The Expression of the PDZ Protein MALS-1/Velis Is Regulated by Calcium and Calcineurin in Cerebellar Granule Cells

Activity-dependent gene expression is thought to be important in shaping neuronal development and in modifying the protein content of neurons. Ca(2+) entry into neurons appears to be one of the key effectors of activity-dependent gene expression. Among the possible downstream targets of calcium, the protein phosphatase calcineurin represents a prime candidate. We hereby report that in cultured cerebellar granule cells the activation of the Ca(2+)/calcineurin pathway via either voltage- or ligand- operated Ca(2+) channels regulates MALS-1 and MALS-2 expression at the transcriptional level. These proteins are integral parts of the post-synaptic density and are also involved in receptor trafficking. MALS regulation is not at the level of mRNA stability and does not require de novo protein synthesis, thereby suggesting a direct pathway. These data suggest that Ca(2+) entry by means of calcineurin is capable of controlling the structure of the post-synaptic density by controlling the expression of key components at the transcriptional level

The Oxidative state of LDL is the major determinant of anti/prooxidant effect of coffee on Cu²⁺ catalysed peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

The Oxidative State of LDL is the Major Determinant of Anti/Prooxidant Effect of Coffee on Cu2+ Catalysed Peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

Development of polymeric microbubbles targeted to prostate-specific membrane antigen as prototype of novel ultrasound contrast agents

Ultrasound-targeted microbubbles (MBs) offer new opportunities to enhance the capabilities of diagnostic ultrasound (US) imaging to specific pathological tissue. Herein, we report on the design and development of a novel prototype of US contrast agent based on polymeric MBs targeted to prostate-specific membrane antigen (PSMA) for use in the diagnosis of prostate cancer (PCa). First, a set of air-filled MBs by a variety of biocompatible polymers were prepared and characterized in terms of morphology and echogenic properties after exposure to US. MBs derived from poly(d,l-lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG) copolymer resulted as the most effective in terms of reflectivity. Such polymer was therefore preconjugated with a urea-based PSMA inhibitor molecular probe (DCL), and the obtained MBs were investigated in vitro for their targeting efficacy toward PSMA positive PCa (LNCaP) cells. Fluorescence microscopy proved a specific and efficient adhesion of targeted MBs to LNCaP cells. To our knowledge, this work reports the first model of polymeric MBs appropriately engineered to target PSMA, which might be further optimized and used for PCa diagnosis and potential carriers for selective drug delivery

Direct and Indirect Interactions between Calcineurin-NFAT and MEK1-Extracellular Signal-Regulated Kinase 1/2 Signaling Pathways Regulate Cardiac Gene Expression and Cellular Growth

MEK1, a member of the mitogen-activated protein kinase (MAPK) cascade that directly activates extracellular signal-regulated kinase (ERK), induces cardiac hypertrophy in transgenic mice. Calcineurin is a calcium-regulated protein phosphatase that also functions as a positive regulator of cardiac hypertrophic growth through a direct mechanism involving activation of nuclear factor of activated T-cell (NFAT) transcription factors. Here we determined that calcineurin-NFAT and MEK1-ERK1/2 signaling pathways are interdependent in cardiomyocytes, where they directly coregulate the hypertrophic growth response. For example, genetic deletion of the calcineurin Aβ gene reduced the hypertrophic response elicited by an activated MEK1 transgene in the heart, while inhibition of calcineurin or NFAT in cultured neonatal cardiomyocytes also blunted the hypertrophic response driven by activated MEK1. Conversely, targeted inhibition of MEK1-ERK1/2 signaling in cultured cardiomyocytes attenuated the hypertrophic growth response directed by activated calcineurin. However, targeted inhibition of MEK1-ERK1/2 signaling did not directly affect calcineurin-NFAT activation, nor was MEK1-ERK1/2 activation altered by targeted inhibition of calcineurin-NFAT. Mechanistically, we show that MEK1-ERK1/2 signaling augments NFAT transcriptional activity independent of calcineurin, independent of changes in NFAT nuclear localization, and independent of alterations in NFAT transactivation potential. In contrast, MEK1-ERK1/2 signaling enhances NFAT-dependent gene expression through an indirect mechanism involving induction of cardiac AP-1 activity, which functions as a necessary NFAT-interacting partner. As a second mechanism, MEK1-ERK1/2 and calcineurin-NFAT proteins form a complex in cardiac myocytes, resulting in direct phosphorylation of NFATc3 within its C terminus. MEK1-ERK1/2-mediated phosphorylation of NFATc3 directly augmented its DNA binding activity, while inhibition of MEK1-ERK1/2 signaling reduced NFATc3 DNA binding activity. Collectively, these results indicate that calcineurin-NFAT and MEK1-ERK1/2 pathways constitute a codependent signaling module in cardiomyocytes that coordinately regulates the growth response through two distinct mechanisms

NAMI-A inhibits the PMA-induced ODC gene expression in ECV304 cells: involvement of PKC/Raf/Mek/ERK signalling pathway

Imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate (NAMI-A) is a new compound active against lung metastasis of solid metastasizing tumours. While its in vivo effect has been studied, the molecular insights that underlie its action are largely unknown. Among the possible pathways responsible for malignant transformation, PKC arose as one of the most promising targets for new antineoplastic drugs. We demonstrated the capability of NAMI-A of inhibiting PMA induced-PKC activity in ECV304 in a dose-dependent fashion. Furthermore, NAMI-A through modulation of PKC activity has been proved capable of reducing the phorbol ester induced expression of ornithine decarboxilase (ODC) gene and to abrogate the activation of the Raf/MEK/ERK pathway. Taken together these results suggest that many of the in vivo outcomes of NAMI-A treatment may be the result of a direct action on PKC

PKC/Raf/MEK/ERK signaling pathway modulates native-LDL-induced E2F-1 gene expression and endothelial cell proliferation

Background and objectives: The interactions of low-density lipoprotein (LDL) with the endothelium are thought to play a major role in the development of atherosclerosis. Due to this reason, the molecular sequelae of events resulting from native LDL (N-LDL) interaction with human endothelial cells (HECs) are largely under investigation. Methods and results: Here, we report that the exposure of serum-free HECs to different concentrations of N-LDL-cholesterol (LDL-chol) elicited a time- and dose-dependent induction of DNA synthesis. The exposure of serum-free HECs to N-LDL was able to elicit a time- and dose-dependent increase of protein kinase C (PKC) activity that, along with the activation of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, leads to an increase in E2F-1 gene expression. In addition, the treatment of HECs with N-LDL was also able to induce both E2F-1 gene transcription and protein expression. These N-LDL-aroused responses were dramatically counteracted by PKC inhibition or down regulation. Similarly to what observed for Raf/MEK/ERK activation and E2F-1 gene expression, the inhibition of PKC as well as its down regulation, significantly lowered the DNA synthesis induced by N-LDL in serum-free HECs. Conclusions: These results suggest that the activation of PKC/Raf/MEK/ERK-mediated events controlling E2F-1 gene expression by N-LDL may represent an important mechanism in the regulation of HECs proliferation during normal and pathological processes

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-<i>myc</i> gene expression and endothelial cell proliferation

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation

The Anti-metastatic agent imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate induces endothelial cell apoptosis by inhibiting the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway

Imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate (NAMI-A) is a new ruthenium compound active against lung metastasis in vivo and tumor cell invasion in vitro. Since angiogenesis was recognized as a key event in the metastasizing process, the manipulation of neo-vessel formation has been developed as a new therapeutic approach. Within this context, a pivotal role for apoptosis in regulating cellular growth has been proposed. In the present study, we exposed to NAMI-A the spontaneously transformed human endothelial cell line ECV304 and assessed a number of apoptosis-related features, including the DNA degradation rate, the activation of caspase-3 protease, the expression of Hsp27, and the release of cytochrome c. Cell treatment with NAMI-A elicited a significant increment in the apoptotic response, as indicated by DNA fragmentation and caspase-3 activation, two classical hallmarks of cellular suicide. Furthermore, NAMI-A was able to down-regulate Hsp27 protein expression and provoke the release of mitochondrial cytochrome c in the cytosol. Here, we analyze the involvement of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signal transduction pathway in the induction of apoptosis elicited by NAMI-A. Such a response was associated with a marked inhibition of MAPK/ERK kinase (MEK) and ERK phosphorylation with a time course and dose dependency overlapping those observed throughout NAMI-A-induced apoptosis. In addition, we report that PD98059, a selective MEK inhibitor, is able to induce apoptosis by itself in the ECV304 cell line. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating an apoptotic event in ECV304

Akt downregulation by flavin oxidase–induced ROS generation mediates dose-dependent endothelial cell damage elicited by natural antioxidants

High intake of natural antioxidants (NA) from plant-derived foods and beverages is thought to provide cardiovascular benefits. The endothelium plays a pivotal role in cardiovascular homeostasis, and for this reason, the molecular events resulting from NA actions on endothelial cells (ECs) are actively investigated. Here, we show the direct impact of two NA, coumaric acid and resveratrol, on intracellular reactive oxygen species levels, protein carbonylation, and cell physiology in human ECs. While at lower doses, both NA promoted antioxidant effects, at moderately high doses, NA elicited a dose-dependent pro-oxidant effect, which was followed by apoptosis, cell damage, and phospho-Akt downregulation. NA-induced pro-oxidant effects were counteracted by N-acetyl cysteine and diphenyleneiodonium (DPI), suggesting a role for flavin oxidases in NA-induced toxicity. DPI also prevented NA-induced phospho-Akt downregulation indicating that Akt can work downstream of flavin oxidases in mediating cellular responses to NA. Stimulation of phospho-Akt by insulin dramatically counteracted NA-induced cell death, an effect abolished by Akt inhibition further suggesting that mechanistically Akt regulates cell survival in response to NA-induced stress. Although further studies are required to better characterize the molecular mechanism of NA-induced cell toxicity, our study is the first to show in a human vascular model that moderately high doses of NA can induce cell damage mediated by flavoproteins and the Akt pathway