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Breast cancer (BC) is the most common malignancy with 1.7 million new diagnoses/year and is responsible for more than 450,000 yearly deaths worldwide. Two thirds of BC express the estrogen receptor (ER) and/or progesterone receptor and are referred to as hormone receptor-positive (HR+) BC. Endocrine therapy (ET) is usually active in these tumors, although drug resistance and side effects limit its benefit. Growth factor signaling through the PI3K/AKT/mammalian target of rapamycin (mTOR) and MAP kinase axes enhances ER activity and is a key mechanism underlying endocrine resistance. Water-only fasting (fasting) or plant-based, low-calorie, carbohydrate- and protein-restricted fasting-mimicking diets (FMDs) reduce circulating growth factors, such as insulin and IGF1 Therefore, we hypothesized that these dietary interventions could be used to enhance the activity of ET and delay the occurrence of resistance. For our in vitro experiments we used the HR+ BC cell lines, MCF7, T47D, and ZR-75-1, as well as metastases-derived organoids from patients with HR+ BC. Our in vivo experiments in mouse xenografts of human BC cell lines, were conducted in six-to-eight-week old female NOD SCID or athymic Nude-FoxN1 mice treated with ET w/ or w/o 48-72 hours of fasting/FMD. We monitored tumor growth and mouse survival and collected tumor masses and blood to detect circulating levels of several growth factors, adipokines and cytokines. In vivo add back experiments with fasting-reduced factors were done with IGF1, insulin and leptin. Circulating growth factors and adipo-cytokines were also detected in blood samples from 36 patients with HR+ BC, who were enrolled in either one of two clinical trials (NCT03595540 and NCT03340935) assessing safety and feasibility of periodic FMD in cancer patients. Patient nutritional status and response to treatment were also monitored in our clinical trials.We found that in HR+ BC models, periodic fasting or FMD enhanced tamoxifen and fulvestrant activity by lowering circulating IGF1, insulin, and leptin levels and by blocking AKT-mTOR signaling via EGR1 and PTEN upregulation. When fulvestrant was combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic FMD cycles promoted long-lasting tumour regressions and reverted acquired resistance to this regime. Moreover, both fasting and FMD prevented tamoxifen-induced endometrial hyperplasia. In HR+ BC patients receiving ET, FMD cycles caused metabolic changes analogous to those observed in mice, including reduced leptin and IGF1 levels, which were found to remain low for extended periods. In mice, these long-lasting effects were associated with carryover anticancer activity. Overall, our results provide the rationale for conducting further clinical studies of fasting-based dietary strategies as an adjuvant to ET w/ or w/o CDK4/6 inhibitors in patients with HR+ BC. Citation Format: Irene Caffa, Vanessa Spagnolo, Pamela Becherini, Francesca Valdemarin, Claudio Vernieri, Min Wei, Sebastian Brandhorst, Chiara Zucal, Else Driehuis, Lorenzo Ferrando, Luca Mastracci, Michele Cilli, Francesco Piacente, Anna Laura Cremonini, Mario Passalacqua, Valerio Vellone, Gabriele Zoppoli, Michele Cea, Giulia Salvadori, Salvatore Cortellino, Hans Clevers, Filippo De Braud, Alessandro Provenzani, Valter D. Longo, Alessio Nencioni. Fasting-mimicking diet and hormone therapy modulates metabolic factors to promote breast cancer regression and reduce side effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT075.

In this manuscript, which appeared in ALTEX 37, 265-274 (doi: 10.14573/altex.1909262), the affiliation of Stefania Vernazza should read: Stefania Vernazza 5# 5 IRCCS-Fondazione Bietti, Rome, Italy and the address for correspondence should read: Stefania Vernazza, PhD, IRCCS, Fondazione Bietti via Livenza 3, 00198 Rome, Italy (stefania.vernazza@yahoo.it).

Engagement, or rather lack thereof has become a major issue because of its negative impact on productivity. Recently, gamification has successfully been implemented into corporate technological interfaces to increase engagement of employees. This paper proposes a theory-driven experiment that examines the impact a gamified interface has on engagement and performance of workers in a warehouse-management task. Specifically, the experiment proposed in this paper compares how the integration of two different types of goal-setting (self-set goals or assigned goals) into a warehouse-employee interface will affect engagement and performance.

The interaction between adenosine A2A and dopamine D2 receptors in striatal neurons is a well-established phenomenon and has opened up new perspectives on the molecular mechanisms involved in Parkinson's disease. However, it has barely been investigated in astrocytes. Here, we show by immunofluorescence that both A2A and D2 receptors are expressed in adult rat striatal astrocytes in situ, and investigate on presence, function, and interactions of the receptors in the astrocyte processes-acutely prepared from the adult rat striatum-and on the effects of homocysteine on the A2A-D2 receptor-receptor interaction. We found that A2A and D2 receptors were co-expressed on vesicular glutamate transporter-1-positive astrocyte processes, and confirmed that A2A-D2 receptor-receptor interaction controlled glutamate release-assessed by measuring the [3H]D-aspartate release-from the processes. The complexity of A2A-D2 receptor-receptor interaction is suggested by the effect of intracellular homocysteine, which reduced D2-mediated inhibition of glutamate release (homocysteine allosteric action on D2), without interfering with the A2A-mediated antagonism of the D2 effect (maintained A2A-D2 interaction). Our findings indicate the crucial integrative role of A2A-D2 molecular circuits at the plasma membrane of striatal astrocyte processes. The fact that homocysteine reduced D2-mediated inhibition of glutamate release could provide new insights into striatal astrocyte-neuron intercellular communications. As striatal astrocytes are recognized to be involved in Parkinson's pathophysiology, these findings may shed light on the pathogenic mechanisms of the disease and contribute to the development of new drugs for its treatment.

Glucagon-like peptide-1 receptors (GLP-1Rs) have been shown to mediate cognitive-enhancing and neuroprotective effects in the central nervous system. However, little is known about their physiological roles on central neurotransmission, especially at the presynaptic level. Using purified synaptosomal preparations and immunofluorescence techniques, here we show for the first time that GLP-1Rs are localized on mouse cortical and hippocampal synaptic boutons, in particular on glutamatergic and GABAergic nerve terminals. Their activation by the selective agonist exendin-4 (1-100 nM) was able to increase the release of either [

Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.

Neuronal adaptation to oxidative stress is crucially important in order to prevent degenerative diseases. The role played by the Nrf2/HO-1 system in favoring cell survival of neuroblastoma (NB) cells exposed to hydrogen peroxide (H2O2) has been investigated using undifferentiated or all-trans retinoic acid (ATRA) differentiated SH-SY5Y cells. While undifferentiated cells were basically resistant to the oxidative stimulus, ATRA treatment progressively decreased cell viability in response to H2O2. HO-1 silencing decreased undifferentiated cell viability when exposed to H2O2, proving the role of HO-1 in cell survival. Conversely, ATRA differentiated cells exposed to H2O2 showed a significantly lower induction of HO-1, and only the supplementation with low doses of bilirubin (0,5–1 μM) restored viability. Moreover, the nuclear level of Bach1, repressor of HO-1 transcription, strongly decreased in undifferentiated cells exposed to oxidative stress, while did not change in ATRA differentiated cells. Furthermore, Bach1 was displaced from HO-1 promoter in undifferentiated cells exposed to H2O2, enabling the binding of Nrf2. On the contrary, in ATRA differentiated cells treated with H2O2, Bach1 displacement was impaired, preventing Nrf2 binding and limiting HO-1 transcription. In conclusion, our findings highlight the central role of Bach1 in HO-1-dependent neuronal response to oxidative stress.

Long-term potentiation (LTP) and the process of memory formation require activation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Notably, recent evidence indicated that both cyclic nucleotides boost the production of amyloid-beta (Aβ) peptides. In particular, cAMP was shown to favor hippocampal LTP by stimulating the synthesis of the amyloid precursor protein APP, whereas cGMP was found to enhance LTP and to improve memory by increasing Aβ levels without affecting the expression of APP. The results of the present study substantiate that cGMP has a role in the endocytic pathway of APP and suggest a scenario where the cyclic nucleotide enhances the production of Aβ by favoring the trafficking of APP from the cell cortex to the endolysosomal compartment.

Background : Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (K ATP ). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac K ATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells. Methods: The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot. Results: Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed. Conclusions : Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin.

Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes’ participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders. This article is protected by copyright. All rights reserved.

The availability of antioxidants is recognized as one of the critical factors able to make cancer cells resistant to chemotherapy. In this context, it has been demonstrated that many chemoresistant cancers display high levels of glutathione (GSH) and consequently, its depletion by L-buthionine sulfoximine (BSO), has been proposed as a chemosensitizing therapy. To investigate the role of GSH and of tumor metabolism in multi-drug resistance (MDR), HTLA-230 neuroblastoma cells were chronically treated with etoposide at a concentration that in vitro mimics the clinically-used dose. The selected cells (HTLA-Chr) were highly tumorigenic and acquired MDR, becoming less sensitive to etoposide or doxorubicin compared to parental cells. Moreover, HTLA-Chr cells, while having an efficient aerobic metabolism, owing to a favourable P/O ratio and a decreased formation of lactic acid, were also characterized by an up-regulation of catalase and higher levels of GSH. BSO treatment of HTLA-Chr cells markedly reduced their tumorigenicity that was, instead, enhanced by N-Acetylcysteine, able to promote GSH synthesis. Collectively, our results show that GSH and the switch of aerobic metabolism collaborate for the acquisition of MDR, providing pre-clinical evidence that may drive future therapeutic approaches for sensitizing neuroblastoma to conventional therapies.

Boosting NAD(+) biosynthesis with NAD(+) intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD(+) biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFβ signaling pathway via increased TGFβ1 production. These findings have implications for the design of therapeutic strategies exploiting NAD(+) biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.

Our patient is a 65-year-old woman presenting with bilateral pes cavus, pronounced distal muscle wasting, weakness and areflexia. Electrophysiological findings included diffuse unrecordable motor and sensory responses. While the CMT phenotype was evident, the lack of family history and the severe, but unspecific electrophysiological impairment, was a challenge for genetic diagnosis. A sural nerve biopsy was performed, showing a severe loss of myelinated fibers with residual axons surrounded by myelin outfoldings. Whereas myelin outfoldings are a pathological hallmark of autosomal recessive CMT4B1 and CMT4B2, due to mutations in myotubularin-related 2 (MTMR2) and 13 (MTMR13) genes respectively, they may also occur in nerve biopsies from CMT1B patients. By direct sequencing, a novel heterozygous transversion c.410G>T in MPZ gene was demonstrated, producing an amino acid change from glycine to valine in position 108 (p.G108V). In HeLa cells the fusion P0G108V-EGFP was normally trafficked to the cell membrane, but with decreased P0 adhesion function, compared with wild-type P0, thus supporting a pathogenic role of the new variant. In conclusion this case highlights the relevance, in selected cases, of sural nerve biopsy to orient the genetic/molecular tests, while in vitro analyses may strengthen the pathogenic role of novel mutations.

High-risk neuroblastoma (NB) is characterized by the development of chemoresistance, and bortezomib (BTZ), a selective inhibitor of proteasome, has been proposed in order to overcome drug resistance. Considering the involvement of the nuclear factor-erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in the antioxidant and detoxifying ability of cancer cells, in this study we have investigated their role in differently aggressive NB cell lines treated with BTZ, focusing on the modulation of HO-1 to improve sensitivity to therapy. We have shown that MYCN amplified HTLA-230 cells were slightly sensitive to BTZ treatment, due to the activation of Nrf2 that led to an impressive up-regulation of HO-1. BTZ-treated HTLA-230 cells down-regulated p53 and up-regulated p21, favoring cell survival. The inhibition of HO-1 activity obtained by Zinc (II) protoprophyrin IX (ZnPPIX) was able to significantly increase the pro-apoptotic effect of BTZ in a p53- and p21-independent way. However, MYCN non-amplified SH-SY5Y cells showed a greater sensitivity to BTZ in relation to their inability to up-regulate HO-1. Therefore, we have shown that HO-1 inhibition improves the sensitivity of aggressive NB to proteasome inhibition-based therapy, suggesting that HO-1 up-regulation can be used as a marker of chemoresistance in NB. These results open up a new scenario in developing a combined therapy to overcome chemoresistance in high-risk neuroblastoma.

// Renata Colla 1 , Alberto Izzotti 2, 3 , Chiara De Ciucis 1 , Daniela Fenoglio 4 , Silvia Ravera 5 , Andrea Speciale 1 , Roberta Ricciarelli 1 , Anna Lisa Furfaro 6 , Alessandra Pulliero 2 , Mario Passalacqua 1 , Nicola Traverso 1 , Maria Adelaide Pronzato 1 , Cinzia Domenicotti 1 , Barbara Marengo 1 1 Department of Experimental Medicine, University of Genova, Genova, Italy 2 Department of Health Sciences, University of Genova, Genova, Italy 3 IRCCS AOU San Martino IST Genova, Genova, Italy 4 Center of Excellence for Biomedical Research, Department of Internal Medicine, University of Genova, Genova, Italy 5 Department of Pharmacy, University of Genova, Genova, Italy 6 Giannina Gaslini Institute, Genova, Italy Correspondence to: Barbara Marengo, email: Barbara.Marengo@unige.it Keywords: neuroblastoma, multi-drug resistance, glutathione, antioxidants, aerobic metabolism Received: March 18, 2016 Accepted: September 13, 2016 Published: September 23, 2016 ABSTRACT Neuroblastoma, a paediatric malignant tumor, is initially sensitive to etoposide, a drug to which many patients develop chemoresistance. In order to investigate the molecular mechanisms responsible for etoposide chemoresistance, HTLA-230, a human MYCN-amplified neuroblastoma cell line, was chronically treated with etoposide at a concentration that in vitro mimics the clinically-used dose. The selected cells (HTLA-Chr) acquire multi-drug resistance (MDR), becoming less sensitive than parental cells to high doses of etoposide or doxorubicin. MDR is due to several mechanisms that together contribute to maintaining non-toxic levels of H 2 O 2 . In fact, HTLA-Chr cells, while having an efficient aerobic metabolism, are also characterized by an up-regulation of catalase activity and higher levels of reduced glutathione (GSH), a thiol antioxidant compound. The combination of such mechanisms contributes to prevent membrane lipoperoxidation and cell death. Treatment of HTLA-Chr cells with L-Buthionine-sulfoximine, an inhibitor of GSH biosynthesis, markedly reduces their tumorigenic potential that is instead enhanced by the exposure to N-Acetylcysteine, able to promote GSH synthesis. Collectively, these results demonstrate that GSH and GSH-related responses play a crucial role in the acquisition of MDR and suggest that GSH level monitoring is an efficient strategy to early identify the onset of drug resistance and to control the patient’s response to therapy.

Although photons have been repeatedly shown to affect the functioning of the nervous system, their effects on neurotransmitter release have never been investigated. We exploited in vitro models that allow effects involving neuron-astrocyte network functioning to be detected (mouse cerebrocortical slices) and dissected these effects at cerebrocortical nerve endings and astrocyte processes. Infrared light proved able to induce glutamate release by stimulating glutamatergic nerve endings.

Cell attachment is provided by cell-matrix and cell-cell bonds, and acts as a regulator of vascular smooth muscle cell (VSMC) survival, activity and homeostasis, as well as of VSMCs response to pathogenic stimuli. In this work we elicited an exclusive cell-cell contact by culturing A7r5 VSMCs on agarose-coated wells to form floating cell clusters, and we demonstrated that a steady state with a reduced response to the vasoactive peptide Angiotensin II (ATII) was induced. We found that clustered VSMCs showed subcortical stabilization of beta-catenin and Caveolin 1 (Cav1), unlike adherent confluent counterparts. We demonstrated that beta-catenin and Cav1 stabilization at the membrane level hampers the molecular cross-talk induced by ATII-activated AT1 receptor (AT1R), thereby impeding the phosphorylation of Cav1 and IGF1R, the NADPH oxidase activity, and counteracting ATII-dependent hypertrophy. Thus, elective cell-cell bond might modulate the proatherogenic activity of ATII, reducing the adverse vascular remodelling associated with AT1R activation.

We show that protein kinase C (PKC) theta localized at the Golgi complex is partially conjugated to monoubiquitin. Using the inactive T538A and activable T538E mutants of PKCtheta, we demonstrate that the presence of an uncharged residue at the 538 position of the activation loop favors both association with the Golgi and monoubiquitination of the kinase. Moreover, the inactive PKCtheta does not translocate from the Golgi in response to a short-term cell stimulation with a phorbol ester and is subjected to different proteolytic degradation pathways compared to the activable cytosolic kinase. These findings highlight the role of T538 as a critical determinant to address the activable and the inactive PKCtheta molecules to different intracellular compartments and to specific post-transductional modifications. The functional relevance of these observations is supported by the impaired cell division observed in phenotypes expressing high levels of the inactive PKCtheta.

A substantial body of evidence indicates that protein kinase C (PKC) is involved in the α-secretory processing of the amyloid precursor protein (APP), an event that reduces the formation of the pathogenic amyloid-β peptide. Recently, we have shown that trafficking and processing of APP are both impaired by knockdown of myosin II-B, one of the major neuronal motor proteins. Here, we provide evidence that the α-secretory processing of APP is mediated by PKC-dependent phosphorylation of myosin II-B. This signaling pathway provides an important link between APP and the neuronal cytoskeleton and might be crucial for the understanding of the biological and pathological roles of APP.—Argellati, F., Domenicotti, C., Passalacqua, M., Janda, E., Melloni, E., Marinari, U. M., Pronzato, M. A., Ricciarelli, R. Protein kinase C-dependent α-secretory processing of the amyloid precursor protein is mediated by phosphorylation of myosin II-B.

The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+-dependent vesicular glutamate release and significant Ca2+-independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X7R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+]i signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R-mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals.

Extracellular high-mobility group box 1 protein (HMGB1) triggers inflammatory events in the brain. We demonstrate that astrocytes, the main glial cells in the brain, acquire a specific reactive phenotype when exposed to HMGB1. This cell activation, which involves the receptor for advanced glycation end-products and the MAPK/ERK1/2 cascade, results in the transcriptional/translational induction of a restricted number of inflammatory mediators, including cyclooxygenase-2, matrix metalloproteinase-9, and several chemokines of the CC and CXC families. The mixture of factors released by HMGB1-reactive astrocytes displays a potent chemotactic activity on human monocytic cells. This study is the first to suggest that HMGB1/astrocyte interaction plays a specific functional role in the progression of inflammatory processes in the CNS by facilitating local leukocyte infiltration.

The Bergmann glia is equipped with Ca2+-permeable AMPA receptors for glutamate, indispensable for structural and functional relations between the Bergmann glia and parallel/climbing fibers-Purkinje cell synapses. To better understand roles for the Bergmann AMPA receptors, herein we investigate on gliotransmitter release and Ca2+ signals in isolated Bergmann glia processes obtained from adult rat cerebellum. We found that: 1) the rat cerebellar purified astrocyte processes (gliosomes) expressed astrocytic and Bergmann markers and exhibited negligible contamination by nerve terminals, microglia, or oligodendrocytes; 2) activation of Ca2+-permeable AMPA receptors caused Ca2+ signals in the processes, and the release of glutamate from the processes; 3) effectiveness of rose bengal, trypan blue or bafilomycin A1, indicated that activation of the AMPA receptors evoked vesicular glutamate release. Cerebellar purified nerve terminals appeared devoid of glutamate-releasing Ca2+-permeable AMPA receptors, indicating that neuronal contamination may not be the source of the signals detected. Ultrastructural analysis indicated the presence of vesicles in the cytoplasm of the processes; confocal imaging confirmed the presence of vesicular glutamate transporters in Bergmann glia processes. We conclude that: a vesicular mechanism for release of the gliotransmitter glutamate is present in mature Bergmann processes; entry of Ca2+ through the AMPA receptors located on Bergmann processes is coupled with vesicular glutamate release. The findings would add a new role for a well-known Bergmann target for glutamate (the Ca2+-permeable AMPA receptors) and a new actor (the gliotransmitter glutamate) at the cerebellar excitatory synapses onto Purkinje cells.

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re-sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [(3)H]d-aspartate form gliosomes in a concentration-dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of [(3)H]d-aspartate was independent of modifications of cytosolic Ca(2+) , but it was blocked by dl-threo-beta-benzyloxyaspartate (dl-TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca(2+)-independent and dl-TBOA-sensitive manner. These findings suggest the involvement of carrier-mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca(2+). Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca(2+). These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.

Clinical progression in hereditary and acquired demyelinating disorders of both the central and peripheral nervous system is mainly due to a time-dependent axonal impairment. We established 90-day dorsal root ganglia (DRG) cultures from a rat model of Charcot-Marie-Tooth type 1A (CMT1A) neuropathy to evaluate the structure of myelin and axons, and the expression of myelin-related proteins and cytoskeletal components, by morphological and molecular techniques. Both wild-type and CMT1A cultures were rich in myelinated fibres. Affected cultures showed dysmyelinated internodes and focal myelin swellings. Furthermore, uncompacted myelin and smaller axons with increased neurofilament (NF) density were found by electron microscopy, and Western blots showed higher levels of nonphosphorylated NF. Confocal microscopy demonstrated an abnormal distribution of the myelin-associated glycoprotein which, instead of being expressed at the noncompact myelin level, showed focal accumulation along the internodes while other myelin proteins were normally distributed. These findings suggest that CMT1A DRG cultures, similarly to the animal model and human disease, undergo axonal atrophy over a period of time. This model may be utilized to study the molecular changes underlying demyelination and secondary axonal impairment. As axonal damage may occur after just 3 months and tissue cultures represent a strictly controlled environment, this model may be ideal for testing neuroprotective therapies.

Previous results of our study carried out on Dictyostelium discoideum showed the presence in single-cell amoebae of both cholinesterase (ChE) activity, able to hydrolyze the propionyl thiocholine iodide substrate, and “true” acetylcholinesterase (AChE) activity, similar to that of Electrophorus electricus . These activities are sensitive to anti-ChE agents, and to the inhibitors classically used to discriminate different ChEs. In this work, we have investigated the presence of ChE activities and of both acetylcholine (ACh) nicotinic and muscarinic receptors, during the developmental cycle of D. discoideum . The spectrophotometric evaluation showed that slugs displayed the highest enzyme activities, AChE being the main activity, while fruiting bodies exhibited the lowest. The presence of AChE activity was cytochemically detected in all the cell populations of the developmental cycle, except in the fruiting bodies where it was localized exclusively to the level of the spores. Molecules recognized by the anti-butyrylcholinesterase antibody were detected in single-cell amoebae, first fingers, slugs, and culminants, except in the fruiting bodies where they were found exclusively in the spore walls. The ACh nicotinic receptors were cytochemically identified in single-cell amoebae, slugs, and spores; however, molecules immunologically related to the ACh muscarinic receptors were not present in the spores.

Neuronal adaptation to oxidative stress is crucial to prevent degenerative diseases. The role of the Nrf2/HO-1 system in cell response to hydrogen peroxide (H2O2) has been investigated using SH-SY5Y neuroblastoma cells as undifferentiated or after differentiation with all-trans retinoic acid (ATRA). We showed that undifferentiated cells resisted to oxidative stimulus and up-regulated HO-1 which was responsible for their survival, since its silencing decreased viability in cells exposed to H2O2. On the contrary, in ATRA-treated cells the viability decreased in response to increasing concentration of H2O2 and HO-1 was not induced. However, bilirubin supplementation restores cell viability, underlining the role of HO-1-derived bilirubin in cell resistance to oxidative stress. Investigating the mechanisms of HO-1 induction, we showed that in undifferentiated cells the nuclear level of Bach1, repressor of HO-1 transcription, decreased as well as its binding to the promoter of HO-1. In the same condition, Nrf2 binding to the same DNA region increased. Yet, in differentiated cells Bach1 nuclear level was not modified by the exposure to H2O2 as well as its binding to HO-1 promoter and, as a consequence, the binding of Nrf2 to HO-1 promoter was not modified by H2O2. In conclusion, our findings highlight the role of Bach1/HO-1/bilirubin in neuronal response to oxidative stress.

The aggregation of amyloid-β (Aβ) peptides plays a crucial role in the onset and progression of Alzheimer's disease. Monomeric form of Aβ, indeed, could exert a physiological role. Considering the anti-oligomerization property of all-trans retinoic acid (ATRA), the involvement of monomeric Aβ1–42 in ATRA-induced neuronal differentiation has been investigated. Four-day ATRA treatment increases β-secretase 1 (BACE1) level, Aβ1–42 production, and receptor for advanced glycation end-products (RAGE) expression. RAGE is a well-recognized receptor for Aβ, and the block of both RAGE and Aβ1–42 with specific antibodies strongly impairs neurite formation in ATRA-treated cells. The involvement of Aβ1–42 and RAGE in ATRA-induced morphologic changes has been confirmed treating undifferentiated cells with different molecular assemblies of peptide: 1 μM monomeric, but not oligomeric, Aβ1–42 increases RAGE expression and favors neurite elongation. The block of RAGE completely prevents this effect. Furthermore, our data underline the involvement of the RAGE-dependent adhesion molecule amphoterin-induced gene and open reading frame-1 as downstream effector of both ATRA and Aβ1–42. In conclusion, our findings identify a novel physiological role for monomeric Aβ1–42 and RAGE in neuronal differentiation.

In this study we demonstrate that the rat pheochromocytoma PC12 cell line expresses the novel protein kinase C isozyme designated PKC-theta;. The isozyme is almost completely localized in the nuclear compartment of proliferating cells. Following stimulation with the nerve growth factor, PKC-theta; is redistributed into the cytoplasm and the outgrowing neurite processes, mostly as a cytoskeletal associated kinase. This event is accompanied by an eightfold increase in the expression level and by the appearance of specific modifications of PKC-theta; molecule. Conversely, the kinase is down-regulated once cells reach the terminally differentiated state displaying a neuron-like phenotype. These data suggest a functional role for the kinase in the regulation of cytoskeletal modeling along the multistage differentiation process of PC12 cells.

Protein kinase C-theta is a member of the n-protein kinase C subfamily that in mitotic cells translocates to centrosomes and kinetochores. Although this kinase is expressed in comparable amounts in murine erythroleukaemia cells during the interphase or metaphase, when localized in the mitotic structures, it selectively phosphorylates a 66 kDa protein, also associated to chromosomes. Moreover, protein kinase C-theta immunoprecipitated from cells at the metaphase results four times more active in the absence of lipid cofactors as compared with the kinase obtained from cells in the interphase. This activation is accomplished by interaction of protein kinase C-theta with a protein factor which also promotes an increased autophosphorylation of the kinase. These findings indicate that in the mitotic phase of the cell cycle, protein kinase C-theta recognizes a protein factor which operates as a positive modulator of the kinase activity in the absence lipids.

In this study we provide evidence that the protein kinase C (PKC)-straight theta isoenzyme is recruited on to the mitotic spindle in dividing murine erythroleukaemia (MEL) cells and associates specifically with centrosome and kinetochore structures. None of the other PKC isoenzymes (-alpha, -delta, -epsilon, -mu and -zeta) expressed by MEL cells shows this localization on the mitotic spindle. An identical subcellular distribution of PKC-straight theta is also observed in dividing murine P3 myeloma cells and human LAN-5 neuroblastoma cells, indicating that this PKC isoenzyme interacts with the mitotic apparatus in mammalian cells. In phorbol-ester-treated non-growing MEL cells, a rapid change in the intracellular distribution of PKC-straight theta occurs. Under these conditions, PKC-straight theta is translocated from the nuclear to the cytosolic cell compartment, an event that is accompanied by phosphorylation of the PKC-straight theta molecule and is followed by its down-regulation. The recovery of cell growth capacity results in the concomitant reappearance of PKC-straight theta. Furthermore, when MEL cells acquire the differentiated non-growing phenotype, the level of PKC-straight theta is reduced to less than 5%, suggesting that this PKC isoenzyme is no longer required. We propose that, unlike other members of the PKC family, PKC-straight theta may play a role in cell proliferation.

In human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) stimulates some important pathways leading to thromboxane B2 formation, calcium intracellular elevation, ATP secretion and actin polymerisation. The aim of the present study was to examine the 2-AG effect on myosin light chain (MLC) phosphorylation and to investigate the mechanisms involved. We demonstrated that 2-AG induced a rapid MLC phosphorylation, stimulating both the RhoA kinase (ROCK) and MLC kinase (MLCK) in a dose and time-dependent manner. In addition MLC phosphorylation was strengthened through the MLC phosphatase inhibition. MLC phosphatase inhibition was accomplished through the RhoA/ROCK and protein kinase C mediated phosphorylation of MLC phosphatase inhibiting subunits MYPT1 and CPI-17. The presence of CB1 receptor in human platelets and the involvement of CB1 receptor in MLC phosphorylation and MLC phosphatase inhibition was shown.

P2X7 receptors trigger Ca(2+) -dependent exocytotic glutamate release, but also function as a route for non-exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full-length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18-amino acid cysteine-rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non-competitive way by extracellular Mg(2+) , did not require the recruitment of pore-forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine-rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca(2+) elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca(2+) -dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non-exocytotic glutamate efflux.

The anti-hyperglycaemic drug metformin has important anticancer properties as shown by the direct inhibition of cancer cells proliferation. Tumor cells avidly use glucose as a source for energy production and cell building blocks. Critical to this phenotype is the production of glucose-6-phosphate (G6P), catalysed by hexokinases (HK) I and II, whose role in glucose retention and metabolism is highly advantageous for cell survival and proliferation. Here we show that metformin impairs the enzymatic function of HKI and II in Calu-1 cells. This inhibition virtually abolishes cell glucose uptake and phosphorylation as documented by the reduced entrapment of (18)F-fluorodeoxyglucose. In-silico models indicate that this action is due to metformin capability to mimic G6P features by steadily binding its pocket in HKII. The impairment of this energy source results in mitochondrial depolarization and subsequent cell death. These results could represent a starting point to open effective strategies in cancer prevention and treatment.

Besides playing a pathogenic role in Alzheimer disease, amyloid-beta peptides are normally produced in low amounts in the brain, and several lines of evidence suggest that they can modulate synaptic plasticity and memory. As cyclic adenosine monophosphate (cAMP) is known to be involved in the same processes and the blockade of its degradation by phosphodiesterase 4 inhibitors has consistently shown beneficial effects on cognition, we investigated the possible correlation between this second messenger and Aβ peptides in neuronal N2a cells overexpressing the amyloid-β precursor protein (APP). We herein report that the elevation of endogenous cAMP by rolipram increased APP protein expression and both its amyloidogenic and nonamyloidogenic processing. The effects of rolipram were reproduced by both the cAMP membrane-permeant analog 8Br-cAMP and the forskolin-induced activation of adenylyl cyclase but were not affected by the PKA inhibitor H-89. Our results demonstrate that, in neuronal cells, APP metabolism is physiologically modulated by cAMP and suggest that this might represent an additional mechanism through which the second messenger could influence memory functions. © 2013 IUBMB Life, 65(2)127–133, 2013

Metformin causes an AMP/ATP ratio increase and AMP-activated protein kinase (AMPK) activation. Since caveolin-1 (Cav-1) plays a role in AMPK activation and energy balance, we investigated whether Cav-1 could participate in metformin's inhibitory effect on IGF1 signaling. The effect of metformin was studied in two non-small-cell lung cancer (NSCLC) cell lines, Calu-1 and Calu-6, expressing higher and lower amounts of Cav-1, respectively. In Calu-1, but not in Calu-6 cells, metformin reduced phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR) substrates Akt and Forkhead transcription factor 3a (FOXO3a), inhibited IGF1-dependent FOXO3a nuclear exit, and decreased IGF1-dependent cell proliferation. Here, we show that sensitivity of NSCLC cells to metformin was dependent on Cav-1 expression and that metformin required Cav-1 to induce AMPK phosphorylation and AMP/ATP ratio increase. Cav-1 silencing in Calu-1 and overexpression in Calu-6 reduced and improved, respectively, the inhibitory effect of metformin on IGF1-dependent Akt phosphorylation. Prolonged metformin treatment in Calu-6 cells induced a dose-dependent expression increase of Cav-1 and OCT1, a metformin transporter. Cav-1 and OCT1 expression was associated with the antiproliferative effect of metformin in Calu-6 cells (IC(50)=18 mM). In summary, these data suggest that Cav-1 is required for metformin action in NSCLC cells.

We report the first case of a missense mutation in MPZ causing a gain of glycosylation in myelin protein zero, the main protein of peripheral nervous system myelin. The patient was affected by a severe demyelinating neuropathy caused by a missense mutation, D32N, that created a new glycosylation sequence. We confirmed that the mutant protein is hyperglycosylated, is partially retained into the Golgi apparatus in vitro, and disrupts intercellular adhesion. By sequential experiments, we demonstrated that hyperglycosylation is the main mechanism of this mutation. Gain of glycosylation is a new mechanism in Charcot-Marie-Tooth type 1B.

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.

In this study the effect of the endocannabinoid anandamide on platelet nitric oxide (NO)/cGMP pathway was investigated. Data report that anandamide in a dose-and time-dependent manner increased NO and cGMP levels and stimulated endothelial nitric oxide synthase (eNOS) activity. These parameters were significantly reduced by LY294002, selective inhibitor of PI3K and by MK2206, specific inhibitor of AKT. Moreover anandamide stimulated both eNOSser1177 and AKTser473 phosphorylation. Finally the anandamide effect on NO and cGMP levels, eNOS and AKT phosphorylation/activation were inhibited by SR141716, specific cannabinoid receptor 1 antagonist, supporting the involvement of anandamide binding to this receptor. Overall data of this report indicate that low concentrations of anandamide, through PI3K/AKT pathway activation, stimulates eNOS activity and increases NO levels in human platelets. In such way anandamide contributes to extend platelet survival. J. Cell. Biochem. 112: 924–932, 2011. © 2010 Wiley-Liss, Inc.

On the basis of the amino acid sequence of isolated tryptic peptides, it has been established that the differentiation enhancing factor, produced and active on murine erythroleukemia (MEL)cells, possesses a unique sequence, with no similarity to that of known proteins. Accordingly, this factor can be defined as a novel biologically active peptide. An antisense oligodeoxynucleotide, deduced from the sequence of a nonadecapeptide (produced by tryptic digestion of the factor), decreases the rate and the extent of MEL cell differentiation, induced by hexamethylenebisacetamide. In these cells the amount of the factor is reduced to one third of that constitutively present in untreated cells. Exogenous addition of the factor restores cell inducibility to normal values. Taken together, these results demonstrate the presence in MEL cells of a new factor, structurally and firnctionally unrelated to any of the known biologically active peptides, and suggest its crucial role in the promotion of an initial signal, in chemically induced erythroid differentiation.

Background: Insulin-like growth factor-I receptor (IGF-IR) is a tyrosine kinase receptor (RTK) associated with caveolae, invaginations of the plasma membrane that regulate vesicular transport, endocytosis and intracellular signaling. IGF-IR internalization represents a key mechanism of down-modulation of receptors number on plasma membrane. IGF-IR interacts directly with Caveolin-1 (Cav-1), the most relevant protein of caveolae. Recently it has been demonstrated that the Polymerase I and Transcript Release Factor I (PTRF/Cavin) is required for caveolae biogenesis and function. The role of Cav-1 and PTRF/Cavin in IGF-IR internalization is still to be clarified. Methodology/Principal Findings: We have investigated the interaction of IGF-IR with Cav-1 and PTRF/Cavin in the presence of IGF1in human Hacat cells. We show that IGF-IR internalization triggers Cav-1 and PTRF/Cavin translocation from plasma membrane to cytosol and increases IGF-IR interaction with these proteins. In fact, Cav-1 and PTRF/Cavin coimmunoprecipitate with IGF-IR during receptor internalization. We found a different time course of co-immunoprecipitation between IGF-IR and Cav-1 compared to IGF-IR and PTRF/Cavin. Cav-1 and PTRF/Cavin silencing by siRNA differently affect surface IGF-IR levels following IGF1 treatment: Cav-1 and PTRF/Cavin silencing significantly affect IGF-IR rate of internalization, while PTRF/Cavin silencing also decreases IGF-IR plasma membrane recovery. Since Cav-1 phosphorylation could have a role in IGF-IR internalization, the mutant Cav-1Y14F lacking Tyr14 was transfected. Cav-1Y14F transfected cells showed a reduced internalization of IGF-IR compared with cells expressing wild type Cav-1. Receptor internalization was not impaired by Clathrin silencing. These findings support a critical role of caveolae in IGF-IR intracellular traveling. Conclusions/Significance: These data indicate that Caveolae play a role in IGF-IR internalization. Based on these findings, Cav-1 and PTRF/Cavin could represent two relevant and distinct targets to modulate IGF-IR function.

Hepatic lesions, experimentally-induced in Fisher 344 (F344) and Brown Norway (BN) rats, respectively, susceptible and resistant to liver carcinogenesis, progress differently to hepatocellular carcinoma (HCC). The mechanisms responsible for the acquisition of the resistant phenotype are not completely clear. Herein, we show that in F344 rats subjected to carcinogenic treatment, angiogenesis and DNA oxidation markers increase in preneoplastic and neoplastic liver lesions. On the contrary, in the HCCs of treated BN rats, angiogenesis and a minor DNA oxidation are accompanied by an attempt of tissue remodelling. This study suggests that DNA oxidation might be an important factor in the initiation and promotion of the events of hepatocarcinogenesis. On the other hand, the enhancement of GSH levels and the down-regulation of superoxide dismutase (SOD) expression in both rat strains suggest that antioxidant response is not involved in the acquisition of resistant phenotype.

Patients treated with low-dose anthracyclines often show late onset cardiotoxicity. Recent studies suggest that this form of cardiotoxicity is the result of a progenitor cell disease. In this study we demonstrate that Cord Blood Endothelial Progenitor Cells (EPCs) exposed to low, sub-apoptotic doses of doxorubicin show a senescence phenotype characterized by increased SA-b-gal activity, decreased TRF2 and chromosomal abnormalities, enlarged cell shape, and disarrangement of F-actin stress fibers accompanied by impaired migratory ability. P16( INK4A) localizes in the cytoplasm of doxorubicin-induced senescent EPCs and not in the nucleus as is the case in EPCs rendered senescent by different stimuli. This localization together with the presence of an arrest in G2, and not at the G1 phase boundary, which is what usually occurs in response to the cell cycle regulatory activity of p16(INK4A), suggests that doxorubicin-induced p16( INK4A) does not regulate the cell cycle, even though its increase is closely associated with senescence. The effects of doxorubicin are the result of the activation of MAPKs p38 and JNK which act antagonistically. JNK attenuates the senescence, p16( INK4A) expression and cytoskeleton remodeling that are induced by activated p38. We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity. In conclusion, this study provides a better understanding of the senescence of doxorubicin-treated EPCs, which may be helpful in preventing and treating late onset cardiotoxicity.

The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2(+) breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs.

Caveolin-1 (Cav-1) regulates both insulin like growth factor receptor (IGF-IR) and integrin beta1 function. However, the role of Cav-1 in IGF-IR/integrin beta1 cross talk remains to be established. In this study, we observed that IGF-I did not induce integrin beta1 internalization but its plasma membrane reorganization. In particular, we found a rapid and transient association between integrin beta1 and Cav-1 followed by the enrichment of integrin beta1 in lipid rafts. To determine the role of Cav-1 in this process, we transfected Hacat cells with small interfering RNA specific for Cav-1 (siRNA-Cav-1) and with a scrambled siRNA as control (siRNA-Ctr). Cav-1 down regulated Hacat cells were then stimulated with IGF-I and analyzed by immunofluorescence and flow cytometry. We found that Cav-1 silencing abolished the recruitment of integrin beta1 to lipid rafts in the presence of IGF-I. These data demonstrate that IGF-IR/integrin beta1 cross talk is followed by integrin beta1 lipid raft compartmentalization and that Cav-1 is required for this process.

Hyperhomocysteinaemia has been associated with increased risk of thrombosis and atherosclerosis. Homocysteine produces endothelial injury and stimulates platelet aggregation. Several molecular mechanisms related to these effects have been elucidated. The study aimed to deeply investigate the homocysteine effect on nitric oxide formation in human platelets. The homocysteine-induced changes on nitric oxide, cGMP, superoxide anion levels and nitrotyrosine formation were evaluated. The enzymatic activity and the phosphorylation status of endothelial nitric oxide synthase (eNOS) at thr495 and ser1177 residues were measured. The protein kinase C (PKC), assayed by immunofluorescence confocal microscopy technique and by phosphorylation of p47pleckstrin, and NADPH oxidase activation, tested by the translocation to membrane of the two cytosolic subunits p47(phox) and p67(phox), were assayed. Results show that homocysteine reduces platelet nitric oxide and cGMP levels. The inhibition of eNOS activity and the stimulation of NADPH oxidase primed by PKC appear to be involved. PKC stimulates the eNOS phosphorylation of the negative regulatory residue thr495 and the dephosphorylation of the positive regulatory site ser1177. GF109203X and U73122, PKC and phospholipase Cgamma2 pathway inhibitors, respectively, reverse this effect. Moreover, homocysteine stimulates superoxide anion elevation and NADPH oxidase activation. These effects are significantly decreased by GF109203X and U73122, suggesting the involvement of PKC in NADPH oxidase activation. Homocysteine induces formation of the peroxynitrite biomarker nitrotyrosine. Taken together these results suggest that the homocysteine-mediated responses leading to nitric oxide impairment are mainly coupled to PKC activation. Thus homocysteine stimulates platelet aggregation and decreases nitric oxide bioavailability.

Angiotensin II exerts its cardiovascular effects mainly through the activation of AT1 receptors. These receptors can be regulated at a post-transcriptional level, that is through the modulation of the mRNA stability. This regulation usually involves proteins which are able to bind the 3'UTR of the mRNA molecule. The experiments of the present paper were performed in order to characterize the RNA-binding proteins interacting with the hAT1 receptor mRNA in human vascular smooth muscle cells. Immunoblot analysis allowed us to identify three different RNA-binding proteins, AUF1, HuR, and hnRNP A1. UV cross-linking and immunoprecipitation experiments demonstrated that AUF1 binds to the hAT1-receptor mRNA radiolabeled probes specifically, but in different ways in relation to the clinically important A/C gene polymorphism. Gel shift experiments using purified recombinant proteins confirmed the specificity of interaction of these proteins with the hAT1-receptor mRNA. In basal conditions the proteins were mainly located in the nuclei, but angiotensin II administration clearly induced their translocation to the cytosol. This observation was confirmed by transfection experiments using both GFP/AUF1 and GFP/HuR fusion proteins. Our findings allow identification of specific RNA-binding proteins possibly involved in the control of the hAT1-receptor mRNA stability and in the regulation of their expressions. Copyright © 2008 John Wiley & Sons, Ltd.

Mutations in the gene MPZ, encoding myelin protein zero (MPZ), cause inherited neuropathies collectively called Charcot-Marie-Tooth type 1B (CMT1B). Based on the age of onset, clinical and pathological features, most MPZ mutations are separable into two groups: one causing a severe, early-onset, demyelinating neuropathy and a second, causing a late-onset neuropathy with prominent axonal loss. To investigate potential pathomechanisms underlying the two phenotypes, we transiently transfected HeLa cells with two late-onset (T95M, H10P) and two early-onset (H52R, S22_W28 deletion) mutations and analyzed their effects on intracellular protein trafficking, glycosylation, cell viability and intercellular adhesion. We found that the two late-onset mutations were both transported to the cell membrane and moderately reduced MPZ-mediated intercellular adhesion. The two early-onset mutations caused two distinct abnormalities. H52R was correctly glycosylated and trafficked to the plasma membrane, but strongly affected intercellular adhesion. When co-expressed with wild-type MPZ (wtMPZ), a functional dominant negative effect was observed. Alternatively, S22_W28 deletion was retained within the cytoplasm and reduced both adhesion caused by wtMPZ and cellular viability. Since the same trafficking patterns were observed in transfected murine Schwann cells, they are not an artifact of heterologous cell expression. Our results suggest that at least some late-onset mutations cause a partial loss of function in the transfected cells, whereas multiple abnormal gain of function pathways can result in early-onset neuropathy. Further characterization of these pathways will lead to a better understanding of the pathogenesis of CMT1B and a rational basis for treating these debilitating inherited neuropathies.

The multifunctional protein high-mobility group box 1 (HMGB1) is expressed in restricted areas of adult brain where it can act as a proinflammatory cytokine. We report here that HMGB1 affects CNS transmission by inducing glutamatergic release from glial (gliosomes) but not neuronal (synaptosomes) resealed subcellular particles isolated from mouse cerebellum and hippocampus. Confocal microscopy showed that gliosomes are enriched with glia-specific proteins such as GFAP and S-100, but not with neuronal proteins such as PSD-95, MAP-2, and beta-tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin-alphaM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several approximately 30-nm nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin or ATP, by mechanisms involving extracellular Ca(2+) and Ca(2+) release from intracellular stores. HMGB1-induced release of the stable glutamate analogue [(3)H]d-aspartate and endogenous glutamate form gliosomes, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of glutamate was independent on modifications of cytosolic Ca(2+) concentration, but it was blocked by dl-threo-beta-benzyloxyaspartate, suggesting the involvement of transporter-mediated release mechanisms. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 does not block the HMGB1 effect, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. HMGB1 bind to gliosomes but not to synaptosomes and can physically interact with GLAST and receptor for advanced glycation end products (RAGE). Taken together, these results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammalian brain.