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Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Autophagy is an essential intracellular process for cellular quality control. It enables cell homeostasis through the selective degradation of harmful protein aggregates and damaged organelles. Autophagy is essential for recycling nutrients, generating energy to maintain cell viability in most tissues and during adverse conditions such as hypoxia/ischaemia. The progressive understanding of the mechanisms modulating autophagy in the vasculature has recently led numerous studies to link intact autophagic responses with endothelial cell homeostasis and function. Preserved autophagic flux within the endothelial cells has an essential role in maintaining their physiological characteristics, whereas defective autophagy can promote endothelial pro-inflammatory and atherogenic phenotype. However, we still lack a good knowledge of the complete molecular repertoire controlling various aspects of endothelial autophagy and how this is associated with vascular diseases. Here, we provide an overview of the current state-of-the-art of autophagy in endothelial cells. We review the discoveries that have so far defined autophagy as an essential mechanism in vascular biology and analyse how autophagy influences endothelial cells behaviour in vascular disease. Finally, we emphasise opportunities for compounds to regulate autophagy in endothelial cells and discuss the challenges of exploiting them to resolve vascular disease.

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol.https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER–endosome contact sites, with the consequent recruitment of endosomal fission factors.

Endothelial cell (EC) proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here we conducted high-content screening of a human miRNA mimic library to identify novel regulators of EC growth systematically. Several miRNAs were nominated that enhanced or inhibited EC growth. Of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human EC types. miR-26b overexpression enhanced EC proliferation, migration, and tube formation, while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of ECs. A combinatory functional small interfering RNA (siRNA) screening of 48 predicted gene targets revealed that miR-26b enhanced EC growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances EC proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease. Keywords: high content screen, miRNA mimic library, miR-26b, endothelial cells, cell growth

Cholesterol is an essential component of eukaryotic cellular membranes. Information about its subcellular localization and transport pathways inside cells are key for the understanding and treatment of cholesterol-related diseases. In this review we give an overview over the most commonly used methods that contributed to our current understanding of subcellular cholesterol localization and transport routes. First, we discuss methods that provide insights into cholesterol metabolism based on readouts of downstream effects such as esterification. Subsequently, we focus on the use of cholesterol-binding molecules as probes that facilitate visualization and quantification of sterols inside of cells. Finally, we explore different analogues of cholesterol which, when taken up by living cells, are integrated and transported in a similar fashion as endogenous sterols. Taken together, we highlight the challenges and advantages of each method such that researchers studying aspects of cholesterol transport may choose the most pertinent approach for their problem.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 (coronavirus disease 2019) pandemic, is a positive strand RNA (+RNA) virus. Like other +RNA viruses, SARS-CoV-2 is dependent on host cell metabolic machinery to survive and replicate, remodeling cellular membranes to generate sites of viral replication. Viral RNA-containing double-membrane vesicles (DMVs) are a striking feature of +RNA viral replication and are abundant in SARS-CoV-2–infected cells. Their generation involves rewiring of host lipid metabolism, including lipid biosynthetic pathways. Viruses can also redirect lipids from host cell organelles; lipid exchange at membrane contact sites, where the membranes of adjacent organelles are in close apposition, has been implicated in the replication of several +RNA viruses. Here we review current understanding of DMV biogenesis. With a focus on the exploitation of contact site machinery by +RNA viruses to generate replication organelles, we discuss evidence that similar mechanisms support SARS-CoV-2 replication, protecting its RNA from the host cell immune response.

Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor α (FRα) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRα selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.

Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease., Traffic of dietary cholesterol from the endocytic pathway to the ER is an essential step in cholesterol homeostasis but the mechanism of this cholesterol transport is not completely understood. Sites of contact between the membranes of endocytic and non‐endocytic organelles are emerging as important regulators of both vesicular and non‐vesicular cholesterol transport. Here, we review current understanding of these membrane contact sites and their importance in redistributing dietary cholesterol from endocytic organelles to other cellular membranes and roles in neurodegenerative disease.

Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome‐dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP‐depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF‐κB inhibition. Moreover, Tchp knock‐out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy‐mediated mechanism., The interaction of TCHP with PCM1 regulates basal autophagy through controlling GABARAP stability. Loss of TCHP function results in impaired autophagy, the accumulation of SQSTM1/p62 and endothelial dysfunction.

Introduction MicroRNAs (miRNAs) are small non-coding RNAs that orchestrate genetic networks by modulating simultaneous gene expression and regulates vascular function. Despite recent accumulation of experimental data and the emergence of functional models, the complexity of miRNA-based regulation is still far from being well understood. By performing high-content phenotypic screening (HCS), miR-26b emerged from the screen as a top candidate for ECs proliferation. The overexpression of miR-26b significantly increased EC proliferation, migration and survival. Furthermore, miR-26b mimics increased EC tube formation and branching morphogenesis. HCS and bioinformatic analysis identified both Phosphatase and Tensin homolog (PTEN), a gene critical in cell survival and Bone Morphogenetic Protein Receptor Type 1B (BMPR1B; ALK6), important in calcium homoeostasis, as direct targets of miR-26b. Because BMPR1B was identified as a direct target of mir-26b, we decided to study the role of miR-26b in vessel calcification. To test our hypothesis, miR-26b CRISPR/Cas9 global knock-out (KO) mice were generated. Objective The aim of this study is to characterize how miR-26b can influence vascular calcification and characterize ALK6 as mir-26b target. Methods In this study, we have used HCS and miR-26b KO mice and artery from patients with critical limb ischaemia to prove our hypothesis Results We confirmed that miR-26b was knocked down in the miR-26b KO mice without effecting the expression of other miR-26 family members and the host gene. To test whether miR-26b KO mice were more susceptible to calcification, we extracted the heart and aorta from 3-month-old mice and cultured for 6 days in a high phosphate media with and without warfarin. Histological analysis with the calcium stains von Kossa and alizarin red confirmed that miR-26b KO mice displayed a higher level and susceptibility to calcifying compared to control mice. Expression of the markers of calcification (RUNX2 & ALK6) supported the histology. Immunohistochemistry and qPCR confirmed that the expression of ALK6 was also increased in the miR-26b KO. To further characterise this phenotype, mice were aged and multiple tissues including the aorta were harvested from 3 and 6 month old mice. The aorta and hearts were then prepared for high resolution PET/CT using the highly calcium specific sodium fluoride (18F) probe. This probe allows for the detection of both micro and macro calcification. PET/CT supported our ex-vivo culture model and confirmed that 6-month-old miR-26b KO mice had a higher level of micro-calcification compared to control mice. To investigate the clinical relevance of miR-26b, we analysed artery and muscles from below the knee amputated limbs from patients with critical limb ischaemia. The levels of miR-26b was decreased in the muscle biopsy from the less perfused section of the amputated leg compared to more perfused counterpart. Moreover, miR-26b was downregulated in the calcified tissue portion of the artery in comparison to the soft tissue. Conclusion Taken together these findings demonstrate an important role for miR-26b in vessel calcification and suggest its effects are through the upregulation of BMPR1B; Alk6. Conflict of interest None

Autophagy is an essential cellular quality control process that emerged critical for vascular homeostasis. Here we describe, the role for Trichoplein (TCHP) protein in linking autophagy with endothelial cells (ECs) function. The depletion of TCHP in ECs impairs migration and sprouting. TCHP directly binds PCM1, to regulate degradation of GABARAP, thus leading to a defective autophagy. Mechanistically, TCHP is indispensable for autophagosome maturation and its depletion resulted in the accumulation of SQSTM1/p62 (p62) and unfolded protein aggregates in ECs. The latter process is coupled to TCHP-mediated NF-kB activation. Of note, low levels of TCHP and high p62 levels were detected in primary ECs from patients with coronary artery disease. In addition, Tchp knock-out mice showed accumulation of p62 in the heart and cardiac vessels and reduced cardiac vascularization. Here, we reveal an autophagy-mediated mechanism for TCHP down-regulation, which poses a plausible target for regulation of endothelial function.

MicroRNAs regulate endothelial function and angiogenesis, but their implication in pericyte biology remains undetermined. A PCR array, covering a panel of 379 human microRNAs, showed microRNA-532-5p to be one of the most differentially modulated by hypoxia, which was confirmed by qPCR in both skeletal muscle and adventitial pericytes. Furthermore, microRNA-532-5p was upregulated in murine muscular pericytes early after experimentally induced ischemia, decreasing below baseline after reperfusion. Transfection of human pericytes with anti-microRNA, microRNA-mimic, or controls indicates microRNA-532-5p modulates pro-angiogenic activity via transcriptional regulation of angiopoietin-1. Tie-2 blockade abrogated the ability of microRNA-532-5p-overexpressing pericytes to promote endothelial network formation in vitro. However, angiopoietin-1 is not a direct target of microRNA-532-5p. In silico analysis of microRNA-532-5p inhibitory targets associated with angiopoietin-1 transcription indicated three potential candidates, BACH1, HIF1AN, and EGLN1. Binding of microRNA-532-5p to the BACH1 3′ UTR was confirmed by luciferase assay. MicroRNA-532-5p silencing increased BACH1, while a microRNA-532-5p mimic decreased expression. Silencing of BACH1 modulated angiopoietin-1 gene and protein expression. ChIP confirmed BACH1 transcriptional regulation of angiopoietin-1 promoter. Finally, microRNA-532-5p overexpression increased pericyte coverage in an in vivo Matrigel assay, suggesting its role in vascular maturation. This study provides a new mechanistic understanding of the transcriptional program orchestrating angiopoietin-1/Tie-2 signaling in human pericytes., Graphical Abstract, Angiopoietin-1 is an important regulator of angiogenesis, yet little is known regarding its transcriptional regulation. Slater et al. (2018) demonstrate angiopoietin-1 induction in pericytes occurs via miR-532-5p regulation of BACH1 and modulation of this pathway promotes changes in endothelial permeability, vascular stability, and angiogenesis.

A experiência de encarceramento precoce, frequentemente agravada por violências impetradas, desde o momento da apreensão, tanto por agentes do Estado quanto por anônimos transeuntes e estendida ao interior de instituições públicas, é relatada por inúmeros adolescentes que cumprem medida socioeducativa em meio fechado no Rio de Janeiro. As estatísticas dessa macabra violência permanecem subestimadas. Neste perverso cenário instituído, como a Psicologia, a Psicanálise e a Arte podem contribuir para a elaboração subjetiva dos socioeducandos que vivenciaram tão poderosos traumas, bem como para a transformação de tão cruel status quo?

Resistance to drugs targeting human thymidylate synthase (TS) poses a major challenge in the field of anti-cancer therapeutics. Overexpression of the TS protein has been implicated as one of the factors leading to the development of resistance. Therefore, repressing translation by targeting the TS mRNA could help to overcome this problem. In this study, we report that the compound Hoechst 33258 (HT) can reduce cellular TS protein levels without altering TS mRNA levels, suggesting that it modulates TS expression at the translation level. We have combined nuclear magnetic resonance, UV-visible and fluorescence spectroscopy methods with docking and molecular dynamics simulations to study the interaction of HT with a region in the TS mRNA. The interaction predominantly involves intercalation of HT at a CC mismatch in the region near the translational initiation site. Our results support the use of HT-like compounds to guide the design of therapeutic agents targeting TS mRNA.

The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [d-Gln(4)]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides' cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.

// Lucia Montorsi 1 , Filippo Guizzetti 1 , Claudia Alecci 1 , Andrea Caporali 2 , Andrea Martello 2 , Claudio Giacinto Atene 1 , Sandra Parenti 1 , Silvia Pizzini 5 , Paola Zanovello 4 , Stefania Bortoluzzi 3 , Sergio Ferrari 1 , Alexis Grande 1, * , Tommaso Zanocco-Marani 1, * 1 Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 2 University/BHF Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, UK 3 Department of Molecular Medicine, University of Padova, Padova, Italy 4 Department of Surgery, Oncology and Gastroenterology (DiSCOG), University of Padova, Padova, Italy 5 Centre for Integrative Biology (CIBIO), University of Trento, Povo (Trento), Italy * These authors contributed equally to this work Correspondence to: Tommaso Zanocco-Marani, email: zanocco@unimore.it Keywords: ZFP36, tristetraprolin, colon cancer, β-catenin, epithelial mesenchymal transition Received: March 21, 2016 Accepted: July 09, 2016 Published: July 24, 2016 ABSTRACT The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/β-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.

Demonstrating a candidate drug’s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.

ZFP36L1 negatively regulates erythroid differentiation of human hematopoietic progenitors by directly binding the 3′ UTR of Stat5b mRNA, thereby triggering its degradation. This study shows that posttranscriptional regulation is involved in the control of hematopoietic differentiation., ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3′-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by posttranscriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3′ untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, because cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.

Critical limb ischaemia (CLI) is a serious form of peripheral artery disease where patients suffer from reduced arterial blood flow and subsequent loss of tissue viability. Therapeutic angiogenesis uses pro-angiogenic factors to stimulate new blood vessels formation to bypass existing blockages. Despite promising pre-clinical data, the outcome of clinical trials using single proangiogenic growth factors remains inconclusive. MicroRNAs (miRNAs) are small non-coding RNAs that orchestrate genetic networks by modulating simultaneous gene expression, thus suitable for therapeutic regulation of post-ischaemic angiogenesis. The aim of this study is to identify novel miRNAs that can influence vascular endothelial cells (EC) function and identify their relevant target genes. High content screening (HCS) of human miRNA mimic library is an emerging approach that utilises robotic microscopy platforms along with unbiased image analysis algorithms to generate quantitative data. Therefore, we used HCS to identify miRNAs that regulate EC proliferation and establish their potential as a therapeutic target for CLI. High throughput screening of over 1500 unique microRNA mimics revealed miR-26b as a top candidate for regulating EC functions. The overexpression of miR-26b significantly increased the proliferation and migration of ECs. Furthermore, it increased cell survival and importantly enhanced EC tube formation and branching morphogenesis. Stimulating ECs with the growth factor VEGFA increased EC proliferation associated with increased miR-26b expression. Expression analysis revealed an increase in endothelial cell tip enrichment genes including FLT4, ESM1, Ang-2 and PDGFB in EC overexpressing miR-26b. In-vivo, Q-PCR analysis confirmed an increase level of miR-26b expression in EC sorted form limb muscles 3 days post-ischaemia. Furthermore, we tested the effects on in vivo angiogenesis using the Matrigel implant model. Histological analysis at day 10 with the implants confirmed miR-26b mimics increased the vessel density. After confirming the important pro-survival and pro-angiogenic role miR-26b has in ECs, we used HCS to identify downstream target genes. We found that Phosphatase and Tensin homolog (PTEN) and phosphatase gene PPP2R2A are direct targets of miR-26b and their regulation determined the phenotype of miR-26b in ECs. MicroRNA-26b was for the first time identified as a pro-survival and pro-angiogenic factor for EC by the use of novel HCS techniques. We believe miR-26b is an excellent therapeutic target for the treatment of CLI and we continue to investigate and characterise its role in post-ischaemic angiogenesis.

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The d-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

RNA binding proteins belonging to the TIS11/TTP gene family regulate the stability of multiple targets. Their inactivation or deregulated expression has recently been related to cancer, and it has been suggested that they are capable of displaying tumor suppressor activities. Here we describe three new targets of ZFP36 (PIM-1, PIM-3 and XIAP) and show by different approaches that its ectopic expression is capable of impairing glioblastoma cell lines viability and invasiveness by interfering with different transduction pathways. Moreover, we provide evidence that compounds capable of inducing the expression of TIS11/TTP genes determine a comparable biological effect on the same cell contexts.

Transcription Factor for Immunoglobulin Heavy-Chain Enhancer 3 (Tfe3) is a transactivator of metabolic genes that are regulated through an EBox located in their promoters. It is involved in physiological processes such as osteoclast and macrophage differentiation, as well as in pathological processes such as translocations underlying different cancer diseases. MAFB is a basic region/leucine zipper transcription factor that affects transcription by binding specific DNA regions known as MARE. It plays a pivotal role in regulating lineage-specific hematopoiesis by repressing transcription of erythroid specific genes in myeloid cells and enhancing expression of macrophage and megakaryocytic genes. Here we have shown MAFB to be highly induced in human hematopoietic cells undergoing macrophage differentiation following Tfe3 ectopic expression, and to be down regulated, compared to the controls, in the same cell population following Phorbol Esters (PMA) dependent differentiation coupled to Tfe3 gene silencing. Electrophoretic mobility shift assays identified a Tfe3-binding site (EBox) in the MAFB promoter region that is conserved in different mammalian species. MAFB promoter was transactivated by co-expression of Tfe3 in reporter gene assays while deletion or mutation of the MAFB EBox prevented transactivation by Tfe3. Both of these genes were previously included in the group of transcription factors able to drive macrophage differentiation. The observation that MAFB belongs to the Tfe3 regulon suggests the existence of a pathway where these two gene families act synergistically to determine differentiation.

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D3 receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D3/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

Background ZFP36 is an mRNA binding protein that exerts anti-tumor activity in glioblastoma by triggering cell death, associated to an increase in the stability of the kinase RIP1. Methods We used cell death assays, size exclusion chromatography, Co-Immunoprecipitation, shRNA lentivectors and glioma neural stem cells to determine the effects of ZFP36 on the assembly of a death complex containing RIP1 and on the induction of necroptosis. Results Here we demonstrate that ZFP36 promotes the assembly of the death complex called Ripoptosome and induces RIP1-dependent death. This involves the depletion of the ubiquitine ligases cIAP2 and XIAP and leads to the association of RIP1 to caspase-8 and FADD. Moreover, we show that ZFP36 controls RIP1 levels in glioma neural stem cell lines. Conclusions We provide a molecular mechanism for the tumor suppressor role of ZFP36, and the first evidence for Ripoptosome assembly following ZFP36 expression. These findings suggest that ZFP36 plays an important role in RIP1-dependent cell death in conditions where IAPs are depleted. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1388-5) contains supplementary material, which is available to authorized users.