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.

Evidence that DeltaNp73 promotes neuronal survival by p53-dependent and p53-independent mechanisms.

The p53 family member, p73, is essential for the survival of sympathetic neurons during the developmental period of naturally occurring neuronal death. Here, we have asked whether DeltaNp73, which is the only p73 isoform expressed in sympathetic neurons, mediates this survival by p53-dependent and/or p53-independent mechanisms. Initially, we used a genetic approach and crossed p53+/- and p73+/- mice. Quantitation of neurons in the sympathetic superior cervical ganglion during the period of naturally occurring cell death revealed that the loss of p53 partially rescued the death of neurons seen in p73-/- animals. Moreover, exogenous expression of DeltaNp73 in cultured p53-/- sympathetic neurons rescued these neurons from apoptosis after NGF withdrawal. Biochemical studies asking how DeltaNp73 inhibited NGF withdrawal-induced apoptosis in wild-type neurons demonstrated that it prevented the upregulation of the direct p53 targets p21 and Apaf-1 as well as cleavage of caspase-3. It also inhibited events at the mitochondrial apoptotic checkpoint, suppressing the induction of BimEL and the release of mitochondrial cytochrome c. Interestingly, DeltaNp73 expression also inhibited one very early event in the apoptotic cascade, the activation of c-Jun N-terminal protein kinase (JNK), likely by binding directly to JNK. Finally, we show that neuronal cell size is decreased in p73-/- mice, and that this decrease is not rescued by the lack of p53, suggesting a role for p73 in regulating cell size that does not involve interactions with p53. Thus, DeltaNp73 promotes neuronal survival via p53-dependent and -independent mechanisms, and it does so at multiple points, including some of the most proximal events that occur after NGF withdrawal.

Signaling mechanisms underlying reversible, activity-dependent dendrite formation.

Neuronal activity and neurotrophins play a central role in the formation, maintenance, and plasticity of dendritic arbors. Here, we show that neuronal activity, mediated by electrical stimulation, KCl depolarization, or cholinergic receptor activation, promotes reversible dendrite formation in sympathetic neurons and that this effect is enhanced by NGF. Activity-dependent dendrite formation is accompanied by increased association of HMW MAP2 with microtubules and increased microtubule stability. Inhibition of either CaMKII or the MEK-ERK pathway, both of which phosphorylate MAP2, inhibits dendrite formation, but inhibition of both pathways simultaneously is required for dendrites to retract. These data indicate that neuronal activity signals via CamKII and the ERKs to regulate MAP2:microtubule interactions and hence reversible dendrite stability, and to provide a mechanism whereby activity and neurotrophins converge intracellularly to dynamically regulate dendritic morphology.