endothelin-1 and Astrocytoma

Endothelin-1 (ET-1) is a potent mitogen for many cells, especially when its levels are elevated under pathological conditions, as seen in tumor cell progression and astroglial activation in neuropathies. While ET-1 is known to cause astroglial proliferation, in the present study, multiple signaling pathways involved in ET-1-mediated astrocyte proliferation were characterized. Treatment with PD98059 and U0126 (MEK inhibitors) inhibited not only ET-1-induced cell proliferation but also ET-1-activated phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in U373MG astrocytoma cells. Whereas the nonselective protein kinase C (PKC) inhibitor chelerythrine attenuated ET-1-induced cell proliferation, it was unable to block ET-1-induced ERK phosphorylation. However, ET-1 did not activate conventional or novel PKCs and did not elevate intracellular calcium. In addition, U73122 (a selective phospholipase C inhibitor), FTI-277 (an H-Ras inhibitor), as well as protein tyrosine kinase inhibitors also did not abolish ET-1-induced ERK1/2 phosphorylation. ET-1 treatment increased the activity of total Ras but not H-Ras. The phosphoinositide 3-kinase (PI3K) pathway appeared to be involved in signal transduction induced by ET-1, but it did not appear to participate in cross talk with the mitogen-activated protein kinase (MAPK) pathway. Activated ET receptors did not propagate signals either through protein tyrosine kinases or transactivation of EGF receptor tyrosine kinases, which typically trigger Ras-Raf-MAPK pathways. The results indicate that ET-1 stimulates cell proliferation by the activation of MAPK-, PKC-, and PI3K-dependent pathways that appear to function in a parallel manner. There is no apparent, direct "cross talk" between these pathways in U373MG cells, but rather, they might act on the independent but necessary components of the mitogenic effects of ET-1.

Topics: Astrocytoma; Calcium Signaling; Cell Line, Tumor; Cell Proliferation; Endothelin B Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Estrenes; Extracellular Signal-Regulated MAP Kinases; Humans; Inositol 1,4,5-Trisphosphate; Isoenzymes; Methionine; Models, Biological; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Pyrrolidinones; Receptor Protein-Tyrosine Kinases; Receptor, Endothelin B; Signal Transduction

Endothelin-1 (ET-1), a vasoactive and mitogenic peptide mainly produced by vascular endothelial cells, may be involved in the progression of several human tumors. Here, we present an immunohistochemical analysis of the expression pattern of ET-1 receptor subtypes (ET(A)-R and ET(B)-R) and a functional study of their potential role in human oligodendrogliomas and oligoastrocytomas. By comparison, we assessed the corresponding expression patterns of glioblastomas. Interestingly, a nuclear localization of ET-1 receptor subtypes (associated or not with a cytoplasmic labeling) was constantly observed in tumor cells from all three glioma types. Moreover, we noted a distinct receptor distribution in the different gliomas: a nuclear expression of ET(B)-R by tumor cells was found to be restricted to oligodendrogliomas and oligoastrocytomas, while a nuclear expression of ET(A)-R was only detected in tumor cells from some glioblastomas. Using primary cultures of oligodendroglial tumor cells, we confirmed the selective expression of nuclear ET(B)-R, together with a plasma membrane expression, and further demonstrated that this receptor was functionally coupled to intracellular signaling pathways known to be involved in cell survival and/or proliferation: extracellular signal-regulated kinase and focal adhesion kinase activation, actin cytoskeleton reorganization. In addition, impairment of ET(B)-R activation in these cells by in vitro treatment with an ET(B)-R-specific antagonist induced cell death. These data point to ET-1 as a possible survival factor for oligodendrogliomas via ET(B)-R activation and suggest that ET(B)-R-specific antagonists might constitute a potential therapeutic alternative for oligodendrogliomas.

Topics: Actin Cytoskeleton; Antihypertensive Agents; Astrocytoma; Brain Neoplasms; Cell Membrane; Cell Nucleus; Cell Survival; Cytoplasm; Endothelin B Receptor Antagonists; Endothelin-1; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glioblastoma; Humans; Immunohistochemistry; Oligodendroglioma; Oligopeptides; Piperidines; Protein-Tyrosine Kinases; Receptor, Endothelin A; Receptor, Endothelin B; Tumor Cells, Cultured

Endothelin-1 (ET-1), a vasoconstrictor and mitogen, has recently been implicated in the pathogenesis of human glioblastoma, neuroblastoma, and meningioma. ET-1, formed by proteolysis of the propeptide big ET-1 by endothelin-converting enzyme-1 (ECE-1), mediates its cellular actions through ETA and ETB receptors. Because only immunoreactive ET-1 has been observed within human astrocytic tumor cells, the authors investigated the localization of the entire ET-1 system (ET-1 mRNA, ET-1, ECE-1, ETA and ETB receptors) in surgical samples of human diffuse astrocytomas WHO Grade II (n = 6).. ET-1 mRNA expression was elucidated by in situ reverse transcriptase polymerase chain reaction (RT-PCR) using synthetic primers. Polyclonal antibodies were used to localize ET-1, ECE-1, ETA and ETB receptors by immunocytochemistry.. All ET components were detected in the six tumor samples. Intense (3+) cytoplasmic ET-1 mRNA labeling was observed in more than 75% of cells in all 6 astrocytomas. Up to 75% of tumor cells displayed intense ET-1 and ECE-1 immunolabeling distributed throughout their cytoplasm. Immunoreactive ETA and ETB receptors, observed in 25% to 75% of astrocytic tumor cells, were of moderate intensity. In addition, all components of the ET system were seen within endothelial cells of tumor blood vessels.. The presence of ET-1 mRNA, ECE-1, and ET-1 within tumor astrocytes suggests local ET synthesis and processing. The mitogenic and antiapoptotic properties of ET-1, as well as the vasodilatory signaling of ETB receptors, may promote tumorigenesis.

Topics: Adult; Aspartic Acid Endopeptidases; Astrocytoma; Endothelin-1; Endothelin-Converting Enzymes; Female; Humans; Immunohistochemistry; Male; Metalloendopeptidases; Receptor, Endothelin A; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

It is demonstrated that endothelin-1 (ET-1) is synthesized by and released from certain malignant tumor cells, and it plays an important role in growth and development of tumor. This study was designed to investigate the expression of ET-1 in astrocytomas and the relationship between the ET-1 quantity and the grades of astrocytomas.. ET-1 expression was determined in 70 astrocytoma specimens using Streptavidin-Peroxidase method and image analysis technology.. The ET-1 expressed in all of astrocytomas, and the ET-1 expression was mainly located in the cytoplasm. The positive rates of ET-1 in grade IV and III astrocytomas (86.67% and 93.33%) were significantly higher than that in grade II, I and normal astrocytes(75.00%, 66.67%, and 37.50%) (P < 0.05). The results of image analysis on astrocytoma (grade IV, III, II, I and normal control: 0.1875 +/- 0.0227, 0.1516 +/- 0.0134, 0.1215 +/- 0.0116, 0.1048 +/- 0.0143, and 0.0717 +/- 0.0074, respectively) showed that the lower differentiation of astrocytoma, the higher ET-1 expression (P < 0.01). The expression of ET-1 was significantly correleted with the tumor grading (r = 0.863, P < 0.01).. The ET-1 quantitative analysis may be used as a monitoring index of astrocytoma growing.

Topics: Adolescent; Adult; Aged; Astrocytoma; Central Nervous System Neoplasms; Endothelin-1; Female; Humans; Immunohistochemistry; Male; Middle Aged

Endothelins (ETs) are a family of potent vasoconstrictor and comitogenic polypeptides consisting of 21-amino acids. Using in situ hybridization, ET-1 mRNA has previously been localized to neuronal cell bodies in fourteen human brain regions. However, because in situ hybridization has a limited detection sensitivity of 20 mRNA copies per cell, ET-1 mRNA may be present in previously undetected areas. Hence, our objective was to localize ET-1 mRNA in specific human brain regions and astrocytic tumours using the more sensitive in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR). Human brain autopsy tissue and surgical cerebral tumour tissue were treated with proteinase K and DNase, followed by RT-PCR using primers specific for ET-1 mRNA and digoxygenin-labelled dUTP in the PCR mixture. The DIG-dUTP was localized with an immunodetection system. We demonstrate ET-1 mRNA labelling in twenty two of the twenty four brain regions studied including those regions in which ET-1 mRNA has been observed by in situ hybridization. In addition, the localization of ET-1 mRNA observed in astrocytomas suggests a role for ET-1 in tumour pathogenesis. In situ RT-PCR has proven to be highly sensitive in its ability to detect low mRNA expression at the cellular level. Our results confirm a role for ET-1 in the human nervous system.

Topics: Astrocytoma; Brain Chemistry; Brain Neoplasms; Endothelin-1; Humans; In Vitro Techniques; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The role of nonproductive infection of astrocytes by human immunodeficiency virus type 1 (HIV-1), characterized by the overexpression of nef, in brain disease progression is largely unknown. We investigated the consequences of stable expression of nef from the HIV-1 strain LAI in the human astrocytic cell line U373. DNA synthesis induced by endothelin-1 (ET-1) was largely decreased by nef. Stable expression of nef did not affect the ET-1-induced tyrosine phosphorylation of focal adhesion kinase, an adhesion-dependent pathway known to participate in DNA synthesis in astrocytes. Conversely, the activation of extracellular signal-regulated kinase (ERK) by ET-1 was largely inhibited in cells stably or transiently expressing nef. A similar inhibitory action of nef on ERK activation was observed after direct stimulation of G proteins. Furthermore, the inhibitory action of nef did not require protein kinase C (PKC) and affected mainly the PKC-independent pathway of ERK activation. Following chemokine receptor CXCR4-mediated infection of U373 cells stably expressing CXCR4 with the T-tropic HIV-1 strain m7-NDK, ET-1-induced activation of ERK was also inhibited. Altogether, these results indicate that intracellular signaling pathways associated with the growth factor activity of ET-1 are impaired in nef-expressing and HIV-1-infected astrocytes, suggesting that infection of astrocytes may play a significant role in the neuropathogenesis of HIV-1 encephalopathy.

Topics: Astrocytes; Astrocytoma; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion Molecules; Cell Line; DNA Replication; Endothelin-1; Enzyme Activation; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Products, nef; HIV-1; Humans; Mitogen-Activated Protein Kinase 1; nef Gene Products, Human Immunodeficiency Virus; Protein-Tyrosine Kinases; Receptors, CCR4; Receptors, Chemokine; Recombinant Proteins; Transfection