Neuronal Differentiation Of SH-SY5Y Cells Research Articles

Altered levels of PP2A regulatory B/PR55 isoforms indicate role in neuronal differentiation

The ubiquitously expressed serine/threonine-specific protein phosphatase 2A (PP2A) is prominent in brain where it serves a wide range of functions under both physiological and pathological conditions. PP2A holoenzymes are composed of a catalytic subunit and a tightly complexed scaffolding subunit. This core enzyme associates with regulatory subunits of the B/PR55, B′/PR56/PR61, B″/PR72 and B‴/PR93/PR110 families. We previously determined distribution and expression levels of the four members of the B/PR55 family in brain, as dysregulation of this subunit family has been specifically implicated in neurodegenerative disorders including Alzheimer's disease. In the present study, we used cell lines widely used in neuroscience research to determine levels of the four PR55 isoforms by qRT-PCR under different experimental conditions. We show that PR55α mRNA levels are highest in both HEK293 cells and SH-SY5Y neuroblastoma cells whereas PR55β levels are lowest. Stepwise neuronal differentiation of SH-SY5Y cells causes the selective upregulation of PR55β, and to some extent PR55γ and PR55δ, but not PR55α mRNAs. In agreement with the qRT-PCR analysis, neuronal differentiation does not alter PR55α protein levels, whereas interestingly, PR55β and PR55γ protein levels are reduced when compared to undifferentiated cells. Our data point at specific roles for distinct regulatory B/PR55 subunits of PP2A in neuron-like cells with PR55α being the major isoform.

Activation of Rac1 by phosphatidylinositol 3‐kinase in vivo: role in activation of mitogen‐activated protein kinase (MAPK) pathways and retinoic acid‐induced neuronal differentiation of SH‐SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.

JNK pathway is required for retinoic acid-induced neurite outgrowth of human neuroblastoma, SH-SY5Y.

Neurite outgrowth is a central event of neuronal differentiation that proceeds in multiple processes requiring various cellular factors. Here we demonstrated that c-Jun N-terminal kinase 1 (JNK1) plays an essential role in RA-induced neurite outgrowth of SH-SY5Y cells. Treatment of SH-SY5Y cells with RA induced a strong activation of JNK1 within 10 min, and the immediate increase of JNK1 activity returned to the basal level in an hour. The second surge of JNK1 activity was observed around 1 day after RA treatment, which coincided with the period of extensive neurite outgrowth. Interestingly, phospho-JNK was concentrated in the nucleus of cells during the early induction, whereas it was distributed into neurite processes during the delayed second activation period. In SH-SY5Y carrying a dominant negative form of SEK1, an upstream kinase of JNK1, both early and late inductions of JNK1 activity were repressed along with RA-induced neurite outgrowth. These results suggest that JNK1 plays an essential role in RA-induced neuronal differentiation of SH-SY5Y cells.

JNK pathway is required for retinoic acid-induced neurite outgrowth of human neuroblastoma, SH-SY5Y

Neurite outgrowth is a central event of neuronal differentiation that proceeds in multiple processes requiring various cellular factors. Here we demonstrated that c-Jun N-terminal kinase 1 (JNK1) plays an essential role in RA-induced neurite outgrowth of SH-SY5Y cells. Treatment of SH-SY5Y cells with RA induced a strong activation of JNK1 within 10 min, and the immediate increase of JNK1 activity returned to the basal level in an hour. The second surge of JNK1 activity was observed around 1 day after RA treatment, which coincided with the period of extensive neurite outgrowth. Interestingly, phospho-JNK was concentrated in the nucleus of cells during the early induction, whereas it was distributed into neurite processes during the delayed second activation period. In SH-SY5Y carrying a dominant negative form of SEK1, an upstream kinase of JNK1, both early and late inductions of JNK1 activity were repressed along with RA-induced neurite outgrowth. These results suggest that JNK1 plays an essential role in RA-induced neuronal differentiation of SH-SY5Y cells. NeuroReport 14:941–945 © 2003 Lippincott Williams & Wilkins.

Regulation of β-amyloid precursor protein and inositol 1,4,5-trisphosphate receptor gene expression during differentiation of a human neuronal cell line

Retinoic acid-induced differentiation of SH-SY5Y human neuroblastoma cells results in the development of extensive neurite processes as well as changes in cell body morphology toward a neuronal phenotype. The authors have examined concurrent regulation of β-amyloid precursor protein (APP) and inositol 1,4,5-trisphosphate receptor (insP3R) gene expression in SY5Y cells during neuronal differentiation. Of the multiple APP mRNA transcripts expressed in this cell line, retinoic acid treatment significantly increased the expression of APP695 transcript while the level of total APP remained unchanged. In the same time course, neuronal differentiation decreased the expression of insP3R at both the mRNA and protein levels. These findings demonstrate an inverse relationship between APP and insP3R gene expression during neuronal differentiation of SH-SY5Y cells and suggest a possible change in intracellular calcium homeostasis.

Tissue Transglutaminase Mediates Activation of RhoA and MAP Kinase Pathways during Retinoic Acid-induced Neuronal Differentiation of SH-SY5Y Cells

All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.

Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma SH-SY5Y cells

Tissue transglutaminase is a normal constituent of the central and peripheral nervous systems and in rats transglutaminase activity in brain and spinal cord is highest during fetal stages when axonal outgrowth is occurring. Further, treatment of human neuroblastoma SH-SY5Y cells with retinoic acid results in the cells withdrawing from the cell cycle and extending neurites, in the same time frame that tissue transglutaminase expression significantly increases. Considering these and other previous findings, this study was carried out to determine whether tissue transglutaminase is involved in neuronal differentiation of SH-SY5Y cells. For these studies SH-SY5Y cells stably overexpressing wild-type tissue transglutaminase, an inactive tissue transglutaminase mutant (C277S) or an antisense tissue transglutaminase construct (which decreased endogenous tissue transglutaminase below detectable levels) were used. SH-SY5Y cells overexpressing wild-type tissue transglutaminase spontaneously differentiated into a neuronal phenotype when grown in low-serum media. In contrast, cells overexpressing inactive tissue transglutaminase or the antisense tissue transglutaminase continued to proliferate and exhibit a flat polygenic morphology even when maintained in low-serum conditions. In addition, increased tissue transglutaminase expression in response to retinoic acid was abolished in the antisense tissue transglutaminase cells, and antisense and mutant tissue transglutaminase expressing cells did not extend neurites in response to retinoic acid. Moreover, wild-type and inactive tissue transglutaminase exhibited differential intracellular localization. These data indicate that tissue transglutaminase is necessary and sufficient for neuronal differentiation of human neuroblastoma SH-SY5Y cells.

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells.

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid–treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIβ and Cα subunits. Since type II protein kinase A and RIIβ have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIβ in SH-SY5Y cells. The growth of RIIβ-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIβ level and further inhibited the growth of RIIβ-overexpressing cells, showing strong correlation between the level of RIIβ and growth inhibition. However, RIIβ-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid–induced growth inhibition through the up-regulation of RIIβ/type II protein kinase A. J. Cell. Physiol. 182:421–428, 2000. © 2000 Wiley-Liss, Inc.

Protein kinase C-alpha and -epsilon are enriched in growth cones of differentiating SH-SY5Y human neuroblastoma cells.

SH-SY5Y cells differentiate into neuronal-like cells and express marker proteins like growth-associated protein (GAP-43) and neuropeptide tyrosine when treated with a low concentration (16 nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of growth factors or serum. Both control and differentiated cells expressed protein kinase C-alpha (PKC-alpha), PKC-epsilon, and PKC-zeta as revealed by Western blot analyses, but the subcellular distribution of the three isoforms was not uniform, indicating specific localized functions of the enzymes. In growth cones prepared from differentiating cells PKC-alpha and PKC-epsilon were enriched. In contrast, PKC-zeta was more evenly distributed within the differentiating cell. Cells treated with a high concentration of TPA (1.6 microM) differentiate poorly and continue to proliferate. In those cells, PKC-alpha and PKC-epsilon were found to be down-regulated while PKC-zeta remained present. Thus, down-regulation of PKC-alpha and PKC-epsilon appears to be incompatible with neuronal differentiation of SH-SY5Y cells. These cells also differentiate when treated with a combination of basic fibroblast growth factor and insulin-like growth factor I. Growth cones isolated from such cells are also enriched in PKC-alpha and PKC-epsilon, but not in PKC-zeta. Based on the subcellular distribution of PKC-alpha and epsilon, and that PKC substrates like GAP-43 and pp60c-src are enriched in SH-SY5Y growth cones, a role during neurite growth is suggested.

Altered ganglioside expression by SH-SY5Y cells upon retinoic acid-induced neuronal differentiation.

SH-SY5Y Neuroblastoma cells were used to study the effect of retinoic acid (RA)-induced differentiation on the expression of gangliosides and neuronal markers. In the presence of 10 microM RA, more than 70% of the cells differentiate to a neuronal phenotype within 8 days. They extend long neuritic processes and show an enhanced immuno-expression of neurone-specific enolase (NSE), neurofilament protein (NF-M), and polysialic acid (PSA). SH-SY5Y cells were found to express at least 12 different gangliosides. RA-induced neuronal differentiation led to a decrease in the content of GM2, GD3, and GD2 and to a 3-7 fold increased concentration of the ganglio-tetraosyl gangliosides GM1, GD1a, GT1a, GD1b, and GT1b. Thus, RA-induced neuronal differentiation of SH-SY5Y cells is accompanied by ganglioside changes similar to those observed during embryonic neuronal differentiation.