A novel role for Gab2 in bFGF-mediated cell survival during retinoic acid–induced neuronal differentiation

Gab proteins amplify and integrate signals stimulated by many growth factors. In culture and animals, retinoic acid (RA) induces neuronal differentiation. We show that Gab2 expression is detected in neurons in three models of neuronal differentiation: embryonic carcinoma (EC) stem cells, embryonic stem cells, and primary neural stem cells (NSCs). RA treatment induces apoptosis, countered by basic FGF (bFGF). In EC cells, Gab2 silencing results in hypersensitivity to RA-induced apoptosis and abrogates the protection by bFGF. Gab2 suppression reduces bFGF-dependent activation of AKT but not ERK, and constitutively active AKT, but not constitutively active MEK1, reverses the hypersensitization. Thus, Gab2-mediated AKT activation is required for bFGF's protection. Moreover, Gab2 silencing impairs the differentiation of EC cells to neurons. Similarly, in NSCs, Gab2 suppression reduces bFGF-dependent proliferation as well as neuronal survival and production upon differentiation. Our findings provide the first evidence that Gab2 is an important player in neural differentiation, partly by acting downstream of bFGF to mediate survival through phosphoinositide 3 kinase–AKT

Towards cardiac cell therapy

Tese de doutoramento em Biologia (Biologia Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 200

New insights into purinergic receptor signaling in neuronal differentiation, neuroprotection, and brain disorders

Ionotropic P2X and metabotropic P2Y purinergic receptors are expressed in the central nervous system and participate in the synaptic process particularly associated with acetylcholine, GABA, and glutamate neurotransmission. As a result of activation, the P2 receptors promote the elevation of free intracellular calcium concentration as the main signaling pathway. Purinergic signaling is present in early stages of embryogenesis and is involved in processes of cell proliferation, migration, and differentiation. The use of new techniques such as knockout animals, in vitro models of neuronal differentiation, antisense oligonucleotides to induce downregulation of purinergic receptor gene expression, and the development of selective inhibitors for purinergic receptor subtypes contribute to the comprehension of the role of purinergic signaling during neurogenesis. In this review, we shall discuss the participation of purinergic receptors in developmental processes and in brain physiology, including neuron-glia interactions and pathophysiology

Cell adhesion and growth factor regulation of Akt

Akt is a protein kinase that contains a C-terminal pleckstrin homology (PH) domain and an N-terminal kinase domain. The activity of Akt is regulated by PI3-kinases that generate 3 phosphoinositides. Akt activation requires the binding of 3 phosphoinositides to the pleckstrin homology (PH) domain and phosphorylation at residues Thr308 and Ser473. Phosphospecific antibodies that recognise the phosphorylated sites and green fluorescent protein (GFP) fusions with mutated or truncated forms of Akt were used to follow the activation of Akt. Confocal microscopy showed that membrane translocation is required for Akt phosphorylation and is dependent on the PH domain but not kinase activity. A GFP fusion with the PH domain of Akt, termed GFP-AH, was assessed and used as a probe for sites of PI3-kinase activity in cells. GFP- AH was used to locate 3 phosphoinositides in epithelial cells that were plated on collagen to investigate extracellular matrix induced cell survival. GFP-AH localised to sites of cell-cell and cell-matrix contact, distinct from focal adhesions. This suggests that the attachment induced, PI3-kinase mediated, survival signal in epithelial cells is generated not only by cell-matrix but also by cell-cell contact. PI3-kinase activity was monitored during the disruption and formation of cell-cell junctions. Disruption of the junctions by calcium removal decreased PI3-kinase activity, whereas the formation of junctions preceded PI3-kinase activity. Akt became phosphorylated 4 h after junction formation was initiated and correlated with the tyrosine phosphorylation of the cell-cell junctional proteins y-catenin and p120 catenin. As p120 catenin is a substrate of activated Src and Src family kinase inhibitors reduced the cell-cell adhesion induced phosphorylation of Akt, this suggests a role for Src family kinases in the regulation of cell-cell adhesion induced PI3-kinase activity

A Small Peptide Modeled after the NRAGE Repeat Domain Inhibits XIAP-TAB1-TAK1 Signaling for NF-κB Activation and Apoptosis in P19 Cells

In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38MAPK and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38MAPK phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/β phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis

Effect of retinoic acid on the expression and function of AP-1 transcription factor in B16 mouse melanoma cells: role of protein kinase

Retinoic acid (RA) induces differentiation of B16 mouse melanoma cells. This differentiation is accompanied by an increase in protein kinase Ca (PKCα) protein level and selective enrichment in nuclear-associated PKCα. PKC is thought to regulate gene expression through the TPA response element (TRE). This element is specifically recognized by the AP-1 transcription factor composed of jun and fos family members. In this study, I have analyzed the effect of RA on the expression and function of AP-1 in B16 mouse melanoma cells. Transient transfection analysis of B16 cells using leuciferase reporter gene constructs with or without AP-1 elements indicated that RA induced a four- to fivefold increase in AP-1 transcriptional activity in a concentration-dependent manner. RA did not change the expression (mRNA and protein) of jun family members while the expression (mRNA and protein) of c-fos was decreased. In contrast, acute phorbol dibutyrate (PDB) treatment increased c-jun and c-fos expression. Analysis of the mobility shift assay by using an oligonucleotide containing the AP-1 element suggested that two of the complexes were negatively regulated by RA. There was no significant change in the binding of the other complexes by RA. Acute PDB treatment increased the binding where as chronic treatment decreased the binding of this complex. Use of specific antibodies indicated that complexes which were decreased by RA and increased by PDB contained fos protein. Down regulation of PKCα by chronic PDB treatment inhibited both the acute PDB and the RA-induced increase in AP-1 activity. However, the potent and selective PKC inhibitor bisindolylmaleimide inhibited the PDB induced increase in AP-1 activity but had no effect on the RA-induced increase in AP-1 activity. Our results suggest that the role played by PKC in RA induced AP-1 activity is independent of its kinase activity. I also determined the role of nuclear retinoid receptors in RA-induced PKCα expression and AP-1 transcriptional activity by using receptor-specific analogs. Results suggest that RARα and RXR play an important role in RA-mediated effect on PKCα expression and AP-1 activity

Distinct Regulatory Functions of Calpain 1 and 2 during Neural Stem Cell Self-Renewal and Differentiation

Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation

Redox Regulation of Differentiation in Neuroblastoma

Neuroblastoma is the most common extracranial solid tumor in childhood, ranking third in prevalence among infant cancers. Despite intensive therapy with surgery, radiation and chemotherapy, there is a high chance of tumor recurrence. These children are therefore given retinoids in the maintenance phase to differentiate the tumor cells and to avoid/lessen this incidence. Although 13-cis retinoic acid (13-cis RA) is currently used to treat neuroblastoma, there is much evidence supporting the prevalence of its metabolite, all-trans retinoic acid (ATRA), to be the main signaling retinoid in vivo. The SK-N-SH neuroblastoma cell line, which is composed of three sub-types: sympatheoadrenal neuroblasts (N-type), substrate-adherent non-neuronal (S-type), and intermediate (I-type), was used as a model for ATRA-mediated differentiation. Through our research, we propose that redox status (i.e. balance of reactive oxygen species (ROS) and antioxidants) plays an important role in the process of neuroblastoma differentiation. More specifically, we first investigated the effects of supplementing ATRA treatment with a known, clinically relevant antioxidant, N-acetyl-L-cysteine (NAC). As opposed to findings in many other neuronal models, we are the first to demonstrate NAC enhancement of ATRA-mediated differentiation. Furthermore, our results suggest that NAC prevents oxidation of either ATRA, or of the nuclear environment in which ATRA binds to retinoic acid receptor (RAR) to induce expression of genes involved in neuronal differentiation. In this work, we also demonstrate that ATRA stimulates superoxide (O2-•) generation, which validates its induction of manganese superoxide dismutase (MnSOD; O2-• scavenger) in this cell model (previously published). Our subsequent studies investigate the role of MnSOD in ATRA-mediated differentiation. Interestingly, ATRA upregulated neuronal differentiation markers (neurofilament M: NF-M and N-methyl-D-aspartate receptor 1: NMDAR1) (48 hrs) prior to induction of MnSOD (72 hrs), which might suggest that this antioxidant is not essential to initiate the differentiation process. Nonetheless, by reducing MnSOD activity (at 48 hrs), we demonstrate enhanced NF-M expression either in the absence or presence of ATRA. Therefore, it is apparent that basal MnSOD activity influences at least one marker of differentiation. Subsequent analysis at 96 hrs demonstrates that by reducing MnSOD activity, there is a concomitant decrease in NF-M expression. Therefore, it is possible ATRA-induction of MnSOD promotes expression of at least one differentiation marker (neurofilament M: NF-M) at this later time point. Furthermore, we provide evidence for the prevalence of MnSOD-generated hydrogen peroxide (H2O2) at 96 hrs, which may play a role in differentiation of the SK-N-SH cells. Our findings show enhanced NF-M expression by either supplementation with an exogenous antioxidant (NAC) or reduction of an endogenous antioxidant (MnSOD). We propose ways in which each of these can be accomplished and provide insight into the redox regulation of neuroblastoma differentiation. Additionally, our work provides a close examination of ROS and antioxidants as they are altered in this process. Because retinoid therapy is often met with unfavorable chemoresistance, there is a need to establish improved therapeutic methods, which may revolve around alteration of redox status

Studies of the Transcriptional Control of the Human c-fos Proto-Oncogene

The c-fos gene is the cellular counterpart of the transforming genes of several retroviruses. Its transcription and expression is increased in many types of different cells following extra-cellular stimuli, and this increase is both rapid and transient in most cases. Many studies have focussed on the transcriptional control of the c-fos proto-oncogene. These studies have characterised an element (the serum response element - SRE) that was initially found to increase transcription from the c-fos promoter following serum stimulation of quiescent cells and subsequently has been shown to activate transcription from the c-fos promoter following a variety of extra-cellular stimuli in a variety of different cell types. A computer-aided search of the human c-fos sequence revealed a sequence directly 3' of the SRE that shows similarity to the DNA binding sites for the transcription factors AP-1 and ATF. These proteins bind to their sequences and confer phorbol ester and cyclic AMP responsiveness upon adjacent promoters. The purpose of this project was to re-examine the role of the SRE in stimulating transcription from the c-fos promoter, to characterise the transcriptional properties of the fosATF/AP-1 binding site that lies adjacent to the SRE, and to investigate any interaction that may take place between these two sequences in controlling transcription from the c-fos promoter. Results obtained confirm that the SRE confers serum and phorbol ester responsiveness upon the c-fos promoter. They also show that the fosATF/AP-1 sequence forms a weaker complex with an AP-l/ATF protein than previously characterised AP-l/ATF binding sites, and that this sequence activates transcription from the c-fos promoter in growing cells. The fosATF/AP-1 binding site also confers serum and phorbol ester responsiveness upon the c-fos promoter. In quiescent cells the juxtaposed SRE and fosATF/AP-1 sequences contribute to a lower level of transcription from the c-fos promoter than does the SRE itself. This suggests an interaction between the proteins binding to these two sequences. Separating the SRE and fosATF/AP-1 sequences resulted in elevated levels of expression in quiescent cells and also resulted in higher levels of transcription from the c-fos promoter following stimulation of quiescent cells with serum or phorbol esters. It is concluded that there is a complex mechanism of interaction between the proteins that bind to the adjacent SRE and fosATF/AP-1 sequences and that there may be several proteins involved in this interaction