Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.     

Marked change in the balance between CYP27A1 and CYP46A1 mediated elimination of cholesterol during differentiation of human neuronal cells

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons.     

Increased expression of Galpha(q/11) and of phospholipase-Cbeta1/4 in differentiated human NT2-N neurons: enhancement of phosphoinositide hydrolysis

The CNS is enriched in phosphoinositide-specific phospholipase C (PLC) and in the G proteins linked to its activation. Although the regional distributions of these signaling components within the brain have been determined, neither their cell type-specific localizations (i.e., neuronal versus glial) nor the functional significance of their high expression has been definitively established. In this study, we have examined the expression of phosphoinositide signaling proteins in human NT2-N cells, a well characterized model system for CNS neurons. Retinoic acid-mediated differentiation of NT2 precursor cells to the neuronal phenotype resulted in five- to 15-fold increases in the expression of PLC-beta1, PLC-beta4, and Galpha(q/11) (the prime G protein activator of these isozymes). In contrast, the expression of PLC-beta3 and PLC-gamma1 was markedly reduced following neuronal differentiation. Similar alterations in cell morphology and in the expression of PLC-beta1, PLC-beta3, and Galpha(q/11) expression were observed when NT2 cells were differentiated with berberine, a compound structurally unrelated to retinoic acid. NT2-N neurons exhibited a significantly higher rate of phosphoinositide hydrolysis than NT2 precursor cells in response to direct activation of either G proteins or PLC. These results indicate that neuronal differentiation of NT2 cells is associated with dramatic changes in the expression of proteins of the phosphoinositide signaling system and that, accordingly, differentiated NT2-N neurons possess an increased ability to hydrolyze inositol lipids.     

CCK processing by pituitary GH3 cells, human teratocarcinoma cells NT2 and hNT differentiated human neuronal cells evidence for a differentiation-induced change in enzyme expression and pro CCK processing

Human teratocarcinoma Ntera2/c 1.D1 (NT2) cells express very low levels of the prohormone convertase enzyme PC1, moderate levels of PC2 and significant levels of PC5. When infected with an adenovirus which expresses rat CCK mRNA, several glycine-extended forms were secreted that co-eluted with CCK 33, 22 and 12. Amidated CCK is not produced because these cells appear to lack the amidating enzyme. Pituitary GH3 cells express high levels of PC2 and PC5. CCK adenovirus-infected GH3 cells secrete amidated versions of the same peptides as NT2 cells. Differentiation of NT2 cells into hNT cells with retinoic acid and mitotic inhibitors increased expression of PC5 and decreased expression of PCI and PC2. CCK adenovirus-infected differentiated hNT cells also secrete glycine extended CCK products and the major molecular form produced co-eluted with CCK 8 Gly. These experiments demonstrate that the state of differentiation of this neuronal cell line influences its expression of PC 1,2, and 5 and its cleavage of pro CCK and suggests that these cells may make an interesting model to study how differentiation alters prohormone processing. These results also support the hypothesis that PC5 in differentiated neuronal cells is capable of processing pro CCK to glycine-extended CCK 8.     

Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells

MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19-23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNAs carrying partially or nearly complementary sequences to the miRNAs in their 3' untranslated regions. Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in human teratocarcinoma NTere2D1, mouse embryonic carcinoma P19, mouse neuroblastoma Neuro2a and rat pheochromocytoma PC12D cells, which can be induced into differentiated cells with long neuritic processes, i.e., after cell differentiation, such that the resultant cells look similar to neuronal cells. The data presented here indicate marked changes in the expression of miRNAs, as well as genes related to neuronal development, occurred in the differentiation of NTera2D1 and P19 cells. Significant changes in miRNA expression were not observed in Neuro2a and PC12D cells, although they showed apparent morphologic change between undifferentiated and differentiated cells. Of the miRNAs investigated, the expression of miRNAs belonging to the miR-302 cluster, which is known to be specifically expressed in embryonic stem cells, and of miR-124a specific to the brain, appeared to be markedly changed. The miR-302 cluster was potently expressed in undifferentiated NTera2D1 and P19 cells, but hardly in differentiated cells, such that miR-124a showed an opposite expression pattern to the miR-302 cluster. Based on these observations, it is suggested that the miR-302 cluster and miR-124a may be useful molecular indicators in the assessment of degree of undifferentiation and/or differentiation in the course of neuronal differentiation.     

Retinoic acid induces neuronal differentiation of embryonal carcinoma cells by reducing proteasome-dependent proteolysis of the cyclin-dependent inhibitor p27.

Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.     

Expression of Cdk5 and its activators in NT2 cells during neuronal differentiation

We have recently developed a rapid protocol involving NT2 cell aggregation and treatment with retinoic acid (RA) to produce terminally differentiated CNS neurons. As a first step to explore the functional roles of cell-cycle regulatory proteins in the process of neuronal differentiation, the expression profiles of cyclin-dependent kinases (Cdks) and their regulators were examined in NT2 cells following treatment with RA. One of the Cdks, Cdk5, has been demonstrated to affect the process of neuronal differentiation and suggested to play an important role in development of the nervous system. We found that the expression of Cdk5 was gradually increased, while its activators (p35 and p39) as well as Cdk5 kinase activity were induced in NT2 cells during the process of neuronal differentiation. Moreover, both p35 and p39 were localized along the axons and varicosity-like structures of differentiated NT2 neurons. Taken together, our results demonstrated that NT2 cells provide a good in vitro model system to examine signaling pathways involved in the regulation of Cdk5 activators and to elucidate the functional roles of Cdk5 in neuronal differentiation.     

Divergent modulation of neuronal differentiation by caspase-2 and -9

Human Ntera2/cl.D1 (NT2) cells treated with retinoic acid (RA) differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2) or -9 (si-Casp9) was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM), microtubule associated protein-2 (MAP2) and tyrosine hydroxylase (TH) mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ～100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved) in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.     

Aicar effect in early neuronal development

The neurological manifestations of Lesch-Nyhan disease (LND) have been attributed to the effect of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency on nervous system development. An increase has been reported in the levels of 5-aminoimidazole-4-carboxamide-1-β-D-ribotide (AICAR) and its triphosphate form ZTP in the red blood cells of patients with LND. AICAR accumulation in the brain has been hypothesized as the cause of some of the neurological symptoms of patients with LND. In this study, we examined the effect of AICAR on the differentiation of neurons in the well-established human NTERA-2 cl.D1 (NT2/D1) embryonic carcinoma neurogenesis model. NT2/D1 cells were differentiated along neuroectodermal lineages after exposure to 10-µM retinoic acid (RA), with or without the addition of 25-µM AICAR to the culture medium. The effect of AICAR on RA differentiation were examined through changes in the expression of genes essential to neuronal differentiation, as well as genes from the Wnt/β-catenin, transforming growth factor beta (TGFβ) and sonic hedgehog (SHH) pathways.

Results: RA-induced differentiation in the NT2/D1 cells significantly increased the expression of MAP2, NRG1, NRP1, NRP2, NEUROG1 and EN1 genes (genes linked to neural differentiation) compared with undifferentiated NT2/D1 cells. We found that AICAR increased the expression of the SHH gene and the WNT2 and WNT7B genes but did not influence the expression of genes whose overexpression characterize early neurodevelopmental processes.

Conclusion: The relevance of the AICAR related changes in the SHH and Wnt/β-catenin pathway genes expression in the physiopathology of LND warrants further exploration.     

Changes in proteasome expression and activity during differentiation of neuronal precursor NTera 2 clone D1 cells

The effect of differentiation of the human neuronal progenitor cell line NTera 2 clone D1 (NT2/D1) by retinoic acid on components of the proteasome system was studied. The chymotrypsin-like and peptidylglutamyl peptide bond hydrolyzing activities of the proteasome increased five weeks after retinoic acid, and following treatment with mitotic inhibitors returned to levels detected in non-differentiated cells. A selective induction of the MHC class II region encoded LMP7 and LMP2 proteasome subunits occurred during differentiation, whereas there were no changes in the expression of the constitutive LMP2 counterpart (delta-subunit) or the constitutive C2 subunit. Immunofluorescence revealed marked LMP7 accumulation in fully differentiated cells, with no changes in the labeling pattern of the constitutive proteasome antigens. The expression of the alpha-subunit of the PA28 proteasome activator was down-regulated in fully differentiated neurons, but was not correlated with changes in enzymatic activity. Changes in proteasome activity and composition may contribute to the processes leading to differentiation of human neurons in vitro and to the properties of fully differentiated neurons.     

Expression and activity of cyclin-dependent kinases and glycogen synthase kinase-3 during NT2 neuronal differentiation

In the presence of retinoic acid undifferentiated NT2 cells turn into terminally differentiated hNT (or NT2N) neurons within 5 weeks. We have used this in vitro cellular model to investigate the changes in expression and activity of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) during this neuronal differentiation process. We here show that CDK1/2 protein level and kinase activity sharply decrease during the NT2-->hNT transition. In contrast, the activity of CDK5/p35 dramatically increases, probably as a result of an enhanced expression of p35 in a stable CDK5 level background. GSK-3 activity increases modestly during the differentiation of hNT cells, and this event correlates with enhanced expression of each of the three GSK-3 isoforms. Pharmacological inhibitors of CDKs and GSK-3 lead to a dose-dependent decrease in cell viability.     

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract. Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NTZ/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/Dl-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.     

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract. Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NT2/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/D1-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.