51 research outputs found
Developing rat brain as well as cultured astrocytes contain H1° mRNA-protein complexes
No full textRNA-binding proteins (RBPs) regulate intracellular transport, pre-localization, stability, and translation of mRNAs [1].
We previously identified a set of proteins which interact with mRNAs encoding H1° and H3.3 histones [2-5]. All these proteins are probably part of a ribonucleoprotein particle [6]. Here we report the results of a more detailed study on the expression and intracellular localization of some of these RBPs, such as hnRNP K and A1, and Hsc70, during rat brain development and in cultured rat astrocytes. We also investigated the presence in the complexes of PIPPin/CSD-C2 protein.
Affinity chromatography was performed as already described [6]. Preparation of total lysates and cellular sub-fractions was done as reported in [3]. Possible co-localization of Hsc70 with CSD-C2 in cultured astrocytes was analysed by immunofluorescence microscopy.
The presence of Hsc70 chaperone in the already identified ribonucleoprotein complex [6] was confirmed by affinity chromatography. We also found that the complex itself is present not only in the nuclear extracts, but also in the cytoplasmic fraction. Moreover, A1 hnRNP, previously found in the complexes, was found to be differentially expressed and localized during rat brain maturation. In particular, we found that nuclear levels of A1 increase in cultured astrocytes grown on a fibronectin-containing substrate, but decrease when cells are fed with Maat medium [7].
We finally report that sumoylated PIPPin, already found in neurons, is also present in the nuclei of cultured astrocytes.
[1] Di Liegro et al. 2014, Int J Mol Med 33:747-62.
[2] Scaturro et al. 1998, J Biol Chem 273:22788-91.
[3] Nastasi et al. 1999, J Biol Chem 274:24087-93.
[4] Sala et al. 2007, Int J Mol Med 19:501-9.
[5] Saladino et al. 2012, Int J Mol Med 29:141-5
[6] Di Liegro et al. 2013, Neuroscience 229:71-6.
[7] Cestelli et al., 1985, Brain Res 354:219-27
Potential roles of extracellular vesicles in brain cell-to-cell communication
No full textPotential roles of extracellular vesicles in brain cell-to-cell communication
Extracellular vesicles (EVs) are released into thè extracellular space from both cancer and normal
brain cells, and are probably able to modify thè phenotypic properties of receiving cells1. EVs
released from astrocytes and neurons contain FGF2 and VEGF2'3 and induce a 'blood-brain
barrier' (BBB) phenotype in cultured brain capillary endothelial cells (BCECs, unpublished results),
On thè other hand, EVs from G26/24 oligodendroglioma induce apoptosis in neurons and
astrocytes4-5. These effects are probably due to Fas Ligand and TRAIL, present in G26/24
vesicles4-5. Moreover, G26/24 EVs contain extracellular matrix remodeling proteases (such as
ADAMTS)6, H1.0 histone protein, and H1.0 mRNA7. In particular, we previously hypothesized that
G26/24 cells, and tumor cells in generai, can escape differentiation cues, and continue to
proliferate by eliminating proteins, such as thè H1° linker histone (and its mRNA)7, which could
otherwise block proliferation.
To study vesicle release in a System that can better resemble in vivo conditions, astrocytes and
BCECs were cultured on poly-L-lactic acid (PLLA) scaffolds and tested for their ability to grow and
survive on this three-dimensional structures. We analyzed in parallel thè celi growth in 2D and 3D
culture systems and observed thè differences in celi morphology by fluorescence analysis: threedimensional
scaffolds have thè ability to guide celi growth, provide support, encourage celi
adhesion and proliferation. Astrocytes8 and BCECs (unpublished results) adapted well to these
porous matrices, not only remaining on thè surface, but also penetrating inside thè scaffolds.
EVs released by astrocytes in these scaffolds are probably exosomes, as suggested by
transmission electron microscopy pictures, and by thè presence of intracellular structures
resembling multivesicular bodies. This 3D celi culture System could be further enriched to host
different brain celi types, in order to set, for example, an in vitro model of BBB, that may be useful
for drug delivery studies, and for thè formulation of new therapeutic strategies for thè treatment of
neurological diseases.
References
[1] Schiera, G., Di Liegro, C.M., Di Liegro I. Int J Mol Sci. 2017, 18(12). pii: E2774.
[2] Schiera, G., Proia, P., Alberti, C., Mineo, M., Savettieri, G., Di Liegro, I., 2007. J Celi Mol Med. 2007,
111(6), 1384-94.
[3] Proia, P., Schiera, G., Mineo, M., Ingrassia, A.M. Santoro, G., Savettieri, G., Di Liegro, I. Int J Mol Med.
2008, 21(1), 63-7.
[4] D'Agostino, S., Salamene, M., Di Liegro, I., Vittorelli, ML, Int J Oncol. 2006, 29(5), 1075-85.
[5] Lo Cicero, A., Schiera, G., Proia, P., Saladino, P., Savettieri, G., Di Liegro, C.M., Di Liegro, I. Int J Oncol.
2011,39(6): 1353-7.
[6] Lo Cicero, A., Majkowska, I., Nagase, H., Di Liegro, I., Troeberg, L., Matrix Biol. 2012, 31(4), 229-33.
[7] Schiera, G., Di Liegro, C.M., Saladino, P., Pitti, R., Savettieri, G., Proia, P., Di Liegro, I. Int J Oncol. 2013,
43(6), 1771-6.
[8] Carfì Pavia, F., Di Bella, M.A., Brucato, V., Blanda, V., Zummo, F., Vitrano, I., Di Liegro, C.M., Ghersi, G.,
Di Liegro, I., Schiera, G. Mol Med Rep. 2019 [Epub ahead of print].
[9] Di Bella MA, Zummo F., Carfì Pavia F., Brucato V., Di Liegro I., Schiera G. 2017, In: Microscopy and
Imaging Science: practical approaches to applied research and education, pp 260-264. Ed: A. Méndez-Vilas
Publisher, Formatex Research Center (Spain), ISBN-13, 978-84-942134-9-6
H1° mRNA-containing complexes in rat brain cells. In: Proceedings of the Abstracts
No full textPost-transcriptional regulation of gene expression depends on RNA-binding proteins (RBPs), which are able to regulate translation, stability and subcellular localization of mRNAs [1]. RNA-protein complexes start to be built up since transcription; some proteins remain then bound to the transcript, while others behave as only transient components. In the developing nervous system of mammals, the postnatal production of the histone variants H1° and H3.3 is mainly regulated at the post-transcriptional level. Synthesis and incorporation into chromatin of the two histone proteins has been suggested to be involved in the epigenetic regulation of gene expression, both in normal brain development and brain cancer.
We previously identified a set of proteins which interact with mRNAs encoding H1° and H3.3 mRNA [2-5]. Recently, we demonstrated that these proteins are probably part of a ribonucleoprotein particle which contains both unspecific and specific RBPs [6]. Here we confirm, by affinity chromatography, the presence of Hsc70 chaperone in the complex and, in addition, we report that the complex is present not only in the nuclear extracts, as previously shown, but also in a membrane-containing cytoplasmic pellet, obtained by centrifugation at 105.000 x g (P105). In parallel, we demonstrated that sumoylated PIPPin, already found in neurons, is present also in the nuclei of cultured astrocytes. Finally, we found that two of the hnRNPs previously found in the complexes show a differential expression and localization during rat brain maturation.
[1] Di Liegro CM et al. 2014, Int J Mol Med 33:747-62.
[2] Scaturro M et al. 1998, J Biol Chem 273:22788-91.
[3] Nastasi T et al. 1999, J Biol Chem 274:24087-93.
[4] Sala A et al. 2007, Int J Mol Med 19:501-9.
[5] Saladino P et al. 2012, Int J Mol Med 29:141-5
[6] Di Liegro CM et al. 2013, Neuroscience 229:71-6
Expression and intracellular localization of H1° mRNA-containing complexes in developing rat brain and astrocytes
No full textINTRODUCTION: Post-transcriptional regulation of gene expression relies on RNA-binding proteins
(RBPs), which regulate intracellular transport, stability, and translation of mRNAs [1]. We previously identified
a set of proteins which interact with mRNAs encoding H1° and H3.3 histones [2-5]. All these proteins
are probably part of a ribonucleoprotein particle [6]. Here we report more details on the expression and
intracellular localization of some of these RBPs, during rat brain development and in isolated rat astrocytes.
METHODS: Affinity chromatography was performed as already described [6]. Preparation of total lysates
and cellular sub-fractions was done as reported in [3]. Possible co-localization of Hsc70 with CSD-C2 in cultured
astrocytes was analysed by immunofluorescence microscopy.
RESULTS: The presence of Hsc70 chaperone in the already identified ribonucleoprotein complex [6] was
confirmed by affinity chromatography. We also found that the complex itself is present not only in the nuclear
extracts, but also in the cytoplasmic fraction. Moreover, A1 and K hnRNPs, previously found in the complexes,
were found to be differentially expressed and localized during rat brain maturation; an increase in A1
expression was also demonstrated in cultured astrocytes grown on a fibronectin-containing substrate.
We finally report that sumoylated PIPPin, already found in neurons, is also present in the nuclei of cultured
astrocytes.
CONCLUSIONS: We confirmed the existence of a group of proteins able to interact with H1°/H3.3 mRNAs.
These proteins are, however, differentially expressed during brain maturation and also show different subcellular
localization.
[1] Di Liegro et al. 2014, Int J Mol Med 33:747-62. ; [2] Scaturro et al. 1998, J Biol Chem 273:22788-91.; [3]
Nastasi et al. 1999, J Biol Chem 274:24087-93. [4] Sala et al. 2007, Int J Mol Med 19:501-9. [5] Saladino et al.
2012, Int J Mol Med 29:141-5 [6] Di Liegro et al. 2013, Neuroscience 229:71-6
Melanoma cells release extracellular vesicles which contain H1° RNA and RNA-binding proteins
No full textG26/24 oligodendroglioma cells produce EVs that contain pro-apoptotic proteins, such as FasL and TRAIL, able to induce neuronal- [1] and astrocytic- [2] death. Cancer cells release EVs [3] through which transferring proteins, such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. By releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used A375 melanoma cells.
EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait [7]. Melanoma cells were found to synthesize H1° and secrete it via EVs. Moreover, EVs also contain H1° mRNA. Interestingly, H1° histone sorted to vesicles seems to be sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot.
[1] D’Agostino et al. 2006, Int J Oncol 29:1075-85.
[2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7
[3] Di Liegro et al. 2015, Biomed Res Int, 2015, Article ID 152926.
[4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33
[5] Schiera G et al. 2013, Int J Oncol 43:1771-6
[6] Scaturro et al. 1998, J Biol Chem 273:22788-91
[7] Scaturro et al. 2003, Int J Mol Med 11:509-51
Melanoma cells release extracellular vesicle which contain H1° linker histone as well as RNA-binding proteins which bind to the H1° mRNA
No full textWe previously demonstrated that G26/24 oligodendroglioma cells release EVs that contain proteins, such as FasL and TRAIL, which induce apoptosis in rat cortical neurons [1] and astrocytes [2]. We also reported that cancer cells use EVs for transferring, into the environment [3], proteins such as extracellular matrix remodelling proteases [4], and H1°, a differentiation-specific histone [5]. In particular, by releasing H1°, cells could escape differentiation cues [5]. To verify the role of EVs in releasing specific proteins and mRNAs, in this study we used as a model A375 melanoma cells.
METHODS
EVs were purified from cell culture media as previously reported [1, 2]. T1 RNase-protection assays were performed on total cell lysates and EVs, as described elsewhere [6]. RNA-binding proteins (RBPs) were isolated by using a biotinylated H1° RNA as a bait.
RESULTS
We found that also melanoma cells synthesize H1° and secrete it via EVs. Interestingly, H1° histone sorted to vesicles is probably sumoylated. By T1 RNase-protection assay, we evidenced in EVs three main H1° RNA-protein complexes, the most abundant of which has a molecular mass of around 65 kDa. By using as a bait biotinylated H1° RNA, we isolated a few proteins, then analyzed by mass spectrometry. The most abundant protein was myelin expression factor-2 (MYEF2), which has a molecular mass of about 60 kDa. Finally, we confirmed MYEF2 presence in EVs by western blot.
CONCLUSIONS
We demonstrated that EVs released from melanoma cells contain the H1° linker histone, which is probably sumoylated before sorting to the vesicles. In addition, EVs contain H1° RNA-binding proteins, one of which seems to be MYEF2.
[1] D’Agostino et al. 2006, Int J Oncol 29:1075-85.
[2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7
[3] Di Liegro et al. 2015, Biomed Res Int, in press
[4] Lo Cicero A et al. 2012, Matrix Biol 31:229-33
[5] Schiera G et al. 2013, Int J Oncol 43:1771-6
[6] Scaturro et al. 1998, J Biol Chem 273:22788-9
3D cultures of rat astrocytes and brain capillary endothelial cells on Poly-L-lactic acid scaffolds
No full textTissue engineering is an emerging multidisciplinary field that aims at reproducing in vitro and/or in vivo
tissues with morphological and functional features similar to the biological tissue of the human body.
In this communication we report setting of three-dimensional structures able to mimic the extracellular
matrix of the nervous system: we prepared Poly-L-Lactic Acid (PLLA) porous scaffolds via thermally
induced phase separation (TIPS), and investigated the parameters that influence porosity, average pore size
and degree of interconnection, i.e. polymer concentration, temperature and time of process. Astrocytes and
brain capillary endothelial cells (BCECs) were cultured on these three-dimensional structures and tested for
their ability to grow and survive on PLLA scaffolds. We analyzed in parallel the cell growth in 2D and 3D
culture systems and observed the differences in cell morphology by fluorescence analysis: three-dimensional
scaffolds have the ability to guide cell growth, provide support, encourage cell adhesion and proliferation.
Astrocytes and BCECs adapted well to these porous matrices, not only remaining on the surface, but also
penetrating inside the scaffolds. This 3D cell culture system could be further enriched to host two or three
different brain cell types, in order to set an in vitro model of blood brain barrier, that may be useful for drug
delivery studies, and for the formulation of new therapeutic strategies, to be used for the treatment of
neurological diseases
Involvement of thyroid hormones in brain development and cancer
No full textThe development and maturation of the mammalian brain are regulated by thyroid hormones (THs). Both hypothyroidism and hyperthyroidism cause serious anomalies in the organization and function of the nervous system. Most importantly, brain development is sensitive to TH supply well before the onset of the fetal thyroid function, and thus depends on the trans-placental transfer of maternal THs during pregnancy. Although the mechanism of action of THs mainly involves direct regulation of gene expression (genomic effects), mediated by nuclear receptors (THRs), it is now clear that THs can elicit cell responses also by binding to plasma membrane sites (non-genomic effects). Genomic and non-genomic effects of THs cooperate in modeling chromatin organization and function, thus controlling proliferation, maturation, and metabolism of the nervous system. However, the complex interplay of THs with their targets has also been suggested to impact cancer proliferation as well as metastatic processes. Herein, after discussing the general mechanisms of action of THs and their physiological effects on the nervous system, we will summarize a collection of data showing that thyroid hormone levels might influence cancer proliferation and invasion
Streptomyces coelicolor secretoma protects astrocytes from oxidative stress
No full textStreptomycetes are gram-positive bacteria that produce about 2/3 of pharmaceutically active secondary
metabolites, such as antibiotics, and anti-tumor, immunosuppressive, antifungal and antiparasitic agents. In
this study, we investigated the possible effects of Streptomyces coelicolor extracts and putative vesicular
fraction on primary cultures of rat astrocytes, in both physiological and stressed conditions, induced by
treatment with hydrogen peroxide. Briefly, crude extracts and putative vesicular fractions were prepared
from two S.coelicolor strains (M145 wild-type strain and bold F166 strain), and used to treat primary
astrocytes, which were then also treated with hydrogen peroxide. Data obtained show that both the general
protein pattern of S.coelicolor and putative vesicular fraction vary at different times of growth. M145 strain
vesicles and the crude extracts from both strains have a protective effect on stressed astrocytes. The
protective effects are higher with the vesicular fraction from bacteria at 18 and 72h of growth. The 166 strain
crude extract has a greater protective effect than M145 strain, especially in the initial stages of growth: 18h
and 24h.
Although they need further studies, these observations are very promising and suggest that, in the future,
molecules produced by these bacteria may offer a protection against the high ROS production observed in
many pathological conditions, such as in neurodegenerative diseases
Extracellular vesicles released from melanoma cells contain H1° mRNA-binding proteins, one of which is (probably) MYEF2.
No full textRelease of extracellular vesicles (EVs) is a process conserved from prokaryotes to eucaryotes. Although EVs are produced from both normal and cancer cells, malignant cells release a much higher amount of EVs, which contain tumour-specific proteins and RNAs.
We previously found that G26/24 oligodendroglioma cells shed EVs that contain the pro-apoptotic factors FasL and TRAIL and are able to inhibit neurite outgrowth, and induce apoptosis in about 75% of rat cortical neurons [1] and 40% of astrocytes [2] in culture. By labelling proteins synthesized in one cell type, we also demonstrated EV-mediated horizontal transfer of proteins among brain cells. Interestingly, G2624 release, via EVs, extracellular matrix remodelling proteases [3], and H1° histone protein [4]. We suggested that by releasing H1°, a differentiation-specific histone, cancer cells may escape differentiation cues [4].
To shed further light on the role of EVs in discarding proteins and mRNAs otherwise able to counteract proliferative signals, we studied a melanoma cell line (A375). We found that also these cancer cells produce H1° and release it into EVs. Interestingly, H1° sorted to vesicles shows a molecular mass higher than expected, and is probably sumoylated. By T1 RNase-protection assay with the H1° RNA, three main complexes were evidenced in EVs, the most abundant of which has a molecular mass of about 65 kDa. By using a biotinylated H1°RNA to fish interacting factors, we isolated from EVs a few proteins which have been then identified by mass spectrometry: the most abundant is a protein of about 60 kDa, recognized as myelin expression factor-2 (MYEF2). Western blot analyses confirmed the presence of MYEF2 in EVs released from A375 melanoma cells.
[1] D’Agostino et al- 2006. Int J Oncol 29:1075-85.
[2] Lo Cicero A et al. 2011, Int J Oncol 39:1353-7
[3] Lo Cicero A et al. 2012, Matrix Biol 31:229-33
[4] Schiera G et al. 2013, Int J Oncol 43:1771-
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