Retinoic Acid-Induced Differentiation of Human Neuroblastoma SH-SY5Y Cells Is Associated with Changes in the Abundance of G Proteins

Abstract: Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH- SY5Y cells: Gaα, G_iα1, G_jα2, G_cα, G_zα, and Gβ. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 μmol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of μ-opioid binding sites, the levels of the inhibitory G proteins G_iα1 and G_jα1 were found to be significantly increased. This coordinate up-reg- ulation is accompanied by functional changes in μ-opioid receptor-stimulated Iow-K_m GTPase, μ-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5′-(βγ-imido)triphosphate [Gpp(NH)p; 10 nmol/ L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prosta- glandin E₁ (PGE₁) receptors and G_sα, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE₁ binding sites and PGE₁ stimulated adenylate cyclase activity, but significantly reduced amounts of G_zα were found. This down- regulation is paralleled by a decrease in the stimulatory activity of G_zα as assessed in S49 cyc- reconstitution assays. However, the reduction in G_aα levels had no effect on both intrinsic and receptor-independent-activated [Gpp(NH)p or forskolin; 100 μtmol/L each] adenylate cyclase, suggesting that the amount of G_zα is in excess over the functional capacity of adenylate cyclase in SH-SY5Y cell membranes. Additional quantitative changes were found for G_zα, G_cα, and Gβ subunits. In contrast, neuronal differentiation in the presence of 12-O-tetradecanoylphor- bol 13-acetate (16 nmol/L; 6 days) failed to affect G protein abundance. Our results provide evidence for a specific RA effect on the abundance of distinct G protein sub- units in human SH-SY5Y neuroblastoma cells. These alterations might contribute to functional changes in transmembrane signaling pathways associated with RA-in- duced neuronal differentiation of the cells.     

Retinoic acid induces gene expression of fibroblast growth factor-9 during induction of neuronal differentiation of mouse embryonal carcinoma P19 cells

We have found that the gene expression of the ninth member of the fibroblast growth factor (FGF) family, FGF9 was induced during retinoic acid(RA)-induced neuronal differentiation of murine embryonal carcinoma P19 cells. We have reported here the nucleotide sequence of the mouse FGF9 cDNA. The murine cDNA showed 92.4% nucleotide sequence homology to the human FGF9 cDNA and 98.2% homology to that of rats. This mouse FGF9 cDNA encoded a polypeptide consisting of 208 amino acids with amino acid sequence identical to that of rats. Only one amino acid was replaced compared to the human homolog. The highly conserved sequence homology of FGF9 suggests its functional importance. FGF9 was originally isolated from a culture medium of a human glioma cell line as a growth-promoting factor for glial cells [5]. Upon induction of neuronal differentiation by forming cell aggregates with 10⁻⁶ M RA, the gene expression of FGF9 was increased biphasically during the first 96 hours when cells were aggregating and from 168 hours to 192 hours followed by plating onto a tissue culture dish as glia-like cells proliferated. Neither undifferentiated P19 cells nor the cells aggregated without RA remaining undifferentiated expressed FGF9. This indicates that RA regulates the gene expression of FGF9 that may play an important role in neuronal differentiation in both early and late developmental process.     

神经钙粘着蛋白在P19神经元分化中的作用

利用ＲＴ－ＰＣＲ技术,我们检测Ｐ１９细胞体外神经元分化过程中神经钙粘着蛋白（Ｎ－ｃａｄｈｅｒｉｎ）的表达模式。结果显示,该基因在上述过程中存在上调和下调过程,与体内中枢神经系统发育过程的表达模式十分相近。在此基础上,我们将神经钙粘着蛋白基因ｃＤＮＡ全长转入Ｐ１９细胞,通过药物筛选,得到稳定表达钙粘着蛋白的细胞株。     

Increase of Adenylate Kinase Isozyme 1 Protein During Neuronal Differentiation in Mouse Embryonal Carcinoma P19 Cells and in Rat Brain Primary Cultured Cells

Abstract: Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.     

Suppression of PTEN expression during aggregation with retinoic acid in P19 mouse embryonal carcinoma cells

Apoptosis is thought to be involved in the maintenance of cellular homeostasis, as well as various pathological processes. However, little information is available about the regulation of apoptosis during the aggregation stage of P19 embryonal carcinoma (EC) cells. Here we report that aggregation-induced apoptosis is markedly attenuated by treatment with retinoic acid (RA). PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression was down-regulated during the aggregation phase of P19 EC cells in the presence, but not in the absence, of RA. Suppression of PTEN expression during the aggregation was accompanied by increased phosphorylation of serine/threonine kinase Akt and glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that RA attenuates the induction of apoptosis during the aggregation phase of P19 EC cells, probably by suppressing PTEN expression.     

Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.     

Glutathione Induces Neuronal Differentiation in Rat Bone Marrow Stromal Cells

It has been reported that rat bone marrow stromal cells (BMSCs) are differentiated into neuronal cells by administration of 2-mercaptoethanol [Woodbury et al (2000) J Neurosci Res 61:364–370]. In this study, we examined the effects of various sulfhydryl (SH) compounds on the differentiation of BMSCs obtained from rat femurs. Neuronal differentiation was detected morphologically and immunocytochemically. It was found that the cells treated with reduced glutathione (GSH) apparently differentiated into neurons, showing extensive processes, and expressing neuron-specific enolase and microtubule-associated protein 2. Glutathione monoethyl ester (GEE), which increased the cellular GSH content, showed no effect on the expression of neuronal markers. It is concluded that the neural differentiation of BMSCs occurs by the administration of GSH. It was suggested that extracellular and not intracellular GSH have effects on the induction of the neuronal differentiation of BMSCs.     

Changes in Arachidonic Acid Levels and Formation and in Lipid Synthesis in the Human Neuroblastoma SK-N-BE During Retinoic Acid-Induced Differentiation

Abstract: We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.     

过量表达Wnt-1基因诱导P19细胞的神经分化

Ｗｎｔ－１基因在小鼠神经发育过程中起着重要的作用。该基因在胚胎性癌细胞Ｐ１９细胞经分化过程中存在瞬时性表达。利用克隆到的Ｗｎｔ－１基因转染Ｐ１９细胞,可使细胞不经视黄酸诱导,自发向神经细胞方向分化。     

二甲基亚砜对P19细胞体外分化为心肌细胞的影响

体外应用不同浓度二甲基亚砜(dimethyl sulfoxide,DMSO)诱导P19细胞向心肌细胞分化,通过免疫细胞化学、流式细胞、RT-PCR方法检测α-横纹肌肌动蛋白(α-sarcomeric actin,α-SA)、心肌肌钙蛋白T(cardiac troponin T,cTnT)及GATA-4、α-肌球蛋白重链(α-myosin heavy chain,α-MHC)mRNA表达。结果显示,DMSO处理结合悬浮培养可诱导部分P19细胞分化为节律跳动的心肌细胞,分化的细胞α-SA、cTnT表达阳性,同时表达心肌特异GATA-4、α-MHCmRNA。1.0%DMSO组α-SA、cTnT阳性细胞免疫荧光强度及GATA-4、α-MHCmRNA表达水平明显高于0.5%及0.8%DMSO组。表明DMSO的诱导作用与其浓度有关。     

Okadaic Acid-Induced Apoptosis in Neuronal Cells: Evidence for an Abortive Mitotic Attempt

Abstract: There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimer's research to increase the degree of phosphorylation of various proteins, such as the microtubule-associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2-N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B₁ and cyclin D₁ markers of the cell cycle, and triggers a time-dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2-N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up-regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.     

The Role of Hypoxia in the Differentiation of P19 Embryonal Carcinoma Cells into Dopaminergic Neurons

Nervous system development at early stage is in hypoxic environment. Very little is known about the role of hypoxia in neuronal development. P19 embryonal carcinoma (EC) cells are a widely used model for studying early neuronal development. In this study we investigated the roles of hypoxia in differentiation of dopaminergic neurons derived from P19 EC cells. Results demonstrate that hypoxia increases the percentage of differentiated neurons, especially neurons of dopaminergic phenotype. To investigate the potential mechanism involved in hypoxia promoted differentiation of dopaminergic neurons, we measured the expression of hypoxia-inducible factor 1α (HIF-1α), based on its characteristic response to hypoxia. The result shows that HIF-1α mRNA level in P19 EC cells increases after hypoxia treatment. It is known that HIF-1α regulates the expression of tyrosine hydroxylase (TH) gene through binding to its promoter. Therefore, we propose that the underlying mechanism for hypoxia promoted differentiation of dopaminergic neurons was mediated by HIF-1α up-regulation under hypoxia. Yue Wang—Co-first author. Special Issue in honor of Dr. Ji-Sheng Han.     

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Galphaq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Galphai/o-protein coupled M2 receptor activity mediated neuronal differentiation.     

全反式维甲酸体外诱导P19细胞向心肌方向分化

目的探讨不同浓度全反式维甲酸(all-trans retinoic acid,atRA)诱导P19细胞向心肌分化的效力。方法细胞分成P19细胞组,2nm/L atRA诱导组,5nm/L atRA诱导组,8nm/L atRA诱导组。各组细胞经过诱导、聚集培养、聚集体贴壁培养10天后,RT-PCR检测GATA-4,α-肌球蛋白重链(α-myosin heavychain,α-MHC)的mRNA表达,免疫荧光双标检测α-sarcomeric actin和cTnT蛋白共表达,Western blot检测cTnT的蛋白表达。结果 atRA可诱导聚集P19细胞表达GA-TA-4、a-MHC mRNA;α-sarcomeric actin和cTnT的表达和共表达增加;5nm/L atRA组,8nm/L atRA组GATA-4、a-MHCmRNA的表达量显著高于P19细胞组;5nm/L atRA组,8nm/L atRA组两种蛋白的表达和共表达量显著高于P19细胞组,以5nm/L atRA组最高,显著高于其它组。结论 atRA可诱导聚集P19细胞向心肌分化,其中5nm/L atRA组效果最好。     

Complexes of gamma-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis.     

Promoter-specific function of the TATA element in undifferentiated P19 cells

P19 embryonal carcinoma cells differentiate into neuronal cells when treated with retinoic acid (RA). To explore the importance of core promoter structures in the regulation of gene expression during neuronal differentiation, the activities of three classes of modified or unmodified model promoters (Spec2a, OtxE, and Ars) were compared in P19 cells before and after RA treatment. The Spec2a promoter was activated in undifferentiated cells specifically when the E-box was located at a proximal position, whereas the OtxE promoter was activated when the E-box was in a distal position. The Ars promoter was only slightly activated by this element. In addition, the TATA element reduced the level of activation provided by the E-box, but only when it was located in the Spec2a core promoter. These results indicate that the core promoter structure may govern, at least in part, the stage-specific expression of endogenous genes involved in the neuronal differentiation of P19 cells.     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.