Therapeutic targeting of g-protein coupled receptor-mediated epidermal growth factor receptor transactivation in human glioma brain tumors.

The epidermal growth factor receptor (EGFR) is the main tyrosine kinase receptor dysregulated or overexpressed in brain cancer types and its expression is directly correlated with tumor malignancy and unfavorable prognosis. Recently, the availability of endogenous EGFR ligands has been reported to be also regulated indirectly by the activation of several G-protein-coupled receptors (GPCRs) in many cancer cell types. This EGFR transactivation mechanism requires the initial activation of a GPCR that in turn induces the cleavage of membrane-bound EGFR ligands precursors via the involvement of the family of disintegrin and metalloproteases (ADAMs). The discovery of ADAMs in this transactivation mechanism led to the development of small molecule inhibitors. In this minireview we describe the expression of GPCR, ADAMs and EGFR ligands in human glioma brain tumors and the characteristics of small molecule ADAMs inhibitors. The addition of ADAM inhibitors to our pharmacological arsenal could enhance the outcome of combination therapies when using EGFR inhibitors against human brain tumors.

Stimulation of endothelin B receptors in astrocytes induces cAMP response element-binding protein phosphorylation and c-fos expression via multiple mitogen-activated protein kinase signaling pathways.

The vasoconstrictor peptide endothelin (ET-1) exerts its physiological and pathological effects via activation of ET(A) and ET(B) receptor (ET-R) subtypes. In this study, we demonstrate that both ET-R subtypes are highly expressed in rat astrocytes in vivo, indicating that these cells are potential targets of the biological effects of ET-1 in the brain. In cultured cortical astrocytes, both ET-R subtypes are expressed, and selective stimulation of ET(B)-R with ET-1 induces phosphorylation of cAMP response element-binding protein (CREB). The signal transduction pathway activated by ET-1 includes the Rap1/B-Raf and the Ras/Raf-1 complexes, protein kinase C (PKC) together with extracellular signal-regulated kinases (ERK), and the ribosomal S6 kinase (RSK) isoforms RSK2 and RSK3, two kinases that lie immediately downstream of ERK and are able to phosphorylate CREB. Moreover, ET-1 activates the p38 mitogen-activated protein kinase (MAPK)-dependent, but not the c-jun N-terminal kinase (JNK)-dependent pathway. By using selective protein kinase inhibitors and expression of dominant-negative Rap1 protein, we also found that the Rap1/PKC/ERK-dependent pathway induces the phosphorylation of activating transcription factor-1, CREB, and Elk-1, whereas the p38MAPK-dependent pathway only causes CREB phosphorylation. ET-1-induced transcription of the immediate early gene c-fos requires the concomitant activation of both the PKC/ERK- and p38MAPK-dependent pathways, because inhibitors of either pathway block the ET-1-induced increase of c-fos mRNA. Our findings indicate that changes in the expression of cAMP response element-dependent immediate and delayed response genes could play a pivotal role in the physiological effects elicited by ET-1 in astrocytes.

cAMP-dependent protein kinase induces cAMP-response element-binding protein phosphorylation via an intracellular calcium release/ERK-dependent pathway in striatal neurons.

Activation of the cAMP-dependent protein kinase A (PKA) pathway may induce cAMP-response element-binding protein (CREB) phosphorylation either directly or via cross-talk mechanisms with other signal transduction pathways. In this study, we have investigated in striatal primary cultures the mechanism by which activation of the cAMP/PKA-dependent pathway leads to CREB phosphorylation via the extracellular signal-regulated kinase (ERK)-dependent pathway. We have found that PKA-induced CREB phosphorylation and CREB-dependent transcription are mediated by calcium (Ca(2+)) release from intracellular stores and are blocked by inhibitors of the protein kinase C and ERK pathways. This mechanism appears to be mediated by the small G-protein Rap1, whose activation appears to be primed by PKA-induced Ca(2+) release but not further induced by direct or indirect PKA- or protein kinase C-dependent phosphorylation. These results suggest that, in striatal neurons, intracellular Ca(2+) release, Rap1, and ERK pathway play a crucial role in the PKA-induced CREB phosphorylation and CREB-dependent transcription.

Pharmacological and molecular evidence for dopamine D(1) receptor expression by striatal astrocytes in culture.

The neurotransmitter dopamine (DA) at a 10 microM concentration elicited a stimulation of intracellular cyclic AMP (cAMP) accumulation in cultured astrocytes derived from embryonic rat striatum. This accumulation was partially blocked by the beta-adrenergic receptors antagonist propranolol, mimicked by the D(1) agonist SKF 38393 and by the mixed D(1)/D(2) agonist apomorphine. A regional heterogeneity in the magnitude of dopamine-induced cAMP accumulation was observed in cultured astrocytes obtained from different brain areas. The maximum effect was observed in striatal astrocytes, a lower effect in cortical astrocytes, and no increase was detected in cerebellar astrocytes. Reverse transcription-polymerase chain reaction (RT-PCR) coupled to Southern blot hybridization demonstrated that striatal astrocytes express only D(1) receptor mRNA and Western blot analysis confirmed the expression of the D(1) receptor protein in striatal astrocytes. In contrast to what found in neurons, the D(1)-dependent cAMP formation in striatal astrocytes is partially reduced by pertussis toxin (PTX) treatment. The stimulation of D(1) receptors or the activation of adenylyl cyclase by forskolin led to an increase of cytosolic and nuclear protein kinase A (PKA) catalytic activity. The presence of dopamine D(1) receptors in cultured striatal astrocytes suggests a role of dopamine in the regulation of cellular processes in striatal astrocytes.

The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells.

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.

Potentiation of dopamine-induced cAMP formation by group I metabotropic glutamate receptors via protein kinase C in cultured striatal neurons.

Metabotropic glutamate receptors have been shown to potentiate the cyclic adenosine monophosphate (cAMP) formation induced by activation of several receptors linked to adenylyl cyclase via Gs-protein. Here we show that, in primary cultures of striatal neurons, group I metabotropic receptors potentiate the cAMP formation induced by activation of D1-like dopamine receptors. Reverse transcription associated with polymerase chain reaction revealed that mGluR5, mGluR3, mGluR4 and mGluR7 are present in striatal cell cultures. The potentiation of cAMP formation is induced by the selective group I mGluRs agonist (S)-3,5-dihydroxyphenylglycine and by other non-selective mGluRs agonists with a typical group I-like pharmacology (quisqualate > ibotenate > 1-aminocyclopentane-1,3-dicarboxylic acid). The rank order potency of mGluRs agonists in potentiating cAMP formation correlates with their ability to induce inositol phosphates production; the potentiation of cAMP formation and the inositol phosphates production are blocked by the group I mGluRs antagonists (S)-4-carboxyphenylglycine and are not affected by group II antagonist 2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)-glycine or group III antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid. The potentiating mechanism involves the activation of protein kinase C, being mimicked by phorbol-12-myristate-13acetate and blocked by the specific protein kinase C inhibitors bisindolylmaleimide I and chelerythrine or by protein kinase C downregulation. Our results indicate that this interaction could have a functional importance in modulating the cAMP-dependent transmission in the striatum.

Coexpression of phospholipase A2 isoforms in rat striatal astrocytes.

The expression and activity of phospholipase A2 (PLA2) isoforms were investigated in primary cultures of striatal astrocytes. The calcium ionophore A23187 together with the protein kinase C activator phorbol ester was the most potent stimulus in eliciting [3H]arachidonic acid release in the extracellular medium. Reverse transcription coupled to polymerase chain reaction (RT-PCR) showed the presence of the 85 kDa cytosolic PLA2 mRNA and the 14 kDa secretory PLA2 mRNA in untreated astrocytes. Immunoblot experiments with isoform-specific antibodies showed the presence of the cytosolic PLA2 in untreated astrocytes, while the secretory PLA2 was detected only in lipopolysaccharide-treated astrocytes. These data suggest that the two PLA2 isoforms expressed in striatal astrocytes might play different roles in cellular processes mediated by astrocytes.

Membrane localization of N-acylphosphatidylethanolamine in central neurons: studies with exogenous phospholipases.

We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [3H]gamma-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A2 (PLA2) (Apis mellifera) and PLC (B. cereus) mobilized [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that approximately 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE.

Formation and inactivation of endogenous cannabinoid anandamide in central neurons.

Anandamide (N-arachidonoyl-ethanolamine) was recently identified as a brain arachidonate derivative that binds to and activates cannabinoid receptors, yet the mechanisms underlying formation, release and inactivation of this putative messenger molecule are still unclear. Here we report that anandamide is produced in and released from cultured brain neurons in a calcium ion-dependent manner when the neurons are stimulated with membrane-depolarizing agents. Anandamide formation occurs through phosphodiesterase-mediated cleavage of a novel phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine. A similar mechanism also governs the formation of a family of anandamide congeners, whose possible roles in neuronal signalling remain unknown. Our results and those of others indicate therefore that multiple biochemical pathways may participate in anandamide formation in brain tissue. The life span of extracellular anandamide is limited by a rapid and selective process of cellular uptake, which is accompanied by hydrolytic degradation to ethanolamine and arachidonate. Our results thus strongly support the proposed role of anandamide as an endogenous neuronal messenger.

Differential effects of CGRP on adenylyl cyclase in adult and embryonic rat brain.

The presence of functional receptors for calcitonin gene-related peptide (CGRP) in the brain of adult rats and on nerve cell cultures was investigated. Neuronal and glial cultures were obtained from mesencephalons of embryos at gestational day 16. The response to CGRP was tested by measuring the adenylyl cyclase (AC) activity on isolated membranes. CGRP binding in adult rat brains was ineffective in activating AC, whereas a dose-dependent stimulation of AC activity was induced by the peptide both in neuronal and glial cultures. This effect was more pronounced in the glial cells where high affinity binding sites for CGRP were detected. The presence of functional CGRP receptors in embryonic mesencephalic cells, suggests a role for CGRP in the development of rat mesencephalon.