Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.

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EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

Medical Immunology (Russia) ◽

10.15789/1563-0625-eom-2276 ◽

2021 ◽

Vol 23 (4) ◽

pp. 677-684

Author(s):

I. M. Rashchupkin ◽

E. Ya. Shevela ◽

E. R. Chernykh

Keyword(s):

Retinoic Acid ◽

Conditioned Medium ◽

Neuronal Differentiation ◽

Macrophage Polarization ◽

Relative Content ◽

M2 Macrophage ◽

Neurite Length ◽

Soluble Factors ◽

Differentiated Cells ◽

Morphological Criteria

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p < 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation.

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Role of down-regulated neutral ceramidase during all-trans retinoic acid-induced neuronal differentiation in SH-SY5Y neuroblastoma cells

The Journal of Biochemistry ◽

10.1093/jb/mvs033 ◽

2012 ◽

Vol 151 (6) ◽

pp. 611-620 ◽

Cited By ~ 15

Author(s):

Kouji Tanaka ◽

Keiko Tamiya-Koizumi ◽

Kazumi Hagiwara ◽

Hiromi Ito ◽

Akira Takagi ◽

...

Keyword(s):

Retinoic Acid ◽

Neuronal Differentiation ◽

Neuroblastoma Cells ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid

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Erratum: Singh A, Rokes C, Gireud M, et al. Retinoic acid induces REST degradation and neuronal differentiation by modulating the expression of SCFβ-TRCP in neuroblastoma cells. Cancer. 2011;117:5189-5202.

Cancer ◽

10.1002/cncr.31377 ◽

2018 ◽

Vol 124 (11) ◽

pp. 2458-2458

Keyword(s):

Retinoic Acid ◽

Neuronal Differentiation ◽

Neuroblastoma Cells

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The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1335 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1335-1341 ◽

Cited By ~ 156

Author(s):

W Poluha ◽

D K Poluha ◽

B Chang ◽

N E Crosbie ◽

C M Schonhoff ◽

...

Keyword(s):

Cell Cycle ◽

Neuronal Differentiation ◽

Kinase Inhibitor ◽

Cyclin E ◽

Neuroblastoma Cells ◽

Protein Kinase Activity ◽

Cyclin Dependent Kinase ◽

Cyclin Dependent Kinases ◽

Cyclin Dependent Kinase Inhibitor ◽

Differentiated Cells

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.

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Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells

Cellular Signalling ◽

10.1016/j.cellsig.2009.01.036 ◽

2009 ◽

Vol 21 (6) ◽

pp. 881-891 ◽

Cited By ~ 50

Author(s):

Pei-Yu Wu ◽

Yu-Chia Lin ◽

Chia-Ling Chang ◽

Hsing-Tsen Lu ◽

Chia-Hsuan Chin ◽

...

Keyword(s):

Retinoic Acid ◽

Neuronal Differentiation ◽

Neuroblastoma Cells ◽

P2x7 Receptors

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Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

Neurology Research International ◽

10.1155/2015/734703 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 15

Author(s):

Kawinthra Khwanraj ◽

Chareerut Phruksaniyom ◽

Suriyat Madlah ◽

Permphan Dharmasaroja

Keyword(s):

Tyrosine Hydroxylase ◽

Differential Expression ◽

Gradual Increase ◽

Cell Model ◽

Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Dopaminergic Cell ◽

Differentiated Cells ◽

Undifferentiated Cells ◽

Cell Plating

The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson’s disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+and evaluate the differential expression of tyrosine hydroxylase (TH) in undifferentiated and retinoic acid- (RA-) induced differentiated cells. The western blot results showed a gradual decrease in TH in undifferentiated cells and a gradual increase in TH in differentiated cells from days 4 to 10 after cell plating. Immunostaining revealed a gradual increase in TH along with neuritic outgrowth in differentiated cells on days 4 and 7 of RA treatment. For the study on cell susceptibility to MPP+and the expression of apoptosis-related genes, MTT assay showed a decrease in cell viability to approximately 50% requiring 500 and 1000 μM of MPP+for undifferentiated and RA-differentiated cells, respectively. Using real-time RT-PCR, treatment with 500 μM MPP+led to significant increases in the Bax/Bcl-2 ratio, p53, and caspase-3 in undifferentiated cells but was without significance in differentiated cells. In conclusion, differentiated cells may be more suitable, and the shorter duration of RA differentiation may make the SH-SY5Y cell model more accessible.

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Evidence of Decreased Activity in Intermediate-Conductance Calcium-Activated Potassium Channels During Retinoic Acid–Induced Differentiation in Motor Neuron–Like NSC-34 Cells

Cellular Physiology and Biochemistry ◽

10.1159/000492653 ◽

2018 ◽

Vol 48 (6) ◽

pp. 2374-2388 ◽

Cited By ~ 11

Author(s):

Pei-Chun Chen ◽

Jing-Syuna Ruan ◽

Sheng-Nan Wu

Keyword(s):

Retinoic Acid ◽

Motor Neuron ◽

Messenger Rna ◽

Single Channel ◽

Slow Component ◽

Delayed Rectifier ◽

Induced Differentiation ◽

Differentiated Cells ◽

Undifferentiated Cells ◽

The Mean

Background/Aims: Intermediate-conductance Ca2+-activated K+ (IKCa; KCa3.1 or KCNN4) channels affect the behaviors of central neurons including motor neurons. The mechanism through which neuronal differentiation is related to the activity of these channels remains largely unclear. Methods: By using various molecular biology tools and electrophysiological measurements, we investigated possible changes in the activity of IKCa channels in a retinoic acid (RA)-induced differentiation process in motor neuron-like NSC-34 cells. Results: The protein and messenger RNA expression of KCa3.1 substantially diminished as NSC-34 cells were differentiated with low serum (1%) and 1 µM RA. In whole-cell current recordings, the density of delayed-rectifier K+ currents obtained from differentiated cells was elevated. However, the density of a ramp pulse-elicited K+ current that was sensitive to blockage by 1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole (TRAM-34)—an inhibitor of IKCa channels—was significantly higher in undifferentiated NSC-34 cells than in differentiated cells. In undifferentiated cells, the activity of IKCa channels was readily detected and the probability of channel openings was resistant to stimulation by diazoxide or suppression by verruculogen. Furthermore, this probability was increased by 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one or 9-phenanthrol and reduced by TRAM-34. The channel-opening probability decreased in RA-induced differentiated cells, whereas the single-channel conductance of IKCa channels did not differ between undifferentiated and differentiated cells. Moreover, the slow component of the mean closed time in these channels was significantly shorter in undifferentiated cells than in differentiated cells; however, the mean open time in the channel remained unchanged as cells were differentiated. Conclusion: RA-induced differentiation in neurons could exert a suppressive effect on the activity of IKCa channels.

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