Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

BACKGROUND: REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. METHODS AND RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's t test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. CONCLUSIONS: If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

Stroke by inducing HDAC9-dependent deacetylation of HIF-1 and Sp1, promotes TfR1 transcription and GPX4 reduction, thus determining ferroptotic neuronal death.

Background: The inhibition of histone deacetylase 9 (HDAC9) represents a promising druggable target for stroke intervention. Indeed, HDAC9 is overexpressed in neurons after brain ischemia where exerts a neurodetrimental role. However, mechanisms of HDAC9-dependent neuronal cell death are not yet well established. Methods: Brain ischemia was obtained in vitro by primary cortical neurons exposed to glucose deprivation plus reoxygenation (OGD/Rx) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcript and protein levels. Chromatin immunoprecipitation was used to evaluate the binding of transcription factors to the promoter of target genes. Cell viability was measured by MTT and LDH assays. Ferroptosis was evaluated by iron overload and 4-hydroxynonenal (4-HNE) release. Results: Our results showed that HDAC9 binds to hypoxia-inducible factor 1 (HIF-1) and specificity protein 1 (Sp1), two transcription activators of transferrin 1 receptor (TfR1) and glutathione peroxidase 4 (GPX4) genes, respectively, in neuronal cells exposed to OGD/Rx. Consequently, HDAC9 induced: (1) an increase in protein level of HIF-1 by deacetylation and deubiquitination, thus promoting the transcription of the pro-ferroptotic TfR1 gene; and (2) a reduction in Sp1 protein levels by deacetylation and ubiquitination, thus resulting in a down-regulation of the anti-ferroptotic GPX4 gene. Supporting these results, the silencing of HDAC9 partially prevented either HIF-1 increase and Sp1 reduction after OGD/Rx. Interestingly, silencing of the neurodetrimental factors, HDAC9, HIF-1, or TfR1 or the overexpression of the prosurvival factors Sp1 or GPX4 significantly reduced a well-known marker of ferroptosis 4-HNE after OGD/Rx. More important, in vivo, intracerebroventricular injection of siHDAC9 reduced 4-HNE levels after stroke by preventing: (1) HIF-1 and TfR1 increase and thus the augmented intracellular iron overload; and (2) a reduction of Sp1 and its target gene GPX4. Conclusions: Collectively, results obtained suggest that HDAC9 mediates post-traslational modifications of HIF-1 and Sp1 that, in turn, increases TfR1 and decreases GPX4 expression, thus promoting neuronal ferroptosis in in vitro and in vivo models of stroke.

Emerging Role of DREAM in Healthy Brain and Neurological Diseases.

The downstream regulatory element antagonist modulator (DREAM) is a multifunctional Ca2+-sensitive protein exerting a dual mechanism of action to regulate several Ca2+-dependent processes. Upon sumoylation, DREAM enters in nucleus where it downregulates the expression of several genes provided with a consensus sequence named dream regulatory element (DRE). On the other hand, DREAM could also directly modulate the activity or the localization of several cytosolic and plasma membrane proteins. In this review, we summarize recent advances in the knowledge of DREAM dysregulation and DREAM-dependent epigenetic remodeling as a central mechanism in the progression of several diseases affecting central nervous system, including stroke, Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and neuropathic pain. Interestingly, DREAM seems to exert a common detrimental role in these diseases by inhibiting the transcription of several neuroprotective genes, including the sodium/calcium exchanger isoform 3 (NCX3), brain-derived neurotrophic factor (BDNF), pro-dynorphin, and c-fos. These findings lead to the concept that DREAM might represent a pharmacological target to ameliorate symptoms and reduce neurodegenerative processes in several pathological conditions affecting central nervous system.

The Transcriptional Complex Sp1/KMT2A by Up-Regulating Restrictive Element 1 Silencing Transcription Factor Accelerates Methylmercury-Induced Cell Death in Motor Neuron-Like NSC34 Cells Overexpressing SOD1-G93A.

Methylmercury (MeHg) exposure has been related to amyotrophic lateral sclerosis (ALS) pathogenesis and molecular mechanisms of its neurotoxicity has been associated to an overexpression of the Restrictive Element 1 Silencing Transcription factor (REST). Herein, we evaluated the possibility that MeHg could accelerate neuronal death of the motor neuron-like NSC34 cells transiently overexpressing the human Cu2+/Zn2+superoxide dismutase 1 (SOD1) gene mutated at glycine 93 (SOD1-G93A). Indeed, SOD1-G93A cells exposed to 100 nM MeHg for 24 h showed a reduction in cell viability, as compared to cells transfected with empty vector or with unmutated SOD1 construct. Interestingly, cell survival reduction in SOD1-G93A cells was associated with an increase of REST mRNA and protein levels. Furthermore, MeHg increased the expression of the transcriptional factor Sp1 and promoted its binding to REST gene promoter sequence. Notably, Sp1 knockdown reverted MeHg-induced REST increase. Co-immunoprecipitation experiments demonstrated that Sp1 physically interacted with the epigenetic writer Lysine-Methyltransferase-2A (KMT2A). Moreover, knocking-down of KMT2A reduced MeHg-induced REST mRNA and protein increase in SOD1-G93A cells. Finally, we found that MeHg-induced REST up-regulation triggered necropoptotic cell death, monitored by RIPK1 increased protein expression. Interestingly, REST knockdown or treatment with the necroptosis inhibitor Necrostatin-1 (Nec) decelerated MeH-induced cell death in SOD1-G93A cells. Collectively, this study demonstrated that MeHg hastens necroptotic cell death in SOD1-G93A cells via Sp1/KMT2A complex, that by epigenetic mechanisms increases REST gene expression.

Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry.

BACKGROUND: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb's epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. METHODS: We generated a mAb against PRAME immunizing mice with PRAME fragment 161-415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. RESULTS: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202-212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. CONCLUSIONS: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody's epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

Development of conformational antibodies targeting Cripto-1 with neutralizing effects in vitro.

Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein. In this work, we report the generation and characterization of murine monoclonal antibodies raised against the synthetic folded CFC [112-150] domain of the human protein. Through subtractive ELISA assays clones were screened for the ability to specifically recognize "hot spot" residues on the CFC domain, which are crucial for the interaction with Activin Type I receptor (ALK4) and GRP78. On selected antibodies, SPR and epitope mapping studies have confirmed their specificity and have revealed that recognition occurs only on a conformational epitope. Furthermore, FACS analyses have confirmed the ability of 1B4 antibody to recognize the membrane-anchored and soluble native Cripto-1 protein in a panel of human cancer cells. Finally, we have evaluated its functional effects through in vitro cellular signaling assays and cell cycle analysis. These findings suggest that the selected anti-CFC mAbs have the potential to neutralize the protein oncogenic activity and may be used as theranostic molecules suitable as tumor homing agents for Cripto-1-overexpressing cancer cells and tissues and to overcome drug-resistance in routine cancer therapies.

Triticum vulgare extract exerts an anti-inflammatory action in two in vitro models of inflammation in microglial cells.

Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The specific extract of Triticum vulgare manufactured by Farmaceutici Damor (TVE-DAMOR) is already present in some pharmaceutical formulations used in the treatment of decubitus ulcers, skin lesions and burns. It has been recently suggested that this Triticum vulgare extract may possess potential anti-inflammatory properties. In the light of these premises the aim of the present paper was to verify the anti-inflammatory role of TVE, using the LPS-stimulated microglia model of inflammation. In particular the effect of different concentrations of TVE on the release of several mediators of inflammation such as nitric oxide, IL-6, PGE2 and TNF alpha was evaluated. More important, the anti-inflammatory effect of TVE was confirmed also in primary rat microglia cultures. The results of the present study show that TVE exerts anti-inflammatory properties since it reduces the release of all the evaluated markers of inflammation, such as NO, IL6, TNF alpha and PGE2 in LPS-activated BV2 microglial cells. Intriguingly, TVE reduced microglia activation and NO release also in primary microglia. Indeed, to verify the pathway of modulation of the inflammatory markers reported above, we found that TVE restores the cytoplasmic expression of p65 protein, kwown as specific marker associated with activation of inflammatory response. The evidence for an inhibitory activity on inflammation of this specific extract of Triticum vulgare may open the way to the possibility of a therapeutical use of the Triticum vulgare extract as an anti-inflammatory compound in certain pathological states such as burns, decubitus ulcers, folliculitis and inflammation of peripheral nerve.

The chemokine scavenging receptor D6/ACKR2 is a target of miR-146a in thyroid cancer.

We have previously shown that miR-146a, a NF-κB-regulated microRNA, is strongly expressed in human specimens and cell lines derived from anaplastic thyroid carcinomas (ATC) where it mediates some of the NF-κB pro-tumorigenic functions. By using a bioinformatic analysis, we identified the chemokine scavenger receptor D6/ ACKR2 as a target of miR146a in human ATC. We found that the expression of D6/ ACKR2 was up-regulated in miR-146a-null ATC cell lines and that the 3' UTR of D6/ ACKR2 mRNA was able to inhibit its expression in parental, but not in miR-146a-null ATC cells. Since human specimens from primary ATC showed a low expression of D6/ ACKR2 compared to normal thyroid tissues, we analyzed the effects of D6/ACKR2 over-expression in ATC cells. Different chemokines added to the conditioned medium of D6/ACKR2 over-expressing ATC cells partially failed to drive in vitro monocyte migration, and tumors derived from the injection of the same cells in nude mice showed a decreased number of infiltrating macrophages. Taken together, these results indicate that ATC cells down-regulate D6/ACKR2 expression through miR-146a activity to sustain leukocyte trafficking inside tumor microenvironment and shed light on a novel mechanism by which NF-κB indirectly inhibits the expression and the function of anti-tumorigenic gene in thyroid cancer.

Monoclonal antibodies against pools of mono- and polyacetylated peptides selectively recognize acetylated lysines within the context of the original antigen.

Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24-39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

UNLABELLED: The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a ß-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE: Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a ß-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

Effects of a novel Nodal-targeting monoclonal antibody in melanoma.

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

New Anti-Nodal Monoclonal Antibodies Targeting the Nodal Pre-Helix Loop Involved in Cripto-1 Binding.

Nodal is a potent embryonic morphogen belonging to the TGF-ß superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44-67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (KD 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.

Oligosaccharidic fractions derived from Triticum vulgare extract accelerate tissutal repairing processes in in vitro and in vivo models of skin lesions.

ETHNOPHARMACOLOGICAL RELEVANCE: Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The aqueous extract of Triticum vulgare (TVE) is currently an active component used by Farmaceutici Damor in the manufacture of certain pharmaceutical products already marketed in Italy and abroad under the brand name Fitostimoline(®), in the formulation of cream and medicated gauze and is commonly used for the treatment of decubitus ulcers, sores, burns, scarring delays, dystrophic diseases, and, more broadly, in the presence of problems relating to re-epithelialization or tissue regeneration. The active components of Fitostimoline(®)-based products determine a marked acceleration of tissutal repairing processes, stimulate chemotaxis and the fibroblastic maturation, and significantly increase the fibroblastic index, which are crucial points in the repairing processes. The aim of the present paper was to identify and characterize the active fractions of TVE responsible for the pharmacological effect in tissutal repairing processes. MATERIALS AND METHODS: Several fractions obtained from TVE by ultrafiltration procedures and HPAE chromatography were tested to measure their growth-enhancing activity on NIH-3T3 fibroblasts. The healing action of the same fractions, prepared as cream formulation, was assessed in rat subjected to two different models of skin lesion, skin scarification and excision. RESULTS: Our results showed a pro-proliferative effect of the fractions ST-98 and K>1000 in NIH-3T3 fibroblasts. Moreover these fractions formulated as cream preparations were effective also in in vivo models of skin lesion. CONCLUSIONS: The results of the present study showed that these active fractions of TVE are responsible for its pro-proliferative effect.

NGAL controls the metastatic potential of anaplastic thyroid carcinoma cells.

CONTEXT: We have previously identified neutrophil gelatinase-associated lipocalin (NGAL) as one of the genes mediating the oncogenic activity of nuclear factor-κB in human anaplastic thyroid carcinomas (ATCs). OBJECTIVES: To further investigate the role of NGAL in thyroid cancer, we established NGAL knocked-down and NGAL overexpressing ATC cell lines. RESULTS: We found that the ability of NGAL knocked-down cells to degrade Matrigel in a transwell invasion assay and to form lung metastasis in nude mice was decreased. Because NGAL binds matrix metalloproteinase-9 (MMP-9), to form a macromolecular complex involved in the regulation of metastatic spread of cancer cells and given the strong expression of both genes in tissue specimens from human ATCs, we analyzed the MMP-9 enzymatic activity in NGAL-null ATC cells. Enzymatic immunoassays show that MMP-9 activity is reduced in NGAL-null ATC cells, even if its expression is not affected by NGAL inhibition. Ectopic expression of NGAL in an ATC cell line not expressing NGAL determines an increase of its metastatic property. The use of a mutated form of NGAL, unable to bind MMP-9, has no positive effect on the invasive potential of ATC cells and does not improve the MMP-9 enzymatic activity. CONCLUSIONS: Our results indicate NGAL as a novel target of nuclear factor-κB prometastatic activity in thyroid cancer through enhancement of MMP-9 enzymatic activity.