Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.     

应用两种方法诱导SH-SY5Y细胞分化的研究

目的：观察全反式维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法：应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果：ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论：维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。     

Changes in inducibility of ornithine decarboxylase activity in differentiating human neuroblastoma cells

Human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), or a combination of these two substances. The phenotypical changes induced by these substances differed, but one effect of both was an inhibition of the cell growth. Addition of TPA or RA to non-treated cells had no effect on the activation of ornithine decarboxylase (ODC, EC 4.1.1.17.), while a change to fresh medium stimulated the ODC to maximum activity after 4-6 h. The activity was not altered by the presence of RA in the fresh medium, but TPA partially inhibited the medium-stimulated ODC activity. Cells treated for 4 or 8 days with TPA or a combination of TPA and RA had a low ODC activity which could not be induced by fresh medium. However, RA-treated (and thus growth-inhibited) cells still responded to a change of medium by exhibiting an ODC activity of the same magnitude and duration as in medium-stimulated control cells. The results seem to suggest that the growth inhibition induced by TPA and RA, respectively, is mediated by different mechanisms.     

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.     

Distinct Mechanisms of Differentiation of SH-SY5Y Neuroblastoma Cells by Protein Kinase C Activators and Inhibitors

Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH-SY5Y differentiates when exposed to 12-tetradecanoyl-13-acetyl-beta-phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron-specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF-H). TPA, 12-deoxy-13-tetradecanoyl-beta-phorbol, and staurosporine, but not DAG or 4-O-methyl-TPA, caused neurite outgrowth. Neuron-specific enolase was expressed in cells treated with TPA and 12-deoxy-13-tetradecanoyl-beta-phorbol but not with DAG, staurosporine, or 4-O-methyl-TPA. NF-H increased in the perikarya of cells treated with DAG and 4-O-methyl-TPA, in processes and to varying degrees in perikarya of TPA- and 12-deoxy-13-tetradecanoyl-beta-phorbol-treated cells, but much more in the processes than in the perikarya of staurosporine-differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH-SY5Y cells by different mechanisms, and that the five-membered/seven-membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.     

Phenotypic differentiation of aphidicolin-selected human neuroblastoma cultures after long-term exposure to nerve growth factor

Human neuroblastoma SH-SY5Y (SY5Y) cultures, exposed to murine 7 S nerve growth factor (NGF) for 5 weeks and selected with aphidicolin (Aph) for 1 week, acquire several properties indicative of mature peripheral nerve cells. The mitotic activity of treated cultures decreases prior to Aph selection and ultimately reaches a level approximately 3% that of untreated cultures by Week 4 of treatment. The measured plasma membrane resting potential of the cells increases from -5 mV for untreated cells to -(45-56) mV for NGF/Aph-treated cells. Intracellular stores of monoamines are increased as determined by histochemical staining, and levels of neuron-specific enolase antigen increase as a result of NGF/Aph treatment. The resulting outgrowth of neurites is extensive and large bundles of processes commonly exceed 300 micron in length. NGF/Aph-treated cells acquire a dependence upon NGF for survival; however, with continued administration of NGF, the cultures appear to be capable of surviving indefinitely. Retinoic acid will also promote certain aspects of a differentiated phenotype under similar culture conditions. As judged by these criteria, cells of the SY5Y human neuroblastoma cell line have the potential for phenotypic and irreversible differentiation in vitro and can survive for prolonged periods under these culture conditions.     

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.     

The Impact of Differentiation on Cytotoxicity and Insulin Sensitivity in Streptozotocin Treated SH-SY5Y Cells

Recently neuronal insulin resistance was suggested playing a role in Alzheimer’s disease. Streptozotocin (STZ) is commonly used to induce impairment in insulin metabolism. In our previous work on undifferentiated SH-SY5Y cells the compound exerted cytotoxicity without altering insulin sensitivity. Nevertheless, differentiation of the cells to a more mature neuron-like phenotype may considerably affect the significance of insulin signaling and its sensitivity to STZ. We aimed at studying the influence of STZ treatment on insulin signaling in SH-SY5Y cells differentiated by retinoic acid (RA). Cytotoxicity of STZ or low serum (LS) condition and protective effect of insulin were compared in RA differentiated SH-SY5Y cells. The effect of insulin and an incretin analogue, exendin-4 on insulin signaling was also examined by assessing glycogen synthase kinase-3 (GSK-3) phosphorylation. STZ was found less cytotoxic in the differentiated cells compared to our previous results in undifferentiated SH-SY5Y cells. The cytoprotective concentration of insulin was similar in the STZ and LS groups. However, the right-shifted concentration–response curve of insulin induced GSK-3 phosphorylation in STZ-treated differentiated cells is suggestive of the development of insulin resistance that was further confirmed by the insulin potentiating effect of exendin-4. Differentiation reduced the sensitivity of SH-SY5Y cells for the non-specific cytotoxicity of STZ and enhanced the relative significance of development of insulin resistance. The differentiated cells thus serve as a better model for studying the role of insulin signaling in neuronal survival. However, direct cytotoxicity of STZ also contributes to the cell death.

     

Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.     

Testosterone and progesterone metabolism in the human neuroblastoma cell line SH-SY5Y

The ability of the human neuroblastoma cell line SH-SY5Y to metabolize androgens and progesterone was studied by incubating the cells in the presence of labeled testosterone (T) or progesterone (P) to measure, respectively, the formation of dihydrotestosterone (DHT) or dihydroprogesterone (DHP) (5-reductase activitiy). The 3-hydroxysteroid dehydrogenase activity was studied by evaluating the conversion of labeled DHT into 5-androstan-3, 17β-diol (3-diol). The results show that undifferentiated neuroblastoma cells posses a significant 5-reductase activity, as shown by the considerable conversion of T into DHT; moreover, this enzymatic activity seems to be significantly stimulated following cell differentiation induced by the phorbol ester TPA, but not after differentiation induced by retinoic acid (RA). The 5-reductase(s) present in SH-SY5Y cells is also able to convert P into DHP. In undifferentiated cells, this conversion was about 8 times higher than that of T into DHT. Under the influences of TPA and RA, the formation of DHP followed the same pattern observed for the formation of DHT. SH-SY5Y cells also appear to possess the enzyme 3-hydroxysteroid dehydrogenase, since they are able to convert DHT into 3-diol. This enzymatic activity is not altered following TPA-induced differentiation and appears to be decreased following treatment with RA. It is suggested that the SH-SY5Y cell line may represent a useful “in vitro” model for the study of the mechanisms involved in the control of androgen and P metabolism in nervous cells.     

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol,Retinoic Acid and Cholesterol

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E₂) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E₂ (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K⁺ depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E₂ treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells’ population growth by inducing maturation and differentiation.     

Translocation and phosphorylation of calcyclin binding protein during retinoic acid-induced neuronal differentiation of neuroblastoma SH-SY5Y cells

For better understanding of functions of the Calcyclin Binding Protein (CacyBP) and exploring its possible roles in neuronal differentiation, the subcellular localization of human CacyBP was examined in retinoic acid(RA)-induced and uninduced neuroblastoma SH-SY5Y cells. Immunostaining indicated that CacyBP was present in the cytoplasm of uninduced SH-SY5Y cells, in which the resting Ca(2+) concentration was relatively lower than that of RA-induced cells. After the RA induction, immunostaining was seen in both the nucleus and cytoplasm. In the RA-induced differentiated SH-SY5Y cells, CacyBP was phosphorylated on serine residue(s), while it existed in a dephosphorylated form in normal (uninduced) cells. Thus, the phosphorylation of CacyBP occurs when it is translocated to the nuclear region. The translocation of CacyBP during the RA-induced differentiation of SH-SY5Y cells suggested that this protein might play a role in neuronal differentiation.     

Differentiation Induces Dramatic Changes in miRNA Profile,Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.     

Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation

Human insulin-like growth factor I and II (IGF-I and IGF-II) in concentrations of 1-30 ng/ml, were shown to stimulate ornithine decarboxylase activity and [3H]thymidine incorporation in human SH-SY5Y neuroblastoma cells. Proliferation of these cells was also stimulated by IGF-I and II when added to RPMI 1640 medium, fortified with selenium, hydrocortisone, transferrin, and beta-estradiol. Labeled IGF-I and II bound to SH-SY5Y cells. The cross-reaction pattern of IGF-I, IGF-II, and insulin in competing with the binding of labeled IGF-I and IGF-II, respectively, indicated that SH-SY5Y cells express both type I and type II IGF receptors. Treatment of SH-SY5Y cells for 4 d with 12-O-tetradecanoylphorbol-13-acetate (TPA), which resulted in morphological and functional differentiation and growth inhibition, abolished the mitogenic response to both IGF-I and II. Concomitantly, the binding of IGF-II disappeared almost totally, which offers a possible explanation for the reduced biological response to IGF-II after TPA treatment. In contrast, the IGF-I binding in TPA-treated cells was only reduced to approximately 70% of the binding to control cells. It is therefore not excluded that the IGF-I receptor could be uncoupled by TPA, with persistent binding capacity for IGF-I.     

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.     

Activation of the kinase activity of ATM by retinoic acid is required for CREB-dependent differentiation of neuroblastoma cells

The ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation.     

A PKCbeta isoform mediates phorbol ester-induced activation of Erk1/2 and expression of neuronal differentiation genes in neuroblastoma cells.

Protein kinase C (PKC) activation induces neuronal differentiation of SH-SY5Y neuroblastoma cells. This study examines the role of PKCbeta isoforms in this process. The PKCbeta-specific inhibitor LY379196 had no effect on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced neurite outgrowth from SH-SY5Y neuroblastoma cells. On the other hand, PKCbeta inhibition suppressed the TPA-stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA-induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCbeta isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells.