered bioactive 31-amino acid endothelins [ETs(1-31)], which can detect as little as 0.16 pg/well of ET-1(1-31), 0.39 pg/well of ET-2(1-31), and 0.16 pg/well of ET-3(1-31). The EIAs showed no crossreactivity with 21-amino acid endothelins [ETs(1-21)] or big ETs at the usual assay concentrations below 1-5 ng/ml. In reversed-phase HPLC, immunoreactive ETs(1-31) in the granulocytes of normal human subjects eluted at the exact positions of authentic ETs(1-31), except for the presence of one additional unknown immunoreactive ET-1(1-31). The results also indicate that ETs(1-31) exist in the granulocytes at levels higher than or similar to those of ETs(1-21). This study is the first to establish EIAs for novel bioactive ETs(1-31). These assays can be utilized to assess the pathophysiological roles of ETs(1-31).

Distribution of endothelin 3-like immunoreactivity in gonadotrophs of the bullfrog (Rana catesbeiana) pituitary.

Immunohistochemical and immunocytochemical techniques were employed to investigate the distribution of endothelin 3 (ET3)-like immunoreactivity in the pituitary of the bullfrog, Rana catesbeiana. ET3-immunoreactive (ET3-IR) cells were scattered all over the pars distalis of the female pituitary; however, only a few ET3-IR cells were observed in the male pituitary. ET3-IR cells were found to correspond to cells immunostained with monoclonal antibodies against the beta-subunit of bullfrog LH (fLH beta) or monoclonal antibodies against the beta-subunit of bullfrog FSH (fFSH beta) at the light microscopic level. However, we could not find ET3-IR cells which were immunoreactive for other pituitary hormones. So far, all ET3-IR cells showed both fLH beta and fFSH beta immunoreactivity. About 24% of the fLH beta-IR cells and about 33% of the fFSH beta-IR cells showed ET3-like immunoreactivity. Immunoelectron microscopic analysis using colloidal gold revealed the coexistence of ET3-like substance(s) and gonadotropins within the same granules. This study demonstrated the presence of ET3-like peptide(s) in bullfrog gonadotrophs, suggesting the possible participation of ET3 in regulating pituitary function as an autocrine and/or paracrine hormone.