Expression of endothelins and their receptors in glioblastoma cell lines.
The endothelins (ETs) are a family of three peptides named ET-1, ET-2 and ET-3 that have been initially isolated as potent vasoactive peptides; ETs are synthesized as precursor proteins (preproETs) and are activated by proteolytic cleavage. ETs exert their biological effects through the activation of two receptors subtypes, ETA and ETB. Recent studies have shown that, besides its vascular effects, ET-1 appears to play a major role also in the growth and progression of various types of cancers and ETA or ETB are alternatively indicated as mediators of the ET-1 biological effects. In this study we have investigated the expression and the amounts of preproET-1, preproET-2, ETA and ETB receptors mRNA by classical RT-PCR and quantitative real-time PCR in one human low grade astrocytoma cell line and eight human glioblastoma cell lines. PCR products corresponding to ETB receptor and preproET-1 were detectable in all the cell lines whilst ETA receptor and preproET-2 were only detected in five cell lines. Quantitative real-time PCR experiments showed wide differences in the amounts of mRNAs among the cell lines examined. Range values were 0.23-4860.51 fg/mug total cDNA for preproET-1; 0.13-3330.18 fg/mug total cDNA for preproET-2; 0.63-286.12 fg/mug total cDNA for ETA and 14.40-6720.67 fg/mug total cDNA for ETB. We measured the ET-1 released in the extracellular medium by an ELISA assay and we found an excellent correlation (correlation coefficient r = 0.9526, P = 0.0003) between the amounts of preproET-1 mRNA and released ET-1 peptide. Finally, in the 1321N1 cell line ETB receptors are functionally coupled to intracellular signaling pathways because the stimulation of ETB receptors by ET-1 induces the phosphorylation of the extracellular regulated kinases (ERKs). Although the majority of glioblastoma cell lines in culture express ET isoforms and ET receptors, we conclude that ET-1 and the ETB receptors are likely to mediate the effects of the ET system in glioblastoma cell lines.
Dipartimento di Farmacologia Sperimentale ed Applicata, Università di Pavia, Viale Taramelli 14, 27100, Pavia, Italy.
Mayra
M
Paolillo
Annalisa
A
Barbieri
Patrizia
P
Zanassi
Sergio
S
Schinelli
D015153:Blotting, Western; D045744:Cell Line, Tumor; D016232:Endothelins / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D017466:Receptors, Endothelin / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016232:Endothelins; D012333:RNA, Messenger; D017466:Receptors, Endothelin; D048049:Extracellular Signal-Regulated MAP Kinases