Sergio Schinelli

Store-Operated Ca2+ Entry Is Expressed in Human Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) may be recruited from the bone marrow to sites of tissue regeneration to sustain neovascularization and reendothelialization after acute vascular injury. This feature makes them particularly suitable for cell-based therapy. In mature endothelium, store-operated Ca(2+) entry (SOCE) is activated following emptying of inositol-1,4,5-trisphosphate-sensitive stores, and controls a wide number of functions, including proliferation, nitric oxide synthesis, and vascular permeability. The present work aimed at investigating SOCE expression in EPCs harvested from both peripheral blood (PB-EPCs) and umbilical cord blood (UCB-EPCs) by employing both Ca(2+) imaging and molecular biology techniques. SOCE was induced upon either pharmacological (ie, cyclopiazonic acid) or physiological (ie, ATP) depletion of the intracellular Ca(2+) pool. Further, store-dependent Ca(2+) entry was inhibited by the SOCE inhibitor, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Real-time reverse transcription-polymerase chain reaction and western blot analyses showed that both PB-EPCs and UCB-EPCs express all the molecular candidates to mediate SOCE in differentiated cells, including TRPC1, TRPC4, Orai1, and Stim1. Moreover, pharmacological maneuvers demonstrated that, as well as in differentiated endothelial cells, the signal transduction pathway leading to depletion of the intracellular Ca(2+) pool impinged on the phospholipase C/inositol-1,4,5-trisphosphate pathway. Finally, blockage of SOCE with BTP-2 impaired PB-EPC proliferation. These findings provide the first evidence that EPCs express SOCE, which might thus be regarded as a novel target to enhance the regenerative outcome of cell-based therapy.

Pharmacological and molecular evidence for dopamine D1 receptor expression by striatal astrocytes in culture

The neurotransmitter dopamine (DA) at a 10 μM 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 β‐adrenergic receptors antagonist propranolol, mimicked by the D1 agonist SKF 38393 and by the mixed D1/D2 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 D1 receptor mRNA and Western blot analysis confirmed the expression of the D1 receptor protein in striatal astrocytes. In contrast to what found in neurons, the D1‐dependent cAMP formation in striatal astrocytes is partially reduced by pertussis toxin (PTX) treatment. The stimulation of D1 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 D1 receptors in cultured striatal astrocytes suggests a role of dopamine in the regulation of cellular processes in striatal astrocytes. J. Neurosci. Res. 58:544–552, 1999.

Opposing Actions of D1 and D2‐Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D1‐and D2‐dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D1 or D2 receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [3H]AA, D2‐receptor stimulation enhanced release of [3H]AA produced by application of the Ca2+ ionophore A23187 or of the purinergic agonist ATP. By contrast, D1‐receptor stimulation inhibited [3H]AA release. This inhibitory effect of D1 receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D2 and D1 receptors in striatum, potentiation and inhibition, respectively, of Ca2+‐evoked AA release.

Small Molecule Integrin Antagonists in Cancer Therapy

Integrins are a large family of dimeric receptors composed by alpha and beta subunits that, once bound to extra-cellular matrix (ECM) proteins, regulate a variety of cellular processes such as cell motility, migration, and proliferation. The integrins transduce signals from inside-out and outside-in the cell, thus representing the cellular link to the external environment. For these properties, integrin activation has been involved in pathological processes like tumor growth and metastasis formation. Recent advances in the elucidation of the crystallographic structures of the alphavbeta3 and alphaIIbeta3 integrins are promoting studies focused to the search of small molecule antagonists that can block the integrin binding to ECM and inhibit the biological effects exerted by these receptors. In this review we will focus on small molecule antagonists of alphavbeta3 and alphavbeta5 integrin as tools for cancer therapy while other integrins will only be briefly mentioned. Cilengitide (cyclic peptidic alphavbeta3 and alphavbeta5 antagonist) is currently in clinical trials for anti cancer therapy. Combination of integrin alphavbeta3 antagonists and other traditional therapeutic approaches may represent a future strategy to inhibit tumor growth and metastasis spreading.

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 RIIβ and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIβ and AKAP75; conversely, granule cerebellar cells express predominantly RIα and RIIα. 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 RIIβ synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIβ 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.

Expression of the new CXCL12 receptor, CXCR7, in gliomas

Gliomas are very invasive brain tumors with poor prognosis and therefore any attempt to limit tumor cell dissemination in the brain is expected to improve glioma treatment. The recent deorphanization of CXCR7 as additional receptor for CXCL12 and CXCL11 has raised key issues on its interaction with the CXCL12/CXCR4 axis as a mechanism to modulate glioma cell migration. In this work we investigated protein and mRNA expression of the two chemokines CXCL12 and CXCL11, together with their receptors CXCR4 and CXCR7 in human glioma specimens and cell lines by immunohistochemistry, flow cytometry and quantitative real-time PCR. The main purpose of this study was to find out whether and at what extent CXCR4 and CXCR7 are differentially expressed in glioma cells. In human glioma specimens the levels of CXCL11 and CXCR4 mRNA were significantly higher in glioblastomas compared to non-tumor controls or low grade gliomas, whilst no difference was found for CXCL12 and CXCR7 mRNA expression. In cell lines, flow cytometry and immunocytochemical experiments showed CXCR4 was mainly expressed irrespective of its membrane or intracellular localization. In contrast, a predominant intracellular localization together with a negligible membrane expression of CXCR7 was found in all cells examined. In in vitro experiments CXCR4 and CXCR7 antagonists and the silencing of CXCR4 showed complete inhibition of glioma proliferation. Our findings, in agreement with previous data, suggest that in human glioma cells the prevalent intracellular localization of CXCR7 might modulate the functionality of CXCL11/12 either acting as a scavenger for these chemokines or interfering with the signaling pathways activated by the stimulation of CXCR4.

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‐13‐acetate 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.

A small-molecule RGD-integrin antagonist inhibits cell adhesion, cell migration and induces anoikis in glioblastoma cells

In cancer cells integrins modulate important cellular events that regulate the metastasic cascade which involves detachment from the tumor mass, dissemination and attachment to the oncogenic niche. The α5β1, αvβ3 and αvβ5 integrins are widely expressed in different cancer types and recognize the tripeptide Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. In human glioblastoma, αvβ3 integrin expression correlates with tumor grade, suggesting that this integrin may play a crucial role in the highly infiltrative behavior of high grade gliomas. However, few selective RGD-like antagonists have been developed and few studies have investigated their effects in in vitro models of human glioblastoma. In this study, we investigated several cellular effects and the underlying molecular mechanisms exerted by a new small-molecule RGD antagonist, 1a-RGD, in the U251 and U373 human glioblastoma cell lines. Treatment with 1a-RGD (20 μM) demonstrated a weak effect on cell viability and cell proliferation but strongly inhibited cell attachment and cell migration together with actin cytoskeleton disassembly. Prolonged 1a-RGD treatment (72 h) induced anoikis, assessed by Annexin staining and nucleosome assay, particularly in the detached cells. When integrin-linked transduction pathways were investigated, 1aRGD was found to exert a marked reduction in focal adhesion kinase (FAK) phosphorylation without affecting the AKT- and ERK-dependent pathways. Our data indicate that 1a-RGD, probably via modulation of the FAK-dependent pathway, inhibits cell migration and attachment and induces anoikis in glioblastoma cells. This novel finding suggests that the development of an RGD-like molecule may represent a promising tool for the pharmacological approach aimed at reducing the malignancy of glioblastoma cells.

Endothelin B receptor antagonists block proliferation and induce apoptosis in glioma cells.

The proliferative and antiapoptotic actions of endothelin (ET)-1 in cancer cells have been documented and ET receptor antagonists have been exploited as potential anticancer drugs. Glioblastoma cell lines express both ETA and ETB receptors and previous works have shown that ETB receptors are involved in the proliferation of different cancer cell types. In this study we have investigated the effects of two structurally unrelated ETB receptor antagonists, BQ788 and A192621, on cell survival, proliferation and apoptosis in 1321-N1, U87 and IPDDCA2 glioma cell lines. BQ788 and A192621 reduced glioma cells viability and proliferation assessed by BrdU incorporation and cell cycle analysis by flow cytometry, while in contrast the ETA receptor antagonist BQ123 had no effect on cell survival. TUNEL assay and immunocytochemical experiments showed that BQ788 and A192621 trigger apoptotic processes mainly via activation of the intrinsic mitochondrial pathway involving caspase-9 activation, AIF release and cytochrome c translocation. Furthermore, treatment with ETB antagonists downregulates ERK- and p38MAPK-dependent pathways but does not affect VEGF mRNA levels. Our findings support the hypothesis that ETB antagonists represent a new promising therapeutic strategy for the treatment of high grade gliomas.